Because nitrogen is one of the major elements limiting growth of plants in agrosystems, large amounts of N fertilisers have been used in the second half of the twentieth century. Chemical fertilisers have contributed to increasing crop yields and food supply, but they have induced environmental damage such as nitrate pollution and wasting fossil fuel. The use of legumes grown in rotations or intercropping is now regarded as an alternative and sustainable way of introducing N into lower input agrosystems. Here we review agricultural practices, measurement methods and biological pathways involved in N cycling. We show that plant roots interact intimately with soil microflora to convert the most abundant but relatively inert form of N, atmospheric N 2 , into biological substrates available for growth of other plants, through two consecutive processes; namely, N 2 fixation and N rhizodeposition. In intercropping, companion plants benefit from biological fixation by legumes and subsequent transfer of N from legumes to non-legumes. This transfer from legumes to the release of N compounds by legume roots, a process named rhizodeposition, then the uptake by the companion crop. The two main rhizodeposition pathways are (i) decomposition and decay of nodules and root cells, and (ii) exudation of soluble N compounds by plant roots. The contribution of root N and rhizodeposited N to the soil-N pool is difficult to measure, particularly in the field. Firstly, root N is often underestimated because root recovery is problematic. Second, assessment of N rhizodeposition is challenging. Several 15 N labelling methods have been performed for different legume species. Rhizodeposition of N, as a percentage of total plant N, varied from 4 to 71%. The high variability of the results illustrates the need for more studies of the environmental and genetic factors influencing the amount of N rhizodeposits released by legumes under field conditions. N rhizodeposition / legumes / N 2 fixation / 15 N / isotopic methods / root exudates / ecological fertilisation

Amino acid concentration in the rhizosphere results from fluxes between plant roots, soil and microorganisms. In this context, root amino acid exudation process, composed of both efflux and influx, remains unclear. One main issue is to understand the selectivity of amino acid exudation resulting mainly in high proportions of glycine and serine in exudates compared to low proportions inside the root. To reach this point, a quantitative analysis of exudation with dissociated measurements of efflux from influx is needed. We measured efflux and influx by supplying 15 N-labelled glycine or serine for a short time of exposure at ecologically relevant concentrations to plants of white clover (Trifolium repens L.), perennial ryegrass (Lolium perenne L.), maize (Zea mays L.), oilseed rape (Brassica napus L.), tomato (Lycopersicon esculentum Mill.) and alfalfa (Medicago sativa L.). Efflux was estimated by the increase of 14 N content of amino acids in root exudates and influx was estimated by the increase of 15 N content in plant tissue. Glycine efflux exceeded influx for all six species and was much higher in Fabaceae than in Poaceae. Serine efflux exceeded influx in alfalfa, white clover and rape. We conclude that presence of glycine and serine in root bathing solutions results from high glycine and serine efflux rates, observed in all six species studied here. The physiological and ecological significances of these high efflux rates are discussed in the context of N metabolism and plantsoil-microorganisms interactions.

The overuse of classical N fertilisers contributes substantially to environmental degradation by pollution of groundwater by nitrates. This leaching of N in waters is also an economic flaw for farmers because only a part of the fertiliser is used by the plants. Here, systems involving mixtures of legumes and grasses represent a sustainable alternative because legumes can fix atmospheric N 2 using symbiotic microbes. N transfer in those mixtures has been thoroughly investigated but little is known concerning the effect of N fertiliser on N transfer between N-fixing legumes and companion grasses. In white clover (Trifolium repens L.) -perennial ryegrass (Lolium perenne L.) associations, N is transferred mostly through rhizodeposition into the soil by clover followed by re-uptake by ryegrass. Rhizodeposition of N occurs through senescence and decomposition of legume tissue or through exudation of N compounds by living cells. Ammonium and amino acids are the main compounds exuded and their exudation is thought to occur by passive diffusion attributed to a concentration gradient from root to soil. In this study, we test the hypothesis that greater N transfer from clover to grass, as seen in N-rich soils or nutrient solutions, is due to greater N rhizodeposition brought about by higher ammonium and amino acid content of roots. The relations between N input, root N content, N net exudation and N transfer between legumes and grasses were investigated using 15 N by growing white clover and perennial ryegrass with increasing N application in axenic microlysimeters or in pots. Ammonium and amino acid concentrations were measured in root tissues, in root bathing solutions and in soils. We found that mineral N application strongly reduced atmospheric N fixation by clover, from 3.0 to 0.9 mg per plant, and root amino acid content, from 164 to 49 nmoles per g dry weight, but had no effect on ammonium and amino acid concentrations in sterile exudates, showing for the first time that amino acid net exudation is independent of root content. In contrast, ammonium and amino acid concentrations in clover soils increased with N fixation, showing the link between N fixation and N rhizodeposition in soils. Nitrate application increased ryegrass root growth by 7-8 times, and transfer of N between clover and ryegrass (by 3 times). It is concluded that N fertiliser does not modify N exudation but decreases N fixation and ammonium rhizodeposition in soil by clover. N fertiliser increases N transfer between clover and ryegrass by increasing soil exploration by ryegrass and giving a better access to different available N sources, including the N compounds exuded from clover.

The mechanism of root amino acid exudation was studied in white clover (Trifolium repens L.) to explain the apparent selectivity of this process resulting in contrasted amino acid profiles between root tissues and root exudates. Asparagine is generally recovered in low proportions in root exudates but represent a major fraction of amino acids in root tissues, and the opposite is found for glycine. Amino acid profiles were studied in two potential sites of intense exudation, root tips and nodules, and a 15 N labelling method was used to compare influxes and effluxes of different amino acids. Metabolic inhibitors were used to test the assumption that amino acid exudation is a passive process. We observed that amino acid profiles were similar between whole roots, root tips and nodules. Influxes of asparagine and glycine were in the same range, but efflux of glycine largely exceeded efflux of asparagine. Metabolic inhibitors strongly reduced glycine influx and modified glycine efflux. It is concluded that the selectivity of amino acid exudation is not explained by the composition of putative intense sites of exudation but is explained by highly contrasted effluxes between amino acids. The effect of metabolic inhibitors shows that glycine efflux occurs not only as a simple passive leakage. These results suggest that amino acid exudation is a plant-controlled process.

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