Induction of nitrate uptake and nitrate reductase activity in trembling aspen and lodgepole pine


Dr Anthony D. M. Glass Fax: (604) 822–6089; e-mail:


13NO3 influx into the roots and in vivo nitrate reductase activity (NRA) in the roots and leaves have been measured in trembling aspen (Populus tremuloides Michx.) and lodgepole pine (Pinus contorta Dougl.) seedlings after exposure to either 0·1 or 1·5 mol m–3 NO3 for varying periods up to 20 d. Both NO3 influx and NRA were inducible in these species and, in trembling aspen, peak induction of nitrate influx and NRA were achieved within 12 h, compared to 2–4 d for influx and 4–12 d for NRA in lodgepole pine. In trembling aspen, ≈ 30% of the total 13N absorbed during a 10 min influx period followed by 2 min of desorption was translocated to the shoot. In lodgepole pine, by contrast, translocation of 13N to the shoot was undetectable during the same time period. Root NRA as well as NO3 influx from 0·1 mol m–3 NO3 were substantially higher in trembling aspen than in lodgepole pine at all stages of NO3 exposure, i.e. during the uninduced, the peak induction, and steady-state stages. In order to examine whether the lower rates of NO3 influx and NRA were related to proportionately fewer young (unsuberized) roots in lodgepole pine, we determined these parameters in young and old (suberized) roots of this species separately. Induction of influx and NRA were initially greater in young roots but at steady-state there were only minor differences between the young and the old roots. However, even the elevated initial rates in the young roots of lodgepole pine were substantially lower than those of aspen. In pine, influx at 1·5 mol m–3 NO3 was ~ 6-fold higher than at 0·1 mol m–3 NO3 and appeared to be mostly via a constitutive system. By contrast, in aspen, steady-state influxes at 0·1 and 1·5 mol m–3 were not significantly different, being similar to the rate attained by pine at only the higher [NO3]. In aspen, leaf NRA was ~ 2-fold higher than that of roots. In lodgepole pine NRA of the needles was below the detection limit. These results show that trembling aspen seedlings are better adapted for NO3 acquisition and utilization than lodgepole pine seedlings.


In soils, nitrogen is available as dissolved organic nitrogen (e.g. amino acids), NH4+, NO3, or any combination of these, for absorption by plants (Glass & Siddiqi 1995). The relative amounts of these various forms of N depend upon a number of biotic and abiotic factors which determine the rates of mineralization, nitrification, denitrification, leaching, relative uptake of different N forms by plants and microorganisms, etc. (Stark & Hart 1997; Eviner & Chapin 1997). In temperate forest ecosystems, soils under undisturbed coniferous communities are typically high in NH4+ and low in NO3 (Van Cleve et al. 1983; Chapin, Van Cleve & Tyron 1986; Lavoie, Vezina & Margolis 1992). At these sites, even when gross nitrification rates are substantial, soil microbes may rapidly assimilate most of the NO3 produced, making it unavailable for plants (Stark & Hart 1997). Following disturbance, characteristically, nitrification rates and soil nitrate concentration ([NO3]) increase substantially (Likens, Borman & Johnson 1969; Rice & Pancholy 1972; Lodhi 1978; Vitousek & Melillo 1979; Van Cleve et al. 1983; Walley, Van Kessel & Pennock 1996; Prescott 1997). In some cases, soil ammonium concentration ([NH4+]) may also increase at these disturbed sites if mineralization exceeds nitrification (e.g. Schmidt, MacDonald & Rothwell 1996).

