Elsevier

Fungal Genetics and Biology

Volume 43, Issue 2, February 2006, Pages 102-110
Fungal Genetics and Biology

GintAMT1 encodes a functional high-affinity ammonium transporter that is expressed in the extraradical mycelium of Glomus intraradices

https://doi.org/10.1016/j.fgb.2005.10.005Get rights and content

Abstract

We report the cloning and characterization of the first NH4+ transporter gene (GintAMT1) in an arbuscular mycorrhizal fungus. GintAMT1 encodes a polypeptide of 479 amino acids sharing high sequence similarity with previously characterized NH4+ transporters from other fungi. Heterologous expression of GintAMT1 in the yeast triple mep mutant complemented the defect of this strain to grow in the presence of less than 1 mM NH4+. As revealed by [14C]methylammonium uptake experiments carried out in yeast, GintAMT1 encodes a high-affinity NH4+ transporter. In mycelia developed in the presence of 0.9 mM NO3, GintAMT1 transcription was increased after the addition of 30 μM NH4+ but decreased after the addition of 3 mM NH4+. However, in mycelia grown in the presence of higher N concentrations, GintAMT1 transcripts decreased after the addition of NH4+, irrespective of the concentration used. These data suggest that GintAMT1 is involved in NH4+ uptake by the extraradical mycelia from the surrounding media when it is present at micromolar concentrations.

Introduction

The roots of most plant species are colonized by arbuscular mycorrhizal (AM) fungi to form mutually beneficial symbiotic associations called mycorrhizas. AM fungi, belonging to the fungal phylum Glomeromycota (Schüßler et al., 2001), are obligate biotrophs which depend entirely on the plant for their carbon supply (Bago and Bécard, 2002). The fungus in return provides the plant with essential mineral nutrients that are taken up from the soil by the extraradical mycelium and translocated to the plant (Smith and Read, 1997). Within the cortical cells of the root the fungus forms finely branched hyphal structures, the arbuscules, which are surrounded by a specialized plant membrane and which are believed to be the site of bi-directional nutrient transfer (Ferrol et al., 2002).

The main benefit of the AM association is an improved P status of the mycorrhizal plant, but there is increasing evidence that AM fungi also contribute to the N-budget of the plant. Different authors have shown the ability of the extraradical mycelium of AM fungi to deplete the inorganic 15N, added as 15NH4+ to the soil of a root-free compartment (Frey and Schüepp, 1993, Johansen et al., 1992, Johansen et al., 1993) and that AM extraradical hyphae were able to transport this N-isotope to a plant which was growing several centimetres away from the nitrogen source. The capability of AM extraradical hyphae to take up (Bago et al., 1996) and transport 15NO3 to the host plant has also been reported (Tobar et al., 1994a, Tobar et al., 1994b). Much less is known about the uptake of organic nitrogen, but Hawkins et al. (2000) showed that AM hyphae are able to take up glycine and glutamic acid and transport nitrogen from these sources to the plant roots in a form that is presently unknown. Moreover, Hodge et al. (2001) reported that the AM symbiosis can both enhance decomposition of and increase nitrogen capture from complex organic material in soil.

Although AM fungi are able to take up both NO3 and NH4+, a clear preference for NH4+ has been demonstrated (Hawkins et al., 2000, Toussaint et al., 2004, Villegas et al., 1996), which is explained, at least in part, by the extra energy the fungus must expend in reducing NO3 to NH4+ before it can be incorporated into organic compounds (Marzluf, 1996). Ammonium is a ubiquitous intermediate in nitrogen metabolism and one of the major nutrients for plants and microorganisms. The whole process of NH4+ assimilation in the AM symbiosis involves NH4+ uptake and assimilation by the AM fungus and transfer to the plant. Several lines of evidence indicate that the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle is responsible for NH4+ assimilation in AM extraradical hyphae (Breuninger et al., 2004, Johansen et al., 1996), although the involvement of glutamate dehydrogenase has not been experimentally excluded. Recently, Govindarajulu et al. (2005) have shown that inorganic nitrogen taken up by the external mycelium is incorporated into amino acids, translocated from the extraradical to the intraradical mycelium as arginine, but transferred to the plant as NH4+.

In most organisms, ammonium uptake is mediated by members of the NH4+ transporter family (AMT/MEP). The first of the genes involved in NH4+ transport were cloned from yeast (Marini et al., 1994) and Arabidopsis (Ninnemann et al., 1994). Since then, these genes have been found in a variety of bacteria (Thomas et al., 2000), fungi (Javelle et al., 2001, Javelle et al., 2003b, Montanini et al., 2002) and homologues have been found in humans (Marini et al., 2000). Aside from their role in NH4+ uptake, NH4+ transporters can also act as NH4+ sensors. In yeast, the high-affinity transporter MEP2 has been considered to act as the sensor for low NH4+ availability and evidence suggests that it is associated with the signal transduction cascades leading to filamentous growth (Lorenz and Heitman, 1998). The objective of the present work was to characterize an NH4+ transporter of Glomus intraradices in order to get some insights into the mechanisms of nitrogen acquisition and sensing in AM fungi.

