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Fluxes, compartmentation and metabolism of nitrate and ammonium in spruce roots Kronzucker, Herbert J. 1995

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F L U X E S , C O M P A R T M E N T A T I O N A N D M E T A B O L I S M O F N I T R A T E A N D A M M O N I U M I N S P R U C E R O O T S b y H E R B E R T J . K R O N Z U C K E R D i p l o m ( B i o l o g y and Chemis t ry ) , Un ive r s i t y o f W i i r z b u r g ( G e r m a n y ) , 1991 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department o f Botany) W e accept this thesis as con fo rming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A Augus t 1995 © Herber t J . K r o n z u c k e r , 1995 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT Techniques of compartmental analysis and kinetic flux analysis with the radiotracer 1 3 N were employed to examine the uptake, compartmentation, and metabolism of N03" and N H 4 + in roots of white spruce (Picea glauca (Moench) Voss.). Efflux analysis, conducted over 22-min periods, resolved the exchange of inorganic N with three subcellular compartments. These were (I) a root-surface film, (II) an adsorptive component of the cell wall, and (III) the root-cell cytoplasm. The identities of the compartments were tested using various approaches. Half-lives of exchange were (for NFL/) « 2 s, 20 s, and 7 min, respectively, and (for N03") 2 s, 30 s, and 14 min, respectively. Under the steady-state conditions assessed by efflux analysis, four to five-fold larger rates of uptake were observed for NIL/ than for N0 3 \ Steady-state N H 4 + influx ranged from 0.3 to 6.5 umol g 1 (FW) h 1 , while it was 0.08 to 1.2 ^mol g 1 h 1 for N 0 3 , at external concentrations from 0.01 to 1.5 mM of the two N sources. Efflux increased with increasing external concentrations of the N sources and ranged from 10% to 30% of influx for N H 4 + and from 1% to 20% for N03". Cytoplasmic concentrations were considerably higher for N H 4 + than for N03'; [NH 4 +] c y t was 2 to 30 mM, whereas [N0 3] c y t was 0.2 to 4 mM under the above conditions. A time-dependent compartmental-analysis study revealed that N03" uptake was inducible by external N03" and that three days were required for maximal uptake to be achieved at 100 uM [N0 3] 0. The dynamics of N03"-flux partitioning to different compartments during induction were characterized. Analysis of nitrate reductase activity under identical conditions confirmed the slow inductive time-scale. Kinetic analysis of influx under perturbation conditions revealed distinct uptake systems for both N species. At [N03"]0 < 1 mM, N03" influx was mediated by a constitutive and saturable high-affinity transport system (CHATS) and by a bisaturable inducible high-affinity transport system (IHATS). Beyond 1 mM, a linear low-affinity ii system (LATS) was evident. NEL,+ influx was not inducible by external NIL/. It was mediated by a constitutive and saturable high-affinity transporter (HATS) at [NH4+]0 < 1 mM, while a linear low-affinity transporter (LATS) operated beyond 1 mM. K,,, values for the initial phase of high-affinity transport were similar for both N species ( « 20 uM), but V,^ was up to 20 times larger for NH 4 + than for N03" when measured in perturbation. Overall, the study establishes a pronounced physiological limitation in spruce roots in transporting and utilizing N03" as compared to NFL/. iii TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables x List of Figures xiii Dedication xv Acknowledgments xvi Chapter 1. General Introduction 1 Chapter 2. Materials and Methods 6 2.1.1. Plant Material and Growth Conditions 6 2.1.2. Root Tissue Heterogeneity 7 2.2. Production of 1 3N Radiotracer 8 2.2.1. Production of 13N03- 8 2.2.2. Production of 1 3 NH/ 9 2.3. Efflux Analysis 11 2.3.1. General Procedure 11 2.3.2. Plant Pretreatments 12 iv 2.3.3. Treatment of Data and Calculation of Fluxes and Pool Sizes 13 2.3.4. Checks of Flux Estimates from Compartmental Analysis 15 2.4. Kinetic Influx Studies 16 2.4.1. General Procedure 16 2.4.2. Kinetic Analysis of Influx Data 18 2.5. Nitrate Reductase Assay 19 Chapter 3. N03"-Efflux Study I: Compartmentation and Flux 21 Characteristics of N03" in Spruce. 3.1. Introduction 21 3.2. Results 22 3.2.1. Phase Regression 22 3.2.2. Nitrate Fluxes 25 3.2.3. Compartmental Nitrate Concentrations 25 3.2.4. Compartment Identification 26 3.3. Discussion 36 3.3.1. Validity of Efflux Calculations 36 3.3.2. Nitrate in the Apparent Free Space 38 3.3.3. Nitrate in the Cytoplasm 41 v 3.3.4. Nitrate Fluxes 44 Chapter 4. N03"-EffTux Study II: Nitrate Induction in Spruce: An 45 Approach Using Compartmental Analysis. 4.1. Introduction 45 4.2. Results 46 4.2.1. Compartment Regression 46 4.2.2. Nitrate Fluxes 47 4.2.3. Compartmental Nitrate Concentrations 50 4.2.4. Root Nitrate Reductase and Unidirectional Flux to the Vacuole 53 4.3. Discussion 53 4.3.1. The Steady-state Assumption in Compartmental Analysis - Is it 53 Violated in the Present Study? 4.3.2. Agreement between Flux Values Estimated from 55 Compartmental Analysis and those Obtained by Independent Methods 4.3.3. Magnitude and Time Profile of Nitrate-induced Nitrate Uptake 56 in Spruce 4.3.4. The Role of Nitrate-flux Partitioning and Negative-feedback 60 Regulation during Induction vi Chapter 5. NH4+-Efflux Study I: Compartmentation and Flux 64 Characteristics of Ammonium in Spruce. * 5.1. Introduction 64 5.2. Results 65 5.2.1. Phase Regression 65 5.2.2. Ammonium Fluxes 67 5.2.3. Compartmental Ammonium Concentrations 68 5.3. Discussion 74 5.3.1. Half-lives of Exchange 74 5.3.2. Ammonium Fluxes 77 5.3.3. Compartmental Ammonium Concentrations 79 Chapter 6. NH4+-Efflux Study II: Analysis of "NH/ Efflux in Spruce 85 Roots: A Test Case for Compartment Identification in Compartmental Analysis. 6.1. Introduction 85 6.2. Results 87 6.2.1. Phase Regression and Half-lives of Exchange 87 6.2.2. Flux Estimations 87 6.2.3. Compartmental Concentrations 99 vii 6.3. Discussion 103 Chapter 7. N03"-Influx Study: Kinetics of Nitrate Influx in Spruce. Ill 7.1. Introduction 111 7.2. Results 113 7.2.1. Time Profile of Induction of Nitrate Influx by External Nitrate 113 7.2.2. Low-concentration Systems for Nitrate Uptake 113 7.2.3. High-concentration System for Nitrate Uptake 119 7.3. Discussion 125 7.3.1. Time Profile of Transporter Induction 125 7.3.2. Constitutive High-affinity Transport System 126 7.3.3. Inducible High-affinity Transport System 126 7.3.4. Low-affinity Transport System 129 Chapter 8. NH4+-Influx Study: Kinetics of Ammonium Influx in Spruce. 131 8.1. Introduction 131 8.2. Results 132 8.2.1. Time Profile of Ammonium Influx 132 8.2.2. Concentration Dependence of Ammonium Influx 134 8.3. Discussion 140 viii 8.3.1. Enhanced Ammonium Influx in N-deprived Plants 140 8.3.2. Steady-state Ammonium Influx 141 8.3.3. Is Ammonium Uptake Inducible by External Ammonium? 142 8.3.4. Concentration Dependence of Ammonium Influx (Transport 143 Systems) 8.3.5. Preference for Ammonium over Nitrate in Spruce 146 Chapter 9. Conclusion and Outlook 148 References 151 Appendix I Determination of Unidirectional Influx into Roots of Higher 170 Plants: A Mathematical Approach Using Parameters from Efflux Analysis. Appendix II A New Method for the Determination of Ion Exchange 175 Parameters in Multicompartmental Plant Systems. ix LIST OF TABLES Table 1 t% values for N03' at different [N03]0 24 Table 2a N03" fluxes at different [N03"]0 28 Table 2b N03" fluxes at 100 [N03]0 (± 75°C or H202) 29 Table 2c N03' fluxes at 1.5. mM [N03]0 (+ 2-chloro-ethanol) 30 Table 2d N03 fluxes at 100 uM [N03]0 with vaying [Pi]0 31 Table 3a Compartmental N03" concentrations at different [N03~]0 32 Table 3b Compartmental N03- concentrations at 100 uM [N03]0 (± 75°C or 33 H202) Table 3c Compartmental N03" concentrations at 1.5 mM [N03"]0 (+ 2-chloro- 34 ethanol) Table 3d Compartmental N03" concentrations at 100 uM [N03~]0 with varying 35 [Pi]0 Table 4 tlA values for N03" at different stages of induction 48 Table 5 N03" fluxes at different stages of induction 49 Table 6 Compartmental N03" concentrations at different stages of induction 51 Table 7 Different methods of N03-flux measurement at 100 fiM [N03"]0 57 Table 8 t,A values for NH 4 + at different [NH4+]0 69 x Table 9 N H 4 + fluxes at different [NH 4 +] 0 70 Table 10 Different methods of NH4+-flux measurement 72 Table 11 Compartmental N H 4 + concentrations at different [NH 4 +] 0 73 Table 12a t,A values for N H 4 + at 100 uM [NH 4 +]C following various 89 pretreatments Table 12b tly4 values for N H 4 + at 100 [NH 4 +]„ with varying [Ca 2 +] 0 90 Table 12c t,A values for N H 4 + at 1.5 mM [NH 4 +] 0 following various 91 pretreatments Table 13a N H 4 + fluxes at 100 uM [NH 4 +]„ following various pretreatments 92 Table 13b N H 4 + fluxes at 100 uM [NH 4 +] 0 with varying [Ca 2 +] 0 95 Table 13c N H 4 + fluxes at 1.5 mM [NH 4 +] 0 following various pretreatments 97 Table 14a Compartmental N H 4 + concentrations at 100 uM tNH 4 +] 0 following 100 various pretreatments Table 14b Compartmental N H 4 + concentrations at 100 uM [NH 4 +]Q with 101 varying [Ca 2 +] 0 Table 14c Compartmental N H 4 + concentrations at 1.5 mM [NH 4 +]D following 102 various pretreatments Table 15 Michaelis-Menten parameters for NCV influx (CHATS) 118 xi Table 16 Michaelis-Menten parameters for N03" influx (component I of 122 IHATS) Table 17 Michaelis-Menten parameters for N03" influx (component II of 123 IHATS) Table 18 Michaelis-Menten parameters for NH 4 + influx (HATS) 136 xii LIST OF FIGURES Fig. 1 Representative efflux plot for N03" at 100 uM [N03"]0 23 Fig. 2 Concentration dependence of net flux, influx, and efflux for N03~ 27 Fig. 3 Overlaid N03-efflux plots at 100 uM [N0 3] o ( ± H20_) 39 Fig. 4 Overlaid N03"-efflux plots at 100 uM [N03 ]0 (uninduced vs. induced) 52 Fig. 5 Derivation of N03" flux to the vacuole from NRA and 4>ttai^ 54 Fig. 6 Representative efflux plot for N H 4 + at 100 uM [NH 4 +]C 66 Fig. 7 Concentration dependence of net flux, influx, and efflux for NIL,"1" 71 Fig. 8 Juxtaposition of cytoplasmic accumulation of N03" and N H 4 + 84 Fig. 9 Representative efflux plot for N H 4 + at 1.5 mM [NH 4 +] 0 88 Fig. 10 Overlaid NH4+-efflux plots at 100 uM [NTi,+]0 ( ± ce-KG) 94 Fig. 11 Overlaid NH4+-efflux plots for phase II (50 uM vs. 5 mM [Ca2+]c) 96 Fig. 12 Overlaid NH4+-efflux plots at 1.5 mM [NH 4 +] 0 (+ MSO) 98 Fig. 13 Time dependence of N0 3' influx at 200 uM [N0 3] 0 (0-72 h) 114 Fig. 14 Time dependence of N0 3' influx at 200 /xM [N0 3] o (0-7 d) 115 Fig. 15 Time dependence of N03" influx at 1.5 mM [N03"]0 (0-7 d) 116 Fig. 16A Concentration dependence of N03" influx (CHATS) 117 Fig. 16B Concentration dependence of N0 3 _ influx (IHATS) 120 xiii Fig. 17 Eadie-Hofstee transformation of data in Fig. 16B 121 Fig. 18 Concentration dependence of N03" influx (LATS) 124 Fig. 19 Time dependence of NH 4 + influx at 100 uM [NH4+]0 (0-7 d) 133 Fig. 20 Concentration dependence of NH 4 + influx (HATS) 135 Fig. 21 Concentration dependence of NH 4 + influx (LATS) 137 Fig. 22 Overlaid NH4+-influx plots (+ N03" pretreatment) 138 Fig. 23 Hanes-Wolf transformation of data in Fig. 22 139 Fig. 24A Representative semi-logarithmic plot of count accumulation (from 176 13N03") in plant tissue versus time of exposure to isotope Fig. 24B as Fig. 24A (+ additional Y-axes drawn for potential compartmental 178 analysis of the three count-accumulation phases) xiv THIS THESIS IS DEDICATED IN DEEP APPRECIATION TO M Y TEACHER ULRICH HEBER ON THE OCCASION OF HIS 65TH YEAR ON T H E PLANET xv ACKNOWLEDGMENTS First and foremost I wish to express my sincere gratitude to Tony Glass, "The Big One", supervisor, colleague and friend. Tony's genuine and almost child-like fascination for science has made graduate life a fun experience. Tony never tried to force me into anything yet was always around for support when it was needed. It is this attitude and the atmosphere created by it which made this work possible. On equal footing, my thanks go to my (unofficial) cosupervisor Yaeesh Siddiqi, whose scientific brilliance, wisdom and humanity are a constant source of inspiration. Yaeesh is a wonderful teacher and I consider myself lucky to also call him a friend. My gratitude further extends to the members of my advisory committee, Drs. Edith Camm, Rob Guy, Paul J. Harrison, and Peter Jolliffe, for their advice and their patience during lengthy presentations at progress meetings. Thanks to Rob also for supplying plants and for many good discussions. Special thanks go to John Worrall, the best teacher and one of the most illustrious personalities I know, for giving me the opportunity to teach trees. Ion-transport work using the radiotracer 1 3N is nerve-racking and always requires teamwork. Many thanks to the members (occasional or constant) of the "fire brigade": J. Bailey, D. Britto, J. Mehroke, P. Poon, B. Touraine, JJ. Vidmar, M.Y. Wang, and D. Zhuo. In this connection, my thanks also go to the team at TRIUMF, UBC, who provided the tracer on a (more or less) regular basis. For support and many good times outside the science realm I wish to thank my Canadian friends, especially Blair, Don & Chris, Jennifer, Jimmy, Jody, John W., Kameron, Kathy and Wayne, Michael and 'the gang', my pals from the 'swim team' and the Vancouver Symphony. Thanks go also to my family and friends back home in Germany. xvi I further owe my gratitude to numerous (mostly European and long-deceased) musical composers, to my guitars and my violin Alfredo. Together, they have made graduate life more tolerable (which, when I come to think of it, wasn't so bad). Sincere (®) appreciation is expressed to the 1,352 seedlings of white spruce who have lost their lives in the course of the author's ridiculous scientific attempts. Lastly, I need to express a special thanks to Prof. Ulrich Heber at Wiirzburg, who dragged me into this plant science stuff in the first place and who, despite my presently expatriated existence, has never lost faith in me. It is to him, his inspiration and guidance, that I wish to dedicate this thesis. xvii 1. G E N E R A L I N T R O D U C T I O N Inorganic nitrogen is available to plants in soil solution either as N03~ or, in the reduced form, as NH 4 +. Well-aerated agricultural or ruderal soils are generally rich in N03" and poor in NH 4 +, while the reverse is true for acidic, cold, dry or waterlogged soils (Smith et al., 1968; Middleton and Smith, 1979; Keeney, 1980; Tills and Alloway, 1981; Stams and Marnette, 1990). These differences are mostly attributable to the activity of nitrifying bacteria in the soil, which is optimal at neutral pH, moderate soil temperatures, and sufficient supply of oxygen (Likens et al., 1969; see also Larcher, 1984, and Vitousek et al., 1989, for references). Despite the preponderance of N03" in agricultural soils, there appears to be an equal ability of most agricultural species to grow on either N source if acidification of the root medium is prevented (Barker et al., 1966; Rufty et al., 1983; Tolley-Henry and Raper, 1986, 1989; Findenegg, 1987; Vessey et al., 1990). Occasionally, even a slight preference for NH 4 + is observed (Goyal and Huffaker, 1986b; Macduff and Jackson, 1991; Botelia et al., 1994). This is in keeping with the assumption that, due to a lesser energy requirement for assimilating NIL,"1" than N03", NH 4 + should be preferred by plants (Reisenauer, 1978; Mengel and Viro, 1978; Middleton and Smith, 1979; Smirnoff and Stewart, 1985; Bloom, 1988). However, if the pH of the root medium is not maintained, proton extrusion associated with NIL/ uptake leads to a pronounced acidification of the medium (Kirkby and Mengel, 1971; Runge, 1983; Peet et al., 1985) and, as a consequence, plant growth is often reduced. Moreover, plants exhibit a typical complex of symptoms, which has been described as the 'ammonium syndrome' (Mehrer and Mohr, 1989; Chaillou et al., 1991). Firstly, lower tissue levels of inorganic cations and organic anions are usually seen in NFL^ -grown compared to N03"-grown plants (Israel and Jackson, 1982; Troelstra et al., 1985; Allen and Smith, 1986; Chaillou et al., 1986; Allen et al., 1988). In addition, 1 increased concentrations of amino acids in roots and shoots as well as of free sugars and starch in shoots have been recorded under NH 4 + nutrition (Magalhaes and Wilcox, 1984; Rosnitschek-Schimmel, 1985; Chaillou et al., 1986, 1991). In certain cases, some of these characteristic physiological changes have also been noted when pH of the growth medium was maintained at 6 to 7 (Blacquiere et al., 1987, 1988; Van Beusichem et al., 1988). Under prolonged application of NIL,"1" and/or at higher external concentrations, a disruption of processes such as photophosphorylation, C02 fixation (Puritch and Barker, 1967; Ikeda and Yamada, 1981), starch synthesis (Matsumoto et al., 1971) and NADP+ reduction (Vernon and Zang, 1960) have been documented, and often severe reductions of growth, followed in some cases by death, are known to result from NIL,"1" nutrition (Magalhaes and Wilcox 1983a, b, c, 1984a, b). For this reason, the term 'ammonium toxicity' has been applied frequently in the literature (Givan, 1979). Certain groups of plants, however, do not appear to suffer the effects of 'ammonium toxicity' under normal circumstances. These include rice (Sasakawa and Yamamoto, 1978; Wang et al., 1993a), several species in the Ericaceae (Ingestad, 1973; Spiers, 1978; Peterson et al., 1988) as well as many conifer species (Krajina et al., 1973; Bigg and Daniel, 1978; Boxman and Roelofs, 1988; Scheromm and Plassard, 1988; Marschner et al., 1991; Lavoie et al., 1992). All of these plants occur naturally on soils where nitrification is almost completely inhibited (cf. Runge, 1983, and Attiwell and Adams, 1993), either due to lack of oxygen, as in rice paddies, or by acidic and/or cold soil conditions, as in many heath lands and coniferous forests (Larcher, 1984). A role of allelopathic chemicals in repressing nitrification has also been documented (Rice and Pancholy, 1972; Baldwin et al., 1983; Turner and Franz, 1985; White, 1986). As a consequence, inorganic N is predominantly available as NH 4 + (Smith et al., 1968; Lodhi, 1978; Klingensmith and Van Cleve, 1993). From an evolutionary perspective, it can therefore be expected that these plant species should be adapted to utilizing reduced N more efficiently than other species, which do not naturally occur on NIL/ soils, and that the ability to successfully deal with even relatively high external NH 4 + concentrations would have been positively selected (Vogt and Edmonds, 1982). While, in the course of this evolutionary adaptation to NIL,"1", the ability to efficiently utilize N03" as an N source has been retained in rice (Malavolta, 1954), ericaceous plants and many conifers appear to have largely lost this ability. In conifers, significantly diminished growth on N03~ as opposed to NH 4 + has been reported by numerous workers (Swan, 1960, van den Driessche, 1971; Etter, 1972; Ingestad, 1973; van den Driessche and Dangerfield, 1975; Ingestad, 1979; Smirnoff and Stewart, 1985; Chapin et al., 1986; Gijsman, 1990). In the case of conifers, this specialized adaptation to NH4+ and the concomitant discrimination against N03~ may have important ecological consequences as well as practical implications. It is known that in most forest soils the availability of the NH 4 + and N03" sources of N can change markedly with the stage of successional development of the forest stand (Smirnoff et al., 1984; Finlay et al., 1998; Donaldson and Henderson, 1990). While NH 4 + is the predominant N form in later successional or climax stands in boreal, sub-boreal, montane or subalpine environments (see earlier references), the scenario changes after forest disturbance, such as fire, landslides, windthrow, insect calamities or clearcut harvesting (cf. Oliver and Larson, 1990). In the wake of such events, soil pH generally rises and a new microbial environment appears, which tends to convert the soil N pool from its predominantly reduced form to mostly N03" (Smith et al., 1968; Likens et al., 1969; Rice and Pancholy, 1972; Lodhi, 1978; Vitousek and Melillo, 1979; Tilman, 1987; Jobidon et al., 1989a, b). As a consequence of these altered soil conditions, later successional conifer species which may have previously inhabited the site become poor competitors for inorganic N, and the site becomes dominated by 'nitrogenous' early successional species whose uptake capacity for N03" is superior to that of the 3 conifers (Smirnoff et al., 1984; Tilman, 1987; Jobidon et al., 1989a, b; Lavoie et al., 1992). Possible limitations in the nitrate uptake system of conifers in the seedling stage may therefore be crucial to the success of reforestation trials. Given the substantial reforestation failure rates in parts of the U.S. and Canada after clearcut harvesting (in the interior of British Columbia alone, more than lxlO 6 hectares of forest land are presently considered 'unsatisfactorily restocked'; B.C. Ministry of Forests Annual Report, 1991), this would appear to be an important aspect for scientific study. Nevertheless, detailed studies of ammonium and nitrate uptake have been carried out almost exclusively in angiosperm and microalgal species (see reviews by: Clarkson, 1986; Glass, 1988; Glass and Siddiqi, 1995), while the physiology of ammonium and nitrate uptake in conifers has been virtually ignored. Only limited information on fluxes and no literature data for cytoplasmic ammonium and nitrate concentrations is available for conifers. Several studies have been undertaken using either depletion methods or the stable isotopic tracer 1 5 N (Rygiewicz et al., 1984; Rygiewicz and Bledsoe, 1986; Boxman and Roelofs, 1988; Scheromm and Plassard, 1988; Marschner et al., 1991; Peuke and Tischner, 1991; Flaig and Mohr, 1992; Lavoie et al., 1992; Kamminga-van Wijk and Prins, 1993; Knoepp et al., 1993). Generally, three to four times higher uptake rates were reported for NH4+ than for N03". Interestingly, this preference for N H 4 + of conifer seedlings seems to also apply to their natural ectomycorrhizal partners (Bowen and Smith, 1981; Alexander, 1983; France and Reid, 1983; Littke et al., 1984; Rygiewicz et al., 1984a, b; Lang and Jagnow, 1986; Plassard et al., 1994). The approaches used by other workers, however, have been limited to long-term estimations of uptake. They failed to distinguish influx and efflux and did not take into account biological rhythms, and longer-term acclimation processes. They further did not provide information on the time profiles of ammonium and nitrate uptake or on exchange kinetics across 4 the root cell plasmalemma, the intracellular compartmentation of ammonium and nitrate, or the magnitude of cytoplasmic accumulation of these ions. Yet it is clear that if the physiological plasticity of conifer seedlings with regard to the two N-forms and possible stress physiological consequences are to be elucidated, research into these aspects of conifer seedling physiology is essential. Using white-spruce seedlings as a model for late successional conifers, it was the purpose of this study to provide necessary physiological information in these much-neglected areas. 5 2. MATERIALS AND METHODS 2.1.1. Plant Material and Growth Conditions. Seedlings of white spruce (Picea glauca (Moench) Voss., BC Ministry of Forests (BCMofF) seed lot 29170, from the Prince George region, British Columbia, Canada) were grown in a peat/perlite (3:1) mixture in styrofoam boxes in an outdoor nursery for a minimum of 3V2 months. Seedlings were then transferred indoors and their roots gently washed under running tap water. They were then placed in 24-dm3 Plexiglas hydroponic tanks containing well mixed and aerated 1/10 strength modified N-free Johnson's solution, the composition of which was (in mM): K H 2 P 0 4 0.2, K 2 S0 4 0.2, MgS0 4 0.1, CaS0 4 0.05, micronutrients and Fe-EDTA (in uM): Cl 5, B 2.5, Mn 0.2, Zn 0.2, Cu 0.05, Fe 2; supply of N 0 3 (as Ca(N03)2) or N H 4 + (as (NH4)2S04) was as indicated in the text. To adjust seedlings to hydroponic culture, minimize post-transfer shock effects and allow for steady-state growth to be established, while keeping the likelihood of microbial and algal contamination small, three weeks preconditioning in hydroponics was found to be ideal. As confirmed by microscopic examination, seedling roots were non-mycorrhizal. Al l seedlings were maintained in a 16h/8h photoperiod, 70% RH, and at 20 ± 2°C. A photon flux of approximately 250 jiimol m"2 s"1 measured at plant level (with a LI-189 light meter and LI-190SA quantum sensor from LICOR, Lincoln, NE, USA) was provided by fluorescent lamps (VITA-LITE/DURO-TEST, U.S. Patent 3,670,193). Solutions were replaced every 3 d and were checked daily for [K +] (using an Instrumentation Laboratory model 443 flame photometer, Lexington, M A , USA) and for pH using a microprocessor-based pocket-size pH meter (pH Testr2 model 59000-20, Cole Parmer, Chicago, IL, USA). [N0 3 ] 0 was measured according to Cawse (1967) and [NH 4 + ] Q was measured according to Solorzano (1969) (using a Philips PU 8820 UV/VIS spectrophotometer). To buffer against pH changes caused by plant-uptake processes, powdered CaC03 was added to the tanks (pH was kept at 6.5 ± 0.3). Typically plants were maintained under steady-state conditions with respect to the composition of the nutrient solutions by regular replenishment of all nutrients in accordance with the depletion of K + . Plants were also maintained under steady-state conditions during the pretreatments immediately prior to loading of tracer and during elution of labelled roots for the compartmental-analysis studies. Experiments to determine influx isotherms required that plant roots be exposed to different concentrations of N03" and NH 4 +, and therefore pretreatment and desorption solutions contained the same N concentrations as was provided in the uptake solution. 2.1.2. Root Tissue Heterogeneity. The root system in the several-months-old spruce seedlings was visibly heterogeneous. Numerous new laterals emerged during the three-week cultivation period in hydroponic tanks (referred to in the text as 'young' roots), which were noticeably different from the root system already existing at the time of transfer. These newly-formed roots attained, on average, lengths of 3 to 5 cm and were exclusively of the 'white-zone' type (Sutton and Tinus, 1983; McKenzie and Peterson, 1995a), i.e. they showed no macroscopic signs of root browning due to either tannin formation or periderm initiation. This white-zone part of the conifer seedling root system is known to largely resemble young roots of herbaceous plants in terms of structure and permeability characteristics (Rudinger et al., 1994; McKenzie and Peterson, 1995a). By contrast, the part of the root system which was already established at the time of transfer to hydroponics was brown in colour (average length: 8-12 cm; referred to in the text as 'old' roots). It corresponded to the 'tannin zone' and the early-periderm region (McKenzie and Peterson, 1995a, 7 b). Deposition of brown-coloured 'tannins' has been causally linked to cortical cell senescence (Sutton and Tinus, 1983), and changes in water and solute permeability characteristics (Kramer, 1969; Rudinger et al., 1994). In addition, a decline in specific ion uptake capacity has been observed (Kamula and Peterson, 1994). Similarly, incipient suberin incrustation in the early-periderm region is expected to reduce ion uptake rates (see section on influx determinations). 2.2. Preparation of 1 3 N radiotracer. 2.2.1. Production of I 3N0 3\ Nitrogen-13 (t1/4 = 9.96 min) was produced by proton irradiation of H20 at the TRIUMF cyclotron on the University of British Columbia UBC campus in Vancouver, Canada, according to the nuclear reaction: 160 + 'H -» 1 3N + 4He (Meeks 1993); A 15-cm3 target volume was loaded remotely and a pressure of 3 atm was applied during irradiation. Irradiation was for 10-15 min with a 20-MeV proton beam and a beam current of 10 fiA. The product of this reaction is principally 13N03". The activity achieved was commonly around 700-750 MBq and the radiochemical purity for ,3N03" > 90%. The target solution was transferred into a 20-cm3 vial and transported in an underground pipeline with transit times of 2-3 min from the particle acceleration facility to the UBC hospital. The time taken from pick-up at the hospital to beginning of the chemical purification processes in the laboratory at the UBC Botany Department was commonly 5-10 min. To remove contaminants, two procedures were used. Both procedures were carried out in a lead-shielded fumehood. Procedure I was as 8 described by Siddiqi et al. (1990). Procedure II was as follows: The irradiated solution was taken up by a 20-cm3 syringe and passed twice through a SEP-PAC Alumina-N cartridge (Waters Associates) to remove the main radiocontaminant 1 8F. The cartridge was rinsed with 5 cm3 of 2.5 uM Ca(N03)2 to remove residual radioactivity. Then the solution was transferred to a 50 cm3 beaker, made strongly alkaline by addition of 100 mm3 of 0.2 M KOH and boiled for 2-3 min to volatilize 13N-ammonia. Then 200 mm3 of 1.2 M H2S04 and 1 cm3 of 10% v/v H202 were added to create an oxidizing and strongly acidic environment, and the solution was boiled for another 2-3 min. This was to oxidize and decompose any residual 13N-nitrite and other nitrous compounds and drive them off as NOx-gases. The solution was then cooled in an ice bath to room temperature, the pH adjusted to 6.5 to 7 with approximately 340 mm3 of 0.2 M KOH (checked with pH paper), and 2 cm3 catalase (2 mg cm3) were added to decompose remaining H202. While about 2 min were given for this reaction to complete, the radioactive solution was transported into a growth room where the experiment was to be carried out and mixed into a prepared solution of the desired composition on a stir-plate behind lead. Thus, approximately 20 min after irradiation of the target, labelled solution was ready to be administered to the experimental plants. To obtain high specific activities, usually no more than 400 cm3 of loading solution were prepared per experimental run. 2.2.2. Production of 1 3 NH 4 + . Since the irradiation procedure used at the UBC cyclotron generated primarily 13N03" (see above), a chemical conversion was necessary to obtain 1 3NH 4 +. The chemical purification and conversion procedure was carried out in a lead-shielded fumehood at the UBC Botany Department and was as follows (in modification of Vaalburg et al. 1975, Meeks et al. 1978, and 9 Wang et al. 1993a): Approximately 15 cm3 of irradiated target solution (see above) were taken up by a 20-cm3 syringe and passed twice through a SEP-PAC Alumina-N cartridge (Waters Associates) to remove the radiocontaminant 1 8F. To remove residual tracer, 5 cm3 of 2.5 uM Ca(N03)2 were used to rinse the cartridge. The sample was then introduced into a 500 cm3 round-bottom flask containing approximately 10 g of Devarda's alloy (50% Cu, 45% Al, 5% Zn). The flask was connected to two 100-cm3 Erlenmeyer flasks arranged in series and containing a total of 100-cm3 'trapping solution' (same composition as in labelling solution) acidified by addition of 1 cm3 1 N HC1. Reduction of 13N03" to 1 3 NH 4 + was then initiated by transferring the round-bottom flask to a 70°C water-bath and adding 20 cm3 of 1 N NaOH, according to the following reaction (shown is the reaction of a putative Na-salt of 13N03" with the Devarda's alloy component Zn; similar hydroxo-complex formation and concomitant , 3NH 3-generation occurs with Cu and Al under alkaline conditions): Na13N03 + 4 Zn + 3 NaOH + 6 H20 ^  4 Na[Zn(OH)3] + 1 3NH 3t; Gaseous 1 3NH 3 was separated from remaining chemical species by steam distillation on the water-bath, and ionic 1 3 NH 4 + was trapped in the acidic solution contained in the Erlenmeyer flasks. These were connected to an aspirator in order to facilitate directional gas flow of 1 3NH 3 into the trapping solution. Under these conditions, most of the 1 3N contained in the original sample solution was transferred to the trapping solution in less than 5 min. The efficiency of transfer was checked by means of a Geiger-Miiller counter. The pH of the "NIL/-labelled trapping solution was then neutralized by adding 1 cm3 of 1 N NaOH and added to the actual loading solution (composition of the latter as described above for growth solutions). 