Plant species differ in their capacities for acquisition and assimilation of particular N forms, which is reflected in their natural distribution (Bledsoe & Rygiewicz 1986; Chapin, Moilanen & Kielland 1993; Kielland 1994). For example, it has been reported that many conifers (e.g. white spruce, lodgepole pine, and western hemlock) grow significantly faster when NH4+, rather than NO3, is provided as the sole source of N, whereas for others (e.g. western redcedar and Douglas fir), the reverse is true (van den Driessche 1971; Krajina, Maddock-Jones & Mellor 1973; see also Bledsoe & Rygiewicz 1986; Knoepp, Turner & Tingey 1993; Kronzucker, Siddiqi & Glass 1997). A comparative study of the utilization of NH4+ versus NO3 showed that white spruce had a distinct preference for NH4+ as a N source (Kronzucker, Glass & Siddiqi 1995a; Kronzucker, Siddiqi & Glass 1995b,c,d, 1996). It was suggested that a limited capacity for the absorption and assimilation of NO3 in white spruce and other late-successional conifers may be an important factor in determining regeneration success at disturbed sites (Kronzucker et al. 1997). Implicit in this hypothesis is the notion that species which more readily colonize disturbed forest sites have superior physiological and biochemical traits for the utilization of NO3 as a N source. To evaluate these assumptions, we have undertaken a comparative study of various aspects of uptake, accumulation and assimilation of NO3 and NH4+ in two early-successional species: trembling aspen (Populus tremuloides Michx.) and lodgepole pine (Pinus contorta Dougl. var. latifolia). The former represents typical fast growing deciduous hardwoods while the latter is a relatively slow growing conifer. In the present paper we describe the patterns of induction of NO3 influx and nitrate reductase activity (NRA) in these species.


Plant culture

Trembling aspen (seedlot 42307) and lodgepole pine (seedlot 3847) seeds were obtained from Tree Seed Centre, Ministry of Forests, Surrey, B.C., Canada. Seeds were sown in a peat/perlite (2: 1 by volume) mixture, containing 4·3 kg m–3 dolomite, in styroblocks. Seedlings were maintained in this medium in an outdoor nursery or a greenhouse for a minimum of 3 months. Subsequently, roots were washed gently under running tap water to remove the medium, and then rinsed with deionized water. Each seedling was then fitted into a Plexiglas disc and placed in a Plexiglas hydroponic tank (40 dm3 capacity) containing 0·1 strength modified Johnson's nutrient solution (Epstein 1972, p. 39) either without N at all, or containing appropriate [NH4+] or [NO3]. The composition of the nutrient solution was as follows: macronutrients (in mol m–3), Pi 0·2, K+ 0·6, Mg2+ 0·1, Ca2+ 0·4, and micronutrients (in mmol m–3): Cl 5, BO3 2·5, Mn2+ 0·2, Zn2+ 0·2, Cu2+ 0·05, Fe-EDTA 2. Nitrate and NH4+ were provided as Ca (NO3)2 and (NH4)2SO4, respectively. Solutions were continuously aerated and mixed by means of immersion circulators (Brinkman, USA). The tanks were maintained in a controlled growth room with 16 h/8 h (light/dark) photoperiod, 70% relative humidity, and 20 ± 2 °C temperature. Light was provided at 250 μmol m–2 s–1 at plant level by fluorescent tubes with spectral composition similar to sunlight (Vita-Lite, Duro-Test, USA). The plants were maintained in the hydroponic tanks for 3 weeks prior to flux measurements and/or nitrate reductase activity assays (see Kronzucker et al. 1995a). Solutions were monitored daily for [K+] (using an Instrumentation Laboratory Model 443 Flame Photometer), for [NO3] (Cawse 1967) and for [NH4+] (Solarzano 1969). Solutions were buffered by adding excess powdered CaCO3 to each tank, and pH was maintained at about 6·4–6·8. Solutions were changed completely every week and 1 d before 13N influx measurement and/or NRA assay.

Time-course of induction of nitrate influx and nitrate reductase activity

For induction of NO3 influx and NRA, seedlings, grown in a solution containing no N or 25 mmol m–3 NH4+, were transferred to a solution containing 0·1 or 1·5 mol m–3 NO3 for various periods of time (0–20 d). In order to measure 13NO3 influx or NRA at the same time for all treatments (exposed to NO3 for different durations), the initiation of NO3 pre-treatment was staggered accordingly.

