Nitrate reductase 15N discrimination in Arabidopsis thaliana, Zea mays, Aspergillus niger, Pichea angusta, and Escherichia coli

Abstract

Stable 15N isotopes have been used to examine movement of nitrogen (N) through various pools of the global N cycle. A central reaction in the cycle involves the reduction of nitrate (NO−3) to nitrite (NO−2) catalyzed by nitrate reductase (NR). Discrimination against 15N by NR is a major determinant of isotopic differences among N pools. Here, we measured in vitro 15N discrimination by several NRs purified from plants, fungi, and a bacterium to determine the intrinsic 15N discrimination by the enzyme and to evaluate the validity of measurements made using 15N-enriched NO−3. Observed NR isotope discrimination ranged from 22 to 32‰ (kinetic isotope effects of 1.022–1.032) among the different isozymes at natural abundance 15N (0.37%). As the fractional 15N content of substrate NO−3 increased from natural abundance, the product 15N fraction deviated significantly from that expected based on substrate enrichment and 15N discrimination measured at natural abundance. Additionally, isotopic discrimination by denitrifying bacteria used to reduce NO−3 and NO−2 in some protocols became a greater source of error as 15N enrichment increased. We briefly discuss potential causes of the experimental artifacts with enriched 15N and recommend against the use of highly enriched 15N tracers to study N discrimination in plants or soils.

Variation in fluxes estimated from nitrogen isotope discrimination corresponds with independent measures of nitrogen flux in Populus balsamifera L.

“…), or even higher (Carlisle et al . ). If E / I is recalculated using 22‰ for both enzymes, E / I using the isotope mass balance decreases and the similarity between the two methods increases.…”

“…4 and 5 assume discrimination factors of 15.4‰ for nitrate reductase (Ledgard et al 1985) and 16.8‰ for glutamine synthetase (Yoneyama et al 1993). However, more recent work has suggested that the discrimination factor of nitrate reductase is closer to 22-26‰ (Needoba et al 2004;Tcherkez and Farquhar, 2006;Karsh et al 2012), or even higher (Carlisle et al 2014). If E/I is recalculated using 22‰ for both enzymes, E/I using the isotope mass balance decreases and the similarity between the two methods increases.…”

Enzyme level N and O isotope effects of assimilatory and dissimilatory nitrate reduction

“…Voss et al ). A recent study, however, reported a more elevated 15 ε for Nar purified from Escherichia coli of 31.6‰ using benzyl viologen as a reductant (Carlisle et al ). This estimate, however, derived from a 2‐point regression on the δ 15 N of the product and is thus subject to considerable uncertainty.…”

Kinetic 15N-isotope effects on algal growth

“…We concur with this recommendation and suggest that any concentration ≥2 at% 15 N is sufficient to introduce alterations in algal metabolism. Moreover, Carliste et al 31. have demonstrated that experiments with enriched 15 N tracers consistently produce lower estimates for N cycling rates than those conducted using natural abundance or non-isotopic methods.…”