10 2.3. Efflux Analysis. 2.3.1. General Procedure. Roots of intact seedlings were equilibrated in non-labelled preloading solution for 5 min before transfer to the 13N-loading solution. Roots were next immersed in 70 to 450 cm3 of "re-labelled loading solution for 35 min (for N03~) to 60 min (for NH/) to bring the cytoplasmic phase to a specific 1 3N activity close to that of the loading solution. Then seedlings were held upright to drain excess surface label, and the roots eluted successively with 60- to 100-cm3 aliquots (volume depending on root mass) for varying time periods. With t=0 as the time of transfer from loading to 'washing' solution and tfmi_=22 min for the last elution, the time periods for the 25 successive 'washes' were: 5 s (2x), 10 s (2x), 15 s (6x), 30 s (4x), 1 min (4x), 2 min (7x). During elution, seedlings were affixed to 1-dm3 plastic funnels, the spouts of which had 5-cm lengths of silicone tubing attached to them; metal spring clips on the tubing served as manually operated drainage valves. Aeration and mixing were provided by glass pipettes taped to the insides of the funnels and connected to valve-controlled air lines. The 60- to 100-cm3 elution aliquots were poured into the funnels at the prescribed times, collected in 100-cm3 plastic beakers, from which 20-cm3 subsamples were pipetted into 20-cm3 scintillation vials. These were then counted in a Packard gamma-counter (Minaxi 5, Auto-7 5000 Series). Roots were excised from the shoots immediately after the final elution, and the roots spun for 45 s to remove excess surface-bound solution. Both plant organs were weighed, introduced into scintillation vials, and counted for 7-activity. In experiments designed to check the charge of the 13N-label eluted from plants, 100 cm3 eluates were collected at 10, 20, and 30 min after plant transfer into non-labelled solution. 20-cm3 subsamples of these eluates were then passed through anion and cation exchange resins 11 (BIO-RAD analytical grade AG 1-X 8 anion exchange resin, 200-400 mesh, acetate form, and BIO-RAD analytical grade AG 50 W-X 8 cation exchange resin, 200-400 mesh, Na+ form), and the proportion of 1 3N going into each fraction was determined by counting the radioactivity of the resins as well as that of the resin filtrates. 2.3.2. Plant Pretreatments. Plants were maintained under steady-state conditions with regard to all environmental parameters, including nutrient concentrations throughout experiments (i.e. throughout growth, pretreatment, prelabelling, labelling and elution; see earlier description of growth conditions). N03" was added as Ca(N03)2 either only during labelling and elution (at 10 or 100 uM, for study of uninduced plants) or else 1, 2, 3, 4 or 5 d prior to (and during) the efflux experiment (at 10 uM, 100 uM, or 1.5 mM, for study of induced plants; normally a 3-d pretreatment was used); NH 4 + was added as (NH4)2S04, usually 4 d prior to (and during) efflux experiments, except in the case of N-deprived plants, where NIL,+ was withheld from growth and pretreatment solutions and only added (at 10 or 100 uM) during labelling and elution to make the monitoring of fluxes and the estimations of compartmental concentrations possible. Even in putative '0 uM' solutions, however, trace amounts of NIL,"1" (< 2 uM) could always be detected by chemical analysis; this was probably due to a chemical equilibration reaction of nutrient solutions with NH3 gas contained in the ambient air. Experiments where growth of plants was on '0 /xM' NH,"1" or N03" and the respective N source was added only for labelling and elution (at 10 or 100 (JM) are referred to in the text as '0-10' or '0-100'. In experiments conducted to test the identity of the compartments, perturbation designs were used for technical reasons. In sodium-dodecyl-sulphate (SDS)-experiments, plant roots were 12 pretreated for 30 min, loaded and eluted in the presence of 1% SDS. In high-temperature experiments, plants were pretreated for 20 min at 75°C. In H202 treatments, H202 (< 0.07% v/v) was present only in the loading solution (from incomplete catalase reactions - see above). In 2-chloro-ethanol experiments, seedling shoots were enclosed in a 4-dm3 air-tight chamber and were exposed to the gas (at 0.075% v/v) for 4 d prior to the efflux experiment (the air-gas mixture was replaced twice daily). In experiments using a-keto-glutarate (a-KG) and L-methionine-DL-sulphoximine (MSO), roots were pretreated with 1 mM a-KG or MSO for 6 h prior to the experiments; a-KG and MSO were also provided during loading and elution. In cation variation experiments, [H+]c was increased by lowering pH to 3.6 with H2S04, [Al3+]c was altered by adding 750 uM A12(S04)3 (at pH 3.6), and [Ca2+]0 was modified with CaS04 (50 juM, 500 jttM, and 1.5 mM). Roots were pretreated for 30 min with the respective cations added to the solutions and were also exposed during loading and elution. 2.3.3. Treatment of Data and Calculation of Fluxes and Pool Sizes. Treatment of data was as outlined by Siddiqi et al. (1991), and essentially followed the theoretical considerations of Lee and Clarkson (1986). All experiments were performed using two replicates and were repeated at least three times. Representative experiments were chosen for semi-logarithmic plots of the rate of 13N-release versus elution time, while all other data shown in tables and graphs represent means of several experiments ± standard error (SE). Since N03" and NIL,"1" are metabolized ions, the commonly employed procedure in efflux analyses of non-metabolized ions, where the logarithm of radioactivity remaining in the plant tissue is plotted versus time of elution (Walker and Pitman, 1976), is not applicable. However, assuming that efflux was constant during the steady-state conditions of the experiment and that 13 specific activity in the plant compartments during elution was declining exponentially, the logarithm of the rate of release of radioactivity from the plant tissue can be plotted versus elution time (Lee and Clarkson, 1986). Linear regression on the semi-logarithmic plots was then used to resolve separate phases. The slopes of the regression lines yielded, after conversion from decadic to natural logarithm (i.e. division by 2.303), kinetic exchange constants (k) for the respective phases, which could be expressed as half-lives of exchange (t1/4 = 0.693/k). The intercept with the ordinate of the regression line for the presumed cytoplasmic phase (i.e. the rate of 1 3N release from the cytoplasm at time zero, R0) divided by s0, the specific activity of the loading solution, yielded the rate of efflux from the cytoplasm 4>co (divided, as all other fluxes, by root fresh weight), while net flux <£net was obtained directly from the accumulation of 1 3N in the plants. Influx <!>„. was calculated as = 0net + #co. Flux to the xylem was obtained from count accumulation in the shoot, and the combined fluxes to reduction (for N03~) or assimilation (for NH4+) and to the vacuole (0^^ and 0ass/vac, respectively) resulted from <£net - (p^^. Because the identity of the chemical species (N03", NH 4 +, amino acids?) translocated to the xylem is unknown in the present studies, it is not possible to calculate the chemical flux for the shoot translocation term, x^yiem therefore represents isotopic flux only; it was, however, calculated using [N03~]o or [NH4+]D as the specific term. Thus, its value is for the derivative calculation of other flux terms (see above) and as an indicator of the relative proportion of 1 3N translocated to the shoot. All fluxes were expressed in nmol (or (imol) g"1 (FW) h"1. In summary, symbols used for fluxes are as follows: 0CO = efflux from the cytoplasm, obtained from the rate of 1 3N release from the cytoplasm at time zero divided by the specific activity of the loading solution; <pnet = net flux, obtained directly from the accumulation of 1 3N in the plants at the end 14 of the elution period; 4>x = unidirectional influx, calculated from <£net + 0CO; ^ x y k m = Aux of 1 3N to the shoot, obtained directly from count accumulation in the shoot at the end of the elution period; 0ass/vac = combined fluxes of ML,"*" to assimilation and to the vacuole, resulting from 0net " x^ylemJ r^eovvac = combined fluxes of N03" to reduction and to the vacuole, resulting from <£net -Cytoplasmic concentrations of N03" or NH 4 + ([N03"]cyt and [NH4+]cyt) were calculated from the quotient of the integrated rate of 1 3N release during five times the half-life of cytoplasmic exchange and the ratio of efflux to all fluxes removing 13N03" or "NIL,"1" from the cytoplasm, and assuming 5% for cell volume occupied by the cytoplasm. Similarly, apparent-free-space concentrations ([N03"]free s p a c e and [NH4+]freespace) were obtained by assuming 10% cell volume for the free space (cf. Lee and Clarkson, 1986, and Siddiqi et al., 1991). 2.3.4. Checks of Flux Estimates from Compartmental Analysis. In order to provide checks on the methodology of compartmental analysis, independent estimates of were determined directly by exposing seedling roots to 13N-labelled solutions of varying [N03~]0 or [NH4+]0 for 10 min. Seedlings were equilibrated in non-labelled 'prewashing' solution of the same composition as the loading solution for 5 min and desorbed in the same solution for a period of 2 min (for N03~) or 3 min (for NH4+) following loading. Prior to desorption, a short 5-s dip in non-labelled solution was used in order to prevent excessive carry-15 over of surface label into the desorption solution. Immediately following desorption, seedlings were cut into roots and shoots, the roots spun for 45 s in a slow-speed centrifuge (International Chemical Equipment, Boston) to remove surface liquid adhering to the roots, and the plant organs then counted in a 7-counter. Concomitantly, ^  was also determined by counting the radioactivities of 1-cm3 samples withdrawn from the labelling solution at various times during the 10-min labelling period. Rates of 13N-depletion, representing of N03" or NH 4 + into seedling roots, were calculated. In separate experiments, <t>ael was measured by monitoring the disappearance of 14N03" or 1 4NH 4 + from uptake solutions over a period of 60 min. Solution samples were taken at different times during this 1-h uptake period. [N03"]0 and [NH4+]C were assayed spectrophotometrically as described earlier. 2.4. Kinetic Influx Studies. 2.4.1. General Procedure. Following purification and, for NIL,"1", chemical conversion of the 1 3N solution, the latter was added to volumes of 4-6 dm3 of vigorously stirred uptake solutions. The nutrient composition of the uptake solution was the same as the hydroponic growth solution (see earlier). It was contained in a double-neck Erlenmeyer flask, which was located on a stir-plate behind lead. The flask was pressurized remotely via a hand-operated pump to deliver the labelled uptake solution to eight to twelve individual 500-cm3 uptake vessels; N03" or NH 4 + had previously been added to these vessels at the desired concentrations (i.e. 2.5 /iM - 50 mM; see influx isotherms). Seedlings were transferred from the hydroponic growth tanks to 'prewash' solutions in 1-dm3 vessels for 5 min prior to immersion of the roots of intact seedlings in the labelled uptake 16 solutions, to minimize perturbation and equilibrate plant roots to the exact solution temperature and to the solution composition used during influx. After the prewash, seedlings were transferred to the uptake vessels for a period of 10 min. Immediately following the 10-min loading, roots were dipped into non-labelled solutions for 5 s to minimize carry-over of label by the root surface to the desorption solution. Roots were then 'postwashed' in non-labelled solution for a period of 2 min (for N03") or 3 min (for NIL/) to desorb tracer contained in the free space. The duration of these steps was based on independent determinations of half-lives of exchange for N03" and NH 4 + for the root surface and the cell-wall free space in efflux experiments (Kronzucker et al., 1995a, c, e). Following desorption, seedling roots were excised from the shoots, the roots spun in a low-speed centrifuge for 30 s to remove surface liquid, and the fresh weights of roots and shoots were determined. The plant organs were then introduced into 20-cm3 scintillation vials, and the radioactivities of roots and shoots were determined in a Packard y-counter (Minaxi 5, Auto-7 5000 Series), measuring the 511-keV positron-electron annihilation radiation generated by recombination of ambient electrons and j8 + particles emitted from 1 3N. Using the value for specific activity (13N/(13N + 14N)) of the loading solution and the total fresh root weight of each seedling, N03" or NH 4 + fluxes were calculated and expressed in jtmol g"1 (FW) h1. In order to determine the influx of 1 3N into young as opposed to old roots, in some selected experiments, labelled roots were excised at the point of transition from the white zone to the tannin/early-periderm zone (see earlier), which was clearly discernible. This was done immediately following desorption in postwash solutions. Fresh weights of young and old roots were recorded, and the accumulation of 1 3N in the excised parts was determined separately. Specific influxes for old, young, or whole roots were then calculated by dividing by the fresh weights of the respective tissues (see text). In our experiments, specific uptake rates for N03" 17 or NH 4 + by old roots were typically only 20-30% of those by young roots (data not shown). However, unless otherwise indicated, integrated influx values, determined on a whole-root basis, were used in Figures and Tables. 2.4.2. Kinetic Analysis of Influx Data. All experiments were replicated at least three times. Each experimental treatment consisted of three seedling samples (minimum root mass was 3 g FW per sample). Data from several experiments were pooled (n > 9) for calculations of means and standard errors (SE). These values were used for plotting representative induction curves and uptake isotherms as well as for calculating V,,^ and values. Four separate data-transformation methods (Cornish-Bowden and Wharton, 1988), based on the Michaelis-Menten formalism, were used to obtain V,,^ and K n , estimates for the saturable isotherm components in the present study. These methods were: (a) linear transformation according to Lineweaver-Burk: 1/v = KJW^ * l/[N03]o + l/V^; (b) linear transformation according to Eadie-Hofstee: v = - K . * v/[N03]0; (c) linear transformation according to Hanes-Wolf: [NCWv = KJV^ + [N03y V,^; 18 (d) least-squares method by Cornish-Bowden: K,,, = (E v 2 * E v/[N0 3] 0 - E v 2/[N0 3] 0 * E v)/(E v 2/[N0 3] 0 2 * E v - E v 2/[N0 3] 0 * E v/[N03]0); = (E v 2/[N0 3] 0 2 * E v2 - (E v2/[N03]0)2)/(Ev2/[N03]0 2 * E v - E v 2/[N0 3] 0 * E v/[N03]0); Student-t tests were used to examine the slopes and Y-intercepts of plots from different linear transformations or transformation plots from separate experiments for significant differences. Estimates of K,,, and V,,^ values obtained by the various transformation-methods (see respective Tables in the text) were not identical. Thus, to avoid subjective data representation, no one specific fit was preferred over another for the Michaelis-Menten phases in isotherm plots. Rather, in isotherms, data points were connected directly. 2.5. Nitrate Reductase Assay. For analysis of root NRA the in vivo assay as described by King et al. (1992) was used. It was modified as follows: 0.1-0.4 g (FW) of root segments were excised and submersed in KPi-buffer (100 uM at pH 7.7) in 10-cm3 test tubes, to each of which was added 0.2 cm3 isopropanol (99% v/v). In some control experiments, test tubes were closed off with air-tight caps, and N2-purging was performed for 10 min. The test tubes were then covered with aluminum foil to keep out light, and the desired nitrate concentration (0.1-50 mM) was added from a 1-M KN0 3 stock solution, so that a final volume of 4 cm3 was achieved in the test tubes. The tubes were then incubated for 30-90 min at 25°C in a waterbath. After incubation, the tubes were transferred to a second waterbath preheated to 95-100°C and were boiled for 10 min to 19 quantitatively extract N02~ from the root tissue. After removal of the tubes from the waterbath, 1.5-cm3 samples were withdrawn and transferred into 5-cm3 test tubes, to which were added 0.25 cm 3 of 0.08% w/v NED (N-(l-naphthyl)-ethylene-diamine-hydrochloride) and 0.5 cm 3 of 2% w/v sulfanilamide. The detection reaction was started by adding approximately 60 mm3 1 % v/v HC1 to each test tube (colour reaction was completed in « 2 min). The tubes were left to stand for 30 min to allow for plant debris to settle. The absorbance was then read at 540 nm in a spectrophotometer (PU 8820 UV/VIS, Philips). 100 nmol N0 271.5 cm 3 in an otherwise identical solution was used as a standard. Activities of root nitrate reductase (NRA) are expressed in /xmol (or nmol) N0 2" produced g"1 (root FW) h"1. 20 3. N0 3-EFFLUX STUDY I: COMPARTMENTATION AND FLUX CHARACTERISTICS OF NITRATE TN SPRUCE. 3.1. Introduction. Detailed studies of nitrate transport have been almost entirely limited to angiosperm species and microalgae, while the physiology of nitrate uptake by conifers has been largely ignored. This is despite the fact that a poor adaptation to nitrate as an N source appears to have important implications for the success of silvicultural practices (see General Introduction). While several net-flux studies based on depletion methods or the stable isotopic tracer 1 5 N have been carried out (Rygiewicz et al., 1984a, b; Marschner et al., 1991; Flaig and Mohr, 1992; Lavoie et al., 1992; Kamminga-van Wijk and Prins, 1993; Knoepp et al., 1993; Plassard et al., 1994; and references therein), no information is available on exchange kinetics of N03" across root cell compartments, the intracellular compartmentation of N0 3\ or the magnitude of cytoplasmic N03" accumulation. The present study represents the first application of efflux analysis and of the short-lived radiotracer 1 3 N to a conifer species. Compartmental analyses for nitrate were conducted at three different ecologically relevant concentrations (Vogt and Edmonds, 1982). Estimates for nitrate influx, efflux, net flux and flux partitioning within the plant are provided as well as for cytoplasmic and cell-wall nitrate concentrations under these conditions. Furthermore, several experimental protocols are presented which were designed to corroborate the validity of compartment identification in the type of efflux analysis employed in the study. While it is recognized that most plant species, including spruce, are mycorrhizal in nature, non-mycorrhizal plants have been used in the present studies, the goals of which were 21 to determine component N03" fluxes and to characterize subcellular compartments for N03" in the plant part of the association. Nevertheless, in reported studies which have specifically investigated the influence of ectomycorrhizal fungi on N03 uptake in spruce, the presence of the fungal partner failed to enhance N03~-uptake rates above values recorded for the non-mycorrhizal counterparts (Littke et al., 1984; Rygiewicz et al., 1984a, b). Hence, the data reported here for non-mycorrhizal spruce roots may directly reflect the situation which applies to mycorrhizal plants in the field. 3.2. Results. 3.2.1. Phase Regression. Figure 1 shows a representative efflux plot for spruce seedlings at 100 uM external N03~ ([N03 ]c). Three different exponential phases, presumably corresponding to physiological N03~-compartments, were identified. Fig. 1 includes linear regression lines for these kinetically distinct 'compartments' in a semi-logarithmic plot. High r2 values (0.82-0.99) were usually found in the linear regression approach. Half-lives of exchange for the 'compartments' were « 2.5 s, 20 s, and 7 min (see Table 1), respectively. Multiple range testing according to Newman-Keuls indicated that there was no significant change in these half-lives with variation in pretreatment (e.g. [N03"]0, SDS, etc.). Compartments tentatively assigned to the exponential phases were the film of surface-adsorbed label carried over from the loading solution (the fastest exchanging phase), the apparent free space (the intermediate phase), and the cytoplasm (the slowest phase) (see Fig. 1). 22 8 2-) , : , 1 1 1 0 5 10 15 20 25 elution time [min] Figure 1. Representative semi-logarithmic plot of the rate of release of 1 3N [log (cpm released) g"1 min"1] versus time of elution for intact roots of white spruce at 100 fiM [N03"]0. The plot includes linear regression lines and equations for the three phases resolved in efflux analysis. 23 Table 1. Half-lives of exchange (t1/4) for NCy of compartments I, II, and III (identified as surface film, apparent free space, and cytoplasm, respectively) at three different external concentrations as estimated from compartmental analysis. Data + SE (n=2-3). (pre)treat- compartment I compartment II compartment III ment: tvi M: t* [s]: t* [min]: 10 uM NCv: 2.11 30.4 7.5 ± 0.13 ± 1.11 + 0.47 100 nM NO3-: 2.62 26.67 8.45 ± 0.16 ± 2.35 ± 1.04 1.5 mM N(V: 2.4 17.45 6.9 ± 0.12 + 2.04 + 0.73 24 3.2.2. Nitrate Fluxes. As illustrated by Figure 2, a clear increase in N03"-influx was evident with increasing [N0 3] 0, from 0.095 itmol g 1 h 1 at 10 i i M to 0.5 itmol g 1 h 1 at 100 uM and 1.2 /xmol g"1 h"1 at 1.5 mM. Efflux rose from 0.004 itmol g"1 h 1 , to 0.03 itmol g"1 h 1 , and 0.3 itmol g"1 h \ constituting 4.3% of influx at 10 itM, 5.6% at 100 itM, and 20.9% at 1.5 mM [NQj]0. Net flux therefore was 0.091 itmol g l h l at 10 uM, 0.48 itmol g 1 h 1 at 100 j t M , and 0.98 /xmol g 1 h 1 at 1.5 mM (Table 2 a). Most of the 1 3 N taken up went to reduction or was sequestered in the vacuole, while only negligible translocation to the shoot (< 1 %) was noted (Table 2 a) over the duration of the experiments, which, including loading and elution, was 47 min. Unfortunately, due to low uptake rates for N03" in spruce combined with high specific activities in the loading solutions, cross-contamination of shoots during plant handling may have resulted in the relatively large errors for the shoot translocation term. 3.2.3. Compartmental Nitrate Concentrations. [N03-]cyt increased with [N03"]o, almost 7-fold from 0.3 mM at 10 uM [NCy]0 to 2 mM at 100/zM [N0 3] o, and a further twofold to 4 mM with [N03']0 at 1.5 mM, while [NCy]^ s p a c e was estimated to be 0.016, 0.173, and 2.2 mM, respectively, at the same [N03~]0 regimes (Table 3 a), and was therefore about 50% higher for all concentrations used than would be expected on the presumption of zero Donnan binding of N0 3' in the free space. 25 3.2.4. Compartment Identification. Since the validity of the calculations of the above parameters depends upon the correct assignment of the compartments identified in efflux plots, several approaches were used (see Materials and Methods for details of protocols) to confirm the assignment of the phases found to the specific subcellular and extracellular compartments. As shown in Tables 2 b-c, all treatments used to perturb the cytoplasm, i.e. H 2 0 2 , 2-chloro-ethanol, and high temperature, led to substantial reduction in count accumulation in the roots, both at 100 uM and at 1.5 mM [N03~]0. Treatment with the detergent SDS gave essentially similar results (data not shown). The reduction in count accumulation was evident in reduced estimates of [N03"]cyt obtained from regression of compartment III (Tables 3 b-c) and of net flux values and was both attributable to depressed influx and to an increase in efflux percentage from the presumed cytoplasmic phase (Tables 2 b-c). [N0 3"] f r e e s p a c e, on the other hand, was significantly affected only in 2-chloro-ethanol pretreatment, and not by any of the other treatments (Tables 3 b-c). Increasing the concentration of orthophosphate in the medium ([Pi]Q) led to a more than twofold increase in N0 3" efflux in the range of 0.2 to 2 mM [PiL. (Table 2 d). Consequently, [N03"]cvt appeared somewhat lower, albeit not significantly, at 2 mM [Pi]Q than at the other concentrations (Table 3 d). However, [N03"] f ree s p a c e was unaffected by these variations in [Pi]c (Table 3 d). 26 1.4-1.2-1-x M 0.8-0 to £ 0.6-c 0.4-0.2-0-net flux 4.28% 20.86% efflux (%) 5.55% 0.01 0.1 1.5 external nitrate concentration [mM] Figure 2. Concentration dependence of net flux, influx and efflux [/*mol g"1 h"1] for N03" in white spruce as determined by efflux analysis. Slanted-line segments in stacked bar graph represent net flux, dark filled segments represent efflux (percentage of influx indicated above bar). Influx results from the sum of the former (see Table 2 a). 27 Table 2 a. N03" fluxes as estimated from compartmental analysis (for symbols see text). Plants were induced for 3 d at the indicated external N03~ concentrations. 'Note that 4^^, unlike the remaining flux terms, represents the flux of 1 3N rather than N03" (see text for explanation). Because of the low magnitude of some flux components and standard errors, fluxes in Table 2 are expressed in [nmol g"1 h"1]. Data + SE (n=2-3). (pre)treat- N03 fluxes [nmol g"1 h"1]: ment: <£oc 4>co $ red/ vac $xylem 10 uM N03: 94.75 4.06 90.7 90.05 0.64 ± 2.64 ± 0.87 + 3.51 ± 3.45 ± 0.06 100 uM N03: 508.39 28.22 480.17 473.43 6.74 + 84.48 ± 7.13 + 81.64 ± 77.66 + 4.67 1.5 mM N03: 1242.39 259.2 983.2 942.02 41.17 ± 61.46 + 25.8 + 87.12 ± 80.34 + 11.05 28 Table 2 b. N03" fluxes as affected by high temperature or H202. Plants were induced at 100 uM [N03-]0 for 3 d (see also Table 2 a). (pre)treat- N03" fluxes [nmol g"1 h"1]: ment: 4>oc 4>co r^ed/vac 100 uM N03" 448.3 38.56 409.74 406.49 3.25 (control): ± 64.11 + 8.44 ± 56.83 + 55.96 ± 2.03 100 uM N037 21.09 18.07 3.03 2.84 0.19 75°C: ± 2.21 ± 2.49 + 0.39 ± 0.35 + 0.09 100 uM N037 45.71 16.96 28.75 28.22 0.53 H202: ± 3.75 + 2.78 + 1.02 ± 1.23 ± 0.26 29 Table 2 c. N03' fluxes at 1.5 mM [N03"]0 as affected by 4 d exposure to 0.075% v/v of the gaseous metabolic poison 2-chloro-ethanol (see also Table 2 a). (pre)treat- NCy fluxes [nmol g 1 h1]: ment: 0net r^ed/vac x^ylem 1.5 mM N03 1347.56 218.67 1128.89 1084.85 44.04 (control): + 105.24 + 29.45 + 134.34 + 117.71 + 15.41 1.5 mM N037 279.71 217.94 61.78 60.09 1.69 2-ChEth: + 16.72 ± 15.95 + 4.39 + 0.42 ± 0.55 30 Table 2 d. NCy fluxes at 100 itM [N0 3] 0 as affected by varying [Pi]0. Plants were induced at 100 uM [N0 3] o for 3 d (see also Table 2 a). (pre)treat- N03" fluxes [nmol g"1 h"1]: ment: 4>co r^ed/vac x^ylem 100 uM NOjV 469.87 21.76 448.1 446.6 1.48 20 itM Pi: ± 44.36 + 2.79 + 46.33 ± 44.57 ± 0.58 100 uM N0 37 514.99 18.61 496.38 490.81 5.57 200 itM Pi: ± 76.98 ± 4.33 ± 76.03 + 73.69 ± 3.58 100 itM NOjV 524.17 52.74 471.43 470.48 0.95 2 mM Pi: ± 32.55 + 4.43 ± 37.62 ± 35.71 + 0.4 31 T a b l e 3 a . Compartment concentrations of N03~ as a function of [N03"]0 as estimated from compartmental analysis. Tissue volume was assumed to be 5% for cytoplasm and 10% for free space. Plants were exposed to the indicated concentrations of external N0 3" for 3 d. Data ± SE (n=2-3). (pre)treatment: [N0 3 ] c y t (/xM): [N0 3 -] f r e e s p a c e (uM): 10 uM N0 3": 328.69 15.79 + 27.07 ± 3.25 100 uM N0 3": 2002 173.47 ± 332.74 ± 30.36 1.5 mM NCv: 4037 2232.33 + 251.44 + 109.8 32 Table 3 b. Compartment concentrations of NCy after disruptive treatment at 75°C or with H 20 2. Plants were induced at 100 uM [N03~]0 for 3 d (see also Table 3 a). (pre)treatment: [N0 3] c y t (itM): [N0 3-] f r e e s p a c e (jiM): 100 LIM N03" 1449.87 164.41 (control): ± 381.9 ± 35.18 100 nM N037 60.22 256 75°C: + 25.78 + 11.03 100 uM N037 148.46 212.04 H 20 2: ± 17.51 + 14.78 33 T a b l e 3 c . Compartment concentrations of N03" as affected by 4 d exposure to the gaseous metabolic poison 2-chloro-ethanol at 0.075% v/v. Plants were induced at 1.5 mM [N03"]0 for 3 d (see also Table 3 a). (pre)treatment: [N03]cyt (JJM): [N03-]freespace (uM): 1.5 mM N03 3575 2020 (control): ± 458.32 + 189.27 1.5 mMN037 685.46 1250 2-ChEth: + 64.71 + 73.63 34 T a b l e 3 d . Compar tment N 0 3 " concentrations as a function o f P i -concent ra t ion i n the m e d i u m . Plants were induced for 3 d at 100 uM N 0 3 \ [ N C y ] 0 and [ P i ] 0 du r ing the exper iment were as indica ted (see also T a b l e 3 a). (pre)treatment: [ N 0 3 ] c v t (fiM): [ N 0 3 ] f r e e s p a c 6 ( i t M ) : 100 i t M N 0 3 7 1737 260 2 0 i t M P i : + 319 .96 ± 36 .32 100 uM N 0 3 7 1462 230 200 uM P i : + 220 .76 ± 45 .93 100 / t M N 0 3 7 1396.1 255 2 m M P i : ± 192.24 ± 36 .41 35 3.3. Discussion. 