Production of 13NO3

Nitrogen-13 was produced by proton irradiation of H2O at the TRIUMF cyclotron on the campus of the University of British Columbia, Vancouver, Canada. The protocol for removal of radiocontaminants was essentially the same as described by Kronzucker et al. (1995a), except that 5 cm3 of 100 mmol m–3 (instead of 2·5 mmol m–3) Ca(NO3)2 was used to elute residual 13NO3 from the SEP-PAC Alumina-N cartridge. We routinely determined the t1/2 of the purified samples which corresponded exactly with that of 13N (9·98 min).

Measurement of influx

Influx of NO3 was measured from modified 0·1 strength Johnson's nutrient solution containing appropriate concentrations of 13N-labelled NO3. Seedlings were transferred from hydroponic tanks to a nonradioactive prewash solution, identical in composition (except for isotope) to the uptake solution, for 5 min; they were then transferred to 13N-labelled uptake solution for 10 min. Immediately following loading, roots were dipped into a non-labelled solution for 5 s, and then transferred to a non-radioactive solution, which was otherwise identical to the uptake solution, for 2 min, to desorb 13NO3 contained in the free space (Kronzucker et al. 1995b). Following desorption, roots were excised from shoots, spun for 45 s in a low-speed centrifuge to remove surface solution, and then fresh weights were determined. The radioactivities of shoots and roots were measured with a Packard-γ-counter (Minaxi, Auto-gamma 5000). NO3 fluxes thus determined are expressed as (μmol g–1 root fresh weight h–1. In determining influx into young (unsuberized) and old (suberized) roots separately, essentially the same procedure was followed as described above except that excised roots were used in these experiments.

Nitrate reductase activity assay

In vivo root NRA assay was carried out using the method described by King, Siddiqi & Glass (1992). Fresh root tissue (0·2–0·5 g) was added to a 10 cm3 test tube containing 4·25 cm3 of 100 mmol m–3 K-PO4 buffer (pH 7·7), 0·25 cm3 isopropanol (99%) and 0·5 cm3 of 0·5 kmol m–3 KNO3 (final concentration 50 mol m–3). The tubes were capped and purged for 10 min with N2 which was introduced through the cap via a 25 gauge/3·5 inch spinal needle. After purging, tubes were incubated for 30 min at 30 °C in a water bath. They were then boiled for 10 min to extract tissue NO2. The solution samples were filtered and 1·5 cm3 aliquots were withdrawn and assayed for NO2 (see below). Nitrate reductase activity was expressed as μmol NO2 g–1 root fresh weight h–1.

The youngest fully expanded leaves were sampled from aspen and lodgepole pine for in vivo NRA assays. The same method as described above for the root NRA assay was used, except that leaf tissue was vacuum-infiltrated for 10 min with NO3 solution prior to N2 purging.

Tissue NO3 and NO2 analysis

Root NO3 was extracted by boiling ≈ 0·5 g tissue in 5 cm3 distilled water for 10 min. The extract was passed through a column packed with insoluble PVPP (polyvinylpolypyrrolidone) to remove phenols and debris. Nitrate was determined spectrophotometrically by the method of Cataldo et al. (1975). The sample (0·05 cm3) was added to 0·2 cm3 of 50 kg m–3 salicylic acid (dissolved in concentrated H2SO4), mixed and incubated for 20 min at room temperature. Then, 4·75 cm3 of 2N NaOH were added and NO3 concentration was determined spectrophotometrically by measuring absorbance at 410 nm in a spectrophotometer (Philips, PU2488 UV/VIS).

Nitrite concentration was determined spectrophotometrically, as described by King et al. (1992). In brief, 0·25 cm3 of 0·8 kg m–3n-1-naphthylene-diamine-dihydrochloride and 0·5 cm3 of 20 kg m–3 sulphanilamide (dissolved in 5 kmol m–3 HCl) were added to 1·5 cm3 of sample in a 10 cm3 test tube and mixed. The sample was incubated at room temperature for 30 min, and absorbance was measured at 540 nm.


