Functional analysis of an Arabidopsis T-DNA "knockout" of the high-affinity NH4(+) transporter AtAMT1;1

Abstract

NH(4)(+) acquisition by plant roots is thought to involve members of the NH(4)(+) transporter family (AMT) found in plants, yeast, bacteria, and mammals. In Arabidopsis, there are six AMT genes of which AtAMT1;1 demonstrates the highest affinity for NH(4)(+). Ammonium influx into roots and AtAMT1;1 mRNA expression levels are highly correlated diurnally and when plant nitrogen (N) status is varied. To further investigate the involvement of AtAMT1;1 in high-affinity NH(4)(+) influx, we identified a homozygous T-DNA mutant with disrupted AtAMT1;1 activity. Contrary to expectation, high-affinity (13)NH(4)(+) influx in the amt1;1:T-DNA mutant was similar to the wild type when grown with adequate N. Removal of N to increase AtAMT1;1 expression decreased high-affinity (13)NH(4)(+) influx in the mutant by 30% compared with wild-type plants, whereas low-affinity (13)NH(4)(+) influx (250 microM-10 mM NH(4)(+)) exceeded that of wild-type plants. In these N-deprived plants, mRNA copy numbers of root AtAMT1;3 and AtAMT2;1 mRNA were significantly more increased in the mutant than in wild-type plants. Under most growth conditions, amt1;1:T-DNA plants were indistinguishable from the wild type, however, leaf morphology was altered. However, when grown with NH(4)(+) and sucrose, the mutant grew poorly and died. Our results are the first in planta evidence that AtAMT1;1 is a root NH(4)(+) transporter and that redundancies within the AMT family may allow compensation for the loss of AtAMT1;1.

Figure 1

Characterization of the plant line with a T-DNA insertion in AtAMT1;1. A, Localization of the T-DNA insert. The diagram illustrates the insertion of the T-DNA 6 bp upstream of the first putative Met in the open reading frame of AtAMT1;1. The bordering nucleotides are presented adjacent to the insertion sites of both the left (LB) and right (RB) borders of the T-DNA insert. In italics, the insertion of three nucleotides of unknown origin is shown at the T-DNA left-border junction as well as the location of primer (P3 P7 P8) binding sites used for selecting tagged and non-tagged AtAMT1;1 alleles. B, Cartoon illustrating the T-DNA insertion into chromosome 4 and the predicted sized fragments after digestion with EcoRI (E) and HindIII (H) not drawn to scale. C, Southern-blot analysis of EcoRI (lane 1) and HindIII (lane 2) digested amt1;1:T-DNA genomic DNA. The blot was probed with a 1.2-kb DIG-labeled β-glucuronidase (GUS) gene and demonstrates a single T-DNA insertion in the amt1;1:T-DNA genome. D, Northern-blot analysis of AtAMT1;1 expression in the mutant and wild-type roots and shoots. Plants were grown vegetatively in liquid culture for 5 weeks in 1 mm NH₄NO₃ and then transferred to nutrient solution without N for 4 d. The blot was probed with DIG-labeled AMT1;1 antisense RNA.

Figure 2

Plant growth in magenta boxes containing sterile nutrient solution including Suc. Stratified seeds were sown directly onto nylon mesh sitting on the floating rafts in nutrient solution containing 1% (w/v) Suc and either 0.5 mm (NH₄)₂SO₄ (A and B) or 1 mm KNO₃ (C). Closed magenta boxes were placed in growth rooms on orbital shakers and plants grown for 21 (A) and 10 (B and C) d.

Figure 3

Cross sections of wild-type and amt1;1:T-DNA leaves. Sections are from leaves of plants grown in a controlled temperature growth chamber in hydroponic tanks (A) or in the greenhouse (B). Plants grown in the greenhouse were on a peat-based potting mix containing a slow-release fertilizer (Osmocote), whereas plants grown in the chamber were identical to those grown on adequate N (1 mm NH₄NO₃) as previously described for the ¹³NH₄⁺ uptake experiments. Cross sections from fully expanded leaves of equal age between lines were taken midway along the blade. Arrows indicate air spaces mainly absent in the amt1;1:T-DNA line. Leaf lengths ranged from 5 to 6 cm.

Figure 4

¹³NH₄⁺ influx by the HATS for both amt1:1:T-DNA and wild-type plants. A, Plants were grown in the presence of 1 mm NH₄NO₃ for 5 weeks and then transferred to nutrient solution with or without 1 mm NH₄NO₃ for 4 d. ¹³NH₄⁺ influx was measured over a 10-min period in plants precultured in 12.5 (25 μm NH₄⁺) and 50 μm (100 μm NH₄⁺; NH₄)₂SO₄. Values are means ± se (n = 5). B, Concentration dependence of ¹³NH₄⁺ influx into plant roots by the HATS. Plants grown as above were starved of N for 4 d. Data represents the averaged values ± se of three independent experiments (n = 14–15 plants). The fitted curve was obtained by direct fit to the Michaelis-Menten equation. Estimated K_M and V_max ± se were 11.3 ± 3.2 and 17.2 ± 4.4 μm (P = 0.2777) and 11.4 ± 0.6 and 8.3 ± 0.5 μmol NH₄⁺ g⁻¹ fresh weight h⁻¹ (P < 0.05) for the wild-type and amt1;1:T-DNA line, respectively.

Figure 5

NH₄⁺ influx by the LATS for both amt1;1:T-DNA and wild-type plants. Both sets of plants were grown on 1 mm NH₄NO₃ for 5 weeks and then transferred to nutrient solution without N for 4 d. A, Concentration dependence of ¹³NH₄⁺ influx by both the HATS and the LATS into plant roots over a 10-min period. Data represent the averaged values of three independent experiments (n = 8–12 plants). B, Predicted NH₄⁺ influx as a result of the LATS solely. V_max values in the high-affinity range (0–250 μm) were subtracted from the individual data points for each concentration and averaged. V_max values used were 8.34 μmol NH₄⁺ g⁻¹ fresh weight h⁻¹ for the mutant and 11.4 μmol NH₄⁺ g⁻¹ fresh weight h⁻¹ for the wild type.

Figure 6

Quantitative AMT gene expression levels estimated using competitive RT-PCR. Plants were grown on 1 mm NH₄NO₃ for 5 weeks and then transferred to nutrient solution with (T = 0) or without (T = 4) N for 4 d before harvest of root tissues. A, A typical multiplex competitive RT-PCR performed using 25 ng of total root RNA mixed with known amounts of competitor cRNA. B, The quantity of AMT transcript present in each sample was determined from the ratio of endogenous and competitor cDNA products. C, Data represent the combined means and se of five to six competitive RT-PCR experiments performed separately from two RNA extractions per treatment. Total RNA extracted from each treatment represented six to eight individual complete root systems. N.D., Not detectable. Bars with different letters are significantly different (P < 0.05).