3.3.1. Validity of Efflux Calculations. The present study is the first to examine N03"-exchange kinetics in conifer roots using the technique of compartmental analysis. Consistent with data on cereal species (Presland and McNaughton, 1984; Lee and Clarkson, 1986; Siddiqi et al., 1991), three distinct phases were found from which N03" was exchanging over a 22-min experimental duration, with half-lives of exchange of 2.5 s, 20 s, and 7 min. Since the slowest exchanging phase detected and used for calculations in these experiments possessed a 7-min half-life, we employed a 35-min loading period to label seedling roots with the tracer. This represents 5 x the half-life for the slowest phase, after which the specific activity of tracer in this compartment theoretically should have reached 96.86% of its value in bulk solution, and, hence, the assumption of steady state with regard to specific activity of the tracer, a prerequisite to using compartmental analysis, can be made with reasonable justification (Walker and Pitman, 1976; Lee and Clarkson, 1986). In order to ensure validity of flux and pool-size calculations, it was also important to ascertain whether the 1 3N effluxing from the root cells was in the form of unconverted N03", and not as N02\ NH 4 + or possibly assimilation products (Lee and Clarkson, 1986; Siddiqi et al., 1991). By passing efflux eluates collected at several different times during the washing period through anion and cation exchange resins and by determining both column-retained and column-eluted radioactivities (see Materials and Methods), it was found that > 98.8% of the 13N-containing species released from the plant roots were negatively charged (data not shown). This rules out confounding effects due to effluxing NIL,"1" or positively charged amino acids. Since the rates of nitrite reductase normally greatly exceed those of nitrate reductase (up to 30-fold higher rates have been reported; Aguera et al. 1990) and N02" therefore does not normally accumulate in 36 plant cells (Solomonson and Barber, 1990; Siddiqi et al., 1992), a significant 13N02" pool available to efflux from the cytoplasm was unlikely under the conditions of the experiments. While the slight possibility of negatively charged 13N-labelled amino acids contributing to efflux remains untested in the present study, the associated error can probably be taken as minimal (Lee and Clarkson, 1986; Siddiqi et al., 1991). Since root cell processing of NH 4 + derived from reduction of N03~, as opposed to NH_,+ absorbed directly from the external solution in the reduced form, appears to be compartmentalized in proplastids and carried out by plastidic glutamine synthetase (GS 2) and ferredoxin-dependent glutamate synthase (Fd-GOGAT) (Redinbaugh and Campbell, 1993), a large cytoplasmic pool of negatively charged 13N-labelled amino acids is considered unlikely in the time-frame of the present experiments. It needs to be stressed that the validity of the calculations used to obtain values for compartmental concentrations and fluxes of N03" crucially rests upon the correct assignment of root cell compartments to the phases seen in regression analysis of the semi-logarithmic efflux plots (see Fig. 1). Therefore, several experiments were undertaken, designed to test the initial tentative interpretation of the regression lines, which took compartment I, II, and III to represent surface adsorption, apparent free space, and the cytoplasm, respectively. In previous studies, independent assessments of [N03~]cyt (Ferrari et al., 1973) or apparent-free-space volume (Lee and Clarkson, 1986) or a phase assignment based on a presumed in-series arrangement of subcellular compartments (Macklon et al., 1990) have been employed to identify the kinetically defined compartments. In the present study, special (pre-)treatments of the plants were employed in an effort to confirm the sub-cellular assignment. Such an approach was first used by Siddiqi et al. (1991) and is expanded in the present study. In the present experiments, seedlings were subjected to H202, SDS, high temperature, or the metabolic poison 2-chloro-ethanol. A consistent reduction of 1 3N accumulation in the plants was found (attributed to both reduced 37 influx and enhanced efflux), which is explained by the destructive effects of the given treatments on membrane integrity and metabolic processes. Compartment III, the presumed cytoplasmic phase, was therefore either partially (H202) or almost completely (other treatments) eliminated, while the presumed 'physical' phases (surface film and free space) were unaffected by the same treatments. To illustrate this finding, two efflux plots were overlaid in Figure 3, one from an experiment conducted in the presence of H202, the other without. The significantly different Y-intercepts of the regression lines demonstrate the specific inhibitory effect on compartment III. The absence of such an effect on the other two compartments is demonstrated by the lack of a significant reduction in calculated [N03"]free s p a c e (see Tables 3 b-c and following section). 3.3.2. Nitrate in the Apparent Free Space. Interestingly, estimates for [N03~]free s p a c e obtained from analysis of compartment II consistently yielded an overestimate of about 50 to 150% with regard to [NCV]0 (Tables 3 a-d), on the presumption that N03" in this phase would be distributed at equilibrium or even at lower concentrations than those of bulk solution. Were N03" in the free space present only in the water free space (i.e. withoutDonnan inclusion), the same or lower concentrations should be expected for free-space N03" as for the external solution. There is the possibility that an error has been introduced into the calculation by the assumption of a value of 10% cell volume for the free space. Yet, similar overestimates have previously been found in completely different plant systems (McNaughton and Presland, 1983; Siddiqi et al., 1991), which suggests that the effect is real rather than an artifact of calculation. Given the surplus of negative charges present in the Donnan free space, which should lead to an exclusion of anions from the free space rather than an enrichment, such overestimates for [N03"]freespace are surprising. Presumably this effect is due 38 Figure 3. Combined semi-logarithmic plots of the rate of release of 1 3 N [log (cpm released) g"1 min1] versus time of elution for intact roots of white spruce at 100 11M [ N Q f L from experiments with (bottom line) and without (top line) H202 present in the loading solution. Plots include linear regression lines for the presumed cytoplasmic phase (compartment III). 39 to the presence of specific N03"-binding proteins, in analogy to the anion-binding proteins found in other species, e.g. for phosphate and sulphate (Siddiqi et al., 1991, and references therein; Arthur Grossmann, Department of Plant Biology, Stanford, CA, personal communication). There are, indeed, indications that the binding is N03"-specific. Varying the external concentration of orthophosphate Pi, which, according to the lyotropic series of ion binding strength, should outcompete N03" at non-specific positive binding sites, failed to alter [N03"]free s p a c e (Table 3 d). The fact that after 4-d exposure to the gaseous metabolic poison 2-chloro-ethanol, the only longer-term disruptive treatment, [N03"]free spac(. actually decreased to that of the external solution may indicate involvement of 'plastic' metabolically produced binding components (Table 3 c). However, in SDS and high-temperature experiments, which should have led to the denaturation of proteins, the overestimation was not abolished. It may therefore be that the presumed apparent-free-space proteins are especially resistant to denaturing treatments, or else that an as yet unidentified non-proteinaceous positively charged cell-wall component is responsible for N03" -binding. Siddiqi et al. (1991) advanced a proposal, according to which binding to the external face of the N03"-transporter proteins may be responsible for the enrichment of N03" in the free space. This proposal seemed attractive, since an increase in the N03~-concentrating effect was seen with induction of N03" transport (McNaughton and Presland, 1983; Siddiqi et al., 1991). Also, in contrast to the studies with spruce, in which a similar overestimate was evident at 1.5 mM [N03"]o as at the other concentrations, these workers did not find the effect to be present at [N03"]Q > 1 mM, which seemed to argue for the phenomenon to be directly connected to the inducible high-affinity transporter (IHATS) active at [N03"]0 < 1 mM. However, on the basis of the peroxide/detergent/high temperature work, this proposal has to be rejected, given the clear disruptive effect of these treatments on membrane proteins as seen in the efflux analyses. At present, it is difficult to advance an explanation that reconciles all the observed 40 phenomena. Possibly, non-proteinaceous cell-wall components are responsible for the concentrating of N03" to a 'baseline' value, while inducible proteins lead to the up to 13-fold increase that has been seen upon induction (McNaughton and Presland, 1983). The latter effect may be somewhat obscured in the several-months-old spruce seedlings used in the present study, due to the presence of large proportions of tannin-impregnated, suberized or lignified roots (see Materials and Methods), which may display non-proteinaceous binding but may not express inducible proteins. Clearly, this warrants further investigation. 3.3.3. Nitrate in the Cytoplasm. The half-life of exchange (7 min) observed for compartment III, the presumed cytoplasm, is in close agreement with the values obtained by others in cereal species (Presland and McNaughton, 1984; Lee and Clarkson, 1986; Siddiqi et al., 1991; Devienne et al., 1994, and references therein). Similar t% values (< 10 min) were found for N03" in control experiments with barley and tomato plants (unpublished results). Macklon et al. (1990), on the other hand, reported a significantly longer tA value (107 min) for the presumed cytoplasmic compartment in their 1 5N studies of onion root segments. This may be a system-specific difference, but given the closeness of values in systems as far apart systematically as grasses, tomato, and conifers, such a drastic difference in onion seems surprising. Rather, it is believed that this apparent discrepancy is attributable to an arbitrary assignment by Macklon and coworkers of the phase displaying the 107 min half-life to the cytoplasm, which was not tested for in their study. The real cytoplasmic compartment may have been incorrectly interpreted as part of the cell-wall fraction. The perturbation experiments of the present study (H202, SDS, 2-chloro-ethanol, high temperature) and those by Siddiqi et al. (1991) provide convincing evidence that the 7-min half-41 life compartment is not of a primarily physical nature (as expected for cell-wall fractions), but is indeed plasmalemma-bound and metabolically dependent, and that therefore estimations of the cytoplasmic pool size of N03" can be undertaken with reasonable confidence on the basis of regressing this compartment. To the author's knowledge, no data on [N03"]cyt in conifers have been published. The present study is the first to provide such values. However, in several other systems [N03"]cyt has been determined using a number of different techniques. Based on compartmental analysis with the tracer 1 3N, as in the present study, [N03"]cyt values of 50 -100 mM were reported for maize roots (Presland and McNaughton, 1984), and 26 mM (Lee and Clarkson, 1986) for barley roots. Siddiqi et al. (1991) examined the dependence of [N03"]cvt on [N03"]o in intact barley roots and found [N03"]cyt to vary from 0.95 to 36.5 mM as [N03~]0 increased from 0 to 1 mM. Macklon et al. (1990), using the stable tracer 1 5N in compartmental analyses of onion root segments, reported higher values in the 40 to 50 mM range for the compartment they identified as the cytoplasm. Devienne et al. (1994) undertook 15N03~-compartmental analyses in intact roots of 30-d-old wheat plants and reported values in the 10 to 20 mM range at [N03"]o from 0.1 to 5 mM. Miller and Smith (1992), using nitrate-specific electrodes, reported 4.9 mM and 2.7 mM at 10 mM [N03"]o in barley for epidermal and for cortical root cells, respectively. These values are lower than those published by the previous workers, but they are still in the millimolar range. Using a method based on an analysis of in vivo and in vitro rates of nitrate reductase, however, Robin et al. (1983), Belton et al. (1985) and King et al. (1992) all reported values for [N03]cyt in the micromolar range. Robin et al. (1983) determined a range of 10 to 100 uM in leaf tissue of barley, corn, rice, alfalfa, pea and soybean. Belton et al. (1985) obtained values of 65 to 140 uM from data gathered on wheat plants and rose cells, also confirming these rather 42 low values by 14N-NMR, although the latter technique provided only low sensitivity for N03" (in contrast to the situation for NIL/). King et al. (1992) found slightly higher values, from 0.66 mM at zero [N03"]0 to 3.9 mM at 20 mM [N03"]0, but likewise significantly lower than estimates obtained by compartmental analysis or microelectrodes. Why these estimates are so widely spread is not entirely clear. It has been advanced that different pools of [N03"]cyt may be measured by the nitrate reductase method as opposed to compartmental analysis (Siddiqi et al., 1991; King et al., 1992), the latter perhaps measuring a (cortical) storage pool with higher [N03~]cyt, while the former would determine a pool rich in nitrate reductase and therefore lower in [N03~]cyt, possibly the epidermis. The [N03"]cyt values obtained in the present study in spruce are in the range of 0.3 to 4 mM at [N03"]0 from 0.01 to 1.5 mM and are therefore closer in magnitude to some of the values reported using the nitrate reductase and microelectrode methods than to other compartmental analysis data. This is, however, not to suggest a methodological reconciliation. It rather needs to be pointed out that a direct comparison of values is only legitimate between data obtained via the same method, because of the unresolved discrepancy of which pools are measured by which method. Given the fact that great care is taken in compartmental analysis to achieve a steady-state labelling condition with the tracer before measurements are undertaken, cross-sectionally (and longitudinally) averaged values for [N03"]cyt in the root cortex are obtained. A possible difference in epidermal cells would certainly be masked by the much larger volume of the cortex and the therefore much stronger cortical 'signal'. It emerges clearly, however, by comparing the present compartmental analysis data to those of other workers (see above), that spruce root cells accumulate significantly less N03" than other species examined by the same method. Interestingly, cytoplasmic concentration estimates for NH 4 + in spruce (see Kronzucker et al., 1995c, d) are much closer in magnitude to the N03 43 values found in these other species and to NH 4 + levels in a plant such as rice which is especially adapted to NH 4 + (Wang et al., 1993a). This apparent discrimination against oxidized N is also seen on the level of N03" fluxes. 3.3.4. Nitrate Fluxes. Fluxes for N03" were found to be much lower than is typical in agricultural species (Clarkson, 1986; Glass, 1988). Interestingly, fluxes of NH 4 + measured by the same technique in the same plant system are in the order of four to five times higher than those for NO/ (Kronzucker et al., 1995c). It is believed that this, along with the lower values for [N03"]cvt, may be a manifestation of a lack of adaptation to N03" as an N source in spruce, and not simply of an inherently lower growth rate (cf. Chapin et al., 1986). Evidence for this apparent lack of adaptation to N03" has also been seen in other conifer species (Marschner et al., 1991; Flaig and Mohr, 1992; Lavoie et al., 1992; Kamminga-van Wijk and Prins, 1993; Knoepp et al., 1993; Plassard et al., 1994; and references therein). In an evolutionary sense, this N-source discrimination may be explained by the fact that later successional conifers, such as white spruce, do not normally encounter appreciable soil N03" concentrations, while for most agricultural species N03" is the predominant N form available in soils (see Introduction for references). Such a specialized adaptation could have important implications for the replanting success of a species like white spruce on disturbed forest sites, where reduced N is in short supply and N03" is the predominant N source (see General Introduction). 44 4. N0 3 -EFFLUX STUDY II: NITRATE INDUCTION IN SPRUCE: AN APPROACH USING COMPARTMENTAL ANALYSIS. 4.1. Introduction. The uptake of nitrate by plants is unusual in that it is considerably enhanced ('induced') by prior exposure to external nitrate (Minotti et al., 1969; Goyal and Huffaker, 1986; Lee and Drew, 1986; Aslam et al., 1993). Following a lag phase of 1-3 h after first exposure to exogenous nitrate, nitrate uptake rates gradually increase from low 'constitutive' levels (Clarkson, 1986) to values as high as 30 times that of the constitutive level (Heimer and Filner, 1971; Jackson et al., 1973; Goldstein and Hunziker, 1985; Dhugga et al., 1988). A similar induction response to external nitrate provision has also been documented for the activity of assimilatory nitrate reductase (Solomonson and Barber, 1990). Rather than serving simply as an N source during induction, it now seems clear that nitrate functions as a signal, triggering both the inductive enhancement of nitrate uptake as well as that of cytoplasmic nitrate reduction (MacKown and McClure, 1988; Tischner et al., 1993). This signal response appears to manifest itself in the specific appearance of several polypeptides, some of which are plasma-membrane-bound and implicated in plasma-membrane nitrate transport (Dhugga et al., 1988; Ni and Beevers, 1994). However, direct participation of these proteins in nitrate transport has yet to be demonstrated. At this point, the most convincing evidence for the inducibility of nitrate transport remains kinetic (Glass and Siddiqi, 1995). In addition, studies of the induction of nitrate transport have been limited to only a select group of 'model organisms', while certain taxonomic groups have been completely neglected. Despite their enormous ecological as well as economic 45 importance, coniferous species belong to the latter. While it is known that conifers normally grow in soils poor in nitrate, it has also been demonstrated that nitrate can become the predominant nitrogen source available to conifers under a variety of ecological conditions (see General Introduction). A concentration-dependent physiological response to external nitrate in terms of N03" fluxes and subcellular concentrations has been shown in white spruce (Kronzucker et al., 1995a). In a previous study the dependence of 13N03~ influx and of cytoplasmic [N03~] on the external concentration of nitrate was characterized in white spruce (Kronzucker et al., 1995a). Here, the first detailed investigation of the process of nitrate induction in a conifer species is presented. Using 1 3N, a time series of compartmental analyses (efflux analyses) was conducted for nitrate, the aim of which was to monitor simultaneously unidirectional flux processes as well as to estimate cytoplasmic and apoplasmic nitrate concentrations within the time-frame of transporter induction. Together with data on changes in root nitrate reductase activity over that time period, this study attempts to further define the limits of nitrate utilization in spruce and - to provide, at the same time, a detailed analysis of the dynamics of nitrate flux partitioning and the relative contributions of efflux and of the fluxes to reduction, vacuole and shoot during induction. 4.2. Results. 4.2.1 .Compartment Regression. In agreement with an earlier study (Kronzucker et al., 1995a), three distinct phases were identified by linear regression of semi-logarithmic plots of 1 3N efflux versus elution time. A representative plot for spruce seedlings, which were induced for 3 d at 100 uM N03" in the 46 external solution ([N03"]0), was shown in Figure 1 (previous chapter). The three phases exhibited half-lives of exchange of 2-3 s (compartment I), 20-30 s (compartment II), and * 7 min (compartment III). Using multiple range testing according to Newman-Keuls, no significant differences in these half-lives of exchange could be found between uninduced seedlings and seedlings which had been induced for varying periods of time (see Table 4). 4.2.2. Nitrate Fluxes. Fluxes and compartmental N03" concentrations were obtained by analyzing the semi-logarithmic efflux plots. As illustrated by Table 5, a clear increase in N03" influx was evident with time of exposure to 100 itM NCV in the external solution, from 0.1 itmol g"1 h"1 in seedlings which had not been previously exposed to N03" (fluxes in these 'uninduced' plants were measured in transition from 0 to 100 itM [N03"]0), to a maximum of 0.5 itmol g"1 h"1 after 3 d of exposure to 100 itM [N03"]0. Thereafter, a decline in influx could be observed to values around 0.3-0.4 itmol g"1 h"1. Most of the 1 3N taken up went to reduction or was sequestered in the vacuole (Table 5), while only negligible translocation to the shoot was measured over the duration of the experiment (47 min). Since efflux remained relatively constant at 0.02-0.04 itmol g"1 h1, net flux exhibited essentially the same pattern as influx over the 5-day time period. Only initially did efflux constitute a high percentage with regard to influx (around 30% in uninduced plants, and 25% in 1-d-induced plants). After only 2 d of exposure to N03", apparent steady-state values for efflux around 4-7% of influx were achieved. 47 Table 4. Half-lives of exchange ( t j for N03" of compartments I, II, and III (believed to represent surface film, apparent free space, and cytoplasm, respectively). Plants were either grown without N03" (experiments a and b; see also Table 5) or induced at 100 uM N03" for the indicated time periods (c-g). Data + SE (n=2-3). (pre)treat- compartment I compartment II compartment III ment: t* [s]: tv. [s]: t% [min]: (a) uninduced 2.14 16.95 7.31 ('0-10'): + 0.27 ± 4.87 ± 2.35 (b) uninduced 2.27 27.96 6.12 ('0-100'): ± 0.08 + 5.71 ± 0.39 (c) 1-d ind.: 2.41 28.98 6.38 ± 0.1 ± 2.37 ± 0.79 (d) 2-dind.: 2.38 21.82 8.44 ± 0.19 ± 3.89 ± 0.28 (e) 3-d ind.: 2.78 28.07 7.49 ± 0.001 ± 3.26 + 0.7 (f) 4-d ind.: 2.45 21.16 7.51 + 0.13 ± 2.09 + 0.71 (g) 5-dind.: 2.64 22.65 6.64 ± 0.2 ± 3.22 ± 0.48 48 T a b l e 5. NCy fluxes as estimated from compartmental analysis. Plants were either grown without N03" (experiments a and b) or induced at 100 i t M N03" for the indicated periods of time. Efflux analyses were undertaken in perturbation (experiments a and b; measurement was at 10 or 100 i t M [N03-]0, indicated as '0-10' and '0-100') and under steady-state conditions at 100 uM [N03]0 (experiments c-g). Data ± SE (n=2-3). (pre)treat-ment: N03" fluxes [nmol g"1 h"1]: </>oc 4>co <r>net r^ed/vac x^ylem (a) uninduced 12.99 4.51 8.49 8.48 0.0089 ('0-10'): ± 1.44 ± 0.55 + 0.89 + 0.89 + 0.0039 (b) uninduced 104.86 28.6 76.26 74.83 1.43 ('0-100'): + 15.09 + 6.54 + 8.56 ± 8.99 + 0.44 (c) 1-d ind.: 165.06 39.76 125.3 124.84 0.45 ± 6.28 ± 3.78 + 10.05 ± 10.21 ± 0.16 (d) 2-dind.: 241.36 17.16 224.2 223.45 0.75 ± 10.92 ± 0.06 + 10.98 ± 10.63 ± 0.42 (e) 3-d ind.: 482.81 17.45 465.36 461.47 3.89 ± 41.37 ± 4.02 ± 37.89 ± 36.7 ± 2.02 (f) 4-dind.: 360.45 26.67 333.78 333.19 0.59 ± 23.57 + 2.49 ± 20.63 + 20.09 + 0.23 (g) 5-dind.: 394.77 24.26 370.51 369.02 1.49 + 30.25 + 3.14 + 27.11 + 26.38 ± 0.73 49 4.2.3. Compartmental Nitrate Concentrations. Cytoplasmic N03~ concentration ([N03 ]cyt) followed a pattern of rise, maximization and decline, comparable to that of influx and net flux. Differences as a function of induction state were substantial. [N03"]cyt increased from virtually undetectable values in the low micromolar range in uninduced plants to maximum values around 2 mM at 100 tiM [N03~]0, and declined thereafter to around 1.2 mM (Table 6). Since rN03"]cvt is calculated from the quotient of the rate of 1 3N release (in a time period of five times the half-life for cytoplasmic exchange) and of the ratio of efflux to all fluxes removing 13N03" from the cytoplasm (Lee and Clarkson, 1986; Siddiqi et al., 1991), the intercept of the presumed cytoplasmic regression line with the Y-axis in an efflux plot (corrected for specific activity in the labelling solution) is an indication of the magnitude of cytoplasmic N03" accumulation, unless differences in N03* accumulation are overcompensated for by high efflux percentages (cf. Kronzucker et al., 1995a). Despite the fact that significant differences in efflux percentage did exist between plants uninduced for N03" and plants induced to a maximum, the differences in [NO/]^ were large enough to manifest themselves in significantly different Y-intercepts. To illustrate this effect, two efflux plots were overlaid (Figure 4), one from an experiment conducted on uninduced seedlings, the other on seedlings fully induced for 3 d at 100 uM \NO{]0, and included regression lines for the presumed cytoplasmic phases. By contrast, N03~ in the cell-wall free space ([N03"]free space) did not change with time of exposure to external N03~. It was dependent only on the magnitude of [N03~]o, and increased from 16 uM at 10 itM [N03"]0 to 160-230 uM at 100 uM, with no significant differences at the different stages of induction (Table 6). 50 Table 6. Compartment concentrations of N03" as a function of N03", estimated from compartmental analysis. Tissue volume was assumed to be 5% for the cytoplasm and 10% for the free space. Plants were either grown without N03" and then measured at 10 or 100 uM [N03"]0 (experiments a and b; see also Table 5), or they were exposed to 100 uM [N03]0 for the indicated time periods (c-g). Data + SE (n=2-3). (pre)treatment: [N03]cyt 0*M): [NCy]free5pace (JJM): (a) uninduced 43.66 16.06 ('0-10'): + 9.3 + 0.44 (b) uninduced 253.25 166.39 ('0-100'): ± 18.21 + 16.21 (c) 1-d ind.: 427.17 226.4 ± 76.9 ± 7.6 (d) 2-d ind.: 978.41 203.29 ± 38.58 + 90.07 (e) 3-d ind.: 1837.29 203.67 + 381.37 ± 39.5 (f) 4-d ind.: 1281.24 206.62 + 225.24 + 19.77 (g) 5-d ind.: 1240 184.39 - ± 10.01 ± 42.9 51 Figure 4. Superimposed semi-logarithmic plots of the rates of release of 1 3N [log (cpm released) g"1 min1] versus time of elution for intact roots of white spruce. Plants were either uninduced (•) or induced for 3 d (*) at 100 uM [N03"]0. Plots include linear regression lines and equations for the presumed cytoplasmic phases ('compartment III'). The significantly different Y-intercepts in the plants at different stages of induction indicate differences in cytoplasmic accumulation of N03". 52 4.2.4. Root Nitrate Reductase and Unidirectional Flux to the Vacuole. NRA was inducible: in vivo NRA, as measured by the in vivo assay at 100 uM [N03~]0, increased from low 'constitutive' levels in uninduced plants (25 nmol N02" g"1 h"1) to maximal values (150 nmol N02" g"1 h"1) after a period of two days of exposure to 100 uM [N03"]0. After this, NRA declined to a steady value of 140 nmol N02" g"1 h"1 at the end of the 5-day period (Figure 5, lower graph). Subtracting these values for NRA at 100 uM [N03"]0 from the values for the combined fluxes to vacuole and reduction as obtained from efflux analyses conducted at the same external N03~ concentration (Figure 5, upper graph) allowed for estimates of the unidirectional flux to the vacuole (see dashed-line connections of the two graphs in Figure 5). This unidirectional flux to the vacuole stayed virtually unchanged at around 50 nmol g"1 h"1 until after 24 h of exposure to external N03", when it slowly started to rise, reaching a maximum of 400 nmol g"1 h"1 at day three of induction. Thereafter, a 20-30% decline was observed. 4.3. Discussion. 4.3.1. The Steady-state Assumption in Compartmental Analysis - Is It Violated in the Present Study? This study represents the first application of compartmental analysis in a time-dependent context to the problem of nitrate induction. As such, this application must be evaluated carefully. It may be argued that the approach of monitoring a time-dependent change in physiological plant activity violates a basic assumption underlying the theory of efflux analysis, which is the requirement of steady state, particularly with respect to compartment sizes and fluxes to and from compartments (Cram, 1968; Walker and Pitman, 1976). However, growing plants rarely, 53 600 600 h500 o O -400 va TJ C to -300 c g '•*-> o T J -200 Q) i_ O X _D -100 •0 time of exposure to 0.1 mM nitrate [d] Figure 5. In vivo rate of root nitrate reductase [nmol g"1 h"1] as measured at 100 uM [N03"]0 (x) and the combined flux to N03" reduction and to the vacuole [nmol g"1 h"1] as estimated from compartmental analysis (A). The difference between the two rates represents the unidirectional flux of N03" to the vacuole [nmol g"1 h"1] (indicated as slashed-line segments connecting the two plots). 54 if ever, achieve a condition of true steady state. Nevertheless, provided that the duration of the efflux analysis is relatively short by reference to the time scale of developmental change and that environmental conditions are held constant during analysis, it can be assumed that the rate of change of compartmental parameters approximates zero during the course of such an analysis. In the present study, nitrate is slowly accumulated in the plants over time by continued exposure to 100 uM N03", and hence, strictly speaking, tissue [N03] is not at steady state. However, during a 57-min experiment, it can be assumed that compartmental parameters remain essentially constant with respect to N03" status of the tissue. Moreover, spruce, even in the seedling stage, is a genus characterized by an extremely low inherent growth rate (Chapin et al., 1986), making it a model organism for compartmental analysis. Likewise, the process of nitrate induction investigated in this study, compared to other plant systems, proceeds quite slowly on a time scale of several days. By contrast, the actual efflux experiment is completed in less than one hour (35-min isotopic loading + 22-min elution). Therefore, the physiological changes occurring during the experimental probing are considered negligible on kinetic grounds, and the thermodynamic prerequisite of steady state is believed to be closely approximated. The validity of these assumptions/approximations regarding steady state is confirmed by the close agreement between influx values calculated on the basis of these assumptions and independent (direct) measurements of influx (see following section). 