Nitrate influx

In both trembling aspen and lodgepole pine, NO3 influx by the high-affinity transport system (HATS) was inducible. In trembling aspen, induction was very rapid. Within 6–12 h of exposure to 0·1 or 1·5 mol m–3 NO3, influx, measured at 0·1 mol m–3 NO3, had increased 10–20-fold, from ≈ 0·2 μmol g–1 h–1 in uninduced plants (constitutive level) to ≈ 2 and 4·3 μmol g–1 h–1, respectively (Fig. 1a, inset). Subsequently, NO3 influx gradually decreased with increasing duration of exposure to NO3 at either [NO3] until 3–4 d, after which influx remained essentially unchanged for up to 20 d (Fig. 1a). However, even this steady-state NO3 influx (≈ 0·8–1·2 μmol g–1 h–1) was several-fold higher than the constitutive influx.

Figure 1.

.13NO3 influx (μmol g–1 h–1± SE) from 0·1 mol m–3 NO3 into intact roots of (a) trembling aspen and (b) lodgepole pine following pre-treatment with 0·1 or 1·5 mol m–3 NO3 for 0–20 d (see text). Inset: data for exposures to 0–48 h in (a) redrawn.

In lodgepole pine pre-treated with 0·1 or 1·5 mol m–3 NO3, induction of NO3 influx from 0·1 mol m–3[NO3] required at least 2 d to fully induce this process, and the influx measured at 0·1 mol m–3 NO3 increased only 2- to 3-fold, from 0·1 μmol g–1 h–1 (constitutive influx) to about 0·2–0·3 μmol g–1 h–1, at either pre-treatment (Fig. 1b). In this species the decline of NO3 influx (down-regulation), so evident in aspen, was not apparent even after 20 d of pre-treatment with either external [NO3]. Nitrate influx into roots of lodgepole pine was much lower than in trembling aspen at all stages of NO3 pre-treatment. Influx was also measured at 1·5 mol m–3 NO3 in lodgepole pine, pre-treated with 1·5 mol m–3 NO3, and found to be much higher (≈ 6-fold) than at 0·1 mol m–3 NO3. Further, when influx was measured at 1·5 mol m–3 NO3, neither induction nor down-regulation of NO3 influx were evident (Fig. 2).

Figure 2.

.13NO3 influx (μmol g–1 h–1± SE) from 1·5 mol m–3 NO3 into intact roots of lodgepole pine following pre-treatment with 1·5 mol m–3 NO3 for 0–20 d (see text).

Translocation of 13N to the shoot

In trembling aspen pre-treated with 0·1 mol m–3 NO3 for periods ranging from 0 to 20 d, 13N translocation to the shoot during 12 min of exposure to the isotope (10 min of 13N loading and 2 min desorption) increased as the duration of pre-treatment was extended (Fig. 3). Translocation, expressed as a percentage of the total 13N absorbed, increased from 2% (0 d pre-treatment) to 35% (20 d pre-treatment). Plants pre-treated with 1·5 mol m–3 NO3 showed a similar pattern (data not shown). In pine, 13N translocation to the shoot was not detectable.

Figure 3.

. Translocation of 13N to the shoot of trembling aspen (% of total 13N absorbed) during 10 min loading with 13NO3 followed by 2 min desorption, in plants pre-treated with 0·1 mol m–3 NO3 for 0–20 d (see text).

