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

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 nitrate (NO3–) reduction to nitrite (NO2–) catalyzed via 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 NO3–. Observed NR isotope discrimination ranged from 22‰ to 32‰ (kinetic isotope effects of 1.022 to 1.032) among the different isozymes at natural abundance 15N (0.37%). As the fractional 15N content of substrate NO3– 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 NO3– and NO2– in some protocols became a greater source of error as 15N enrichment increased. We briefly discuss potential causes of artifactual results with enriched 15N and recommend against the use of highly enriched 15N tracers to study N discrimination in plants or soils

Bulk and Compound-Specific Stable Isotope Analysis for the Authentication of Walnuts (Juglans regia) Origins.

Walnuts are grown in various countries, and as product origin information is becoming more important to consumers, new techniques to differentiate walnut geographical authenticity are needed. We conducted bulk stable isotope analysis (BSIA) and compound-specific stable isotope analysis (CSIA) on walnuts grown in seven countries. The BSIA consisted of δ13Cbulk, δ15Nbulk, and δ34Sbulk, and CSIA covered δ2Hfatty acid, δ13Cfatty acid, δ13Camino acid, δ15Namino acid, and δ2Hamino acid. Analysis of variance (ANOVA) and linear discriminant analysis (LDA) were used for statistical analysis to compare samples from the USA and China. Parameters that yielded significant variations are δ2HC18:1n-9, δ13CC18:2n-6, δ13CC18:3n-3, δ13CGly, δ13CLeu, δ13CVal, δ2HGlu, δ2HIle, δ2HLeu, and δ2HThr. Our findings suggested that CSIA of fatty acids and amino acids can be useful to differentiate the geographical provenance of walnuts

Interlaboratory assessment of nitrous oxide isotopomer analysis by isotope ratio mass spectrometry and laser spectroscopy: current status and perspectives

RATIONALEIn recent years, research and applications of the N2O site-specific nitrogen isotope composition have advanced, reflecting awareness of the contribution of N2O to the anthropogenic greenhouse effect, and leading to significant progress in instrument development. Further dissemination of N2O isotopomer analysis, however, is hampered by a lack of internationally agreed gaseous N2O reference materials and an uncertain compatibility of different laboratories and analytical techniques.METHODSIn a first comparison approach, eleven laboratories were each provided with N2O at tropospheric mole fractions (target gas T) and two reference gases (REF1 and REF2). The laboratories analysed all gases, applying their specific analytical routines. Compatibility of laboratories was assessed based on N2O isotopocule data for T, REF1 and REF2. Results for T were then standardised using REF1 and REF2 to evaluate the potential of N2O reference materials for improving compatibility between laboratories.RESULTSCompatibility between laboratories depended on the analytical technique: isotope ratio mass spectrometry (IRMS) results showed better compatibility for δ15N values, while the performance of laser spectroscopy was superior with respect to N2O site preference. This comparison, however, is restricted by the small number of participating laboratories applying laser spectroscopy. Offset and two-point calibration correction of the N2O isotopomer data significantly improved the consistency of position-dependent nitrogen isotope data while the effect on δ15N values was only minor.CONCLUSIONSThe study reveals that for future research on N2O isotopocules, standardisation against N2O reference material is essential to improve interlaboratory compatibility. For atmospheric monitoring activities, we suggest N2O in whole air as a unifying scale anchor. Copyright © 2014 John Wiley & Sons, Ltd