4.3.2. Agreement between Flux Values Estimated from Compartmental Analysis and those Obtained by Independent Methods. In the present study, N03" fluxes were found to be very low in white spruce, even after full induction. This is in agreement with an earlier study (Kronzucker et al., 1995a). However, 55 three independent approaches were employed to conf i rm the magnitude o f these f luxes . F i r s t l y , measurements o f un id i rec t iona l N 0 3 " in f lux were undertaken based o n the accumula t ion o f 1 3N after a 1 0 - m i n exposure o f roots to label led solut ion. Second ly , the 'quasi-s teady' f l ux to the vacuo le was determined based on the accumulat ion o f 1 3N over 30-60 m i n per iods (cf. C r a m , 1968), and , th i rd ly , net f lux , based o n the deplet ion o f 1 4 N 0 3 " over per iods o f 4-6 h was measured. S ince i t was par t icular ly c r i t i ca l to ascertain the va l id i t y o f the use o f compar tmenta l analysis i n a t ime-dependent study o f nitrate induct ion (see previous paragraph), a l l o f the above approaches were appl ied to seedlings at v a r y i n g stages o f induc t ion (uninduced, 1-d-induced, and 3-d- induced seedlings). F l u x values obtained f rom these independent assessments were i n close agreement w i t h values der ived f rom compartmental analysis (see T a b l e 7 ) , w h i c h provides conf i rmat ion o f the methodology o f compartmental analysis and, i n par t icular , its use i n the present study. In addi t ion , w h i l e unidi rect ional N 0 3 " f luxes i n conifers have remained undetermined b y other workers , the 'steady-state' values for net f lux at a round 0 .3 i t m o l g" 1 h" 1 as seen i n our experiments after 4-5 d o f seedling exposure to external N 0 3 " (see T a b l e 5) agree w e l l w i t h values reported f rom longer- term net uptake studies i n other con i fe r species (see K r o n z u c k e r et a l . 1995a, for references). S i m i l a r agreement w i t h w o r k publ i shed b y other groups was found for values o f root nitrate reductase act iv i ty (Peuke and T i schner , 1991; S c h m i d t et a l . , 1991; and references therein). 4.3.3. Magnitude and Time Profile of Nitrate-induced Nitrate Uptake in Spruce. B o t h i n terms o f magnitude and t ime prof i le , the induc t ion response i n spruce differs m a r k e d l y f r o m what i s seen i n other species. Const i tu t ive levels o f N 0 3 " i n f l u x as l o w as 0.1 i t m o l g 1 h 1 (very close to the observed values i n spruce) have been p rev ious ly recorded i n 56 Table 7. N03" influx and net flux at 100 uM [N03"]0 as determined by methods independent of compartmental analysis. Influx was measured concomitantly by the accumulation of 1 3N in intact roots of white spruce seedlings after immersion in isotopic solution for 10 min (a) and by the depletion of , 3 N from solution during this time (b). Net flux was determined from the depletion of 14N03" over a period of 4 - 6 h (c). An additional estimate of influx (d) was obtained from the sum of 1 4N depletion values and 1 3N efflux estimates (see Table 5). Seedlings were either uninduced, or induced at 100 uM [N03"]0 for 1 or 3 d, as indicated. Data ± SE (n=3-4). method of measurement: induction state of seedlings: uninduced: 1-d induced: 3-d induced: (a) 13N-accumulation 1 0 9 . 5 3 2 9 5 . 0 1 4 0 3 . 1 7 ( < U : ± 1 0 . 5 9 ± 2 0 . 4 1 ± 6 7 . 4 9 (b) 13N-depletion 1 3 4 . 5 8 3 2 6 . 0 2 5 9 4 . 6 8 (0oc): + 1 1 . 3 + 9 . 6 + 2 7 . 1 5 (c) 14N-depletion 9 4 . 0 1 3 0 9 . 0 1 4 9 7 . 0 2 (4w): ± 3 1 . 6 9 + 2 9 . 5 9 ± 4 8 . 8 1 (d) 14N-depl. + 13N- 1 2 2 . 6 1 3 4 8 . 7 7 5 1 4 . 4 7 effl. ( 4 > J : ± 1 9 . 1 2 + 1 6 . 6 9 + 2 6 . 4 2 57 wheat (Goyal and Huffaker, 1986) and in the barley variety 'Klondike' (Siddiqi et al., 1989). Nevertheless, constitutive levels of NCy influx in particular varieties may be more than ten times higher than this (Lee and Drew, 1986; King et al., 1993). Estimates by other workers commonly fall between these extremes (Clarkson and Liittge, 1991; Aslam et al., 1992; Aslam et al., 1993). It is generally assumed that constitutive uptake is mediated by a low-capacity uptake system ('CHATS') and serves the function of 'NO/ sensing' (King et al., 1993). It is conceivable that the considerable differences reported between species and varieties for the activity of 'CHATS' in the uninduced state simply indicates genetic variability, but it is equally possible that the 'true' values for 'CHATS' are indeed quite similar in different plant systems, and that the differences observed are instead attributable to varying degrees of internal N03~ formation. This might be achieved via oxidative processes in the plant tissue (see Clarkson and Liittge, 1991, and Aslam et al., 1993, for references), or it might be attributable to endogenous seed N03~ reserves, varying with the nature of the seed and the age of the plant material, or to trace N03" contamination in chemicals or growth media (Mack and Tischner, 1986). Given that the spruce seedlings used in this study were several months old, that they were starved for N03 for a period of three weeks prior to experiments, and that both analytical-grade chemicals and high-purity water were used in growth media and in induction experiments, the constitutive level of N03" uptake determined is probably a realistic value for this species, and not different from values for certain varieties of barley (see above). Unlike 'CHATS' activity, however, the magnitude of induced N03" fluxes (commonly attributed to an 'IHATS', i.e. an induced high-affinity transport system; see King et al., 1993) is decidedly lower than in any other plant system so far investigated. Upon exposure to external N03", increases in the amplitude of N03 influx of at least five to ten times are normally observed in other species (Siddiqi et al., 1989; Warner and Huffaker, 1989; King et al., 1993; 58 Glass and Siddiqi, 1995). In 'Klondike' barley, with its constitutive fluxes around 0.1 umol g"1 h1, the increase of 'IHATS' was as high as 30-fold (Siddiqi et al., 1989). With its maximum influx around 0.5 umol g"1 h"1 at full induction, spruce sets a 'record low' in terms of inductive flux enhancement. White spruce seedlings do not normally encounter appreciable quantities of N03" in their environment, and NH 4 + serves as the main inorganic source of nitrogen (see General Introduction). This inherently low rate of inducible N03" uptake may therefore represent an evolutionary adaptation to these particular environmental conditions. Since it is now believed that constitutive and inducible transporters (i.e. 'CHATS' and IHATS') are separate protein entities and appear to be regulated and coded for by different genes (Clarkson and Liittge, 1991; Aslam et al., 1992), an adaptive modification toward reduced expression (atrophy?) of the inducible but not of the constitutive transport component in spruce appears plausible from an evolutionary perspective. The limited capacity of white-spruce seedlings to use N03" as a nitrogen source is also seen in the time profile of induction. It is known that the time of exposure necessary for maximum fluxes to be achieved varies between species. In intact roots of higher plants, it ranges from 4-6 h in corn (Jackson et al., 1973) to 6-8 h (Warner and Huffaker, 1989) and 24 h in different varieties of barley (Siddiqi et al., 1989; King et al., 1993). By comparison, the inductive response in spruce is much slower. The use of N03" pulses has provided strong evidence for a 'signal' function of exogenous N03" (MacKown and McClure, 1988; Tischner et al., 1993). It has been postulated that quite low concentrations of N03~ can be effective in inducing N03" uptake, and that once the induction process has been initiated, the presence of exogenous N03" is no longer necessary (Tischner et al., 1993). However, Siddiqi et al. (1989) demonstrated that the time to peak induction varied inversely with [N03"]Q in barley, when exogenous N03" was supplied continuously. In the present experiments with white spruce, there 59 appeared to be no significant differences, with the time resolution used, in the kinetics of the induction response at different [N03"]0. Three days of exposure to exogenous N03" were necessary at 10 uM, at 100 uM and at 1.5 mM [N03"]0 (Kronzucker et al. 1995a). Clearly, with its requirement for a 3-d exposure to external N03" to maximize NCy fluxes, white spruce is the slowest responding amongst the species investigated. With a view to the typical transient appearance of N03" in forest soils, as in the form of seasonal N03" flushes (Vitousek and Melillo, 1979), such a slow induction response would appear to put spruce seedlings at a further disadvantage in a plant competition scenario on soil habitats poor in other N sources such as NH 4 + or organic N (see General Introduction). Thus, combined with the extremely low inductive enhancement of N03" influx, additional evidence is seen in the slow kinetics of the induction response to external nitrate for the previously advanced physiological classification of white spruce as an 'NIL,"1" species' rather than an 'N03" species' (Kronzucker et al., 1995a). This basic conclusion is also shared by other workers (see Lavoie et al., 1992). As discussed before, the practical implications of this inability to utilize N03" efficiently may be considerable with a view to large-scale reforestation of such species on disturbed (i.e. N03"-rich) forest soils. 4.3.4. The Role of Nitrate-flux Partitioning and Negative-feedback Regulation during Induction. The fact that, kinetically, the induction of N03" uptake by external N03" is so slow in spruce seedlings offers the interesting possibility of exploring the dynamics in flux partitioning and of monitoring the development of positive and negative feedback on N03~ influx during the process of induction. The mechanisms of negative feedback on N03" uptake have been much 60 discussed in the literature but few details have been resolved (Breteler and Siegerist, 1984; Lee and Rudge, 1986; Cooper and Clarkson, 1989; Siddiqi et al., 1989; Lee et al., 1992; King et al., 1992, 1993). The discussion has particularly centered around the agent responsible for the negative feedback effect on N03~ uptake (Siddiqi et al., 1989), seen in the decline of flux values following inductive rise to a maximum. Based on work with the glutamine synthetase inhibitor MSO (methionine sulphoximine), which appeared capable of overcoming this negative feedback effect, Breteler and Siegerist (1984) and Lee et al. (1992) concluded that the effect arose from a product of ammonium assimilation rather than from nitrate or possibly nitrite (see also Lee and Rudge, 1986). However, King et al. (1993) pointed out some of the difficulties in interpreting results from experiments with MSO, and presented data of their own, which showed that a 24-h exposure of 'Steptoe' barley to 250 /xM MSO failed to prevent negative feedback. Thus, either nitrate or nitrite would have to initiate negative feedback rather than reduced N products emanating from ammonium assimilation. This notion was further supported by the fact that barley mutants deficient in both the NADH-specific and the NAD(P)H-bispecific nitrate reductases displayed essentially the same pattern of induction and subsequent negative feedback as the respective wild type (Warner and Huffaker, 1989; King et al., 1993). Similar evidence comes from studies with Lemna gibba (Ingemarsson et al., 1987), where inactivation of nitrate reductase activity was achieved by adding the nitrate-reductase inhibitor tungstate (W042~), yet negative feedback was unaffected (Mattson et al., 1991). It is believed that some of the data presented here may contribute to our understanding of the nature of induction and negative feedback. Firstly, the relatively constant absolute value of efflux (Table 5) during the entire course of induction in the present study unequivocally identifies unidirectional influx as the target of negative feedback, not efflux, which would be a theoretical possibility for both up- and downregulation of net flux values. Only the percentage 61 of efflux with respect to influx changes significantly, from high initial values to rather low steady-state values (Table 5). This high initial efflux percentage presumably signifies an initial imbalance of N03" fluxes into the different compartments, and therefore a low efficiency in N03" utilization. Interestingly, the unidirectional flux to the vacuole remained constant at around 50 nmol g"1 h"1 during this time of poor N03"-utilization efficiency (i.e. up to.day two of the induction treatment), while both nitrate reduction and cytoplasmic accumulation of nitrate were positively correlated with influx (see Table 6 and Figure 5). Apparently, a low flux to the vacuole accounted for the relatively high amounts of N03~ lost to the ambient solution through efflux. Only after day two, when <£vac clearly increased, did the efficiency of N03" utilization improve and efflux percentage assume a lower value. Thus, the enhancement of influx during induction appeared to be partitioned mainly to nitrate reduction initially (see the parallel rise of NRA and 4>KAI^ m Figure 5). During this time, the efficiency of N03" utilization, as judged from the percentage of efflux, seemed to be low (see Table 5). Corresponding to the development of <£vac and the downregulation of NRA, <f>co as a proportion of influx was much reduced. In spruce, <£v_c also appeared to be the factor determining the onset of negative feedback. While influx continued to increase until day three, the rate of nitrate reduction had achieved its maximum at day two and was immediately followed by a decline. It seems unlikely, therefore, that either nitrite or products of ammonium assimilation were responsible for the downregulation of influx. Were this to have been the case, influx should have been at a minimum as the production of nitrite and the flux to GS/GOGAT were at their maxima, i.e. at day two. When negative feedback began to take effect, NRA had already been down-regulated to a steady-state level (Figure 5). This agrees with the conclusion arrived at by Mattson et al. (1991) and King et al. (1993), who considered nitrate itself to be an important candidate for the feedback effector. These workers were, however, unable to determine whether the cytoplasmic or the vacuolar 62 nitrate pool was ultimately responsible (King et al., 1993). Data in the present study suggest that cytoplasmic nitrate concentration may be an unlikely candidate as the negative feedback agent, because the peaks of influx and of cytoplasmic accumulation coincided at day three (Tables 5 and 6). In the down-regulated state, [N03"]cyt was also lowered compared to the value at maximal induction. It seems more plausible that the signal originates from the vacuole, which also represents the largest sink for N03". This is, however, not intended to suggest that under different conditions other agents may not play a role in negative feedback upon N03" influx. For instance, the cycling of amino-N through the whole plant (Cooper and Clarkson, 1989; Muller and Touraine, 1992) will almost certainly exert a regulatory effect on N03" uptake. The author's concern here is that before definitive evidence for a role of amino-N in the downregulation of N uptake has been established, many workers appear to have rejected the potential role of N03" itself in this process. 63 5. NH/-EFFLUX STUDY I: COMPARTMENTATION AND FLUX CHARACTERISTICS OF AMMONIUM IN SPRUCE. 5.1. Introduction. Species indigenous to later successional forest habitats are believed to preferentially utilize ammonium as their inorganic nitrogen source, whereas species indigenous to agricultural, perturbed or early successional forest soils are believed to prefer nitrate (see General Introduction). While the assumption that a poor utilization capacity for nitrate is a characteristic trait of woody as opposed to herbaceous species represents an incorrect generalization (Stadler and Gebauer, 1992), a pronounced preference for ammonium over nitrate does appear to be systemic in many coniferous species (Kronzucker et al., 1995a, and references therein). This preference seems to be shared by the ectomycorrhizal fungi normally colonizing the roots of these species in the field (Littke, 1982; Littke et al., 1984; Lang and Jagnow, 1986; Rygiewicz etal., 1984a, b). Previously, the physiological nature of this poor adaptation to nitrate in non-mycorrhizal white spruce seedlings has been investigated by exploring nitrate fluxes and compartmentation in the roots using the technique of efflux analysis (Kronzucker et al., 1995a, b). It was shown that rates of the very first steps in N03" utilization, i.e. N03" influx and N03" reduction to N02", are inherently low and undoubtedly limiting for growth when N03" is the sole N source available to the plants (Kronzucker et al., 1995a, b). An inherent physiological preference for ammonium over nitrate in certain conifer species may have considerable ecological and economic consequences, given that major reforestation failures of such 'NH4 + species' have been observed on 'N03" soils' (see General Introduction). 64 Since physiological information on NH_,+ fluxes in conifer roots is sparse, the technique of compartmental analysis was applied to investigate the flux characteristics and the subcellular compartmentation of ammonium in roots of intact white spruce seedlings. With a view to the ecological implications advanced above, the design of these experiments on ammonium was identical to that of the earlier nitrate study (Kronzucker et al., 1995a), to allow for direct comparisons in terms of N-source adaptation. The study provides estimates for cytoplasmic and cell-wall ammonium concentrations as well as values for unidirectional influx and efflux of ammonium under varying external ammonium concentrations. In juxtaposition to the earlier study on nitrate, the work demonstrates that the capacities for ammonium and nitrate utilization differ very substantially in white spruce at the levels of ion uptake across the plasmalemmata, intracellular accumulation and physiological processing. 5.2. Results. 5.2.1.Phase Regression. Figure 6 shows a representative semilogarithmic plot of 1 3N efflux versus elution time for white spruce seedlings, determined at 100 uM external NH 4 + ([NH4+]0). Assuming first-order kinetics for the loss of tracer from subcellular compartments, three kinetically distinct phases with high r2 values (0.91-0.99) were identified in linear regression of elution data such as seen in Figure 6. The plot shown includes linear regression lines for each of the three phases as well as the respective linear equations. By analyzing the slopes of these lines (i.e. the kinetic constants k), half-lives of exchange (tA) for the phases could be derived. These were 2.3 s (for the fastest-exchanging phase), 33.7 s (for the intermediate phase), and 14.1 min (for the slowest-exchanging phase) (Table 8). The subcellular and/or extracellular compartments that were 65 F i g u r e 6. Representative semi-logarithmic plot of the rate of release of 1 3 N [log (cpm released) g"1 min"1] versus time of elution for intact roots of white spruce at 100 uM [ N I L / ] , , . The plot includes linear regression lines and equations for the three phases resolved in efflux analysis. 66 tentatively assigned to these phases were the film of liquid adhering to the root surface, the cell-wall free space, and the cytoplasm, respectively. No significant difference was found in these t,A values as [NH4+]0 was varied. It must be emphasized that [NH4+]0 values were held constant for 4 d so that steady state was established prior to efflux analysis. The one exception to this was in N-starved plants which required no N until the 13NH4+-loading period (see Materials and Methods). Analyses of the electrical charge of the 1 3N species released from seedling roots by means of anion and cation exchange columns (as outlined in Materials and Methods) revealed that > 96.3% of the radioactive compounds effluxing from the roots were positively charged (data not shown). Since the concentration of positively charged amino acids in the root-cell cytoplasm of plants is usually negligible (see Wang et al., 1994, for references), 1 3N release was assumed to be in the form of NFL,"1". 5.2.2. Ammonium Fluxes. NH 4 + fluxes, as determined by compartmental analysis, changed significantly as a function of [NH4+]0. In N-starved plants ([NH4+]0 = 0 iiM, prior to compartmental analysis), <!>„. was measured at 10 itM [NIL/h (indicated in Figures and Tables as '0-10') and was determined to be 0.96 iimol g"1 h"1. Under steady-state conditions, ^ was 0.21 /imol g"1 h"1 at 10 iiM [NH4+]0, 1.96 ^mol g1 h"1 at 100 itM [NH4+]0, and 6.46 iimol g1 h 1 at 1.5 mM [NH4+]0 (Table 9). All plants were exposed to the respective concentrations for 4 d. Under the same conditions, 0CO was 0.08 /xmol g"1 h1 in N-starved plants and was 0.02 iimol g"1 h"1 at 10 itM [NH4+]G, 0.55 iimol g1 h1 at 100 uM [NH4+]0, and 2.25 /imol g 1 h1 at 1.5 mM [NH4+]0. Thus, expressed in percent of influx, efflux increased from 8.7% to 10%, 28.1% and 34.8%, respectively (Figure 7). <j>net therefore was 0.88 iimol g"1 h"1 in N-starved plants, 0.19 itmol g"1 67 h 1 at 10 ttM [NH4+]OJ 1.41 /xmol g1 h1 at 100 uM [NH4+]0 and 4.21 /xmol g"1 h 1 at 1.5 mM [NH4+]0. Most of the 1 3N taken up by the roots went to NIL,+ assimilation or to the vacuoles in the root cells ((p^*/™ m Table 9), while only small rates of translocation to the stele were observed over the duration of the experiments, which, including labelling and elution, totalled 67 min in most experiments. (frxyiem (probably of assimilation products, not 1 3NH 4 +; cf. Wang et al., 1993a) was less than 0.2% of ^  in N-starved plants, while it constituted 2.1-3.8% in plants grown and measured under steady-state conditions with regard to [NH4+]0 (see Table 9). Flux estimates from methods independent of compartmental analysis were close in magnitude to the ones arrived at by analyzing efflux plots (Table 10). <px, as determined by 1 3N accumulation in seedling roots after exposure to isotopically labelled solution for 10 min, was 0.57 /xmol g1 h"1 in N-starved plants, 0.41 /xmol g"1 h"1 at 10 uM [NH4+]0, 1.28 /xmol g"1 h 1 at 100 /xM [NH4+]0 and 6.72 ttmol g"1 h"1 at 1.5 mM [NH4+]C. By contrast, 4v estimates based on 1 3N depletion over the same time period tended to be somewhat higher. They were 1.09 umol g"1 h 1 in N-starved plants, 0.49 /xmol g1 h1 at 10 uM [NH4+]0, 2.34 /xmol g1 h1 at 100 uM [NH4+]0 and 6.26 /xmol g"1 h"1 at 1.5 mM [NH4+]0. A direct measurement of <paet based on the chemical depletion of 1 4NH 4 + from solution over a time period of 60 min yielded values very similar to </>net estimates from efflux analysis (see Tables 9 and 10). <f>nei obtained by these measurements was 0.25 /xmol g4 h 1 at 10 uM [NH4+]0, 1.44 /xmol g"1 h 1 at 100 uM [NH4+]0 and 3.42 /xmol g1 h"1 at 1.5 mM [NH4+]0. 5.2.3. Compartmental Ammonium Concentrations. [NH4+]cyt increased substantially with increasing [NH4+]0 (Table 11). It rose almost eightfold from 1.75 mM at 10 uM [NH4+]0 to 13.7 mM at 100 uM [NH4+]0 and, by more than 68 Table 8. Half-lives of exchange (t,J for N H 4 + of compartments I, II, and III (assumed to represent surface film, cell-wall free space, and cytoplasm, respectively) at four different external concentrations of NIL/ as estimated from compartmental analysis. Data ± SE (n=3-9). (pre)treat- compartment I compartment II compartment HI ment: ta [s]: t* [s]: tvi [min]: 0 uM N H 4 + 1.67 36.24 13.67 ('0-10'): ± 0.37 ± 5.82 ± 1.5 10 uM N H 4 + : 2.61 33.11 17.47 ± 0.17 ± 4.84 + 1.41 100 uM N H 4 + : 2.15 34.41 14.74 ± 0.31 ± 3.56 + 1.41 1500 fiM N I L / : 2.67 28.53 10.53 ± 0.05 ± 1.25 ± 0.39 69 Table 9. NFL/ fluxes as estimated from compartmental analysis (for symbols see Materials and Methods). Plants were grown in N-free solution for 3 weeks and then exposed to the indicated NH 4 + concentrations for 4 d. Efflux analysis was conducted at the same NH4+-concentration, with the exception of '0 uM NIL/ plants', which were grown and pretreated at 0 /xM [NH4+]0, but measured at 10 uM [NH4+]0. Data + SE (n=3-9). (pre)treat- NH 4 + fluxes [iimol g1 h1]: ment: <t>oc <£co *Aass/vac <£xylem 0 uM NH 4 + 0.9639 0.0839 0.8786 0.8771 0.0015 ('0-10'): + 0.0814 + 0.0132 ± 0.095 ± 0.0944 + 0.0011 10 uM NH 4 +: 0.2129 0.0214 0.1915 0.1848 0.0068 + 0.0171 + 0.0009 ± 0.0179 + 0.0149 + 0.003 100 uM NH 4 +: 1.9626 0.5512 1.4114 1.3698 0.0418 ± 0.2091 ± 0.1699 + 0.1226 ± 0.1126 ± 0.0133 1500 itM NIL/: 6.4584 2.2458 4.2126 3.9671 0.2455 + 0.2589 ± 0.4295 + 0.6133 + 0.6125 ± 0.001 70 '0-10' 10 100 1500 ammonium concentration [uM] Figure 7. Concentration dependence of net flux, influx and efflux [/xmol g"1 h"1] for NH 4 + in white spruce as determined by efflux analysis. Slanted-line segments in stacked bar graph represent net flux, dark filled segments represent efflux (percentage of influx indicated above bar). Influx results from the sum of the former (see Table 9). 71 Table 10. N H 4 + influx and net flux as determined by methods independent of compartmental analysis. Influx was measured at the same time by accumulation of 1 3 N in roots after loading in labelled solution for 10 min and by depletion of 1 3 N from the loading solution during this time. Net flux was determined from depletion of 1 4 N H 4 + over a period of 60 min. Data + SE (n=3-4). (pretreat-ment: method of measurement: 13N-accumulation: 13N-depletion: 14N-depletion: 0 ttM N H 4 + 0.57 1.09 N.D. ('0-10'): ± 0.03 + 0.04 10 uM N H 4 + : 0.41 0.49 0.25 ± 0.13 ± 0.06 ± 0.05 100 uM N H 4 + : 1.28 2.34 1.44 ± 0.02 ± 0.37 ± 0.27 1500 itM NIL/: 6.72 6.26 3.42 ± 0.47 ± 0.58 ± 0.54 72 Table 11. Compartment concentrations of N H / as a function of [NH 4 +] 0 as estimated from compartmental analysis. Tissue volume was assumed to be 5% for cytoplasm and 10% for free space. Treatment of plants was as indicated in Table 9. Data + SE (n=3-9). (pre)treatment: [NH 4 + ] c y t o p l a s m (mM): [NH 4 +] f r e e s p a c e(txM): 0 uM N H 4 + 6.02 84.01 ('0-10'): ± 1.01 ± 17.61 10 uM N H 4 + : 1.75 50.06 ± 0.26 ± 2.33 100 uM N H / : 13.66 690.03 ± 1.77 ± 159.57 1500 itM NH4+: 32.7 8098.01 ± 2.31 ± 793.72 73 a further twofold, to 32.7 m M at 1.5 mM [NH 4 +] 0. In N-starved seedlings when measured at 10 uM ('0-10'), [NH 4 +] c y t was 6.02 mM, significantly higher than in seedlings measured at 10 uM [NH 4 +] 0 in the steady state. [NH4+]freeSpaCe was estimated to be 84.01 uM in N-starved plants in the '0-10' design, and 50.1 / t M at 10 uM [NH/L, in the steady state. At 100 uM [NH 4 +] 0 the estimate was 690 uM, and at 1.5 mM [NH 4 +] 0 it was 8.1 mM (Table 11). 5.3. Discussion. 5.3.1. Half-lives of Exchange. As in previous studies on N03~-exchange kinetics in the roots of white spruce (Kronzucker et al., 1995a, b), three kineticaily distinct phases were found for the exchange of N H 4 + in this plant system by means of compartmental analysis. The mean half-lives of exchange for these phases were 2.3 s, 33.7 s, and 14.1 min. These phases are believed to represent a film of superficial N H 4 + adhering to the root surface (possibly including the water free space; see later discussion), the Donnan free space, and the cytoplasm, respectively. This interpretation is based on the close agreement of these values with those found for N03" exchange in barley (Siddiqi et al., 1991) and in white spruce (Kronzucker et al., 1995a, b). In these latter studies, extensive tests, including treatment of seedling roots with high temperature, sodium-dodecyl-sulphate (Siddiqi et al., 1991), H 20 2 , and 2-chloro-ethanol (Kronzucker et al., 1995a), were conducted to distinguish intracellular versus extracellular compartments. These tests, in combination with the assumption of an in-series arrangement of root cell compartments, were taken as strong evidence that compartments I and II (i.e. the fastest exchanging and the intermediate phase), which were unaffected by the perturbational treatments, represented a film of solution adhering to the root surface and the apparent free space, respectively. Compartment 74 Ill, which was significantly affected by the treatments, was interpreted as corresponding to the cytoplasm. Analogous results were obtained in compartment identification tests for NIL,"1" exchange in white spruce (Kronzucker et al., 1995e). Half-lives of similar magnitude to the ones reported in this study have also been found by other workers for N03" exchange in corn (Presland and McNaughton, 1986; Cooper et al., 1989; Devienne et al., 1994), and in barley (Lee and Clarkson, 1986), as well as for NH 4 + exchange in rice (Wang et al., 1993a). Interestingly, there appeared to be no significant difference in t,A for exchange with the cell wall of NIL,"1" versus N03", while the quantity of the NH 4 + bound was in the order of three- to five-fold larger than that of NO/ (see Kronzucker et-al., 1995a). Due to the vast excess of negative charges in the cell-wall free space strong Donnan binding can be expected for NH 4 + but not for NO/. The lack of a difference in the 1 3NH 4 +-release kinetics for the two N species seems to argue for specific free-space binding also of NO/ which may be similar in nature to that of NFL/ (see Siddiqi et al., 1991, and Kronzucker et al., 1995a). In fact, compartment II, as evident in this type of efflux analysis, is assumed to represent 'adsorptive' binding for both N species, rather than it being a combination of water free space and Donnan free space, as suggested by Wang et al. (1993a). It appears more reasonable that the water free space may exchange with a t% closer to that of the surface film (i.e. compartment I) and may therefore be hidden in the latter phase in efflux analysis, rather than being part of compartment II. Notably, tA values for NH 4 + exchange with the cytoplasm were longer in white spruce («14 min) than they were in rice in the study by Wang et al. («8 min). This difference may be species-specific. However, in studies of NIL/ exchange in barley (unpublished results), t,A for the cytoplasm was in the 14-18 min range, i.e. closer to the values in white spruce than to those in rice. Macklon et al. (1990), in studies of 1 5NH 4 + exchange in excised root segments of 75 onion, reported hA values for the cytoplasm of 68.6 to 82.4 min, which is significantly longer than any of the estimates reported by other workers, including the present study. Because of the large discrepancy between values reported by Macklon and those obtained by others, and since there were no tests of compartment identity conducted by Macklon and coworkers, the validity of the assignment of efflux phases to subcellular compartments as advanced in that study is questionable (see also Kronzucker et al., 1995a, and later discussion). Knowledge of t,A values, particularly for the adsorptive component of the cell wall matrix and exchange of the cytoplasm, is important in designing proper protocols for influx experiments (Cram, 1968). In fact, the choice of a 10 min loading time as well as that of 5 min for prewashing and 3 min for desorption in the present study was arrived at on the basis of our tA estimates. For the loading period, 10 min was chosen, because of the necessity of allowing for statistically reliable count accumulation in the tissue. Also, if the loading time is kept too short, the error associated with any counts remaining in the free space would be magnified significantly when calculating fluxes on a per hour basis. On the other hand, since the goal is to measure unidirectional influx, the specific activity in the cytoplasm has to be kept as low as is compatible with accurate detection. The reason for this is that if the specific activity in the cytoplasm rises significantly during the loading period then, inadvertently, some counts will be released due to efflux during this time of loading and lead to an underestimate of influx. However, in the present system the error associated with this efflux remains minimal, since even after the loading period was complete the specific activity in the cytoplasm had reached only 39% of the specific activity in the outside solution. Therefore, even in the case of plants grown at 1.5 mM [NH4+]0, where efflux constituted close to 35% of influx (see Figure 7), the underestimate of influx due to efflux would come to a maximum of 14% at the end of the 10-min period. Since this error increases in an exponential fashion, the underestimate, integrated over the entire loading 76 duration, would therefore be significantly less than 10%, even under these conditions of high efflux percentage. For desorption, 3 min was chosen, since, this period represents six times the half-life of exchange for the free space (t,A of « 3 0 s) and almost one fifth of that for the cytoplasm (tA « 1 4 min). Such a time period allows for a virtually complete release of the 1 3 N H 4 + associated with the cell wall to the ambient solution (98.44% of the exchange should be complete after 6 x t j , while facilitating only marginal efflux of tracer from the cytoplasmic compartment during desorption. Even in the case of plants grown at 1.5 mM [NH 4 +] 0, the underestimate due to efflux during desorption would be less than 6%. In fact, it is more likely that, due to continued uptake and the presence of still not quantitatively desorbed tracer in the free space, an error leading to an overestimate for would be introduced during the desorption period (see Wang et al. 1993). 