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Nitrogen (N) is one of the main limiting factors for plant growth and ultimately for the production of harvestable plant material used for animal and human food. In most plant species examined so far, the plant life cycle with regard to the management of N can be roughly divided into two main phases occurring successively. During the first phase, i.e. the vegetative phase, young developing roots and leaves behave as sink organs for the assimilation of inorganic N and the synthesis of amino acids. These amino acids are further used for the synthesis of enzymes and proteins mainly involved in building up plant architecture and the different components of the photosynthetic machinery. Notably, the enzyme Rubisco can alone account for up to 50% of the total soluble leaf protein content (Mae et al., 1983). Later, at a certain stage of plant development generally starting after anthesis, the remobilization of N takes place. At this stage, shoots and/or roots start to behave as sources of N by providing amino acids released from protein hydrolysis, which are subsequently exported to reproductive and storage organs represented, for example, by seeds, bulbs or trunks (Masclaux et al., 2001). In maize, 45–65% of the grain N is provided from pre-existing N in the stover before silking, a process that is strongly dependent upon the environmental conditions and/or the genotype (Weiland & Ta, 1992; Gallais & Coque, 2005). The remaining 35–55% of the grain N originates from postsilking N uptake (Ta & Weiland, 1992; Bertin & Gallais, 2000; Gallais & Coque, 2005). Therefore, two distinct N fluxes must be considered during the grain-filling period: N translocation from the stover (stalks + leaves + sheaths + cob) and N uptake (Crawford et al., 1982; Rendig & Crawford, 1985; Ta & Weiland, 1992).
However, from the point of view of N management at the whole-plant or organ level, the arbitrary separation of the plant life cycle into two phases is rather simplistic, as it is well known that, for example, N recycling can occur before anthesis for the synthesis of new proteins in developing organs (Lattanzi et al., 2005). In addition, during the assimilatory phase, the ammonium incorporated into free amino acids is subjected to a high turnover, as a result of photorespiratory activity, as it needs to be immediately re-assimilated into glutamine and glutamate (Hirel & Lea, 2001; Novitskaya et al., 2002). Therefore, the photorespiratory flux of ammonium, which, at least in C3 plants, can be 10 times higher than that originating from nitrate reduction, is mixed with that channelled through the inorganic N assimilatory pathway (Novitskaya et al., 2002). Although in C4 plants photorespiration is low, this process is likely to occur in maize (Zea mays) (Taiz & Zeiger, 2002; Ueno et al., 2005). Furthermore, Liu & Jagendorf (1984) and Malek et al. (1984) found that in pea (Pisum sativum) approx. 30% of the labelled amino acids newly incorporated into proteins by isolated chloroplasts were lost during the next 30 min, after a chase with cold amino acids. Therefore, the occurrence of protein turnover concomitantly with the two fluxes of ammonium (from assimilatory and photorespiratory fluxes) introduces another level of complexity in the exchange of N within the pool of free amino acids. The coexistence of these different N fluxes has led us to reconsider the mode by which N is managed from the cellular level to the level of the whole plant (Hirel & Gallais, 2006).
Despite the complexity of the N fluxes, a number of attempts have been made to estimate globally the amount of N remobilized to the grain. To achieve this, methods based on the calculation of N budgets, termed the ‘balance method’, or techniques using 15N-labelling have been used. The ‘balance method’ allows estimation of the amount of N remobilized by calculating the difference between total plant N (stalks + leaves) at silking and total N in the stover at grain maturity. This method is based on the assumption that all the newly synthesized amino acids originating from N uptake are directly allocated to the grain (Moll et al., 1982; Di Fonzo et al., 1993; Rajcan & Tollenaar, 1999; Bertin & Gallais, 2000). However, this method does not take into account the possibility that a significant amount of the newly synthesized amino acids may be allocated to the stover before leaf N remobilization, as already shown by Ta & Weiland (1992). The other techniques, which use 15N-labelled fertilizer, provide an elegant way of investigating how N originating from postsilking uptake is directed towards the kernels in the grain. Methods based on short- or long-term labelling have been developed using plants grown either under hydroponics or in the field. Hydroponics allows 15N application during a well-defined period, either short (with pulse chase experiments) or long (with quasi steady-state labelling), whereas in the field only long-term labelling is possible, with the problem that some residual 15N can remain in the soil long after the labelling period. However, the benefit of field experiments employing long-term labelling near natural abundance is that they allow the separation of newly assimilated N from that taken up earlier, as discussed by Deléens et al. (1994). Furthermore, a large number of genotypes can be studied under agronomic conditions.
N recovery from fertilizer has been studied in 15N-labelling experiments in the field (for example, in wheat (Triticum aestivum) by Broadbent & Carlton, 1978, and in maize by Ma & Dwyer, 1998). The reallocation of N to the different organs has been analysed following application of the isotope at different plant developmental stages in maize in the field (Ta & Weiland, 1992; Ma & Dwyer, 1998) or in hydroponics (Weiland, 1989; Cliquet et al., 1990a; Deléens et al., 1994). More recently, Sheehy et al. (2004a,b) have used 15N-labelling in irrigated plots to determine the temporal origin of N in the rice (Oryza sativa) grain.
In maize, application of 15N-nitrate at the beginning of stem elongation allows the determination of N remobilization (Cliquet et al., 1990b). Indeed, when 15N tracer is applied during the vegetative growth period, just before the beginning of rapid growth resulting from stem elongation, both leaves and stalks are labelled, whereas kernels are labelled only as a result of N remobilization. Furthermore, if the proportion of 15N uptake after silking is negligible (which is easier to arrange in a hydroponic experiment than in the field), N remobilization from the stover to the kernels can then be estimated from the percentage of whole-plant 15N uptake allocated to the kernels. Conversely, when the 15N tracer is applied just after silking, the allocation of recent N uptake to the stover and to the kernels can be estimated. Therefore, the use of two labelling techniques at different times is desirable, in order to obtain a complete picture of N management and recycling during the entire developmental cycle of the maize plant.
With plants grown in the field, the originality of our study was to use two 15N-labelling techniques (labelling at the beginning of stem elongation and after silking), in order to investigate the different N fluxes occurring within the plant. A detailed analysis and exploitation of the 15N-labelling data, taking into account the potential of the technique, were performed by estimating the relative 15N-specific allocation (RSA), in order to follow the fate of N assimilated after silking and the N remobilization flux. A predictive model depicting the 15N fluxes occurring between the stover and the grain was then developed to explain and predict the contribution of the various N fluxes to the amount of N in the stover protein and to grain N protein deposition.