Section snippets

Arbuscular mycorrhizal monoxenic cultures and N treatments

Arbuscular mycorrhizal monoxenic cultures were established as described by St-Arnaud et al. (1996). Briefly, clone DC2 of carrot (Daucus carota L.) Ri-T DNA transformed roots were cultured with the AM fungus G. intraradices Smith & Schenck (DAOM 197198, Biosystematic Research Center, Ottawa, Canada) in two-compartment Petri dishes. Cultures were initiated in one compartment (“root compartment”) of each plate, which contained M medium (Chabot et al., 1992). Fungal hyphae, but not roots, were

Sequence analysis of GintAMT1

The full-length GintAMT1 cDNA contains a 1437 nucleotide-long open reading frame with 142 nucleotides before the putative ATG start codon and a tail of 46 bp. The translation product of this open reading frame was 479 amino acids long, with a predicted molecular weight of 50 kDa. The alignment shown in Fig. 1A indicates that the predicted sequence from cDNA shows high homology to the functionally characterized NH4+ transporter AMT1 of Tuber borchii (61% identity) and to the Hebeloma cylindrosporum

Discussion

We have identified the first NH4+ transporter in an AM fungus. The identification has been based on homology to members of the MEP/AMT protein family and on the functional complementation of the yeast triple mutant strain 31019b (mep1Δ mep2Δ mep3Δ). MEP/AMT proteins have now been identified and characterized from a range of organisms and a number of similarities and conserved properties have been observed for these important proteins. Members of the MEP/AMT family are predicted to exist as

Acknowledgments

We thank Dr. Anne-Marie Marini (Institut de Biologie et de Médecine Moléculaire, Université Libre de Bruxelles, Gosselies, Belgium) for providing the S. cerevisiae strains used in this study and Dr. José Miguel Barea for critical reading of the manuscript. This research was supported by the EU project Genomyca (QLK5-CT-2000-01319) and by CICyT (project AGL2003-01551), Spain.

References (52)

  • A. Schüßler et al.

    A new fungal phylum, the Glomeromycota: phylogeny and evolution

    Mycol. Res.

    (2001)
  • M. St-Arnaud et al.

    Enhanced hyphal growth and spore production of the arbuscular mycorrhizal fungus Glomus intraradices in an in vitro system in the absence of host roots

    Mycol. Res.

    (1996)
  • B. Bago et al.

    Bases of the obligate biotrophy of arbuscular mycorrhizal fungi

  • B. Bago et al.

    Nitrate depletion and pH changes induced by the extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic culture

    New Phytol.

    (1996)
  • B. Bago et al.

    Differential morphogenesis of the extraradical mycelium of an arbuscular mycorrhizal fungus grown monoxenically on spatially heterogeneous culture medium

    Mycologia

    (2004)
  • D. Becker et al.

    Expression of the NH4+-transporter gene LEAMT1;2 is induced in tomato roots upon association with N2-fixing bacteria

    Planta

    (2002)
  • J.A. Coffman et al.

    Gat1p, a GATA family protein whose production is sensitive to nitrogen catabolite repression, participates in transcriptional activation of nitrogen-catabolic genes in Saccharomyces cerevisiae

    Mol. Cell. Biol.

    (1996)
  • S. Chabot et al.

    Life cycle of Glomus intraradix in root organ culture

    Mycologia

    (1992)
  • Felstein, J., 1993. PHYLIP, version 3.5. Department of Genetics, University of Washington,...
  • N. Ferrol et al.

    Mechanisms of nutrient transport across interfaces in arbuscular mycorrhiza

    Plant Soil

    (2002)
  • B. Frey et al.

    Acquisition of nitrogen by external hyphae of arbuscular mycorrhizal fungi associated with Zea mays L

    New Phytol.

    (1993)
  • S. Gazzarrini et al.

    Three functional transporters for constitutive, diurnally regulated and starvation-induced uptake of ammonium into Arabidopsis roots

    Plant Cell

    (1999)
  • M. Govindarajulu et al.

    Nitrogen transfer in the arbuscular mycorrhizal symbiosis

    Nature

    (2005)
  • H.J. Hawkins et al.

    Uptake and transport of organic and inorganic nitrogen by arbuscular mycorrhizal fungi

    Plant Soil

    (2000)
  • T. Hirokawa et al.

    SOSUI: classification and secondary structure prediction system for membrane proteins

    Bioinformatics

    (1998)
  • A. Hodge et al.

    An arbuscular mycorrhizal fungus accelerates decomposition and acquires nitrogen directly from organic material

    Nature

    (2001)
  • Cited by (152)

    • Plant nitrogen nutrition: The roles of arbuscular mycorrhizal fungi

      2022, Journal of Plant Physiology
      Citation Excerpt :

      AM fungi are thought to have a preference to acquire NH4+ in comparison with NO3− from soil, probably because NO3− needs to be reduced to NH4+ before it can be incorporated into organic compounds, with the process requiring extra energy (Tobar et al., 1994; Tanaka and Yano, 2005). The molecular evidence for the capacity of AM fungi in taking up NH4+ and NO3− could be traced through the identification of several fungal NH4+ and NO3− transporter genes, such as GintAMT1, 2, and 3, and GiNT, from Rhizophagus intraradices (Table 1) (Lopez-Pedrosa et al., 2006; Tian et al., 2010; Pérez-Tienda et al., 2011; Tisserant et al., 2013; Koegel et al., 2015; Calabrese et al., 2016). With no exception, all these transporters showed a high abundance of transcripts in ERM when the AM fungus was associated with a carrot root.

    • Use of microbial inoculants against biotic stress in vegetable crops: physiological and molecular aspect

      2022, Sustainable Horticulture: Microbial Inoculants and Stress Interaction
    View all citing articles on Scopus
    1

    The two first authors have contributed equally to this work.

    View full text