Phytochemistry Reviews 2: 133–144, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 133 Prospects for improving nitrogen use efficiency: Insights given by 15 N-labelling experiments † Bertrand Hirel 1,∗ & Anis M. Limami 2 1 Unité de Nutrition Azotée des Plantes, I.N.R.A., R.D. 10, 78026 Versailles Cedex France; 2 U.M.R. Physiolo- gie Mol´ eculaire des Semences. U.F.R. Sciences, 2 Bd. Lavoisier. 49045 Angers Cedex 01, France; ∗ Author for correspondence (Tel: (33) 1 30 83 30 96; Fax: (33) 1 30 83 30 89; E-mail: hirel@versailles.inra.fr) Key words: breeding, genetic manipulations, nitrogen assimilation, 15 N-labelling, transport Abstract In higher plants, recent advances in plant molecular genetics, combined with modern physiological and biochem- ical studies, have expanded our understanding of the regulatory mechanisms controlling the primary steps of inorganic nitrogen assimilation and the subsequent biochemical pathways involved in nitrogen supply and recycling for higher plant metabolism, growth and development. In this presentation, we describe improvements in our understanding of the molecular controls of nitrogen assimilation through the use of transgenic plants and the study of genetic variability in model and crop species. To illustrate this research programme, the physiological impact of modified gene expression, using either transgenic plants or different genotypes, was studied using 15 N-labelling experiments in order to monitor the influx of nitrate or ammonia and its subsequent incorporation into amino acids. Abbreviations: cHATS – constitutive high affinity transport system; iHATS – inducible high affinity transport system; LATS – Low affinity transport system; GS – Glutamine synthetase; GOGAT – Glutamate synthase; N – nitrogen; NR – Nitrate reductase; NUE – Nitrogen use efficiency; RILs – Recombinant inbred lines. Introduction In higher plants, recent advances both in molecular physiology and genetics have helped to expand our un- derstanding of the regulatory mechanisms controlling the primary steps of inorganic nitrogen assimilation and the subsequent biochemical pathways involved in nitrogen supply to allow optimal growth and develop- ment (Figure 1). Nitrate is the principal nitrogen source for most wild and crop species. Following its uptake by means of specific transporters located in the root cell mem- brane (Orsel et al., 2002), the assimilation of nitrate is a two-step process. First, the enzyme nitrate reductase (NR) catalyses the reduction of nitrate to nitrite. Sub- sequently, the enzyme nitrite reductase mediates the † This paper is dedicated to Dr Eliane Del´ eens, deceased in March 2003, in memory to her outstanding scientific contribution to plant physiology and agronomy through the use of heavy isotopes. reduction of nitrite to ammonium. Root-specific trans- porters can allow the direct absorption of ammonium when it is available in the soil or under particular en- vironments, such as rice paddy fields or acidic forest soils (Salsac et al., 1987; Mae, 1997). In addition to nitrate reduction, ammonium can be generated inside the plant by a variety of metabolic pathways such as photorespiration, phenylpropanoid metabolism, util- isation of nitrogen transport compounds and amino acids catabolism, from symbiotically fixed nitrogen (Hirel and Lea, 2001) and from insect digestion in carnivorous plants (Shulze et al., 1997). In particular. terrestrial ecosystems, where the rate of mineralisation of organic nitrogenous compounds is low, plants can directly take up amino acids or through mycorrhizal symbiotic associations (Näsholm et al., 1998; Chalot and Brun, 1998). Ammonia, which is the ultimate form of inorganic nitrogen available to the plant, is then incorporated