Nitrate reductase activity in the roots and leaves

In trembling aspen, NRA in the roots was fully induced within 6–12 h of exposure to 0·1 or 1·5 mol m–3 NO3. NRA increased from ≈ 0·5 μmol NO2 g–1 h–1 in uninduced roots to ≈ 1·6 μmol NO2g–1 h–1 (Fig. 4a, inset). However, in contrast to the pattern of NO3 influx, NRA failed to decline at longer exposures and remained at the maximum level under steady-state conditions. There were no significant differences in the rates of induction or NRA between exposures to 0·1 and 1·5 mol m–3 NO3 (Fig. 4a). In lodgepole pine, root NRA was lower and its induction slower than that of aspen (Fig. 4b). The peak induction was achieved after 8–12 d of exposure to NO3 and the rates of NRA were dependent on pre-treatment concentration: the rate increased from ≈ 0·02 μmol g–1 h–1 to ≈ 0·4 μmol g–1 h–1 following pre-treatment with 0·1 mol m–3 NO3 and to ≈ 0·8 μmol g–1 h–1 at 1·5 mol m–3 NO3. In the latter pre-treatment, however, NRA declined to ≈ 0·5 μmol g–1 h–1 after 20 d, whereas at 0·1 mol m–3 it remained at the fully induced level (Fig. 4b).

Figure 4.

. Nitrate reductase activity (NRA) (μmol g–1 h–1± SE) in the roots of (a) trembling aspen and (b) lodgepole pine following pre-treatment with 0·1 or 1·5 mol m–3 NO3 for 0–22 d (see text). Inset: data for exposures to 0–24 h in (a) redrawn.

In trembling aspen, leaf NRA, similar to that of roots, was fully induced within 1 d. It increased from ≈ 0·1 μmol NO2 g–1 h–1 (constitutive value) to ≈ 3 μmol NO2 g–1 h–1 after one day of exposure to 0·1 mol m–3 NO3 (Fig. 5). However, no clear pattern of down-regulation was subsequently evident. In lodgepole pine leaves, by contrast, NRA was not detectable even after 20 d of pre-treatment at either external [NO3].

Figure 5.

. Nitrate reductase activity (NRA) (μmol g–1 h–1± SE) in the leaves of trembling aspen following pre-treatment with 0·1 mol m–3 NO3 for 0–18 d.

Nitrate influx and NRA in young and old roots of lodgepole pine

At the time of flux determinations and NRA assays, trembling aspen seedlings had proportionately more young roots (50% or more of the total root mass) than lodgepole pine (10–20%). In selected experiments, we determined NO3 influx and NRA in lodgepole pine in young and old roots separately. In the young roots, induction was more rapid and both influx and NRA at peak induction were much higher (≈ 6 and ≈ 3 times, respectively) than in the old roots (Fig. 6a,b). Subsequently, in young roots, influx and NRA declined gradually with increasing duration of exposure to NO3. On the other hand, influx and NRA in the old roots remained at the maximum level so that after 20 d of pre-treatment influx in young roots had declined to only 1·7 times that of old roots, while NRA was actually similar in young and old roots (Fig. 6a,b). Nevertheless, even the higher values of NO3 influx and NRA in these young roots of lodgepole pine remained substantially lower (≈ 25 and 50%, respectively, for influx and NRA) than average rates (young plus old roots) for trembling aspen (Figs 1a, 4a).

Figure 6.

. (a) 13NO3 influx (μmol g–1 h–1± SE) and (b) nitrate reductase activity (NRA) (μmol g–1 h–1± SE) in the young and old roots of lodgepole pine following pre-treatment with 0·1 mol m–3 NO3 for 0–20 d (see text).

Nitrate concentration of root cells

In the roots of trembling aspen, NO3 accumulation was rapid (Fig. 7a). At 0·1 mol m–3 NO3, root [NO3] reached a steady value in 1 d, from ≈ 0·2 μmol g–1 FW to about 6 μmol g–1 FW. Root [NO3] at 1·5 mol m–3 external NO3 was higher than at 0·1 mol m–3 NO3 (Fig. 7a). Lodgepole pine roots did not show any change in internal [NO3] after exposure to either external [NO3] for up to 20 d (Fig. 7b).

