The effect of manuring on cereal and pulse amino acid δ(15)N values.

Amino acid δ(15)N values of barley (Hordeum vulgare) and bread wheat (Triticum aestivum) grains and rachis and broad bean (Vicia faba) and pea (Pisum sativum) seeds, grown in manured and unmanured soil at the experimental farm stations of Rothamsted, UK and Bad Lauchstädt, Germany, were determined by GC-C-IRMS. Manuring was found to result in a consistent (15)N-enrichment of cereal grain amino acid δ(15)N values, indicating that manuring did not affect the metabolic routing of nitrogen (N) into cereal grain amino acids. The increase in cereal grain δ(15)N values with manuring is therefore due to a (15)N-enrichment in the δ(15)N value of assimilated inorganic-N. Greater variation was observed in the (15)N-enrichment of rachis amino acids with manuring, possibly due to enhanced sensitivity to changes in growing conditions and higher turnover of N in rachis cells compared to cereal grains. Total amino acid δ(15)N values of manured and unmanured broad beans and peas were very similar, indicating that the legumes assimilated N2 from the atmosphere rather than N from the soil, since there was no evidence for routing of (15)N-enriched manure N into any of the pulse amino acids. Crop amino acid δ(15)N values thus provide insights into the sources of N assimilated by non N2-fixing and N2-fixing crops grown on manured and unmanured soils, and reveal an effect of manure on N metabolism in different crop species and plant parts.

Cereal grain, rachis and pulse seed amino acid δ15N values as indicators of plant nitrogen metabolism.

Natural abundance δ(15)N values of plant tissue amino acids (AAs) reflect the cycling of N into and within plants, providing an opportunity to better understand environmental and anthropogenic effects on plant metabolism. In this study, the AA δ(15)N values of barley (Hordeum vulgare) and bread wheat (Triticum aestivum) grains and rachis and broad bean (Vicia faba) and pea (Pisum sativum) seeds, grown at the experimental farm stations of Rothamsted, UK and Bad Lauchstädt, Germany, were determined by GC-C-IRMS. It was found that the δ(15)N values of cereal grain and rachis AAs could be largely attributed to metabolic pathways involved in their biosynthesis and catabolism. The relative (15)N-enrichment of phenylalanine can be attributed to its involvement in the phenylpropanoid pathway and glutamate has a δ(15)N value which is an average of the other AAs due to its central role in AA-N cycling. The relative AA δ(15)N values of broad bean and pea seeds were very different from one another, providing evidence for differences in the metabolic routing of AAs to the developing seeds in these leguminous plants. This study has shown that AA δ(15)N values relate to known AA biosynthetic pathways in plants and thus have the potential to aid understanding of how various external factors, such as source of assimilated N, influence metabolic cycling of N within plants.

Practical considerations in the determination of compound-specific amino acid δ15N values in animal and plant tissues by gas chromatography-combustion-isotope ratio mass spectrometry, following derivatisation to their N-acetylisopropyl esters.

RATIONALE: Stable nitrogen isotope (δ(15)N) values of bone collagen are routinely used to inform interpretations of diet and trophic positions within contemporary and ancient ecosystems, yet the underlying physiological and biochemical factors which contribute to the bulk collagen δ(15)N value remain little understood. Determination of individual amino acid (AA) δ(15)N values in animal and plant proteins can help to elucidate the cycling of nitrogen and inform predictions of palaeodiet and ecology. METHODS: In this study we present a methodology for the measurement of amino acid δ(15)N values using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Amino acid standards of known δ(15)N values were derivatised to their N-acetylisopropyl (NAIP) esters and purified through Dowex ion-exchange resin to determine any isotopic fractionation associated with derivatisation and ion-exchange chromatography. The effect of starch on AA δ(15)N values was also determined by hydrolysing bone collagen with and without the presence of starch. RESULTS: The amino acids derivatised to their NAIP esters give values within ±0.8‰ of their δ(15)N values measured separately by elemental analyser (EA)-IRMS, with a precision of better than 0.8‰. The δ(15)N values of AAs after Dowex ion-exchange chromatography were within ±0.9‰ of their values prior to ion-exchange chromatography. The AA δ(15)N values of bone collagen hydrolysed with and without starch were within ±0.8‰. CONCLUSIONS: Hydrolysis of lipid-extracted plant material followed by purification of AAs using Dowex ion-exchange resin and derivatisation to their NAIP esters is a suitable protocol for the accurate determination of individual plant and animal AA δ(15)N values by GC-C-IRMS.