5.3.2. Ammonium Fluxes. Fluxes of N H / in white spruce estimated by compartmental analysis were considerably higher than fluxes of N03" measured under comparable conditions by the same technique (Kronzucker et al., 1995a, b). In fact, </>TO of NIL/ at 10 uM [NH 4 +] 0 in the steady state was « 2-fold higher than of N03" after exposure to 10 itM [N03"]0 for 3 d (i.e. in a state of full induction for N03" transport; see Kronzucker et al., 1995a). It was ~4-fold higher when the external concentration was 100 uM for the two N species, and ~ 5-fold higher at 1.5 mM. Since N03" influx is substrate-inducible, i.e. it is considerably enhanced by the presence of external N03" (see Kronzucker et al., 1995b, for references), while N H 4 + influx in most plant systems is down-regulated rather than enhanced by the presence of external N H 4 + (see Wang et al., 1993b, for references), the most dramatic difference for ^ for the two N species was found in 77 the '0-10' i t M design, i.e. with plants not previously exposed to their respective N source and then measured at an external concentration of 10 / t M . Under those conditions, <f>x for NFL/ was almost 75 times larger than ^  for N03", and, due to a low efficiency of N03" utilization in white spruce in this state (evident as a relatively high efflux percentage; see Kronzucker et al., 1995b), 0n e t was even 100-fold lower for N03". Clearly, the previously advanced proposal that white spruce, as a later successional species normally occurring on soils rich in N H 4 + and poor in N03" (see General Introduction), be physiologically much better adapted to utilizing N H 4 + as an N source than N03" seems to be substantially supported by these differences in flux values. Similar differences in the magnitude of fluxes for N H 4 + and N03" were also observed when methods independent of compartmental analysis were used in the same plant system (see Table 10, and Kronzucker et al., 1995b), and they are in general agreement with results of longer term net flux estimations by other workers (see General Introduction). Very good agreement was found between flux estimates from compartmental analysis and those determined by three independent methods. Firstly, unidirectional NIL/ influx was measured, based on the accumulation of 1 3 N tracer after a 10-min loading period of seedling roots in labelled solution. Secondly, the 'quasi-steady' flux to the vacuole (cf. Cram, 1968) was determined, and, thirdly, the depletion of 1 4 N H 4 + over a 60-min period was measured to estimate <paet (Table 10). The close agreement between estimates arrived at by these different means provides good support for the methodology of compartmental analysis and the validity of the calculations involved in deriving the various parameters reported here. The fluxes for NFL/ showed a clear increase with increasing [NFL/]0 (see Table 9). Interestingly, estimates for <£co and <£net were « 4 . 5 times higher in the '0-10' treatments than under steady-state conditions of 10 uM [NFL/]0 (Figure 7). This contrasts with the situation for N03", where in the 10-itM scenario ^ was more than 7-fold and $n e t even close to 11-fold 78 higher than in the '0-10' design (Kronzucker et al., 1995a, b). This phenomenon seen with N03" is attributable to the induction of N03" uptake by external N03~ (see above). With NH 4 +, on the other hand, such an induction of transport was not observed in spruce. Rather, a down-regulatory response was evident (Figure 7). The ratio of (pj^ (i.e. the percentage of efflux) appeared to be relatively unaffected in this downregulation after prolonged presence of external NH 4 +, but, in absolute terms, the reduction in <px (and </>net) was almost 5-fold. Since [NH4+]cyt was also significantly higher in the '0-10' experiments (see Table 11 and Figure 8), the factor responsible for the observed negative feedback upon NIL,"1" uptake is probably not the cytoplasmic NH 4 + pool under these conditions, but rather one or several down-stream assimilation product(s) produced by the flux through GS/GOGAT. This conclusion agrees with that by other workers (see Wang et al. 1993b, for references). 5.3.3. Compartmental A m m o n i u m Concentrations. Few studies have been undertaken to determine [NH4+]cyt in plant roots, and no data are available for [NH4+]cyt in roots of tree species. The only higher plant studies the author is aware of have relied on compartmental analyses with the isotopes 1 5N and 1 3N (Cooper et al., 1989; Macklon et al., 1990; Wang et al., 1993a), on mathematical modelling of 15N-labelling data (Fentem et al., 1983a), on 14N-NMR (Lee and Ratcliffe, 1991) and on a combination of 31P- and 13C-NMR (Roberts and Pang, 1992). However, a direct comparison of the [NH4+]cyt values presented in this study with those reported by other groups proves problematic. Using in vivo 14N-NMR, Lee and Ratcliffe (1991) determined values of 3-8 mM for [NH4+]cyt for maize root segments grown at 1.5 mM [NH4+]0) and values as high as 90 mM after pretreatment of plants with the GS/GOGAT inhibitor methionine sulphoximine (MSO). There 79 are several uncertainties in NMR determinations of this nature. While the NIL / ion is easily detected in vivo by analyzing 1 4 N-NMR spectra (Belton et al., 1985), a distinction of the cytoplasmic versus the vacuolar NIL / pool is not directiy possible, because of the lack of pH-dependence in the chemical shift of NIL,"1". Only by simultaneously monitoring the 14N-signal and the signal(s) emanating from ! H atoms contained in the NIL / ions can such a distinction between compartments of different pH be made, based on the pH-dependent H + exchange between NIL / and H 2 0 . Lineshape analysis of the 'H-coupled NMR spectra then shows a quintet resonance at low (vacuolar) pH, while at neutral (cytoplasmic) pH a singlet resonance is observed (Lee and Ratcliffe, 1991). Yet, this distinction is only clear at exceptionally high signal-to-noise ratios and when [NH 4 + ] c y t is sufficiently high so as not to be masked by the vacuolar signal (Lee and Ratcliffe, 1993). Therefore, estimates for [NFL/Lyt in the study by Lee and Ratcliffe (1991) remained afflicted by large errors, unless the cytoplasmic NIL / pool was increased substantially by pretreatment with MSO. Interestingly, the estimates for [NH 4 + ] C y t of « 9 0 mM under these conditions are very close to estimates in spruce obtained after 6 h pretreatment with 1 mM MSO and at 1.5 mM [NH 4 + ] 0 , which were in the 50 to 70 mM range (Kronzucker et al., 1995e). Additional problems in estimations using NMR lie in the difficulty of simulating cytoplasmic and vacuolar conditions when defining chemical compositions of the solutions against which the pH and concentration dependence of the N IL / signal are calibrated as well as in the sometimes long data acquisition times required to allow for a satisfactory signal-averaging procedure (Fourier analysis). The latter problem inherent to any NMR study particularly compromises the physiological relevance of [NrL/] c y t estimates reported by Roberts and Pang (1992). These workers used NMR signals originating from 3 1 Pi- and from 13C-malate to monitor the pH changes accompanying NIL/ uptake and derived [NH 4 + ] c y t of 3 to 438 uM at [NH 4 + ] 0 up to 10 mM. The study employed excised root tips of maize, which were perfused 80 with an NH4 +-free solution for 3 h. This time period appears quite long for use with excised tissue. In combination with the fact that NH4+-free perfusion solution was used, which may have led to substantial N H 4 + efflux from the root tissue, this might explain the much lower values for [NH 4 + ] c y t than were seen in the study by Lee and Ratcliffe (1991). Fentem et al. (1983a) used a different approach to derive values for [NH 4 + ] c y t . Their method was based on mathematical modelling of 15N-labelling data. By examining the kinetics of 15N-label incorporation into barley roots, these workers were able to identify several distinct phases, presumably representing separate subcellular ' N H 4 + pools'. By defining kinetic constants for 1 5 N exchange with each of these pools, called 'transfer coefficients' by the authors (expressed in Ijumol g"1 min"1]), a 'metabolic' N H 4 + pool of 1.37 /xmol g"1 was obtained for basal root segments and one of < 0.1 itmol g"1 was found for root tips. These pool sizes, if confined to the cytoplasm, would correspond to values for [NH 4 + ] c y t of 27.4 mM in basal root cells (containing mature vacuoles) and «200 uM in apical (only partially vacuolated) root cells, if one assumes conservatively that the cytoplasm occupies ~ 5 % of the tissue volume in the former and «50% in the latter. Considering that these pool size estimates were obtained at 40 itM [NH 4 + ] C , the values, especially for basal root cells, appear high by comparison to other literature data and to the values communicated in the present study. However, it is clear that the approach by Fentem and coworkers is highly derivative, and therefore the potential for error could be considerable. Compartmental analysis, similar to the present study, was used to provide [NH 4 + ] c y t estimates by Cooper et al. (1989), Macklon et al. (1990) and Wang et al. (1993a). The work by Cooper et al. (1989) is difficult to evaluate, since, in its published form as a conference abstract, it withholds details of methodology. The researchers used 1 3 N , as in our study, and their reported steady-state values of 40 mM for [NH 4 + ] c y t at 500 itM [NH 4 + ] 0 for mature roots of 81 wheat plants are very close in magnitude to the values for spruce. Macklon et al. (1990) reported a value of 75.8 mM for [NH4+]cvt at 2 mM [NH4+]0 in onion slices. This estimate was, however, based on a speculative (i.e. untested) assignment of phases found in 15N-efflux analyses to actual subcellular compartments. Without rigorous testing of compartment identification as attempted for N03" by Siddiqi et al. (1991) and Kronzucker et al. (1995a), and for NFL/ by Kronzucker et al. (1995e), calculations of compartmental pool sizes are highly questionable. Since there is a considerable discrepancy in the hA values reported for the presumed cytoplasmic compartment in the study by Macklon et al. and those reported by others using 13N-efflux analysis (Lee and Clarkson, 1986; Siddiqi et al., 1991; Wang et al., 1993a; Kronzucker et al. 1995a, b), including the present study, a direct comparison of the results obtained in the different laboratories is probably not valid. In NH4+-efflux analyses in white spruce seedlings, extensive tests were used to confirm compartment identity, including pretreatment of seedling roots at high temperature and sodium-dodecyl-sulfate (SDS), as well as addition of a-ketoglutarate and MSO to ascertain the identity of the presumed cytoplasmic compartment. In addition, varying concentrations of Ca2 + (and Al3+) were applied to identify the presumed Donnan free space (cf. Kronzucker et al., 1995e). For methodological reasons, only the study by Wang et al. (1993a) seems directly comparable to the present study. As in the present study, Wang et al. used 13N-efflux analysis and non-excised root material. The values reported for roots of intact rice plants under steady-state nutritional conditions were 3.72 mM at 2 uM [NH4+]0, 20.55 mM at 100 uM [NH4+]0, and 38.08 mM at 1 mM [NH4+]0, and are thus slightly higher than, but in the same range as, the values in white spruce. Since rice is a species which occurs naturally in soils where NFL/ is the most readily available form of N and is known to clearly prefer NIL/ over N03" as an inorganic N source (see Wang, 1994, and references therein), the fact that spruce seedlings accumulate 82 NH 4 + in the cytoplasm to levels similar to those in rice supports the proposal that spruce constitutes an 'NH4 + plant', not a 'N03~ plant' (Kronzucker et al., 1995a, b). Interestingly, barley, a typical 'N03~ plant' (Huffaker and Rains, 1978), accumulates N03" in the cytoplasm to values very similar to [NH4+]cyt in rice and spruce (Siddiqi et al., 1991). [N03~]cyt in the latter study was estimated at approximately 1 mM for N-starved plants, 12 mM at 10 itM [NQj"]0, 20 mM at 100 uM [N03"]0, and 37 mM at 1 mM [N03"]o after 4 d of exposure to these respective concentrations (i.e. under steady-state conditions). Values for [N03"]cyt in spruce under conditions relatively comparable to the ones used in the present NH 4 + study were, by contrast, much lower than both the reported [N03"]cyt levels in barley (Siddiqi et al., 1991) and the [NH4+]cyt levels in rice and in spruce (Wang et al., 1993a), despite the fact that the [NCvLyt estimates in spruce represent maximal values that were obtained in a state of full induction of N03" transport 2(Kronzucker et al., 1995b). A direct graphical juxtaposition of the cytoplasmic accumulation data for N03" versus NH 4 + in spruce illustrates the dramatic preference for NH 4 + over N03" in this species (Figure 8). 83 '0-10' 10 100 1500 nitrate or ammonium concentration [JJM] Figure 8. Juxtaposition of the differential magnitude of cytoplasmic accumulation in mM of NH 4 + (horizontal-line bars) and N03" (slanted-line bars) in roots of intact white spruce seedlings at 4 different regimes of external concentration of the respective N-species. N03"-data are redrawn from Kronzucker et al. (1995a, b). 84 6. NH 4 +-EFFLUX STUDY II: ANALYSIS OF 1 3 NH 4 + EFFLUX IN SPRUCE ROOTS: A TEST CASE FOR COMPARTMENT IDENTIFICATION IN COMPARTMENTAL ANALYSIS. 6.1. Introduction. Efflux analysis is widely used to determine unidirectional ion fluxes, kinetic exchange constants of subcellular compartments, as well as ionic concentrations within compartments. In plants, compartmental analyses have been undertaken for a variety of ions, including Na+, K + , Mg 2 +, Ca 2 +, NH 4 +, N03", Pi, S042, Cl\ and Br (see Wang, 1994, for references). The majority of efflux studies have been limited to non-metabolized ions and were mostly performed on excised tissues or suspension-culture systems, mainly because of the (presumed) absence of complicating factors like metabolism and long-distance transport to the shoot (Pitman, 1963; Cram, 1968; Poole, 1971a, b; Macklon, 1975a, b; Macklon and Sim, 1976; Pfruner and Bentrup, 1978; Macklon and Sim, 1981; Macklon et al., 1990). For use with intact plant material, a detailed treatise on parameter extraction was presented by Jeschke and coworkers (Jeschke and Jambor, 1981; Jeschke, 1982). More recently, compartmental analysis has also been applied to metabolized ions, including S042" (Thoiron et al., 1981; Cram, 1983; Bell et al., 1994), Pi (Lefebvre and Clarkson, 1984; Macklon and Sim, 1992), N03" (Presland and McNaughton, 1984; Lee and Clarkson, 1986; Macklon et al., 1990; Siddiqi et al., 1991; Devienne et al., 1994; Kronzucker et al., 1995a, b) and NH 4 + (Presland and McNaughton, 1986; Cooper et al., 1989; Macklon et al., 1990; Wang et al., 1993a; Kronzucker et al., 1995c). Despite this widespread use of the technique, workers have typically neglected to conduct physiological tests to verify the subcellular identities of the phases revealed in efflux data. As 85 a consequence o f this omis s ion , the assignment o f par t icular k ine t ica l ly -def ined phases to their cor responding subcel lular compartments has not a lways been unequ ivoca l (see M a c k l o n et a l . , 1990). O n l y i n studies by C r a m (1968), L e e and C l a r k s o n (1986) and S i d d i q i et a l . (1991) as w e l l as i n a p rev ious study o f NO3" exchange i n spruce ( K r o n z u c k e r et a l . , 1995b) was the assignment o f compartments substantiated. U s u a l l y phase assignment has been based on the assumption o f an in-series arrangement o f ce l l compartments, i . e . c e l l w a l l , cy top lasm, and vacuo le (P i tman , 1963; C r a m , 1968, 1975). Thus , the first ( rapidly exchanging) phase has been assumed to represent the c e l l w a l l and the last (slowest exchanging) phase assumed to represent the vacuo le . H o w e v e r , the der ivat ion o f f lux components as w e l l as o f p o o l sizes f rom eff lux data is o n l y v a l i d i f subcel lular compartments are assigned correct ly to their cor responding efflux phases. In the eff lux analyses reported i n the present study a combina t ion o f strategies was e m p l o y e d to d is t inguish between membrane-bound and metabol ica l ly dependent ( intracellular) compartments and those that are non-membrane-bound and apparently independent o f metabol i sm (extracel lular) . Intact seedlings o f whi te spruce were used as the m o d e l system, since detailed studies o n both N 0 3 " and N I L / exchange have been performed p rev ious ly i n the same species us ing the same technique ( K r o n z u c k e r et a l . , 1995a, b , c ) . A s i n these ear l ier studies, the tracer 1 3 N was used, s ince its l o w detection l im i t s a l l o w for excellent t ime resolut ion i n short-duration eff lux experiments (see S i d d i q i et a l . , 1991; W a n g et a l . , 1993a; K r o n z u c k e r et a l . , 1995a). It i s be l i eved that the combined use o f perturbational and non-perturbat ional treatments i n the present study prov ides good evidence that the compartments seen i n this type o f eff lux analysis are (I) a f i l m o f solut ion adhering to the root surface, (II) the adsorpt ive component o f the c e l l w a l l (the Donnan free space), and (III) the cy toplasm. 86 6.2. Results. 6.2.1. Phase Regression and Half-lives of Exchange. Three kinetically distinct efflux phases were distinguishable by linear regression of semi-logarithmic plots of the 13N-efflux rate versus elution time. Figure 9 shows a representative plot obtained with intact spruce seedlings at 1.5 mM [NIL/],,. The plot includes regression lines (r2 = 0.91-0.99) for the three phases. Half-lives of exchange (tt/i) for these phases were determined from the slopes of the regression lines after data transformation to natural logarithm. Mean t,A values were 2.4 s (phase I), 26.7 s (phase II), and 14.7 min (phase III). No significant difference was found in these half-lives for seedlings grown at 100 itM or 1.5 mM [NH4+]0 pr after pretreatment with MSO, a-KG or with different levels of external Ca2+ or pH (Tables 12 a, b, c). However, a significant decline in tA values, by as much as 60-80%, was noted for phase III following the treatments with SDS, 75°C (Table 12 a), and Al 3 + (Table 12 c). 6.2.2. Flux Estimations. For seedlings grown and measured at 100 ttM [NH4+]0, unidirectional NH 4 + influx (0TC) was determined to be ~ 1.9 itmol g"1 h"1 (Table 13 a) by compartmental analysis. Efflux of NIL/ from root tissue under those conditions was close to 25% of influx (see <t>co). No more than 3% of the total 1 3N taken up by the plants was translocated to the shoot within the time-frame of the experiments (see ^xykJ, while as much as 97% of the incoming NIL/ was either channeled into metabolism or was sequestered in the vacuole (#ass/vac). The denaturing treatment of plant roots at 75 °C and with solutions containing 1% SDS led to substantial decreases in all parameters, 87 6.5 6H 2-| , , , , 1 0 5 10 15 20 25 elution time [min] Figure 9. Representative semi-logarithmic plot of the rate of release of 1 3N [log (cpm released) g"1 min1] versus time of elution in roots of intact white spruce seedlings. Seedlings were maintained at 1.5 mM [NH4+]0. The plot includes linear regression lines and equations for the three phases resolved in efflux analysis. 88 Table 12 a. Half-lives of exchange (tlj4) for NH.+ of phases I, II, and III (assumed to represent surface film, Donnan free space, and cytoplasm, respectively) in roots of spruce seedlings grown at 100 ttM [NH4+]0 and following various (pre)treatments as indicated. Data + SE (n=3-9). (pre)treatment: phase I phase II phase III t.A [s]: t,A [s]: tA [min]: 100 ttM NH 4 + 2.22 30.84 15.08 (control): + 0.38 + 2.5 ± 1.25 100 ttM NH 4 +/ 2.31 28.64 12.72 + 1 mM MSO: ± 0.55 ± 2.71 + 0.75 100 ttM NH 4 +/ 2.05 24.7 19.82 + 1 mM a-KG: + 0.67 ± 5.7 ± 4.11 100 ttM NH 4 +/ 2.24 43.73 6.49 + 1% SDS: ± 0.49 + 12.54 ± 2.78 100 ttM NH 4 +/ 3.34 29.68 3.29 + 75°C: ± 0.78 + 3.09 ± 2.99 89 Table 12 b. Half-lives of exchange (t,A) for NH 4 + of phases I, II, and III (assumed to represent surface film, Donnanfree space, and cytoplasm, respectively) in roots of spruce seedlings grown at 100 uM [NH4+]0 and (pre)treated at various external Ca2+ concentrations. Data ± SE (n=3-9). (pre)treatment: phase I phase II phase III t.A [s]: 1* [s]: tvi [min]: 100 uM NH 4 +/ 2.26 22.39 15.48 + 50 t t M Ca2+: ± 0.57 ± 6.6 ± 0.05 100 t t M NH 4 +/ 1.24 14.27 17.71 + 500 i t M Ca2+: ± 0.83 ± 3.61 ± 3.39 100 i t M NH 4 +/ 2.48 20.18 15.12 + 5 mM Ca2 +: + 0.13 ± 2.5 + 0.43 90 T a b l e 12 c . H a l f - l i v e s o f exchange (t1 / t) for N H 4 + o f phases I , I I , and III (assumed to represent surface f i l m , Donnan free space, and cy toplasm, respectively) i n roots o f spruce seedlings g r o w n at 1.5 m M [ N H 4 + ] 0 and f o l l o w i n g var ious (pre)treatments as indica ted . D a t a + S E (n=3-9 ) . (pre)treatment: phase I phase II phase III 1* [s]: t% [s]: tvi [m in ] : 1.5 m M N H 4 + 2 .66 29 .78 10.92 (control) : ± 0 .08 ± 0 .19 ± 1.67 1.5 m M N H 4 + / 3.1 29 .44 14.65 + 1 m M M S O : + 0 .65 + 2 .79 + 2 .16 1.5 m M N H 4 + / 2 .31 23 .14 10.42 + p H 3 .6 : ± 0.11 + 12.54 + 2 .35 1.5 m M N H 4 + / 1.99 23.01 6 .19 + 1.5 m M A l 3 + : ± 0 .94 + 5.7 + 1.17 91 Table 13 a. NH 4 + fluxes as estimated from compartmental analysis (for symbols see Materials and Methods). Plants were maintained and measured at 100 uM [NH4+]0. (Pre)treatments were as indicated. *<£xyiem is expressed in [nmol g"1 h"1] and represents the flux of 1 3N rather than NH 4 +. Data + SE (n=3-9). (pre)treat-ment: NH 4 + fluxes |>mol g1 h1]: <t>co- ^ass/vac 0 xylem : control 1.89 0.46 1.42 1.38 44.8 (100 fiM): ± 0.14 ± 0.05 ± 0.14 + 0.13 ± 17.9 + MSO: 1.51 0.64 0.87 0.87 1.2 + 0.11 ± 0.07 ± 0.11 + 0.11 ± 0.3 + a-KG: 1.11 0.19 0.91 0.9 15.6 ± 0.12 ± 0.03 ± 0.09 ± 0.09 ± 3.1 + SDS: 0.51 0.42 0.09 0.08 9.7 ± 0.19 ± 0.06 + 0.01 ± 0.01 + 2.1 + 75 °C: 0.59 0.5 0.09 0.08 10.1 + 0.22 + 0.06 + 0.01 + 0.01 + 2.6 92 except <£co, which was apparently unaffected by these treatments (Table 13 a). Exposure of seedling roots to a 1 mM solution of the GS/GOGAT-inhibitor MSO for a period of 6 h prior to loading led to a slight depression of ^  (about 20%), while <f>co was enhanced by almost 40%. <£net, therefore, was significantly lower than in control plants. (p^i^a was almost 40 times lower in MSO-pretreated plants than in controls. Treatment of seedling roots with 1 mM of the carbon source a-KG for 6 h caused an even larger (about 40%) depression of 4>octnan w a s seen with MSO (Figure 10). However, in this case, <pco was depressed proportionately even more (about 60%) than which led to a higher <px to $c0 ratio than in control plants (5.8 versus 4.1; with MSO treatment this ratio was 2.4). Interestingly, cp^^ was also lowered by a-KG treatment, almost threefold compared to control plants. By contrast, in statistical analyses of slopes and Y-intercepts of efflux plots obtained in experiments on seedlings (pre)treated with varying concentrations of external Ca2+, no significant differences were found for transmembrane flux parameters at the 0.05 level of probability (Table 13 b). However, 1 3NH 4 + efflux from the free space to bulk solution and estimates of cell-wall [NIL,"1"] were diminished as [Ca2+]0 was increased (Figure 11). Plants grown and measured at 1.5 mM [NH4+]0 exhibited <px values around 6.6 ttmol g"1 h"1 (Table 13 c). Treatment of these plants with 1 mM MSO for 6 h had no significant effect on 4>x, but 4>co increased more than twofold (Figure 12). Consequently, $net was decreased by about 35% with respect to control plants. 4>xy\tm w a s decreased by as much as 27-fold. Even more pronounced reductions in (p^^ were observed after treating seedling roots at pH 3.6 or at 1.5 mM Al 3 + (which also lowered solution pH to 3.6). These latter treatments also effected substantial reductions in (p^ and <£co. ^ depression was around 86% at pH 3.6 and «91 % with Al 3 + (Table 13 c). Because of differential effects on <£co in the two treatments, $net was depressed to a similar extent in both cases, to a 'residual' rate of approximately 0.3 ttmol g"1 h"1. 93 Figure 1 0 . Combined semi-logarithmic plots of the rate of release of 1 3N [log (cpm released) g"1 min1] versus time of elution in roots of intact white spruce seedlings. Seedlings were maintained at 100 uM [NH4+]C. Experiment (1) represents plants (pre)treated with 1 mM a-KG for 6 h, experiment (2) represents control plants. Plots include linear regression lines and equations for the presumed cytoplasmic phases (phase HI). 94 Table 13 b. NH 4 + fluxes as estimated from compartmental analysis. Plants were maintained and measured at 100 iiM [NH4+]0 and were (pre)treated at the indicated external Ca2+ concentrations. Data + SE (n=3-9). See also Table 13 a. (pre)treat- NH 4 + fluxes [iimol g1 h1]: ment: Geo' <r>net: ^ass/vac- ^ xylem • + 50 iiM 1.82 0.33 1.5 1.44 58.3 Ca2+: ± 0.12 ± 0.06 ± 0.12 ± 0.12 ± 23.1 + 500 iiM 1.64 0.53 1.11 1.08 32.8 Ca2+: ± 0.13 + 0.06 + 0.11 ± 0.1 + 7.2 + 5 mM: 1.6 0.42 1.18 1.11 71.7 Ca2+: ± 0.33 ± 0.08 ± 0.26 ± 0.24 + 12.2 95 Figure 11. Combined semi-logarithmic plots of the rate of release of 1 3N [log (cpm released) g"1 min"1] from phase II versus time of elution in roots of intact white spruce seedlings. Seedlings were maintained at 100 iiM [NFL/],,. Experiment (1) represents plants (pre)treated at 5 mM [Ca2+]0 for 6 h, experiment (2) represents plants (pre)treated for 6 h at 50 uM [Ca2+]0. Plots were obtained after subtraction of counts eluting from phase III and include linear regression lines and equations for the presumed Donnan free space (phase II). Phase I is not included. 96 T a b l e 13 c . NIL," 1" f luxes as estimated f rom compartmental analysis for plants maintained and measured at 1.5 m M [ N H 4 + ] 0 . (Pre)treatments were as indicated (see also T a b l e 12 c ) . D a t a ± S E ( n = 3 - 9 ) . See also Tab le 13 a. (pre)treat- N H 4 + f luxes [/tmol g 1 h 1 ] : ment: <r>c0: 4>nei- *r^ ass/vac- ^xylem : con t ro l 6.63 1.82 4.81 4 .56 246 .4 (1.5 m M ) : + 0 .34 + 0 .08 + 0 .26 ± 0 .26 + 0 .9 + M S O : 6.99 3.84 3.15 3.14 9.01 + 0 .15 + 0.18 ± 0.2 ± 0 .2 ± 2 .8 + p H 3 .6 : 0 .93 0 .62 0.31 0.3 2 .03 ± 0 .27 + 0.13 + 0.04 ± 0 .04 + 0.8 + A l 3 + : 0 .58 0.25 0.33 0 .32 1.6 + 0 .13 + 0.1 + 0.001 + 0 .002 + 0 .2 97 6.5-x [• 6-< 5.5H CD (n CO 0 CD c o o o CD CD 4.5H • control (1) x + 1 mM M S O (2) >x • C3< O 3.5-Y (2) = X (-0.02) + 4.28 Y (1) = X (-0.02) + 4.09 10 15 elution time [min] 20 25 Figure 12. Combined semi-logarithmic plots of the rate of release of 1 3N [log (cpm released) g"1 min"1] versus time of elution in roots of intact white spruce seedlings. Seedlings were maintained at 1.5 mM [NH4+]0. Experiment (1) represents control plants, experiment (2) represents plants (pre)treated with solutions containing 1 mM MSO for 6 h. Plots include linear regression lines and equations for the presumed cytoplasmic phases (phase III). 