Regulation of the Nitrogen Transfer Pathway in the Arbuscular Mycorrhizal Symbiosis: Gene Characterization and the Coordination of Expression with Nitrogen Flux

The arbuscular mycorrhiza (AM) brings together the roots of over 80% of land plant species and fungi of the phylum Glomeromycota and greatly benefits plants through improved uptake of mineral nutrients. AM fungi can take up both nitrate and ammonium from the soil and transfer nitrogen (N) to host roots in nutritionally substantial quantities. The current model of N handling in the AM symbiosis includes the synthesis of arginine in the extraradical mycelium and the transfer of arginine to the intraradical mycelium, where it is broken down to release N for transfer to the host plant. To understand the mechanisms and regulation of N transfer from the fungus to the plant, 11 fungal genes putatively involved in the pathway were identified from Glomus intraradices, and for six of them the full-length coding sequence was functionally characterized by yeast complementation. Two glutamine synthetase isoforms were found to have different substrate affinities and expression patterns, suggesting different roles in N assimilation. The spatial and temporal expression of plant and fungal N metabolism genes were followed after nitrate was added to the extraradical mycelium under N-limited growth conditions using hairy root cultures. In parallel experiments with ¹⁵N, the levels and labeling of free amino acids were measured to follow transport and metabolism. The gene expression pattern and profiling of metabolites involved in the N pathway support the idea that the rapid uptake, translocation, and transfer of N by the fungus successively trigger metabolic gene expression responses in the extraradical mycelium, intraradical mycelium, and host plant.The arbuscular mycorrhizal (AM) symbiosis brings together the roots of the majority of land plant species and fungi of the phylum Glomeromycota to great mutual advantage (Smith and Read, 2008). AM fungi improve the acquisition of phosphate, nitrogen (N), sulfur, and trace elements such as copper and zinc (Clark and Zeto, 2000; Allen and Shachar-Hill, 2008) and increase the biotic and abiotic stress resistance of their host (Smith et al., 2010). In return, the host transfers up to 20% of its photosynthetically fixed carbon to the AM fungus (Jakobsen and Rosendahl, 1990), which depends on its host plant for its carbon supply (Bago et al., 2000).N is the nutrient whose availability most commonly limits plant growth in natural ecosystems. AM fungi can take up NO₃⁻NH₄⁺ and can also increase access to organic N sources from the soil (Ames et al., 1983; Johansen et al., 1993; Bago et al., 1996; Hodge et al., 2001). The translocation by the fungus can represent a significant route for N uptake by the plant (Johansen and Jensen, 1996). For example, Toussaint et al. (2004) showed that in an in vitro mycorrhiza at least 21% of the total N uptake in the roots came from the fungal extraradical mycelium (ERM); for other mycorrhizal systems, even larger proportions have been described (more than 30% and 50%; Govindarajulu et al., 2005; Jin et al., 2005). Tanaka and Yano (2005) reported that 75% of the N in leaves of mycorrhizal maize (Zea mays) was taken up by the ERM of Glomus aggregatum.A mechanism of N transfer from the fungus to the plant has been proposed (Bago et al., 2001) that involves the operation of a novel metabolic route in which N was translocated from the ERM to the intraradical mycelium (IRM) as Arg but transferred to the plant without carbon as inorganic N. This mechanism has been supported by labeling experiments (Johansen et al., 1996; Govindarajulu et al., 2005; Jin et al., 2005), enzyme activity analysis (Cruz et al., 2007), and limited gene expression data (Govindarajulu et al., 2005; Gomez et al., 2009; Guether et al., 2009). Nevertheless, our molecular knowledge of the metabolic and transport pathways involved and how they are regulated is still rudimentary. A better understanding of the mechanism and regulation of N uptake assimilation, translocation, and transfer to the host is important for potential applications of AM fungi as biofertilizers, bioprotectors, and bioregulators in sustainable agriculture and restoration as well as for understanding the role of AM fungi in natural ecosystems (Bruns et al., 2008).In this study, we postulate that the uptake, translocation, and transfer of N by the fungus triggers the metabolic gene expression responses successively in the ERM, IRM, and host plant, which will result in the synthesis and accumulation of Arg in the ERM, the turnover of Arg to release ammonium in the IRM, and the assimilation of ammonium by the host plant via the glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway inside the root (Fig. 1). To test these predictions, 11 genes involved in the N primary assimilation and metabolism were cloned and verified from Glomus intraradices; six enzymes with full-length coding sequences (CDSs) were functionally characterized by yeast knockout mutant complementation. Two GS proteins were found to have different substrate affinities and expression patterns, suggesting that they have different roles in N assimilation. The time courses of gene expression and N movement in fungal and host tissues were analyzed following nitrate supply to the ERM of a mycorrhiza grown under N-limited conditions. The results substantially increase our knowledge of the identity and regulation of most of the metabolic and transport genes involved in N movement through the AM symbiosis.Open in a separate windowFigure 1.Working model of N transport and metabolism in the symbiosis between plant roots and arbuscular mycorrhizal fungi. N moves (black arrows) from the soil into the fungal ERM, through a series of metabolic conversion reactions into Arg, which is transported into the IRM within the root (host) and there is broken down; N is transferred to and assimilated by the host as ammonia. Red circles refer to the sites of action of the genes identified and analyzed in this study. Blue arrows indicate mechanisms hypothesized to regulate gene expression by N metabolites involved in the pathway.