Figure 7.

. Root [NO3] (μmol g–1 h–1± SE) in the roots of (a) trembling aspen and (b) lodgepole pine.


It is well established that both NO3 influx and NRA are substrate-inducible processes. Plants grown in the absence of NO3 have low rates of NO3 uptake and NRA. Exposure to NO3 increases the rates of these processes to some peak values (induction), followed by a gradual decline (down-regulation) until a steady value is attained. While this overall pattern is common among plants, species differ greatly in the time scale over which these events occur as well as in the magnitude of these processes. Crop species such as maize, which have high growth rates and generally have a preference for NO3 as a N-source, show maximum induction of NO3 influx and NRA within a few hours of exposure to NO3 (e.g. Oaks 1994; Glass & Siddiqi 1995 for review). By contrast, slow growing conifers such as white spruce, which prefer NH4+, may require several days to achieve peak induction (Kronzucker et al. 1995a).

In both trembling aspen and lodgepole pine, as in most plant species investigated with respect to this effect, NO3 influx as well as NRA were inducible (Figs 1, 4). However, there were substantial differences between these species in the rapidity of induction and the absolute rates of these processes. Trembling aspen appeared to be, by far, the superior species in the utilization of root-available NO3. In this species, induction of both NO3 influx and NRA were rapid, with the maximum induction achieved within 6–12 h of exposure to NO3. Lodgepole pine roots, by contrast, required much longer exposure (several days) for maximum induction, which is similar to another conifer, white spruce (Kronzucker et al. 1995a). Not only was induction more rapid in trembling aspen, but its rate of NO3 uptake (particularly in the range of HATS) and potential rates of NRA were several-fold higher than those of lodgepole pine (Fig. 1,4 ) and those reported for other conifers (e.g. Kronzucker et al. 1995a,bc). Clearly, these differences between the two species are genetic and are not the result of proportionately more young roots in trembling aspen than lodgepole pine at the time of determination of these parameters. Both NO3 influx and NRA in the former, determined on a whole-root basis, were much higher than those of young roots of lodgepole pine (Fig. 6).

In trembling aspen, substantial amounts of NO3 and/or amino acids were translocated to the shoot; in 0·1 mol m–3 pre-treated plants, as much as 35% of the total 13N absorbed was recovered in the shoot within 12 min of exposure to the isotope (10 min 13NO3 loading followed by 2 min desorption). This, coupled to the high rates of leaf NRA, obviously increases the capacity for utilization of NO3 in this species. The rates of leaf NRA we have observed in trembling aspen agree well with those reported for other Populus spp. (Dykstra 1974). By comparison, in lodgepole pine, NRA in the leaves was below the limit of detection. This is in agreement with the results reported for red spruce (Yandow & Klein 1986). In fact, conifers in general appear to have very low leaf NRA (Smirnoff & Stewart 1985). In some conifers, detectable NRA has been found in the needles, but it was much lower than the root NRA (e.g. Peuke & Tischner 1991). Indeed, it is generally recognized that in conifers most of the NO3 is reduced within the roots (e.g. Sarjala 1991 and references therein). This is consistent with the fact that, in lodgepole pine, 13N was not detectable in shoots. Even in experiments which involved exposures to 13NO3 for 35 min followed by 22 min desorption, only traces of the isotope were found in shoots (our unpublished results). Translocation of N to the shoot may be as NO3 and/or as amino acids; generally very little NH4+per se is translocated to the shoot (Wang et al. 1993; Kronzucker et al. 1995d; Glass & Siddiqi 1995). It follows then that in conifers either rates of translocation to the shoot are low not only for NO3 but also for amino acids or there is a substantial delay in the appearance of the isotope in the shoot.