98 6.2.3. Compartmental Concentrations. Assuming 5% tissue volume for the average root cell cytoplasm and 10% for the tissue volume occupied by the cell-wall free space (see Kronzucker et al. 1995a, b, c), NH 4 + concentrations for these two compartments were calculated from the 1 3NH 4 + contents in phases II and III (Tables 14 a-c). [NH4+]cyt for spruce seedlings grown at 100 uM [NH4+]Q was ~ 13 mM, while [NH4+]free s p a c e was approximately 1.2 mM. Interestingly, (pre)treatment of seedling roots with MSO and a-KG both led to a decrease in [NH,"1"]^  of about 30% (see Figure 10), whereas [NH4+]free s p a c e was not altered significantly (Table 14 a). Addition of 1 % SDS or pretreatment at 75 °C dramatically reduced the value for 1 3NH 4 + effluxing from the presumed 'cytoplasm', to 12% of control values in SDS-treated roots and as little as 7% after high temperature treatment. By contrast, [NH4+] freespace was not changed by these manipulations (Table 14 a). Increasing external Ca2+ had no significant effect on [NH4+]cyt, but reduced estimated [NH4+] f reespace (Figure 11) by approximately 30% when [Ca2+]0 was increased from 50 uM to 500 itM and by almost 80% at 5 mM [Ca2+]Q with respect to the control at 50 ttM (Table 14 b). Plants grown at 1.5 mM [NH4+]0 accumulated NH 4 + to levels around 35 mM in the cytoplasm and to about 8.8 mM in the free space (Table 14 c). An approximate 40% increase in [NH4+]cyt was observed in these plants after exposure of roots to 1 mM MSO for 6 h (Figure 12). [NH4+]frec s p a c e appeared to be slightly enhanced, but this increase was statistically insignificant. Lowering solution pH from 6.5 to 3.6 decreased [NH4+]cyt by 92% and reduced free-space NH 4 + binding by «30% (Table 14 c). This presumed free-space binding was decreased even further (to less than one fourth of the control value) by addition of 1.5 mM Al 3 + to pretreatment and loading solutions. The Al 3 + treatment decreased [NH4+]cyt to less than 2 mM (Table 14 c). 99 T a b l e 14 a . Compar tmen ta l concentrations o f NFL,"1" i n spruce root compartments as estimated b y compar tmenta l analysis at 100 uM [ N F L / h and after var ious (pre)treatments (for details see M a t e r i a l s and M e t h o d s ; see also Tab le 12 a). D a t a + S E (n=3-9). (pre)treatments: compartmental N H 4 + concentrations [ N H 4 + ] f r e e s p a c e ( m M ) : [ N H 4 + ] c y t o p l a s m ( m M ) : con t ro l (100 itM): 1.22 13.44 ± 0.23 + 1.51 + M S O : 1.51 9.24 + 0.16 + 1.35 + a-KG: 1.41 9.91 + 0.18 + 0.89 + S D S : 1.39 1.59 ± 0.34 ± 0.78 + 75°C: 1.22 0.93 + 0.2 + 0.47 100 Table 14 b. Compartmental concentrations of NFL,"1" in spruce root compartments as estimated by compartmental analysis at 100 itM [NFL,"1"], and at various external Ca2+ concentrations (for details see Materials and Methods). Data + SE (n=3-9). (pre)treatments: compartmental NH 4 + concentrations [NH4+] freespace (mM): [NH4+]cytoplasm (mM): 100 uM NH 4 +/ 1.16 13.52 50 uM Ca2+: ± 0.2 ± 2.42 100 uM NH 4 +/ 0.84 11.35 500 uM Ca2+: ± 0.19 + 1.74 100 itM NH 4 +/ 0.25 11.63 5 mM Ca2+: ± 0.02 ± 2.53 101 Table 14 c. Compartmental concentrations of NIL/ in spruce root compartments as estimated by compartmental analysis at 1.5 mM [NH4+]0 and after various (pre)treatments (for details see Materials and Methods; see also Table 12 c). Data + SE (n=3-9). (pre)treatments: compartmental NH 4 + concentrations [NH4+] freespace (mM): [NH4+]cytoplasm (mM): control (1.5 mM): 8.82 34.71 ± 0.59 ± 1.99 + MSO: 10.47 48.59 ± 2.63 ± 6.84 + pH 3.6: 6.42 2.86 ± 1.67 ± 0.36 + Al 3 + : 2.04 1.73 + 1.31 ± 0.06 102 6.3. Discussion. It is w e l l recognized that data extraction f rom eff lux analysis can have serious shor tcomings (Cheeseman, 1986; Z i e r l e r , 1981). In addi t ion to prerequisites w h i c h are c o m m o n to a l l systems w h i c h have been explored by this methodology (see b e l o w ) , the conifer roots w h i c h have been used i n the present study are characterized b y somewhat greater tissue d ivers i ty than i s t yp ica l of , for example , young cereal roots (see Rt id inger et a l . , 1994; M c K e n z i e and Peterson, 1995a, b ; K r o n z u c k e r et a l . , 1995d). In studies us ing s ingle-cel led organisms, the idea l si tuation i s met and parameters obtained f rom analysis represent means for re la t ive ly homogeneous ce l l s . B y contrast, when used to analyze compartmental characterist ics i n complex organs such as roots, der ived parameters must reflect the means o f several c e l l types. Y e t , i f t ime constants for eff lux differed substantially among c e l l types, eff lux curves w o u l d be expected to reflect this heterogeneity. A failure to dis t inguish addi t ional phases i n the present analyses leads to the conc lus ion that t ime constants do not differ s ignif icant ly among c e l l types (see also po in t V be low) but o n l y among the ' in-ser ies ' compartments discussed. Est imates o f k inet ic constants and tissue concentrations should therefore be taken as representing average values for the system under study. In addi t ion, the f o l l o w i n g prerequisites for eff lux analysis should be satisfied un iversa l ly : (I) T h e tissue must be at steady state for the duration o f the exper iment . (II) T h e specif ic ac t iv i ty o f the tracer i n the subcellular compartment(s) to be invest igated must be the same as o r ve ry close to that o f the outside solut ion p r i o r to the onset o f e lu t ion ; load ing w i t h tracer must therefore occur for at least four to f ive times the tA values o f the respective compartment(s) ( C r a m , 1968). 103 (III) The tracer for the ion under study must be taken up at the same rate as the non-labelled ion (i.e., isotope discrimination at the uptake step, as known for several tracers, must be taken into account; West and Pitman, 1967; Jacoby, 1975; Behl and Jeschke, 1982). (IV) Incoming tracer must be well mixed in the compartment(s) under study to make calculations of compartmental concentrations possible. (V) If several compartments are under study, these must be arranged in series (Pitman, 1963; Cram, 1968, 1975), and their t,A values must be sufficiently different to allow resolution by linear regression of a semi-logarithmic efflux plot (Cheeseman, 1986). (VI) If metabolized ions are to be investigated, metabolism needs to be taken into account, either by subtraction of the metabolized fraction when using a standard plot of tissue tracer content versus elution time (see, e.g., Bell et al., 1994) or by plotting the rate of tracer release from the tissue versus elution time (Lee and Clarkson, 1986). (VII) The assignment of linear phases in semi-logarithmic efflux plots to actual subcellular compartments must be tested. While most of these issues are usually adequately addressed in efflux studies, the importance of testing for compartment identity (prerequisite VII) has been largely ignored, with exceptions of a few preliminary attempts (Cram, 1968; Lee and Clarkson, 1986; Siddiqi et al., 1991; Kronzucker et al., 1995a). An untested a priori approach has been taken in most studies, leading, in some cases, to considerable discrepancies in the literature concerning parameters derived from efflux analysis (see e.g. the summary of t,A values for N03" by Devienne et al., 1994, or for NH 4 + by Wang et al., 1993a). Yet it is clear, if any of the derivative flux or pool size calculations are to be valid, that a knowledge of compartment identity is imperative. For this reason, the purpose of the experiments described in this section of the thesis was to 104 substantiate the assignment of logarithmic phases seen in 13NH4+-efflux data to the corresponding subcellular compartments. As in our earlier study of NIL,"1" exchange in white spruce (Kronzucker et al., 1995c), three kinetically distinct phases were found. These were tentatively assigned to a film of solution adhering to the root surface, to binding sites in the cell wall, and to the cytoplasm (see Figure 9). This interpretation was consistent with ealier studies from this laboratory (Siddiqi et al., 1991; Wang et al., 1993a; Kronzucker et al., 1995a, b). In the present study, treatments were designed to selectively influence NFL/ exchange in either the (presumed) cytoplasmic phase, or the (presumed) cell-wall phase. For example, pretreating plant roots at 75° C or in 1 % (w/v) SDS solution was anticipated to selectively reduce plasma membrane fluxes and thus cytoplasmic [NH4+]. The same rationale dictated the choice of MSO and a-KG exposures. In contrast, varying external cation concentrations could be expected to selectively affect cell-wall-exchange properties. These treatments included varying concentrations of Ca2 +, H + , and Al 3 + . The latter two treatments also affected cytoplasmic parameters. However, together with results from the Ca2+ treatments, confirmation of the cation exchange nature of phase II was possible. High-temperature pretreatment of plant roots as well as treatment with SDS have been previously used by Siddiqi et al. (1991) and by Kronzucker et al. (1995a) in 13N03"-efflux studies of barley and spruce, respectively. Both of these studies used a 22-min elution time, and reported a substantial decrease of I 3N release from phase III; phases I and II remained unaffected. Since high-temperature and detergent treatments disrupt the lipid bilayer of the plasma membrane, and denature or solubilize membrane proteins, respectively, it was concluded that phase III probably represented the cytoplasm. Similarly, in an early study of Cl" exchange in carrot-root tissue, Cram (1968) used chloroform-killing of cells to distinguish between 'intracellular' and 'extracellular' binding. In an earlier study on N03" exchange in spruce 105 (Kronzucker et al., 1995a), seedlings were also treated with H202 and 2-chloro-ethanol to perturb a membrane-bound and metabolically-dependent compartment. The results were in agreement with the assumption that phase III corresponded to the cytoplasm and that therefore, by elimination, phases I and II represented extracellular binding. In the present NIL/ study in spruce a similar reduction was found in 1 3N exchange of phase III following applications of high temperature or SDS, yet no effect on phases I and II. These results are consistent with a cytoplasmic identity for compartment III. More subtle explorations of the identity of compartment III were achieved by exposures to MSO and a-KG. MSO is a known inhibitor of glutamine synthetase (GS) (see, e.g., Wedler and Horn, 1976; Meister, 1981; Monselise and Kost, 1993), the primary NH4+-assimilating enzyme located in the cytoplasm. Significant increases in cytoplasmic NIL/ concentrations (up to 90 mM) after MSO pretreatment in root cells of other plant systems have been observed by nuclear magnetic resonance (NMR) (Lee and Ratcliffe, 1991) and inferred from labelling kinetics (Fentem et al. 1983a, b). It has also been determined that MSO does not influence exchange characteristics with the cell wall (Lee and Ayling, 1993). Figure 12 illustrates that the effect of a 6-h MSO pretreatment of seedling roots was limited to phase III. However, while at 1.5 mM [NIL/],, an expected increase in [NH4+]cyt was observed after exposure to MSO (Table 14 c), [NH4+]cyt actually decreased by approximately 30% (Table 14 a) at 100 ttM [NIL/],,. This apparent discrepancy is explained by a discrete effect of MSO not only at the enzyme level, but also at the level of plasma membrane NH 4 + transport. Given the chemically analogous structure of the MSO molecule to that of glutamine, a 'recognition' of MSO as a glutamine analogue by either influx or efflux transport proteins is conceivable. While an effect consistent with this assumption has normally not been observed (see Lee and Ayling, 1993), both depression of NFL/ influx and enhancement of NFL/ efflux, apparently caused by direct MSO effects on NFL/ transport, have 106 been documented in Sorghum bicolor L. (Feng et al., 1994). On the enzyme level, it is known that MSO can act as an inhibitor not only of glutamine synthetase, but also of glutamate synthase (GOGAT) (Gauthier, 1983; Takashi et al., 1983) as well as asparagine synthetase (Monselise and Kost, 1993). The present results (Tables 13 a and c) show that the rates of unidirectional influx and efflux of NFL,"1" were clearly altered by MSO. Efflux was significantly enhanced both in plants maintained at 1.5 mM [NH4+]0 and at 100 uM [NH4+]0. This is seen clearly in the marked increase of the Y-intercept of the 'cytoplasmic' regression line in an MSO (pre)treatment experiment compared to a control experiment without MSO (Figure 12). In addition to an enhancement of <pco, was depressed following MSO pretreatment in plants maintained at 100 uM [NH4+]0, an effect not observed at 1.5 mM [NH4+]C. This led to a marked increase in the 4>J4><K ratio, and to the observed decrease in [NH4+]cyt in the roots of these plants (Table 14 a). These findings, while initially surprising, are in keeping with the widely documented negative-feedback role of glutamine upon NH 4 + uptake (see Wang et al., 1993b, and Kronzucker et al., 1995b, for references), if MSO is able to mimic the feedback role of glutamine on the transporter level. It is not inconsistent with these assumptions that the putative negative-feedback effect caused by this glutamine analogue be different at 100 uM than at 1.5 mM [NH4+]0, since distinct high-affinity (HATS) and low-affinity (LATS) transport systems operate at these two concentrations (Wang et al., 1993b; Kronzucker et al., 1995e). Such discriminating effects of MSO on separate systems of NFL/ uptake and on the concentration of free NFL/ in phase III, combined with the fact that the characteristics of phases I and II are not affected, provides strong support for the assumption that phase III indeed represents the cytoplasmic compartment. Pretreatment of seedling roots with a-ketoglutarate (a-KG) led to similar results in terms of phase identification. a-KG should alleviate possible carbon limitation to the assimilation of NH 4 + and thereby alter [NH4+]cyt and plasmalemma fluxes of NH 4 +, while having no effect on 107 the binding of NH 4 + in the cell wall. The provision of a-KG failed to alter exchange characteristics of phases I or II, but decreased the concentration of NIL/ in phase III (Figure 10), the putative cytoplasmic phase. While this was most certainly attributable to enhanced rates of NIL,"1" assimilation, a depression of NIL/ influx also contributed to the effect (Table 13 a). It is well established that the availability of carbon skeletons to the roots, both endogenous in the roots as well as supplied via the shoot, affects rates of N uptake (Michael et al., 1970; see also Monselise and Kost, 1993, and Rideout et al., 1994). In the present study, an additional 'regulatory' role of a-KG was evident in an effect on the partitioning of NFL/-assimilation products between root and shoot. Significantly reduced rates of (Table 13 a) indicate that transport of products of 1 3N assimilation to the shoot was reduced under these conditions. Changes in the allocation of NIL/- assimilation products to root and shoot in response to changing carbon abundance in the roots have been documented by others (see Talouizte et al., 1984; Champigny and Talouizte, 1986; Tolley-Henry and Raper, 1986; Henry and Raper, 1991). Both the observed effects on 0^  and on (p^^ in the present study argue for a possible feedback role of a-KG on the uptake and assimilation of NFL/, as well as the allocation pattern of assimilation products. It remains undetermined, however, whether the 'a-KG effect' is, in fact, caused by a-KG directly or whether it is mediated via some down-stream NFL/-assimilation product, such as glutamate or glutamine. More importantly, however, from the perspective of compartment identification, the results of the a-KG experiments appear to substantiate the assignment of phase III to the root-cell cytoplasm. In previous attempts to characterize compartment III as membrane-bound and metabolically active, the identities of the remaining two phases were deduced by elimination to correspond to extracellular compartments. In the present study of NIL/ exchange, the assumptions regarding phases I and II were tested more directly, by varying the cation 108 composition of the preloading and loading solutions, utilizing the different capacities for ionic binding to cation exchange matrices exhibited by Al 3 + , H + , and Ca2+ compared to NH 4 +. Since the previous tentative interpretation of phase II in NFL/-exchange kinetics was that of the Donnan free space in the cell wall, cations capable of competing with NH 4 + for these binding sites were anticipated to selectively affect 1 3NH 4 + exchange with phase II. According to the lyotropic series for cations (the 'Hofmeister' series), the strength of adsorptive binding to non-specific cation exchange matrices should decrease in the order: Al 3 + > H + > Ca2+ > NFL/ (see, e.g., Brady, 1974). The above cations significantly reduced NH 4 + binding in phase II. Figure 11 shows this effect on phase II for Ca2+ in an overlay graph, which was obtained after correction for specific activity and subtraction of counts eluting from phases I and III. While phase I remained unaffected by all manipulations, treatment at low pH and with Al 3 + also affected characteristics of phase III. This was evident in changes in the plasmalemma fluxes of NFL/ and in [NH4+]cyt (see Tables 13 c and 14 c). The results from H + and Al 3 + experiments are therefore only useful in combination with results obtained in experiments using Ca2 +, which, for the range of 50 itM to 5 mM, did not seem to perturb phase III (Tables 13 b and 14 b). While the half-lives of exchange for phase II remained statistically unchanged with the cation treatments (Tables 12 b and c), significant differences in Y-intercepts indicated that the NH 4 + content of that phase was significantly altered by cation competition. Displacement of NFL/ from phase II by Al 3 + , H + and Ca2 + was exactly consistent with expectations of the lyotropic series. Furthermore, as [Ca2+]Q increased, the [NFL/] of phase II decreased correspondingly (Table 14 b). Thus, the data are in excellent agreement with the assumption that phase II represent a cation-exchange matrix. Together with the earlier findings that phase II was neither membrane-bound nor metabolically dependent, the interpretation of phase II as the Donnan free space in the cell wall is supported 109 substantially. Phase I was neither affected by the earlier treatments designed to alter phase III nor by the cation variations. Rather, the NH 4 + exchanging with that phase seemed to be exclusively dependent upon the specific activity of the loading solution. This suggests, along with the very short half-life of exchange for that phase, that it reflects a film of solution adhering to the root surface, possibly also including the water free space (W.F.S.) of the cell wall. The absence of a phase which might be identified as the water free space suggests that either the latter is kinetically indistinguishable from the Donnan free space (D.F.S.) or that it is indistinguishable from the rapidly exchanging surface film. A priori, the former seems unlikely, whereas a half-life of 2-3 s for the W.F.S. may appear too short. However, Siddiqi et al. (1991) were able to detect 13N03" in the shoots of barley after root exposure to labelled solution for as little as 10 s. Such rapid (apparently apoplasmic) movements of ions may indicate that equilibration of the W.F.S. is faster than might have been formerly anticipated. 110 7. N03-INFLUX STUDY: KINETICS OF NITRATE INFLUX TN SPRUCE. 7.1. Introduction. Nitrate uptake in higher plants is well characterized on the kinetic level (Clarkson, 1986). There is general agreement in the literature that the dependence of N03" uptake on [N03"]o can be resolved into at least two kinetically distinct systems (Clarkson and Luttge, 1991; Glass and Siddiqi, 1995). Most kinetic experiments on N03" uptake have been performed in cereal species and have been based on measurements of chemical depletion rates of N03~ from nutrient solutions, i.e. the determination of N03" net flux (Neyra and Hageman, 1975; Rao and Rains, 1976; Doddema and Telkamp, 1979; Breteler and Nissen, 1982; Pace and McClure, 1986; Warner and Huffaker, 1989; Aslam et al., 1992). While kinetic inferences from net flux studies have to be approached with caution, studies using radiotracers to determine the unidirectional influx of N03" into root tissue have also confirmed a (biphasic) pattern for N03~ uptake (Siddiqi et al., 1990). In the majority of studies, N03" uptake appeared to be mediated by a high- affinity transport system (HATS), which operated in a Michaelis-Menten-type fashion at [NO/],, < 1 mM, and by a low-affinity transport system (LATS), which operated in a linear fashion at [N03']0 > 1 mM. Only a few workers reported apparently different or more complex patterns (Doddema and Telkamp, 1979; Breteler and Nissen, 1982). The N03"-uptake system in higher plants is unusual in that it is subject to induction by external nitrate (Minotti et al., 1969; see also Kronzucker et al., 1995b, for references), i.e. N03" influx and net flux are considerably enhanced following initial exposure of roots to solution N03". Several workers have compared the concentration-dependence of N03" uptake in induced and uninduced states. Uninduced plants exhibited a constitutive high-affinity transport system 111 (CHATS) with saturable kinetics (Lee and Drew, 1986; Behl et al., 1988; Klobus et al., 1988; Siddiqi et al., 1989; Hole et al., 1990; Siddiqi et al., 1990). Because of significant differences in K,,, (Lee and Drew, 1986; Warner and Huffaker, 1989; Aslam et al., 1992) and response to metabolic poisons (Jackson et al., 1973), CHATS was considered a genetically discrete system from the saturable high-affinity transport system (IHATS) apparent in the induced state (Clarkson, 1986). In some plants, the CHATS and IHATS were found to operate concurrently in the induced state (Warner and Huffaker, 1989; Aslam et al., 1992), while in others this was not obvious (Siddiqi et al., 1990). In contrast to the large body of experimental work on cereal species, our knowledge of N03~-uptake kinetics in conifer species is rudimentary, in spite of the enormous ecological and economic importance of such species. Only a small number of N03"-depletion studies in conifers have been reported (Peuke and Tischner, 1991; Kamminga-van Wijk and Prins, 1993; Plassard et al., 1994) and have provided no conclusive results as to the identity of the transport systems or their operation at different states of induction. It is possible that the poor growth response of many conifers on soils with N03" as the predominant source of nitrogen (see General Introduction) can be attributed to a lower capacity of the N03" transport system in conifers than usually is characteristic of the more-studied cereals. Thus, the purpose of this section of the thesis is to characterize the kinetics of the N03" uptake system in white spruce. The radiotracer 1 3N was used to conduct direct measurements of unidirectional N03" influx as functions of [N03"]0 and the duration of exposure to external N03". 112 7.2. Results. 7.2.1. Time Profile of Induction of Nitrate Influx by External Nitrate. Measured N03" influx of N-deprived seedlings was enhanced with increased time of exposure to external N03" for up to 72 h (Figs. 13 and 14). In the uninduced state, influx measured at 200 tiM was 0.1-0.15 timol g"1 h"1. Peak influx (0.6-0.7 timol g"1 h"1) was reached after 3 d of induction (Fig. 14), after which point influx declined to a value of 0.3 timol g"1 h"1 by 7 d. This value at 7 d corresponded to the net flux of NCV measured by chemical depletion under steady-state conditions (data not shown). This time course of nitrate influx was evident on a whole-root basis as well as in separately analyzed young root material (Fig. 14). In plants induced by exposure to 1.5 mM [N03~]0 (Fig. 15), the induction pattern of N03" influx (measured at 1.5 mM [N03"]o) was similar. Influx ranged from a constitutive level of 0.3 ttmol g"1 h"1 to a fully induced value of 1.2 timol g"1 h"1. Maximal fluxes occurred earlier, at day 2, for plants exposed to 1.5 mM than for plants at 200 ttM [N03"]0 (cf. Figs. 14 and 15). 7.2.2. Low-Concentration Systems for Nitrate Uptake. In uninduced seedlings, a saturable N03"-influx system was apparent in the concentration range from of 2.5 to 500 11M [N03"]0 (Fig. 16 A), yet at 1 mM [N03"]0 influx had increased to almost double the saturated rate. The saturable low-concentration phase conformed to Michaelis-Menten kinetics. The kinetic parameters of and K,,, for this system were determined by several data transformations and an additional least-squares method (Table 15; see also Materials and Methods). Estimates for K,,, obtained via these methods ranged from 13.6 to 21 tiM, while V,,^ estimates were between 0.11 to 0.13 umol g"1 h"1. 113 Figure 13. Nitrate influx into roots of white spruce as a function of shorter-term exposure to external N03". Seedlings were cultivated in N-free solutions for 3 weeks and then supplied with 200 uM [N03"]0 for the indicated time periods and during the 10-min flux measurements. Data ± SE (n > 9). 114 2 18H time of exposure to 0.2 mM nitrate [d] Figure 14. Nitrate influx into roots of white spruce as a function of longer-term exposure to external N03" at 200 uM (see Fig. 13). Influx was determined both on a young-root basis (upper curve) and on a whole-root basis (lower curve). Data + SE (n > 9). 115 1.4 time of exposure to 1.5 mM nitrate [d] Figure 15. Nitrate influx into roots of white spruce as a function of time of exposure to external N03- at 1.5 mM (flux measurements were also at 1.5 mM [N03"]0). Data ± SE (n > 9). 116 Figure 16 A. Nitrate influx into roots of uninduced white-spruce seedlings as a function of [N03-]o in the low-concentration range (2.5-1000 uM). Data ± SE (n > 9). 117 Table 15. K,,, and V,,,^  values for the constitutive high-affinity transport system (CHATS) for N03" in roots of white spruce as estimated by different mathematical methods. Seedlings were cultivated hydroponically without N for three weeks and exposed to external N03" only during the 10-min influx period. An influx isotherm constructed from data pooled from several experiments was used as the basis for calculation of the kinetic parameters (see Materials and Methods). calculation method K.O1M] [/xmol g1 h"1] r2 Lineweaver-Burk 13.63 0.11 0.91 Eadie-Hofstee 15.85 0.11 0.81 Hanes-Wolf 21.04 0.13 0.98 Cornish-Bowden 16.96 0.12 — 118 In seedlings that had been fully induced for N03" influx by exposure of the roots to 100 itM [N03"]0 for a period of 3 d, the low-concentration response of influx was more complex (Fig. 16 B). Rather than consisting of a single saturable phase, low-concentration N03 influx appeared to result from two saturable components, one operating at [N03~]0 < 75 itM and another operating in the 100 to 750 itM range of [N03~]0. Beyond 750 itM influx again increased beyond the saturated levels. Despite the fact that this biphasic pattern in the low-concentration range was already evident in the influx isotherm, it was confirmed by significance testing of the slopes and Y-intercepts of the regression lines for the presumed different components in linear-transformation plots of the influx data according to Lineweaver-Burk (p < 0.01 for slopes; p < 0.025 for intercepts), Eadie-Hofstee (p < 0.005 for slopes; p < 0.0005 for intercepts; see Fig. 17), and Hanes-Wolf (p < 0.005 for slopes; p < 0.05 for intercepts). Kinetic analyses of the two saturable phases yielded K m values of 11.1 to 16.8 itM for the first phase and of 98.8 to 153.2 fiM for the second phase (Tables 16 and 17). V,^ estimates were 0.27 to 0.32 itmol g"1 h 1 for the first component and 0.7 to 0.82 itmol g"1 h"1 for the second. 7.2.3. High-Concentration System for Nitrate Uptake. In both uninduced and induced seedlings an additional linear uptake system operated at higher [N03"]0. It was evident at [N03"]0 of 1 mM in uninduced plants (Fig. 16 A) and at [N03"]0 > 750 itM in plants fully induced for N03" uptake (Fig. 16 B). The system was additive to the low-concentration systems. The concentration response of the system between 1 and 50 mM [N03"]0 showed linearity for the entire range (r2 = 0.97; Fig. 18). 119 Figure 16 B. Nitrate influx into roots of white spruce seedlings as a function of [NCVL in the low-concentration range (2.5-1000 uM). Seedlings were induced for N03" uptake by 3-d exposure to 100 itM [N03]o. Data ± SE (n > 9). 120 0.8 6 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 influx/concentration [L/g/h] Figure 17. Eadie-Hofstee transformation of the data for induced high-affinity transport of NCy in white spruce (Fig. 16 B) in the 2.5-750 itM range of [N03"]0. Regression lines and linear equations are included for components I and II (see text). The slopes of the two lines (-KJ and the intercepts with the Y-axis ( V ^ were significantly different as evaluated by Student's t-test (p < 0.005 for slopes; p < 0.0005 for intercepts). 121 Table 16. K,,, and V,^ values for component I of inducible high-affinity influx of NO3" into roots of white spruce as estimated by different mathematical methods. Seedlings were induced for N03 uptake by exposure to 100 uM [N03"]0 for 3 d and were then exposed to various [N03~]0 in concentration-dependence experiments (10-min influx periods). See also Table 15. calculation method K ^ M ] [/xmol g1 h1] r2 Lineweaver-Burk 16.83 0.32 0.97 Eadie-Hofstee 12.89 0.28 0.75 Hanes-Wolf 11.12 0.27 0.97 Cornish-Bowden 14.73 0.3 — 122 Table 17. K,,, and values for component II of inducible high- affinity influx of N03" into roots of white spruce as estimated by different mathematical methods (see Tables 15 and 16 for details). calculation method K ^ M ] [ttmol g1 h1] r2 Lineweaver-Burk 153.17 0.82 0.9 Eadie-Hofstee 101.06 0.7 0.75 Hanes-Wolf 98.8 0.7 0.99 Cornish-Bowden 112.39 0.73 — 123 nitrate concentration [mM] Figure 18. Nitrate influx into roots of uninduced white-spruce seedlings as a function of [NCVL in the high-concentration range (1-50 mM). The results of linear regression are included in the graph (r2 = 0.97). Data ± SE (n > 9). 124 7.3. Discussion. 7.3.1. Time Profile of Transporter Induction. Rates of N03" uptake are considerably enhanced by exposure to external N03~, a process usually referred to as nitrate induction (Minotti et al., 1969; Goyal and Huffaker, 1986; Behl et al., 1988). Flux increases of at least five to ten times are typically observed (Warner and Huffaker, 1989), but enhancements by as much as 30-fold have been recorded (Siddiqi et al. 1989). Most workers have recorded a lag phase of several hours before induction was apparent (Clarkson, 1986; Warner and Huffaker, 1989), although in some studies a more or less immediate response was found (see Tischner et al., 1993). In the present experiments with spruce, induction of influx was clearly apparent after 3 h (Fig. 13); however, the induction response was unusually slow in that up to 3 d were required for maximal induction. This was obvious both at the level of young as well as whole root material (Fig. 14). This finding of slow induction kinetics in spruce, which is in agreement with earlier compartmental-analysis results in spruce (Kronzucker et al., 1995b), contrasts sharply with the time required for maximal inductive flux by other species (Glass and Siddiqi, 1995). In barley, however, the time required for maximal induction decreases as [N03"]0 increases (Siddiqi et al., 1989). Similarly, the induction response in spruce appeared to be accelerated at higher [N03"]0 (cf. Figs. 13, 14, 15). Nevertheless, spruce remains the slowest responding species so far investigated. This, together with the rather low inductive enhancement factor (only about five-fold) for V,,^ in the high-affinity range, may be a competitive disadvantage for spruce seedlings compared to nitrophilous species, in soil habitats poor in N sources other than N03" (see Kronzucker et al. 1995a, b). 