A mycorrhizal-specific ammonium transporter from Lotus japonicus acquires nitrogen released by arbuscular mycorrhizal fungi

Abstract

In mycorrhizal associations, the fungal partner assists its plant host by providing nitrogen (N) in addition to phosphate. Arbuscular mycorrhizal (AM) fungi have access to inorganic or organic forms of N and translocate them via arginine from the extra- to the intraradical mycelium, where the N is transferred to the plant without any carbon skeleton. However, the molecular form in which N is transferred, as well as the involved mechanisms, is still under debate. NH(4)(+) seems to be the preferential transferred molecule, but no plant ammonium transporter (AMT) has been identified so far. Here, we offer evidence of a plant AMT that is involved in N uptake during mycorrhiza symbiosis. The gene LjAMT2;2, which has been shown to be the highest up-regulated gene in a transcriptomic analysis of Lotus japonicus roots upon colonization with Gigaspora margarita, has been characterized as a high-affinity AMT belonging to the AMT2 subfamily. It is exclusively expressed in the mycorrhizal roots, but not in the nodules, and transcripts have preferentially been located in the arbusculated cells. Yeast (Saccharomyces cerevisiae) mutant complementation has confirmed its functionality and revealed its dependency on acidic pH. The transport experiments using Xenopus laevis oocytes indicated that, unlike other plant AMTs, LjAMT2;2 transports NH(3) instead of NH(4)(+). Our results suggest that the transporter binds charged ammonium in the apoplastic interfacial compartment and releases the uncharged NH(3) into the plant cytoplasm. The implications of such a finding are discussed in the context of AM functioning and plant phosphorus uptake.

Figure 1.

A, An unrooted phylogenetic tree for the amino acid sequences of AMT1/AMT2 plant AMTs. The dendrogram was generated by Mega 4.0 software using ClustalW for the alignment and the neighbor-joining method for the construction of the phylogeny (Tamura et al., 2007). Bootstrap tests were performed using 1,000 replicates. The branch lengths are proportional to the phylogenetic distances. Abbreviations for plant species: At, A. thaliana; Le, Lycopersicon esculentum; Lj, L. japonicus; Os, O. sativa; Ptr, P. trichocarpa; Ec, E. coli; Ne, N. europaea. The mycorrhiza-specific LjAMT2;2 is marked with a black arrowhead. The accession numbers for the AMTs used are as follows: AtAMT1;1 (1703292); AtAMT1;2 (4324714); AtAMT1;3 (5880355); AtAMT1;4 (7450345); AtAMT1;5 (5672513); AtAMT2 (3335376); OsAMT1;1 (AAL05612.1); OsAMT1;2 (AAL05613.1); OsAMT1;3 (AAL05614.1); OsAMT2;1 (BAB87832.1); OsAMT2;2 (NP_915334); OsAMT2;3 (NP_915337.1); OsAMT3;1 (BAC65232.1); OsAMT3;2 (AAO41130); OsAMT3;3 (AK108711); OsAMT4 (AAL58960); LeAMT1;1 (P58905); LeAMT1;2 (O04161); LeAMT1;3 (Q9FVN0); LjAMT1;1 (Q9FSH3); LjAMT1;2 (Q7Y1B9); LjAMT1;3 (Q70KK9); LjAMT2;1 (AAL08212); P. trichocarpa AMTs (Couturier et al., 2007); EcAmtB (NP_286193); and NeRh-1 (NP_840535). B, Intron-exon structure of the LjAMT2;2 gene; exons, black.

Figure 2.

Relative expression of LjAMT2;2 assessed by quantitative RT-PCR in L. japonicus roots after 28 d of mycorrhization by Gi. margarita and 35 d after nodulation by Mesorhizobium loti. The C_t values (threshold cycles) of the samples are corrected against the C_t values of the housekeeping gene ubiquitin (LjUBQ10 [UBI]). Data for each condition are presented as mean + sd and were obtained from three biological and three technical replicates. −N = 10 μm KNO₃; +N = 4 mm KNO₃; −P = 20 μm PO₄³⁻; +P = 500 μm PO₄³⁻.

Figure 3.

RT-PCR analysis of LjAMT2;2 in three different cell-type populations: ARB, arbusculated cells; MNM, noncolonized cortical cells from mycorrhizal roots; and C, cortical cells from nonmycorrhizal roots. LjEF1α amplicons, obtained from parallel control reactions and loaded in the same order as above, were used as internal standards. The sizes of the LjAMT2;2 and LjEF1α amplicons are indicated. Reactions were performed allowing both 37 and 40 cycles of amplifications.

