Identification and characterization of a PutAMT1;1 gene from Puccinellia tenuiflora

Abstract

Nitrogen is one of the most important limiting factors for plant growth. However, as ammonium is readily converted into ammonia (NH3) when soil pH rises above 8.0, this activity depletes the availability of ammonium (NH4(+)) in alkaline soils, consequently preventing the growth of most plant species. The perennial wild grass Puccinellia tenuiflora is one of a few plants able to grow in soils with extremely high salt and alkaline pH (>9.0) levels. Here, we assessed how this species responds to ammonium under such conditions by isolating and analyzing the functions of a putative ammonium transporter (PutAMT1;1). PutAMT1;1 is the first member of the AMT1 (ammonium transporter) family that has been identified in P. tenuiflora. This gene (1) functionally complemented a yeast mutant deficient in ammonium uptake (2), is preferentially expressed in the anther of P. tenuiflora, and (3) is significantly upregulated by ammonium ions in both the shoot and roots. The PutAMT1;1 protein is localized in the plasma membrane and around the nuclear periphery in yeast cells and P. tenuiflora suspension cells. Immunoelectron microscopy analysis also indicated that PutAMT1;1 is localized in the endomembrane. The overexpression of PutAMT1;1 in A. thaliana enhanced plant growth, and increased plant susceptibility to toxic methylammonium (MeA). Here, we confirmed that PutAMT1;1 is an ammonium-inducible ammonium transporter in P. tenuiflora. On the basis of the results of PutAMT1;1 overexpression in A. thaliana, this gene might be useful for improving the root to shoot mobilization of MeA (or NH4(+)).

Figure 1. Sequence and phylogenetic analysis of PutAMT1;1.

(A) Amino acid sequence of PutAMT1;1 and phylogenetic trees analysis of AMT families; Sequences were obtained from the GeneBank database. The accession numbers are listed in the Material and Methods section. (B) An alignment of the amino acid sequence of PutAMT1;1 (GenBank NO. JQ279059) with TaAMT1;1 (GenBank NO. AY525637), OsAMT1;1 (GenBank NO. AF289477), and AtAMT1;1 (GenBank NO. NM_-117425); the colored box indicates conserved amino acid residues. (C) The transmembrane domains in PutAMT1;1 were predicted by the TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM), and are underlined in black in (B).

Figure 2. Expression analysis of the PutAMT1;1 gene.

Expression was analyzed using semi-quantitative RT-PCR (A) and real-time PCR in the roots (B) and shoots (C) using actin and tubulin mRNA as reference material, respectively. The expression of PutAMT1;1 is given relative to tubulin mRNA levels, and is the mean of 3 replicates ±S.E. PutAMT1;1 expression in control plants (-N) and plants exposed to different nitrogen sources: (NH₄)₂SO₄, Ca(NO₃)₂, and NH₄NO₃ at 2 and 24 h after treatment.

Figure 3. Subcellular localization of PutAMT1;1.

(A to C) Using the petiole epidermis of transgenic plant with pBI121-PutAMT1;1-GFP. (A) is GFP, (B) is the green GFP fluorescence merged with the chloroplast fluorescence, (C) is DIC; (D to G) GFP fusion proteins of PutAMT1;1 expressed in yeast (D, E) and Puccinellia tenuiflora cells (F, G). All images were created using a laser-scanning confocal imaging system (Olympus Fluoview, FV500). GFP fluorescence was excited using an argon laser (488 nm). Chloroplast fluorescence was detected at 620 nm. The size of the scale bars are shown on the images.

Figure 4. Immunogold localization of PutAMT1.1-GFP.

Overview of an immunogold labeled cryosection of a root tip cortex cell expressing PutAMT1.1-GFP (A and B). Arrows indicate the gold particles that specifically labeled a thin layer of endomembrane. Scale bar is 1 µm. B is a magnified view of A.

Figure 5. Growth complementation of the ammonium uptake-defective yeast strain 31019b by PutAMT1;1.

The triple mep yeast mutant 31019b was transformed with the empty vector pYES2, pYES2-PutAMT1;1 growth; (A) on YNB medium containing either 0 mM, 1 mM, 5 mM, 10 mM, or 20 mM NH₄ ⁺ as the sole nitrogen source in the presence of 2% (w/v) galactose, and (B) on different pH medium at 4.0, 5.0, 6.0, 7.0, and 8.0 in the presence of 1 mM NH₄ ⁺ and 2% (w/v) galactose.

Figure 6. (A) RNA gel blot analysis of T3 transgenic plants expressing PutAMT1;1.

WT: Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0; #1, #2, and #3. T3 transferred Columbia-0 with PutAMT1;1. (B to F) Growth of wild-type and PutAMT1;1 transgenic lines on different amounts of nitrogen sources. Growth of WT plants and transgenic PutAMT1;1 plants (lines 1, 2, and 3) on agarose containing (B)1/2 MS, (C) 0 µM, (D) 25 µM, and (E) 250 µM ammonium succinate for 7 days after pre-culture on half-strength MS medium for 7 days. (F) Plot showing root growth vs. the amount of ammonium succinate. Bars indicate the means ± SE (n = 12). Statistical significance was determined using the Student’s t-test. * represents p<0.05 and ** represents p<0.01.

Figure 7. Growth of wild-type and PutAMT1;1 transgenic lines on different amounts of nitrogen sources.

Root growth of WT plants and transgenic PutAMT1;1 plant (lines 1, 2, and 3) were plotted after incubating plants on agarose containing different nitrogen sources, including 5 mM KNO₃, 250 µM NH₄NO₃, and 125 µM (NH₄)₂SO₄ for 7 days, with plants being pre-cultured on half-strength MS medium for 7 days. Bars indicate means ± SE (n = 12). Statistical significance was determined using the Student’s t-test. * represents p<0.05 and ** represents p<0.01.

Figure 8. Growth of wild-type and PutAMT1;1 transgenic lines on ammonium toxic analog methylammonium (MeA).

(A) Growth of WT plants and transgenic PutAMT1;1 plant (lines 1, 2, and 3) on agarose containing 0 mM, 10 mM or 20 mM MeA for 10 days after being pre-cultured on half-strength MS medium for 7 days. (B) Root length of the same plants as shown in (A). Bars indicate means ± SE (n = 12). Bars represent 1 cm. Statistical significance was determined using the Student’s t-test. * represents p<0.05 and ** represents p<0.01.