125 7.3.2. Constitutive High-Affinity Transport System. Plants that have not been exposed to N03" prior to flux measurement, i.e. are not induced for N03" transport, typically display low rates of N03" uptake. This uptake in the uninduced state has been termed constitutive and is believed to be mediated by a specific saturable high-affinity transport system (Behl et al., 1988; Warner and Huffaker, 1989; Hole et al., 1990; Siddiqi et al., 1990). Reported I^ s for CHATS in higher plants fall into a range from 1 itM (Breteler and Nissen, 1982) to 20 uM (Siddiqi et al., 1990). The K,,, estimates of « 15 uM for white spruce are within this range of literature values. Despite the low capacity of the CHATS, it is clearly essential in order to absorb (catalytic?) N03" in sufficient quantities to induce the inducible HATS (IHATS) (Glass and Siddiqi, 1995). 7.3.3. Inducible High-Affinity Transport System. Most studies of N03" uptake have focused on the inducible N03"-transport system operating in the low-concentration range of external nitrate (< 1 mM [N03"]0) in induced plants. The system active in that range has typically been found to exhibit saturable kinetics from about 200 to 500 itM [N03]0 and has been referred to as the inducible high-affinity transport system (IHATS) for nitrate and has been demonstrated in numerous plant systems (see Glass and Siddiqi, 1995). So far, the existence of a saturable IHATS has been demonstrated in Arabidopsis (Doddema and Telkamp, 1979), barley (Rao and Rains, 1976; Bloom, 1985; Lee and Drew, 1986; Konesky et al., 1989; Siddiqi et al., 1990; Aslam et al., 1992), buckwheat (Paulsamy and Chrungoo, 1994), corn (van den Honert and Hooymans, 1955; Neyra and Hageman, 1975; Pace and McClure, 1986; Hole et al., 1990), rice (Youngdahl et al., 1982), ryegrass (Lycklama, 126 1963), wheat (Goyal and Huffaker, 1986; Botelia et al., 1994), sunflower (Aguera et al., 1990) and squash (Wieneke, 1992). The K^ s reported for IHATS in these species range from 7 to 187 tiM (Bloom, 1985; Aslam et al., 1992). In conifers, kinetic analyses of N03" uptake in the high-affinity or the low-affinity range are scarce. From concentration-dependence data obtained through chemical depletion protocols, Peuke and Tischner (1991) calculated a K,,, value of 200 tiM and a Vm a x of 18 timol g"1 d"1 (approximately 0.7-0.8 timol g"1 h"1) for N03" uptake in Norway spruce, and Kamminga-van Wijk and Prins (1993) reported values of 17 tiM for K„, and » 0.5 to 1 timol g"1 h"1 (our calculations) f° r Vmax m Douglas-fir. Plassard et al. (1994), also using depletion data, communicated a K,,, value of 120 tiM and a V,,^ of 0.55 timol g"1 h"1 for N03 uptake in non-mycorrhizal maritime pine. Since plants in all three studies were exposed to external N03" for some time before uptake rates were determined, the reported values do not represent kinetic parameters of unidirectional influx. Nevertheless, these published values of V,,^ are in reasonably close agreement with the present estimates (Table 16). The estimates for IC^ , however, are not in close agreement. Only Lineweaver-Burk transformations were used in the above studies to provide estimates for the kinetic parameters. Since Lineweaver-Burk plots may yield biased results, in particular regarding K,,, (Dowd and Riggs, 1965; Cornish-Bowden and Wharton, 1988), in the present study several mathematical methods were employed for the derivation of K,,, and values. The relatively good agreement between the values obtained by these different methods helped to confirm the validity of the Michaelis-Menten formalism in our data. The different methods of data transformation establish that N03" influx in induced spruce consisted of two distinct saturable components in the low-concentration range. The possibility that this pattern could arise from different populations of roots cells was ruled out by separately analyzing the kinetic patterns of young, old, and whole root tissues (see Materials and Methods). 127 T h e double-saturat ion response, albeit w i t h l o w e r V , ^ values i n o l d than i n young roots, was apparent i n both root tissues (data not shown). B o t h saturable components confo rmed to M i c h a e l i s - M e n t e n kinet ics (Tables 16 and 17), and therefore separate values for K m and V , ^ are g i v e n . W h i l e such a pattern o f induced in f lux has usual ly not been observed (see, e . g . , S i d d i q i et a l . , 1989, 1990), the simultaneous operation o f a low-K, , , and a h i g h -Kn , system i n the induced high-aff in i ty state has been recently demonstrated through a c lea r ly b i m o d a l L i n e w e a v e r - B u r k t ransformation o f net uptake data i n barley ( A s l a m et a l . , 1992). for component I was 7 uM, and was 36 uM for component II . S i m i l a r results cou ld be seen i n ear l ier data b y Brete ler and N i s s e n (1982) for d w a r f bean and W a r n e r and Huf fake r (1989) for 'Steptoe ' bar ley . Interest ingly, the apparent affinities o f both components for N 0 3 " were m u c h greater i n bar ley and bean than i n spruce, po in t ing to a re la t ively poorer adaptation to this N source i n the conifer (see Gene ra l Introduct ion) . In the present study, a l l l inear- t ransformation methods used con f i rmed that the assumed differences between the two saturable components were statistically s ignif icant (see F i g . 17). H o w e v e r , un l ike the study by A s l a m et a l . (1992) , the double-saturation pattern o f in f lux was evident even i n the isotherms wi thout data t ransformation ( F i g . 16 B ) . It i s poss ib le that, because o f much larger in f lux enhancement f o l l o w i n g induc t ion i n cereal species than i n spruce (see K r o n z u c k e r et a l . , 1995b), the l ow-K, , , component may be h idden i n the former but m u c h more c lear ly v i s ib l e i n the latter. A l s o , i f data resolut ion i n the range o f component I is not sufficient, indiscr iminate data regression o f the entire range may reflect the k ine t ic parameters o f the more dominant component II (see P lassard et a l . , 1994). It has been argued that the l o w - K n , component may correspond to the const i tut ive system ( C H A T S ) be ing expressed simultaneously wi th the induc ib le system ( I H A T S ) ( A s l a m et a l . , 1992; Glass and S i d d i q i , 1995). In agreement w i t h A s l a m et a l . (1992), the present data show that even the l o w - K n , component o f in f lux appeared to be induced s igni f icant ly . W h i l e the K„, 128 estimates for CHATS and component I of induced high-affinity influx are very close (» 15 itM), suggesting that the transport proteins may be identical, was about three times as high as the constitutive value after induction. Similarly, Aslam et al. (1992) found that for CHATS increased from 0.82 itmol g"1 h"1 in the uninduced state to 3 itmol g"1 h"1 after exposure to N03" in barley. Therefore, while component II of induced high-affinity influx undoubtedly represents an induction response, component I is also clearly inducible, notwithstanding the fact that it may represent the same transport system earlier identified as CHATS. 7.3.4. Low-Affinity Transport System. The present results establish that, beyond the saturable high-affinity components at lower [N03"]0, N03" influx across the root plasmalemma of spruce followed a linear pattern at [N03"]0 > 500-750 i t M (Fig. 18). This linear system was present in both uninduced and induced seedlings (data only shown for uninduced seedlings) and appeared to be additive to the high-affinity components, as in other plant systems (Siddiqi et al., 1990; Aslam et al., 1992). In the present data (cf. Figs. 4 A and B), the transition from the saturable to the linear phase was visible earlier (at « 500 i t M [N03]0) in uninduced plants than in induced plants (at « 750 i t M [N03"]0). This is in agreement with studies in cereal species (Siddiqi et al., 1990; Aslam et al., 1992). A dual pattern of N03" uptake (i.e. saturable and linear), as observed in the present study for spruce, has been recorded in several species, including the diatom Skeletonema costatum (Serra et al., 1978), Arabidopsis thaliana (Doddema and Telkamp, 1979), several varieties of barley (Siddiqi et al., 1990; Aslam et al., 1992) and corn (Pace and McClure, 1986). By contrast, it has been claimed that N03~ uptake in spruce is limited to a saturable system and that 129 a linear system is not expressed even at [N03~]0 up to 10 mM (Peuke and Tischner, 1991). Plassard et al. (1994) also reported a lack of substantial increases of N03" uptake rates beyond the saturable low-concentration component in maritime pine. It has to be noted, however, that the studies from which these conclusions have been drawn were based on the measurement of chemical depletion of N03" from solution and that the plants employed in these studies were grown in N03"-containing media for extended periods of time. As pointed out earlier, such measurements can only provide estimates of N03" net flux rather than influx. Influx at high [N03"]0 may be obscured by efflux of N03" from the root tissue and by physiological changes occurring in the plants during the extended durations of measurement. Moreover, it is known that the linear low-affinity transport system (LATS) responds markedly to tissue N-status and that its slope is significantly depressed in plants grown at high compared to low [N03"]Q (Siddiqi et al., 1990). In white spruce, this effect was evident at different stages of induction, i.e. LATS was more pronounced in uninduced plants than in plants previously exposed to external N03" for 3 d. It is conceivable, therefore, that in plants cultivated under conditions of high [N03"]Q, LATS may be indistinguishable from the saturation plateau of HATS. However, the present study establishes clearly the presence of a low-affinity transport system, which under perturbation conditions mediates a linear increase in N03" influx up to 50 mM [N03"]0 (Fig. 18). 130 8. N i l / - I N F L U X STUDY: KINETICS O F A M M O N I U M INFLUX IN S P R U C E . 8.1. Introduction. Early studies on NFL/ uptake in corn (Becking, 1956; van den Honert and Hooymans, 1961), wheat (Tromp, 1962), and ryegrass (Lycklama, 1963) established that net uptake of NFL/ could be described using the Michaelis-Menten formalism of enzyme kinetics. The existence of such a Michaelis-Menten-type uptake system, operating at [NFL/]0 < 1 mM, was later confirmed in barley (Bloom and Chapin, 1981; Mack and Tischner, 1994), corn (Vale et al., 1988), wheat (Goyal and Huffaker, 1986; Botelia et al., 1994), rice (Youngdahl et al., 1982; Wang et al., 1993b), Lemna (Ullrich et al., 1984), tomato (Smart and Bloom, 1988; Kosola and Bloom, 1994), Phalaris and Glyceria (Brix et al., 1994), as well as in several algal systems (see Glass and Siddiqi, 1995, for references). This saturable uptake component has been termed the high-affinity transport system (HATS) for NIL/ (Wang et al., 1993b). K m values for this system in the various species commonly range from approximately 10 to 170 uM (see Glass and Siddiqi, 1995), but values as low as 1.6 uM have been reported (Brix et al., 1994). At higher [NH4+]C (> 500 itM), the operation of a linear system was also observed in barley (Mack and Tischner, 1994), corn (Vale et al., 1988), rice (Wang et al., 1993b) and Lemna (Ullrich et al., 1984). This linear system has been referred to as the low-affinity transport system (LATS) for NIL/ and was found to be additive to the saturable HATS component (see Wang et al., 1993b). In soybean, however, a multiphasic NH4+-uptake system was reported to operate over the entire range of [NH4+]0, with three distinct saturable phases, but no linear component (Joseph et al., 1975). In previous studies (Kronzucker et al., 1995a, b, c, e) a marked preference for NIL/ over N03" was established in white spruce, and it was pointed out that the ecological as well as 131 practical implications in deforestation and reforestation of boreal forests may be considerable (cf. Kronzucker et al., 1995a). Yet, in conifers, a detailed kinetic characterization of NIL,"1" uptake is lacking. The author is aware of only one study, which reported kinetic parameters for net NFL,"1" uptake in Douglas-fir (Kamminga-van Wijk and Prins, 1993). The purpose of the present study was to kinetically define NFL/ influx into seedling roots of a conifer species. The radiotracer 1 3 N was employed to conduct direct measurements of NFL/ influx as a function of [NFL/] 0 . Moreover, the time profile of the response of NFL,"1" influx to external N H 4 + provision was recorded, and changes in kinetic parameters of NFL/ influx with previous exposure of seedling roots to external N0 3" were examined. The present study further defines the pronounced preference for NFL/ over N0 3" in spruce. 8.2. Results. 8.2.1. Time Profile of Ammonium Influx. Measured N H 4 + influx was substantially higher in seedlings which had been deprived of N for a period of three weeks than those maintained under steady-state conditions of external N H 4 + supply (Fig. 19). Upon first exposure of seedling roots to 100 uM [NFL/] 0, influx was 1.6 jtimol g"1 h 1 and declined to a steady value around 1 jumol g"1 h"1 after approximately 4 d of continued exposure to the same concentration of external NFL/. This steady rate of influx was maintained until 7 d of exposure, the longest exposure period in the present experiments. Only at day one did the plants exhibit a rate of influx which was approximately 12% higher than the initial rate measured in the N-deprived state. However, this increase was transient, and influx measured at day two was already below the initial rate. 132 2 0.8H 1 1 1 1 1 1 -i 1 1 -1 0 1 2 3 4 5 6 7 8 time of exposure to 0.1 mM ammonium [d] Figure 19. Ammonium influx into roots of white spruce as a function of exposure to external N H 4 + for up to 7 d. Seedlings were cultivated in N-free solutions for 3 weeks and then supplied with 100 uM [NH 4 +] 0 for the indicated time periods and during the 10-min flux measurements. Data ± SE (n > 9). 133 8.2.2. Concentration-Dependence of Ammonium Influx. In N-deprived seedlings, a single saturable NH^-infiux system was apparent in the concentration range from 2.5 to 350 uM [ N H / L (Fig- 20). At 500 /xM [NH 4 + ] 0 , influx had increased 20 to 30% beyond the saturated rate, and at 1 mM [NH 4 + ] 0 influx was two to three times that rate. The saturable low-concentration system was constitutive and conformed to Michaelis-Menten kinetics. The kinetic parameters of V, ,^ and K,,, for this influx system were determined by several linear data-transformation and a least-squares method as described in Materials and Methods. The estimates for K,,, ranged from 19.8 uM to 41 uM, while V , ^ estimates were between 1.86 to 2.44 /xmol g"1 h"1 (Table 18). The lowest estimates for both Km and V, ,^ were obtained by Lineweaver-Burk transformations, whereas Hanes-Wolf transformations consistently yielded estimates biassed towards higher values. Notwithstanding a commonly lower r 2 value in regressions of Eadie-Hofstee plots compared to either Lineweaver-Burk or Hanes-Wolf plots (Table 18), the Eadie-Hofstee estimates were closest to those arrived at by the least-squares method according to Cornish-Bowden and Wharton. At [ N H 4 + ] 0 > 1 mM, a separate constitutive high-concentration influx system was evident. It was apparently additive to the saturable low-concentration system and showed linearity up to 50 mM [NH 4 + ] 0 (Fig. 21). In seedlings which had been previously exposed to external N03~ for 3 d, NH4+ influx was also resolved into two kinetically distinct systems, a Michaelis-Menten-type low-concentration system and a linear high-concentration system (Fig. 22). However, influx in the low-concentration range (up to 500 /xM [NH 4 +] 0) was 20 to 40% higher in N03~-supplied as opposed to N-deprived plants. The difference for was highly significant (p < 0.005), as illustrated for Hanes-Wolf-transformed data (Fig. 23). By contrast, influx beyond 1 mM [NH 4 + ] D 134 6 0-1 1 1 1 1 1— 1 1 1 " 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 ammonium concentration [mM] Figure 20. Ammonium influx into roots of N-deprived white-spruce seedlings as a function of [NH 4 + ] 0 in the low-concentration range (2.5 uM to 1 mM). Data ± SE (n > 9). 135 Table 18. and values for the high-affinity transport system (HATS) for NIL,"1" in roots of white spruce as estimated by different mathematical methods. Seedlings were cultivated hydroponically without N for three weeks and exposed to external NIL,+ only during the 10-min influx period. An influx isotherm constructed from data pooled from several experiments was used as the basis for calculation of the kinetic parameters (see text). calculation method Ka.t/xM] r^ mol g1 h1] r2 Lineweaver-Burk 19.79 1.86 0.93 Eadie-Hofstee 24.42 2.07 0.86 Hanes-Wolf 41.00 2.44 0.99 Cornish-Bowden 25.81 2.14 — 136 45-r 40-ammonium concentration [mM] Figure 21. Ammonium influx into roots of N-deprived white-spruce seedlings as a function of [NH 4 + ] 0 in the high-concentration range (1-50 mM). Regression line included (r2 = 0.96). Data ± SE (n > 9). 137 C H 1 1 1 1 • — i 0 0.2 0.4 0.6 0.8 1 1.2 ammonium concentration [mM] Figure 22. Overlay plot of ammonium influx in the low-concentration range (2.5 uM to 1 mM) for N-deprived white-spruce seedlings (empty symbols) and seedlings which were exposed to 100 uM [N03"]0 for 3 d prior to NH -^flux measurements (filled symbols). Data ± SE (n > 9). 138 -0.1 0 0.1 0.2 0.3 0.4 0.5 ammonium concentration [mM] Figure 23. Hanes-Wolf transformation of the data for high-affinity NE!/ transport of N-deprived (1) and N03-supplied (2) white-spruce seedlings (Fig. 22) in the 2.5-500 itM range of [NH4+]0. Regression lines and linear equations are included for the two data sets (see text). The slopes of the two lines (1/V^ were significantly different as evaluated by Student's t-test (p < 0.005), while the intercepts with the Y-axis (J^/V,,^ were not significantly different. 139 was substantially lower in N03"-supplied than in N-deprived plants. Already at 1 mM [NH4+]0 influx, after subtraction of the respective values for of the low-concentration systems, was 2.7 times larger in N-deprived than in NCy-supplied seedlings (2.56 iimol g"1 h"1 compared to 0.96 fimol g"1 h1, respectively). 8.3. Discussion. 8.3.1. Enhanced Ammonium Influx in N-deprived Plants. The results show that rates of NH 4 + uptake in spruce were considerably larger when seedlings were first exposed to external NH 4 + after three weeks of N-deprivation than steady-state rates of uptake (Fig. 19). After a transient maximum of influx at day 1 of NFL/ provision (see section 8.3.3.), influx subsequently declined (days 2-7) to a steady value. This finding is in agreement with several other studies in higher-plant species (Ivanko and Ingversen, 1971; Lee and Rudge, 1986; Morgan and Jackson 1988a, b; Henriksen et al., 1992; Jackson and Volk, 1992; Mack and Tischner, 1994). Typically, initial fluxes are in the order of two to three times the steady rates (Bowman and Paul, 1988; Lee et al., 1992). This increased absorptive capacity is commonly observed before growth rates are affected by the N-deprivation. Similar responses have been documented for potassium, phosphorus, sulphur, chloride and bromide, when plants were deprived of these ions (cf. Lee and Rudge, 1986, for references). Following the initial maximum in NH 4 + uptake, its rate usually declines progressively with continued time of exposure to external NH 4 +. This gradual repression of NFL/ uptake is most likely due to a build-up of negative feedback effectors on NFL/ uptake, such as cytoplasmic and vacuolar NFL/ as well as assimilation products (Ullrich et al., 1984; Lee et al., 1992; Wang et al., 1993b; Kronzucker et al., 1995c). Likewise, the initially high rate of uptake can probably be explained 140 by release from negative feedback in N-deprived plants as well as initially low efflux of NH/. It has been suggested (Mack and Tischner, 1994) that the initially high rate of uptake corresponds to the filling of the apparent free space. However, the pronounced temperature dependence of this flux (Jackson and Volk, 1992) is at variance with this hypothesis, as is its disappearance in N-pretreated plants, regardless of whether N was supplied as N03" or as NIL/ (Volk and Jackson, 1992; Mack and Tischner, 1994). 8 . 3 . 2 . S teady-s ta te A m m o n i u m I n f l u x . Following the initially high NFL/ influx in N-deprived plants when NH 4 + is resupplied, a steady rate of uptake is commonly achieved within 5 to 10 h of NFL/ resupply in cereals and soybean (Goyal and Huffaker, 1986; Morgan and Jackson, 1988b; Jackson and Volk, 1992; Mack and Tischner, 1994). In white spruce, with the exception of a peak at day 1 of NH/ provision (see following section), NFL/ influx gradually declined following provision of external NH 4 + at 100 ttM to previously N-deprived seedlings (Fig. 19). Compared to other species where this has been investigated (see above), the initially high influx value was only marginally enhanced over that achieved at steady state following down-regulation (»1.6 times higher than steady state as opposed to 2-4 times in other systems). Also, the decline in influx to the lower steady value around 1 itmol g"1 h"1 required 3 to 4 d of exposure to external NFL/. A slower and smaller response to changes in N supply compared to other higher-plant species has also been reported for the induction of N03" transport in spruce (Kronzucker et al., 1995b). Nevertheless, as in the case of N03" induction, the basic form of the time-dependent NH4+-influx response in previously N-deprived spruce seedlings closely resembles that documented in other species. 141 8.3.3. Is Ammonium Uptake Inducible by External Ammonium? In the present experiments, a transient peak of NIL/ influx was apparent after 24 h of resupplying external NH 4 +, before fluxes began to decrease towards a steady level (Fig. 19). In several other studies, such an additional increase in the NH4+-uptake rate was noted in the first hours of resupplying NFL/ (Goyal and Huffaker, 1986; Morgan and Jackson, 1988a; Jackson and Volk, 1992; Mack and Tischner, 1994). In these systems, NFL/ uptake reached a new maximum after approximately 5 h. The increase was either visible as a transient peak followed by a more or less immediate decline of uptake rates (Goyal and Huffaker, 1986; Morgan and Jackson, 1988a; Jackson and Volk, 1992) or was sustained for at least 3 h (Mack and Tischner, 1994). This observation has been taken as evidence for an 'inductive' effect of external NIL/ on NH 4 + uptake, analogous to the induction of N03" uptake by external N03" (cf. Kronzucker et al., 1995b). However, the enhancement of NFL/ uptake which has been observed by those workers was considerably less than the increase of N03" influx associated with the induction of N03" uptake, where up to 30-fold increases have been observed in some species (Siddiqi et al., 1989; see also Kronzucker et al., 1995b, for references). Moreover, Jackson and Volk (1992) have demonstrated that exposure to N03" also evoked the transient increase of NIL/ uptake. Therefore the response is not specific for NH 4 +, in contrast to the induction of N03" uptake by external N03" (cf. Kronzucker et al., 1995b). Wang (1994) also found that K + influx was enhanced in rice as NIL/ influx reached its peak. It appears more plausible that plants transiently respond positively to the increased N availability, before negative-feedback effectors accumulate to appreciable levels and lead to a subsequent repression of NIL/ transport (see previous paragraph). In this regard it is interesting that glutamine, a popular candidate for the feedback effector (Clarkson and Liittge, 1991), has been shown to reach peak levels 142 approximately 5 to 10 h following NH^-resupply to previously N-deprived cereals (Amancio and Santos, 1992; Lee and Lewis, 1994), whereas in conifers this accumulation is more gradual and more than 24 h are commonly required (Vezina et al., 1992; Lavoie et al., 1992). It is well-known that the magnitude of NIL,+ uptake correlates negatively with tissue NIL,"1" (Wang et al., 1993b), glutamine and other NH4+-assimilation products (Lee and Rudge, 1986; Morgan and Jackson, 1988b; Lee et al., 1992). The present evidence therefore appears to argue against the ' N H 4 + induction' hypothesis and rather for an improved N status prior to the establishment of negative-feedback control as an explanation for the transient flux increase. 8.3.4. Concentration-Dependence of A m m o n i u m Influx (Transport Systems). Compared to the large number of higher-plant influx studies for other macronutrient ions, such as N03" (see Kronzucker et al., 1995e), the kinetics of N H 4 + uptake have remained understudied (Wang et al., 1993b; Kosola and Bloom, 1994). Moreover, with only a few exceptions (see Glass and Siddiqi, 1995), previous kinetic studies of NFL,"1" uptake have been largely based upon the determination of depletion of NFL/ from solution. Data obtained by such experiments fail to distinguish between influx and efflux and may be confounded by biological acclimation occurring during the relatively long measurement times. Particularly when measurements occur over hours or even days, the application of Michaelis-Menten kinetics is questionable (see Kronzucker et al., 1995d). Furthermore, fluxes measured during long-term studies represent the averages of diurnal changes as well as responses to other environmental variables and to changes of internal nutrient status. Therefore, derivations of kinetic parameters from such experiments must be approached with caution. At best, such values have descriptive rather than mechanistic meaning. 143 The kinetic pattern of NH 4 + influx in white spruce was clearly resolved into two distinct phases. As has been observed in several other higher-plant species (see Introduction), influx displayed saturable kinetics at [NH4+]0 < 500 ttM (Figs. 20 and 22). The system could be described using the Michaelis-Menten formalism of enzyme kinetics (Table 18), and its calculated K,,, and V,^ values are within the range of reported literature values (Glass and Siddiqi, 1995). In accordance with other studies it has been termed the high-affinity transport system (HATS) for NH/ (Wang et al., 1993b). Since it was fully expressed in N-deprived seedlings (Fig. 20), HATS was constitutive in white spruce, and clearly responded to tissue N status. This was evident as a 30% increase in and a 20 to 30% increase of influx values at any given concentration below 500 ttM [NH4+]0, when seedlings had been exposed to 100 ttM N03" for 3 d prior to NH4+-influx determinations. Statistically, this increase was highly significant (Fig. 22). Similar to the transient peak of NFL/ influx which was observed in the present time-dependence study (see above), this flux increase is believed to result from an improved N status of the plants after N03" provision compared to the N-deprived state. Resupplying N-deprived plants with N03" may lead to a recovery of photosynthetic activity in the shoot, which in turn may lead to increased synthesis of NFL,"1" transporters or enhanced supply of carbohydrates to the roots, thereby increasing NH 4 + influx (see Saravitz et al., 1994). Obviously, negative feedback upon NH 4 + influx, which is evident when N is resupplied in the form of NIL/ (see Fig. 19, and Kronzucker et al., 1995c), is not occurring when N is administered in the form of N03". Since it is well established that the reduction of N02" and the assimilation of N02~-derived NH 4 + in roots of higher plants is compartmentally sequestered in proplastids (see Kronzucker et al., 1995b, for references), it is possible that negative feedback on NH 4 + influx may not be manifest even after 3 d of N provision. While cytosolic N03" is known to rise substantially in spruce after 3 d of N03" supply, the accumulation of the 144 presumably critical negative-feedback effectors for NH 4 + uptake, i.e. cytosolic NH 4 +, glutamine, or possibly other reduced N compounds (see above), may have been bypassed or at least delayed. In addition, total N uptake from external N03" was much lower than from external NH 4 +, thus requiring much longer for negative-feedback effects to be expressed. At [NH4+]0 > 500 itM, an additional NH4+-influx system was evident in white spruce. Like HATS, it was constitutive (Figs. 20 and 21) but followed a linear pattern, with no indication of saturation even at 50 mM [NH4+]0 (Fig. 21). A similar linear high-concentration system has also been documented in Lemna (Ullrich et al., 1984) as well as in several cereal species (Vale et al., 1988; Wang et al., 1993b; Mack and Tischner, 1994) and has been termed the low-affinity transport system (LATS) for NFL/. Unlike HATS, LATS in spruce was down-regulated in N03'-supplied as opposed to N-deprived seedlings. In fact, this down-regulation was so pronounced that already at 1 mM [NH4+]0, NIL/ influx was lower in seedlings which had been exposed to 100 itM [N03~]0 for 3 d than in N-deprived ones. This difference was visible even without subtraction of the respective Vm a x values for HATS (Fig. 22). The finding is similar to what has been reported for N03" in barley and spruce (Siddiqi et al., 1990; Kronzucker et al., 1995d), but different from the results by Wang et al. for NFL/ in rice (1993b), who found that LATS activity was enhanced by prior exposure to elevated levels of [NFL/]0. However, the rather long N-deprivation periods used in the latter study render a direct comparison with the present results difficult. Evidently, in spruce, HATS and LATS appear to be affected differentially in terms of negative feedback when N03" is used as an N source. While HATS responds positively to increased N availability, LATS was repressed. Since cytosolic NIL/ or amino acids cannot be expected to increase substantially upon N03" feeding (see earlier and Kronzucker et al., 1995b), the nature of the negative-feedback signal acting upon LATS is unclear. Possibly, shoot-to-root communication plays a role which does not affect HATS, at least 145 not at this 'early' stage of N resupply. 