Fluxes, compartmentation and metabolism of nitrate and ammonium in spruce roots

Techniques of compartmental analysis and kinetic flux analysis with the radiotracer ¹³N were employed to examine the uptake, compartmentation, and metabolism of NO₃⁻ and NH₄⁺ 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 NH₄⁺) ≈ 2 s, 20 s, and 7 min, respectively, and (for NO₃⁻) 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 NH₄⁺ than for NO₃⁻ Steady-state NH₄⁺ influx ranged from 0.3 to 6.5 μmol g⁻¹ (FW) h⁻¹ , while it was 0.08 to 1.2 μmol g⁻¹ h⁻¹ for NO₃⁻, 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 NH₄⁺ and from 1% to 20% for NO₃⁻. Cytoplasmic concentrations were considerably higher for NH₄⁺ than for NO₃⁻; [NH₄⁺][sub cyt] was 2 to 30 mM, whereas [NO₃⁻][sub cyt] was 0.2 to 4 mM under the above conditions. A time-dependent compartmental-analysis study revealed that NO₃⁻ uptake was inducible by external NO₃⁻ and that three days were required for maximal uptake to be achieved at 100 μM [NO₃⁻]₀. The dynamics of NO₃⁻-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 [NO₃⁻]₀ ≤ 1 mM, NO₃⁻ 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 system (LATS) was evident. NH₄⁺ influx was not inducible by external NH₄⁺. 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[sub m] values for the initial phase of high-affinity transport were similar for both N species (≈ 20 μM), but V[sub max] was up to 20 times larger for NH₄⁺ than for NO₃⁻ when measured in perturbation. Overall, the study establishes a pronounced physiological limitation in spruce roots in transporting and utilizing NO₃⁻ as compared to NH₄⁺.Science, Faculty ofBotany, Department ofGraduat