Figure 4.

A, Growth of NH₄⁺ uptake-deficient yeast (31019b; ΔΔΔmep1;2;3) transformed with NeRh-1, AtAMT1;2, AtAMT2;2, LjAMT2;2, and the empty control plasmid pDR199 on 3 mm ammonium as sole N source at different pH. Shown are serial dilutions (dil.) of cell suspensions ranging from 1 to 1 × 10⁻⁴. B, [¹⁴C]MeA uptake rates of LjAMT2;2 and NeRh-1 at pH 4.5 and 6.5. Data for each condition are presented as mean + SEM and were obtained from four technical replicates.

Figure 5.

Growth of yeast AMT-deficient strain 31019b on YNB medium supplemented with 80 mm MeA⁺ and Arg as the sole metabolizable N source. The strain was transformed with NeRh-1, AtAMT1;2, AtAMT2;2, LjAMT2;2, and control plasmid pDR199.

Figure 6.

Concentration-dependent kinetics of [¹⁴C]MeA uptake by yeast strain 31019b transformed with the pDR199 LjAMT2;2. Yeast strain 31019b transformed with insert-free pDR199 were used to measure background transport activity, and results are reported as net transport.

Figure 7.

A, Expression of LjAMT2;2 and AtAMT1;2 in oocytes. Shown are inward currents by 3 mm NH₄Cl at −100 mV from oocytes injected with equal amounts of cRNA. Data are from three to six oocytes. Similar data were obtained in three independent experiments. Data for each condition are presented as mean + sd and were obtained from more than eight biological replicates. B, [¹⁴C]MeA uptake rate of the same oocytes injected with LjAMT2;2 cRNA and of water-injected controls. Data for each condition are presented as mean + sd and were obtained from eight biological replicates for AtAMT1;2 and 20 biological replicates for LjAMT2;2.

Figure 8.

Homology model of LjAMT2;2 (A) and illustration of important residues in the crystal structure of EcAmtB (B). Key residues that are thought to be important for NH₄⁺ binding, subsequent deprotonation, and NH₃ translocation in EcAmtB are also found in LjAMT2;2.

Figure 9.

The scheme illustrates N, phosphorus, and carbohydrate exchanges at the mycorrhizal interface according to previous works and the present results. a, NH₃/NH₄⁺ is released in the arbuscules from Arg, which is transported from the extra- to the intraradical fungal structures (Govindarajulu et al., 2005). NH₃/NH₄⁺ is then translocated by so far unknown mechanisms (transporter, diffusion, or vesicle mediated) into the periarbuscular space, where, due to the acidic environment, its ratio shifts toward NH₄⁺ (>99.99%). b, The acidity of the interfacial apoplast is established by plant and fungal H⁺-ATPases (Hause and Fester, 2005; Balestrini et al., 2007), thus providing the energy for H⁺-dependent transport processes. c, The NH₄⁺ ion is deprotonated prior to its transport across the plant membrane via the LjAMT2;2 protein and released in its uncharged NH₃ form into the plant cytoplasm. The NH₃/NH₄⁺ acquired by the plant is either transported into adjacent cells or immediately incorporated into amino acids. d, Phosphate is released by so far unknown transporters into the interfacial apoplast. e, The uptake of phosphate on the plant side then is mediated by mycorrhiza-specific Pi transporters (Javot et al., 2007b; Guether et al., 2009). f, AM fungi might control the net Pi release by their own Pi transporters, which may reacquire phosphate from the periarbuscular space (Balestrini et al., 2007). g, Plant-derived carbon is released into the periarbuscular space probably as Suc and then cleaved into hexoses by Suc synthases (Hohnjec et al., 2003) or invertases (Schaarschmidt et al., 2006). AM fungi then acquire hexoses (Shachar-Hill et al., 1995; Solaiman and Saito, 1997) and transport them over their membrane by so far unknown hexose transporters. It is likely that these transporters are proton cotransporters as the GpMST1 described for the glomeromycotan fungus Geosiphon pyriformis (Schuessler et al., 2006). Exchange of nutrients between arbusculated cells and noncolonized cortical cells can occur by apoplastic (h) or symplastic (i) ways.