8.3.5. Preference for Ammonium over Nitrate in Spruce. Interestingly, measured NH 4 + influx in white spruce was substantially higher than N03" influx in the same species measured under comparable conditions (cf. Kronzucker et al., 1995a, b, c, d). In fact, when influx was determined in N-deprived seedlings upon first exposure to either NH 4 + or N03", V,,^ for NH 4 + influx was approximately 20 times larger than that for N03" influx (cf. Kronzucker et al., 1995d). The difference is particularly pronounced under these perturbation conditions, since NH 4 + influx is maximal in the N-deprived state (see above), whereas N03" influx requires induction by prolonged exposure to the external substrate prior to flux maximization. It was established previously that white-spruce seedlings required 3 d of exposure to external N03" (at 100 uM [N03"]Q) before an inductive influx maximum was reached (Kronzucker et al., 1995b, d). For this reason, the 3-d pretreatment of seedling roots at 100 uM [N03"]Q was included in the present study and NH4+-influx isotherms were determined under those conditions. When such rates were compared directly with fully induced rates for N03" influx (Kronzucker et al., 1995d), four- to five-fold higher V,^ values were still obtained for NH 4 + influx (« 3 /xmol g"1 h"1 as opposed to 0.7 /tmol g"1 h"1), while IC^  values were only marginally different. Therefore, the capacity for synthesizing transport systems appears much higher for NH 4 + than for N03". A preference of a similar magnitude for NH 4 + over N03" in conifers has also been documented by others (see General Introduction, and Kronzucker et al., 1995a and c, for references), and it was shown previously that this apparent discrimination against N03" in spruce is seen not only on the level of transport across the root plasmalemma, but also extends to the capacity for N03" reduction and subsequent metabolic processing 146 (Kronzucker et al., 1995a, b). Interestingly, mycorrhization of the roots, as would be expected for spruce seedlings in the field, has been shown to enhance NFL,"1" absorption rates in conifers but does not appear to significantly alter N03" uptake in spruce (see Kronzucker et al., 1995a, for references). The preference for.NH4+, as observed in non-mycorrhizal plant material in these studies, may therefore be even further accentuated in the field. It is, indeed, well established that many conifers exhibit a significantly better growth response on NH 4 + than on N03~ (see General Introduction). It is, however, common silvicultural practice in many parts of North America to outplant seedlings of conifer species like white spruce onto disturbed sites, where N03" is the predominant N source and NH 4 + is in short supply. At times considerable reforestation failures on such sites have occurred and have given rise to concern (cf. Lavoie et al., 1992, and Kronzucker et al., 1995a, c). It is clear from the present as well as the previous studies (Kronzucker et al., 1995a, b, c, d) that, for reasons of pronounced inherent differences in physiological utilization capacity for the NH 4 + and N03" sources of N, reforestation problems with species such as white spruce on disturbed sites are to be expected and should not surprise the forest practitioner. 147 9. CONCLUSION AND OUTLOOK The present studies have focused on the time and concentration-dependence profiles of N03" and NH 4 + transport as well as on the subcellular compartmentation of these ions in hydroponically grown white spruce in the seedling stage. Using the highly sensitive radiotracer 1 3N, detailed kinetic analyses of N03" and NH 4 + uptake into spruce roots were possible. In summary, the results encompass: I. Identification of three kinetically distinct transport systems for N03" in spruce: a. ) a constitutive high-affinity transport system (CHATS), operating in a saturable pattern up to 500 uM [N03"]0 in the uninduced state, i.e. without prior exposure of the plants to N03; b. ) an inducible high-affinity transport system (IHATS), operating in a bisaturable pattern at [N03"]0 < 1 mM, in the induced state, i.e. after prior exposure to N03"; c. ) a low-affinity transport system (LATS), operating in a linear pattern at [N03"]0 beyond 1 mM in both uninduced and induced plants; II. Identification of two distinct transport systems for NIL,"1": a. ) a high-affinity transport system (HATS), operating at [NH4+]0 < 1 mM in a saturable pattern; b. ) a low-affinity transport system (LATS), operating in a linear pattern at [NH4+]0 beyond 1 mM; HI. Characterization of the short- and long-term time profiles of N03" and NH 4 + uptake, establishing: a.) the existence of an induction response of spruce to external N03" like in other species; however, the induction response was slow (maximal fluxes were observed after 3 d of 148 exposure to external N03" as opposed to several hours in cereal species); this was also confirmed using in vivo nitrate reductase measurements; b.) the absence of such an induction response for NH 4 + (influx, with the exception of a small initial peak following resupply of NFL,"1" to N-deprived plants, did not increase but rather declined with increased exposure time to external NFL/; this appears to be a manifestation of negative feedback); Furthermore, the technique of compartmental analysis (efflux analysis) was applied to spruce seedlings, which made it possible to estimate the magnitude of N03~ versus NH 4 + accumulation in the root cytoplasm and the free space. Consistent with the flux data, it was found that: a. ) N03" accumulation in the cytoplasm under steady-state was very low (2 mM at 100 uM [N03"]0, compared to around 20 mM in barley at the same concentration of external N03); b. ) NH 4 + accumulation by contrast was about seven to ten-fold higher (15 mM at 100 iiM [NH4+]0, which is similar to what has been reported for rice; cf. Wang, 1994); In general, the technique of compartmental analysis allowed the concurrent monitoring of the unidirectional components of influx and efflux and the changing magnitudes of these components under varying conditions of external concentration and metabolic conditions. More specifically, also using efflux analysis, it was shown that spruce seedlings are an ideal model system to study the dynamics of nitrate-flux partitioning during the process of nitrate induction and to identify the nature of the exchange 'compartments' for N03" and NH 4 + which are evident in efflux data of the sort presented in this work. 149 In sum, beyond some more fundamental aspects, the present work has demonstrated that a clear preference for NFL,"1" over N03" as a nitrogen source exists at the uptake level (up to 20 times higher influxes under perturbation for NH 4 +, with a difference of four to five times remaining under steady state), as seen both in uptake isotherms (i.e. measurement of unidirectional influx) as well as in compartmental analysis for the two N species. It is believed that this pronounced preference for NH 4 + may constitute a significant stress factor to spruce seedlings if planted on soils poor in NH 4 + and rich in N03" (see General Introduction). It should be a future research goal, therefore, to investigate the possible (and likely) physiological implications of the stress associated with this N-source preference. Specifically, the response of N fluxes to varying light conditions should be examined and the stress status of seedlings grown on N03" or NH 4 + sources of N under different photon fluxes should be assessed. Since the increase in average and peak photon fluxes incident on freshly outplanted conifer seedlings upon complete removal of forest overstory, e.g. after clearcutting, represents perhaps the most critical ambient environmental change in addition to the discussed changes in the soil-N pool (Leibundgut, 1973; Mayer, 1984; Gnojek, 1992), a study of re-source/light interactions would be of particular importance. 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Trees 6: 236-240 Stams ATM, Marnette ECL (1990) Investigation of nitrification in forest soils with soil percolation columns. Plant and Soil 125: 135-141 Stewart GR, Gracia CA, Hegarty EE, Specht RL (1990) Nitrate reductase activity and chlorophyll content in sun leaves of subtropical Australian closed-forest (rainforest) and open-forest communities. Oecologia 82: 544-551 Sutton RF, Tinus RW (1983) Root and root system terminology. For Sci Monogr, No. 24 Swan HSD (1960) The mineral nutrition of Canadian pulpwood species. I. The influence of nitrogen, phosphorus, potassium and magnesium deficiencies on the growth and development of white spruce, jack pine and western hemlock seedlings grown in a controlled environment. Pulp and Paper Res Inst of Canada (Montreal) Tech Rep 168 Takashi T, Sung HC, Shinji W, Tatsurokuro T (1983) Purification and some properties of glutamate synthase from Gluconobacter suboxydans grown on glutamate as a nitrogen source. J Ferment Technol 61: 179-184 Talouizte A, Champigny ML, Bismuth E, Moyse A (1984) Root carbohydrate metabolism associated with nitrate assimilation in wheat previously deprived of nitrogen. Physiol Veg 22: 19-27 Thoiron A, Thoiron B, Demarty M, Thellier M (1981) Compartmental analysis of sulfate transport in Lemna minor L. taking plant growth and sulfate metabolism into consideration. Biochim Biophys Acta 644: 24-35 Tilman D (1987) Secondary succession and the pattern of plant dominance along experimental nitrogen gradients. Ecol Monogr 57: 189-214 Tills AR, Alloway BJ (1981) The effect of ammonium and nitrate nitrogen sources on copper uptake and amino acid status of cereals. Plant and Soil 62: 279-290 Tischner R, Waldeck B, Goyal SS, Rains WD (1993) Effect of nitrate pulses on the nitrate-166 uptake rate, synthesis of mRNA coding for nitrate reductase, and nitrate-reductase activity in the roots of barley seedlings. Planta 189: 533-537 Tolley-Henry L, Raper Jr CD (1986) Expansion and photosynthetic rate of leaves of soybean plants during onset of and recovery from nitrogen stress. Bot Gaz 147: 400-406 Tolley-Henry L, Raper CD Jr (1986) Utilization of ammonium as a nitrogen source. Effects of ambient acidity on growth and nitrogen accumulation in soybean. Plant Physiol 82:54-60 Tolley-Henry L, Raper CD Jr (1989) Effects of root-zone acidity on utilization of nitrate and ammonium in tobacco plants. J Plant Nutr 12: 811-826 Troelstra SR, Van Dijk K, Blacquiere T (1985) Effects of N source on proton excretion, ionic balance and growth of Alnus glutinosa (L.) Gaertner: comparison of N2 fixation with single and mixed sources of N03" and NH 4 +. Plant and Soil 84: 361-385 Tromp J (1962) Interactions in the absorption of ammonium, potassium, and sodium ions by wheat roots. Acta Bot Neerl 11: 147-192 Turner DP, Franz EH (1985) The influence of western hemlock and western redcedar on microbial numbers, nitrogen mineralization and nitrification. Plant and Soil 88: 259-267 Ullrich WR, Larsson M, Larsson C, Lesch S, Novacky A (1984) Ammonium uptake in Lemna gibba gl, related membrane potential changes, and inhibition of anion uptake. Physiol Plant 61: 369-376 Vaalburg W, Kamphuis JAA, Beerling-van der Molen HB, Reiffers S, Rijiskamp A, Woldring MG (1975) An improved method for the cyclotron production of 13N-labelled ammonia. Int J Am Rad Isot 26: 316-318 Vale FR, Volk RL, Jackson WA (1988) Simultaneous influx of ammonium and potassium into maize roots: kinetics and interactions. Planta 173: 424-431 Van Beusichem ML, Kirkby EA, Baas R (1988) Influence of nitrate and ammonium nutrition on the uptake, assimilation, and distribution of nutrient in Ricinus communis. Plant Physiol 86: 914-921 van den Driessche R (1971) Response of conifer seedlings to nitrate and ammonium sources of nitrogen. Plant and Soil 34: 421-439 van den Driessche R, Dangerfield J (1975) Response of Douglas-fir seedlings to nitrate and ammonium nitrogen sources under various environmental conditions. Plant Soil 42 : 685-702 van den Honert TH, Hooymans JJM (1955) On the absorption of nitrate by maize in water culture. Acta Bot Neerl 4: 376-384 167 van den Honert TH, Hooymans JJM (1961) Diffusion and absorption of ions by plant tissue. I. Observations on the absorption of ammonium by cut discs of potato tubers as compared to maize roots. Acta Bot Neerl 10: 261-273 Vernon LP, Zang WS (1960) Photoreduction by fresh and aged chloroplasts: Requirements for ascorbate and 2, 6-dichlorophenol indophenol with aged chloroplasts. J Biol Chem 235: 2728-2733 Vessey JK, Henry LT, Chaillou S, Raper Jr CD (1990) Root-zone acidity affects relative uptake of nitrate and ammonium from mixed nitrogen sources. J Plant Nutr 13: 95-116 Vezina L-P, Lavoie N, Jow KW, Margolis HA (1992) The fate of newly absorbed ammonium and nitrate ions in roots of jack pine seedlings. J Plant Physiol 141: 61-67 Vitousek PM, Gosz JR, Grier CC, Melillo JM, Reiners WA, Todd RL (1979) Nitrate losses from disturbed ecosystems. Science 204: 469-474 Vitousek PM, Matson PA, Van Cleve K (1989) Nitrogen availability and nitrification during succession: Primary, secondary and old-field seres. Plant and Soil 115: 229-239 Vitousek PM, Melillo JM (1979) Nitrate losses from disturbed forests: Patterns and mechanisms. Forest Sci 25: 605-619 Vogt M, Edmonds RL (1982) N03" and NH 4 + levels in relation to site quality in Douglas-fir soil and litter. Northwest Sci 56: 83-89 Walker NA, Pitman MG (1976) Measurement of fluxes across membranes. In U Liittge, MG Pitman, eds, Encyclopedia of plant physiology. Vol. 2, part A, Springer Verlag, Berlin, pp 93-126 Wang MY (1994) Ammonium uptake by rice roots. PhD thesis. University of British Columbia, Vancouver, Canada Wang MY, Siddiqi MY, Ruth TJ, Glass ADM (1993a) Ammonium uptake by rice roots. I. Fluxes and subcellular distribution of , 3NH 4 + . Plant Physiol 103: 1249-1258 Wang MY, Siddiqi MY, Ruth TJ, Glass ADM (1993b) Ammonium uptake by rice roots. II. Kinetics of 1 3NH 4 + influx across the plasmalemma. Plant Physiol 103: 1259-1267 Warner RL, Huffaker RC (1989) Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings. Plant Physiol 91: 947-953 Wedler G (1985) Lehrbuch der Physikalischen Chemie. 2nd ed. VCH Publishers, Deerfield Beach, Florida Wedler FC, Horn BR (1976) Catalytic mechanisms of glutamine synthetase enzymes. J Biol Chem 251: 7530-7538 168 West KR, Pitman MG (1967) Rubidium as a tracer for potassium in marine algae Ulva lactuca L. and Chaetomorpha darwinii (Hooker) Kuetzing. Nature 214: 1262-1267 White CS (1986) Volatile and water-soluble inhibitors of nitrogen mineralization and nitrification in a ponderosa pine ecosystem. Biol Fertil Soils 2: 97-104 Wieneke J (1992) Nitrate fluxes in squash seedlings measured with 1 3N. J Plant Nutr 15: 99-124 Wilson FJ, Skeffington RA (1994) The effects of excess nitrogen deposition on young Norway spruce trees: Part II. The vegetation. Environ Poll 86(2): 152-160 Youngdahl LJ, Pacheco R, Street JJ, Vlek PLG (1982) The kinetics of ammonium and nitrate uptake by young rice plants. Plant Soil 69: 225-232 Zierler K (1981) A critique of compartmental analysis. Ann Rev Biophys Bioeng 10: 531-562 169 APPENDIX I: DETERMINATION OF UNIDIRECTIONAL INFLUX INTO ROOTS OF HIGHER PLANTS: A MATHEMATICAL APPROACH USING PARAMETERS FROM EFFLUX ANALYSIS. In the recent literature the validity of measurements of unidirectional influx by means of tracers has been questioned (see Lee and Ayling, 1993). In this light, a new flux equation has been developed which is based on parameters obtained from efflux analysis. The mathematical approach presented here considers the influence of efflux on 'influx' determinations and allows for subtraction of the contribution of tracer bound in the adsorptive component of the cell wall. The latter consideration theoretically eliminates the need for a desorption period following the actual uptake step. Since the relative contributions of efflux and reabsorption of tracer specifically during desorption are difficult to determine and are therefore not usually quantified yet may significantly confound the results of uptake experiments (cf. Walker and Pitman, 1976), the possibility of modelling mathematically the error potential inherent in this step is believed to be of particular importance. The following parameters are used in the equation: (1) the kinetic exchange constant k (corresponding to 0.693/t,A) for the water free space (WFS) of the cell wall, obtained from efflux analysis and designated as k(WFS) (believed to be identical to that of the surface film; see Chapters 3 to 6); (2) the kinetic exchange constant for the more slowly exchanging adsorptive (Donnan) component of the cell-wall free space (AFS), obtained from efflux analysis and designated as k(AFS); 170 (3) the kinetic exchange constant for the cytoplasm, obtained from efflux analysis and designated as k(c); (4) the percentage of efflux with respect to influx, obtained from efflux analysis and designated as p e f f; (5) the concentrating factor of ions in the adsorptive phase of the cell-wall free space compared to the concentration in bulk solution, obtained from efflux analysis and designated as f^; (6) the percentage of root-tissue volume of the adsorptive-free-space component, obtained from literature values (cf. Lee and Clarkson, 1986) and designated as V ^ ; Derivation of the flux equation: The flux of an ion from an external compartment (o), such as bulk solution or free space, to an internal compartment (i), such as the cytoplasm of a cell, can be described as the quotient of the change of the ion content (Q) in the internal compartment over time (t). Such an 'influx' ( 0 o i ) can therefore be written as: 0oi = Q/t [/xmol g"1 h"1] (if Q is expressed on a mass rather than area basis); If a tracer (stable or radioactive) is used to measure the change in the internal ion content Q, the content of tracer in the internal compartment i (Q*) at any time t has to be used, and the chemical content Q is then obtained by dividing Q* by the specific activity of the tracer in the external solution (s0): <£oi = Q7(fs0) [timol g1 h-1]; 171 T h i s i s v a l i d for a unid i rec t ional f lux process i n the d i rec t ion o->i, and can be rearranged as: Q * - = 4>oi#s0#t [cpm g 1 ] ; (the subscript -* denotes that the internal tracer content is the result o f a un id i rec t iona l f lux process) ; M o r e rea l i s t i ca l ly , however , over the t ime periods usual ly used for ' i n f l u x ' determinations (e .g. 10 m i n ) , the f lux process i s , due to an efflux term (<j>io), at least b id i r ec t iona l . T h u s , by subtracting eff lux f r o m the internal compartment (wi th internal specif ic ac t iv i ty s-j, the express ion for Q * as a result o f b id i rec t iona l f lux becomes: Q / ~ = ( « o W t ) - .fowl) [cpm g" 1]; H e r e i n , eff lux can be expressed as a percentage o f in f lux ( p e f f # $ o i ) , w h i c h can be determined exper imenta l ly . Therefore: Q ~ = 4>oi-t#(s0 - Peff#Si) [cpm g 1 ] ; In i n f l u x exper iments , the internal compartment most often o f p h y s i o l o g i c a l interest is the cy top lasm (c) . I f load ing o f the cytoplasm w i t h tracer is assumed to occu r v i a the water free space ( W F S ) o f the c e l l w a l l , the equation can be rewri t ten: Q*c = </>oc#t#(SwFs - Peff#Sc) [cpm g 1 ] ; 172 Unidirectional influx from the external solution (or more exactly the water free space) into the cytoplasm can therefore be calculated from: 0oc = Q*~/((SWFS " Peff#Sc)#t) [/xmol g1 h1]; The specific activities of tracer in the cytoplasm and the water free space show an exponential increase with time (cf. Walker and Pitman, 1976), and the kinetics of their rise is determined by the respective exchange constants k for the two compartments (k = 0.693/t,^ ): Swps = s0«(l - e-^") [cpm janol-1]; s0 = W ( l - ek(c),t) = W ( l - e-k(WFS)>(l - e^O [cpm itmol1]; Thus, the influx equation becomes: = QV((s0-(l - e**"9") - peff*s0.(l - e-k(WFS>>(l - e-^ ")).t); = QV(s 0«(l - e - w ^ l - peff«(l - e-*(e)n))»t) [ttmol g"1 h1]; The tracer content (Q*det) which is determined in the root tissue after a loading period in an uptake experiment (without subsequent desorption of the roots) is the result not only of 'physiological' uptake into the cytoplasm, but also of tracer bound to the cell wall (if tracer carried over by the surface film is desorbed by a quick 10-s dip in non-labelled solution after the loading, the contribution to this will only be from the adsorptive component of the free space, AFS). By knowing the tissue volume V A F S of that component and the factor of 173 accumula t ion o f the g i v e n i o n w i t h respect to its concentrat ion i n the external so lu t ion ( i .e . the quotient o f i o n concentrat ion i n the free space and o f that i n b u l k solut ion) , Q * A F S can be ca lcula ted: Q'AFS = 8^(1 - e - k < A F S ) > V A F S » f A F S [cpm g- 1 ]; S ince Q*« = Q * d e t - Q * A F S , the ove ra l l equation for in f lux becomes: = (Q/det - ( V A F S - f A F S * s 0 « ( l - e- k < A F S ) "))) / (s 0 <»(l - c " * ™ > ( l - p e f f « ( l - e « ^ ) ) « t ) [ i tmo l g 1 t r 1 ] ; S ince every f lux i s a time-dependent process ( i .e . dQ/dt), the above set o f equations are inherent ly different ia l (even i f not specif ical ly wri t ten out as such). It is be l i eved that this mathemat ical approach to quant i fying in f lux , by taking into account both the eff lux term and the c e l l - w a l l component , may present a useful too l for compar ing i n f l u x values obtained i n the ' c l a s s i c a l ' w a y (wi th a desorption per iod f o l l o w i n g l abe l l ing w i t h a tracer, see previous Chapters) w i t h the mathematical ly 'expected ' values. It i s , o f course, acknowledged that this is poss ib le o n l y under certain (representative) condit ions for w h i c h the needed parameters have been determined by efflux analysis . A l s o , i f l abe l l ing times m u c h longer than the convent iona l 10 m i n are used i n an uptake experiment, other f lux terms, such as f lux to the x y l e m or the vacuo le (and i n the opposite di rect ion) , may become significant , and the equat ion w o u l d have to be expanded accord ing ly . 174 APPENDIX H: A NEW METHOD FOR THE DETERMINATION OF ION EXCHANGE PARAMETERS IN MULTICOMPARTMENT AL PLANT SYSTEMS. A large part o f the w o r k reported i n this thesis has been based o n eff lux analysis and the der iva t ion o f parameters such as kinet ic exchange constants and compar tmenta l concentrations f r o m that method. F o r this reason, a method independent o f eff lux analysis was developed w h i c h a l l o w s the determinat ion o f half- l ives o f exchange for several subcel lular compartments and, under cer tain condi t ions , even o f compartmental i o n concentrations. U n l i k e the e lu t ion o f root tissue p rev ious ly labe l led w i t h tracer, as i n efflux analysis , this new method i s based o n the k ine t ic analysis o f the time-dependent appearance o f tracer i n p rev ious ly unlabel led root tissue upon first exposure to tracer ( in this case 1 3 N 0 3 ~ ) . T h e procedure used here was essentially as out l ined i n Ma te r i a l s and M e t h o d s for k inet ic i n f l u x measurements, w i t h the except ion that l abe l l ing w i t h tracer was not standardized at 10 m i n , but v a r y i n g t imes o f exposure to isotope were chosen. In the present p r e l imina ry experiments these t imes were: 10 s, 20 s, 30 s, 1 m i n , 1.5 m i n , 2 m i n , 2 .5 m i n , and 3 , 4 , 5, 6, 7 , 8, 9 , 10, 12, 14, 16 m i n . F o l l o w i n g 2 -min desorption, 30-s low-speed centrifugation and fresh-weight determinat ion (see Mate r i a l s and Methods ) , the count accumula t ion o f 1 3N i n the plants (roots + shoots) was measured by means o f a 7-counter. T h i s count accumula t ion ( in [cpm g 1 ] ) was then plotted logar i thmica l ly against the t ime o f exposure to the tracer ( F i g . 24) . B y means o f l inear regression, as i n the analysis o f eff lux plots , dist inct k ine t ic phases o f tracer f i l l i n g c o u l d be reso lved . F i g u r e 24 A shows a representative p lo t for whi te-spruce seedlings at 10 ttM [ N 0 3 " ] 0 . A t least three phases were evident i n the present studies. A s i n ef f lux analysis , ha l f - l ives o f exchange for these phases cou ld be der ived f rom the slopes o f the regression l ines . These were » 20 to 30 s, 2 to 2.5 m i n , and 6 to 7 m i n , respect ively . B y ana logy to the efflux 175 0 2 4 6 8 10 12 14 16 time of exposure to isotope [min] Figure 24 A . Semi-logarithmic plot of the 1 3 N accumulation in roots of intact white-spruce seedlings as a function of time of exposure to 1 3N0 3". The solution contained 10 uM [N03~]0 ( 1 3N0 3" was added as the tracer). The plot includes linear regression lines and equations for the three phases. Half-lives of exchange (t1A) for the resolved phases were calculated from the X -coefficients of the respective regression lines after converting to natural logarithm (i.e. division by 2.303 gives the corresponding k values; then: t% = 0.693/k). 176 -analysis findings (see Chapters 3 to 6 of this thesis), phase I likely represents the adsorptive component of the cell wall, and phase III the average root cytoplasm, while phase II is of unknown origin. It is possible that the latter phase is evident in this analysis as a result of sequential labelling of different root-cell populations (epidermal versus cortical?), which would not be visible in an efflux analysis, where roots are cross-sectionally as well as longitudinally labelled to a steady state prior to elution. Clearly, a check of compartment identity is warranted. A disadvantage of this new method as opposed to efflux analysis (see Materials and Methods) is that relatively large numbers of plants are needed to obtain a sufficient time resolution. However, the use of larger numbers of plants also provides for an improved statistical approach to kinetic phase determination. Moreover, the method has the advantage that it directly describes the kinetics of filling rather than emptying (important in the prediction of specific-activity terms in uptake experiments; see previous appendix). Perhaps even more importantly, less detection sensitivity is required than in efflux analysis (since tracer appearing in the tissue is analyzed rather than tracer eluates), and therefore the method is amenable to use with other (less sensitive) tracers (e.g. 15N) and can be conducted at higher external concentrations of the investigated ion, where efflux analysis can no longer be applied (e.g. experiments at 10 mM [N03"]0 were quite feasible). It is furthermore believed that the method could be adapted to allow for the determination of compartmental contents. If the 2-min desorption step was eliminated and replaced by a brief 10- to 15-s dip (to remove surface-bound tracer), cell-wall contents could be derived from examining intercepts of the regression line for the corresponding phase (phase I) with a Y-axis drawn at approximately five times the half-life of exchange for this compartment. This approach is illustrated in Figure 24 B. Contents in the other (presumably cytoplasmic) compartments could be derived similarly, if non-metabolized ions are being investigated (Fig. 24 B). For metabolized ions, the extent to which the ions are 177 4-1 1 1 1 1 1 1 1 \ 1 0 5 10 15 20 25 30 35 time of exposure to isotope [min] Figure 2 4 B . Semi-logarithmic plot as in Fig. 24 A. Additional Y-axes have been drawn at five times the respective t,A values for each of the three phases resolved by linear regression. By knowing the specific activity of tracer in the external solution and by subtraction of the respectively preceding Y-intercepts from a Y-intercept with any given axis, ion contents or concentrations (if compartmental tissue volumes are known) in the corresponding compartments could be determined (with the restrictions outlined in the text). 178 metabolized as well as other fluxes removing these ions from the cytoplasm would have to be quantified separately (cf. Bell et al., 1994). A calibration of this type of count-accumulation plot against efflux plots obtained under comparable conditions may be a possible means for evaluation of the feasibility of the approach with metabolized ions. This needs to be investigated further. 179 dura t ion , w o u l d therefore be s ignif icant ly less than 1 0 % , even under these condi t ions o f h igh ef f lux percentage. F o r desorpt ion, 3 m i n was chosen, since, this pe r iod represents s ix t imes the hal f - l i fe o f exchange for the free space (t,A o f ~ 30 s) and almost one fifth o f that for the cy top la sm (t^ « 1 4 m i n ) . S u c h a t ime pe r iod a l lows for a v i r tua l ly complete release o f the 1 3 N H 4 + associated w i t h the c e l l w a l l to the ambient solut ion (98 .44% o f the exchange should be comple te after 6 x tA), w h i l e faci l i ta t ing o n l y marg ina l efflux o f tracer f rom the cy top lasmic compartment dur ing desorpt ion. E v e n i n the case o f plants g r o w n at 1.5 m M [ N H 4 + ] 0 , the underestimate due to efflux dur ing desorpt ion w o u l d be less than 6%. In fact, i t is more l i k e l y that, due to cont inued uptake and the presence o f s t i l l not quantitatively desorbed tracer i n the free space, an e r ror leading to an overest imate for w o u l d be introduced dur ing the desorption pe r iod (see W a n g et a l . 1993). 5 . 3 . 2 . Ammonium F l u x e s . F l u x e s o f N F L / i n whi te spruce estimated b y compartmental analysis were considerably h igher than f luxes o f N 0 3 " measured under comparable condi t ions b y the same technique ( K r o n z u c k e r et a l . , 1995a, b) . In fact, o f N H / at 10 uM [ N H 4 + ] C i n the steady state was « 2 - fo ld h igher than o f N 0 3 " after exposure to 10 uM [ N 0 3 " ] 0 for 3 d ( i .e . i n a state o f fu l l i nduc t ion for N 0 3 " transport; see K r o n z u c k e r et a l . , 1995a). It was « 4 - f o l d h igher w h e n the external concentrat ion was 100 i i M for the two N species, and « 5- fo ld h igher at 1.5 m M . S ince N 0 3 " i n f l u x i s substrate-inducible, i . e . i t is considerably enhanced b y the presence o f external N 0 3 " (see K r o n z u c k e r et a l . , 1995b, for references), w h i l e N H 4 + i n f l ux i n most p lant systems is down-regula ted rather than enhanced by the presence o f external N H 4 + (see W a n g et a l . , 1993b, for references), the most dramatic difference for for the t w o N species was found i n 77 

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