Clearly, translocation of 13N to the shoot of trembling aspen was ‘inducible’ (see Siddiqi, Glass & Ruth 1991; Kronzucker et al. 1995a; D. Zhou et al. unpublished results); it increased gradually from ≈ 2% in uninduced plants to ≈ 35% after 20 d of pre-treatment (Fig. 2). The rapidity and the magnitude of induction of leaf NRA suggest that a substantial amount of NO3per se was translocated to the shoot since NO3 itself is the known effector of induction of the NR enzyme. It has been demonstrated that translocation of NO3 as well as the enzymes responsible for its metabolism leading to amino acids, i.e. NR, NiR, GS, GOGAT, are all inducible (e.g. Oaks 1994 for review). However, the time-scale for induction of these processes (a few hours) is much too rapid to explain the observed pattern of 13N translocation to the shoot. It appears that filling of vacuoles in root cells takes precedence (Glass 1978; Kronzucker et al. 1995a) and as the [NO3] in the vacuole increases, flux to the vacuole declines and proportionately more incoming NO3 becomes available for translocation to the shoot as well as for assimilation (Siddiqi et al. 1990). The half-life of exchange for vacuolar NO3 in some crop species has been reported to be ≈ 16–21 h (Lee & Clarkson 1986), which broadly corresponds to the pattern we have observed (Fig. 3).

Interestingly, in lodgepole pine, when influx was measured from 1·5 mol m–3[NO3], induction was not evident, i.e. influx remained unchanged following pre-treatment with 1·5 mol m–3 NO3 (Fig. 2). In this species, particularly in uninduced plants, the activity of a low-affinity transport system (LATS) was evident at relatively low (0·1–0·2 mol m–3) external [NO3] (our unpublished results). At 1·5 mol m–3 NO3, the contribution of LATS, which is constitutive (Siddiqi et al. 1990; Kronzucker et al. 1995b), may have been so large as to mask changes of HATS activity. Indeed, influx from 1·5 mol m–3 NO3 was several-fold higher than the Vmax for HATS (our unpublished results), irrespective of the duration of pre-treatment of lodgepole pine roots with NO3. Thus, the capacity for NO3 influx in lodgepole pine roots increased substantially as external [NO3] increased. In trembling aspen, by contrast, steady-state influxes from 0·1 mol m–3 and 1·5 mol m–3 were similar (0·94 ± 0·06 and 1·33 ± 0·09 μmol g–1 h–1, respectively) (our unpublished results).

In trembling aspen, NO3 influx showed typical negative-feedback effects as a result of prolonged exposures to NO3 (Fig. 1, see Glass & Siddiqi 1995 for review). These effects were absent from old lodgepole pine roots. In neither species, however, was influx correlated with root [NO3] or pre-treatment [NO3]. There is evidence at both physiological and molecular levels, that the main effector(s) of negative feedback inhibition is/are some metabolite(s) downstream from NH4+, e.g. glutamine, and/or glutamate, rather than NO3per se (Lee & Rudge 1986; Lee et al. 1992; D. Zhou et al. unpublished results). It is noteworthy that even the down-regulated, steady-state NO3 influx into aspen roots was ≈ 4-fold greater than that into lodgepole pine roots (Fig. 1). In the latter species, there was no clear indication of down-regulation of influx; in fact, influx slightly increased after 16–20 d of exposure to NO3 (Fig. 1b). We speculate that this time-dependent pattern of influx may be the result of increased growth rate at this stage. We have noticed a marked increase in the production of young roots after ≈ 12–16 d of exposure to NO3. Also, in contrast to trembling aspen, there was no accumulation of NO3 in the roots of lodgepole pine after exposure to 0·1 or 1·5 mol m–3 NO3 for as long as 20 d (Fig. 7).

In trembling aspen, potential NRA showed no evidence of negative-feedback effects after prolonged exposures to NO3, and there was no significant difference in the NRA between the two levels of NO3 pre-treatments. In lodgepole pine, by contrast, potential NRA showed evidence of negative feedback inhibition (Figs 4b, 6b). Interestingly, in 0·1 mol m–3-pre-treated lodgepole pine seedlings, NO3 influx as well as NRA were induced to much higher levels in the young roots (Fig. 6). However, in these same young roots, both influx and NRA were down-regulated after prolonged exposures to NO3 and, at steady state, there were only minor differences between the young and the old roots. Note that the peak influx and NRA levels are similar in young roots pre-treated with 0·1 mol m–3 NO3 (Fig. 6a,b) to those measured in whole roots (≈ 80–90% old roots) pre-treated with 1·5 mol m–3 NO3 (Figs 2, 4b). Thus, these observations suggest that 0·1 mol m–3 NO3 was not adequate to fully induce NO3 influx and NRA in the old roots. While root [NO3] determined on a whole-root basis (≈ 80–90% old roots) was similar between the two levels of NO3 pre-treatment, cytoplasmic [NO3] was ≈ 20-fold higher in plants pre-treated with 1·5 mol m–3 than in those pre-treated with 0·1 mol m–3 (our unpublished results; see also Kronzucker et al. 1995c for white spruce). It is possible that in the young roots of 0·1 mol m–3-pre-treated plants, cytoplasmic [NO3] was higher, perhaps comparable to the 1·5 mol m–3-pre-treated plants. Indeed, variations in tissue [NO3], NRA and transport activity have been proposed both transversely and longitudinally along the roots and the leaves (see Siddiqi et al. 1991 and references therein). In this regard, it is interesting to find that in trembling aspen NRA (Fig. 3a) and cytoplasmic [NO3] (our unpublished results) were both similar between the two pre-treatments.

In the present study, plants were grown in a hydroponic system so that the composition of the pre-treatment nutrient media could be rigorously controlled. These conditions were not conducive to the growth of mycorrhizal fungi and, consequently, the plants used in this study were nonmycorrhizal (Kronzucker et al. 1995a,b,c). Furthermore, the aim of our study was to examine genetic differences in the acquisition and utilization of NO3 between the two tree species studied. In the case of certain nutrients, notably Pi, mycorrhizae may enhance the uptake capacity of plants. However, mycorrhizae are reported generally not to have any significant effect on the uptake, accumulation and/or reduction of NO3 (Rygiewicz et al. 1984; Marschner, Häussling & George 1991; Sarjala 1991; Eltrop & Marschner 1996).

In summary, the present study demonstrates that trembling aspen is highly efficient in the acquisition and metabolism of NO3. These adaptations for NO3 utilization may be important for the success of this species as an early colonizer of disturbed sites where available N is predominantly NO3. This applies equally to regeneration from seed or from vegetative organs. Generally, coniferous species with a relatively low capacity for NO3 utilization would be at a disadvantage under such conditions. On the other hand, conifers appear to be efficient in utilizing NH4+. The adaptive significance of a high capacity for NH4+ utilization in the late successional or so called ‘climax’ species (e.g. white spruce) is apparent: soils under these communities are typically high in NH4+ and low in NO3 (Kronzucker et al. 1997 for review). Lodgepole pine, however, is an early successional species, particularly adapted to colonize forest sites following wildfire. Clearly, at low external [NO3] its limited capacity to utilize NO3 might place lodgepole pine at a considerable disadvantage in competition with nitrophiles such as aspen. However, at high external [NO3] the capacity of lodgepole pine for NO3 utilization increases substantially; in fact, at 1·5 mol m–3 NO3 its rate of NO3 uptake was similar to that of trembling aspen.


We wish to thank the following: Dev Britto, Anshuman Kumar, Mamoru Okamoto, John Vidmar and Degen Zhuo, of the Botany Department, UBC for assistance during the experiments involving 13NO3, and Mike Adam, Tamara Hurtado and Tom Ruth of TRIUMF, for production of the isotope. 13NO3 was provided as a gift by TRIUMF. Financial support to R.D.G. and A.D.M.G. from Forest Renewal British Columbia (FRBC) is gratefully acknowledged.