Functional characterization of the sugarcane (Saccharum spp.) ammonium transporter AMT2;1 suggests a role in ammonium root-to-shoot translocation

Abstract

AMMONIUM TRANSPORTER/METHYLAMMONIUM PERMEASE/RHESUS (AMT) family members transport ammonium across membranes in all life domains. Plant AMTs can be categorized into AMT1 and AMT2 subfamilies. Functional studies of AMTs, particularly AMT1-type, have been conducted using model plants but little is known about the function of AMTs from crops. Sugarcane (Saccharum spp.) is a major bioenergy crop that requires heavy nitrogen fertilization but depends on a low carbon-footprint for competitive sustainability. Here, we identified and functionally characterized sugarcane ScAMT2;1 by complementing ammonium uptake-defective mutants of Saccharomyces cerevisiae and Arabidopsis thaliana. Reporter gene driven by the ScAMT2;1 promoter in A. thaliana revealed preferential expression in the shoot vasculature and root endodermis/pericycle according to nitrogen availability and source. Arabidopsis quadruple mutant plants expressing ScAMT2;1 driven by the CaMV35S promoter or by a sugarcane endogenous promoter produced significantly more biomass than mutant plants when grown in NH₄ ⁺ and showed more ¹⁵N-ammonium uptake by roots and nitrogen translocation to shoots. In A. thaliana, ScAMT2;1 displayed a K_m of 90.17 µM and V_max of 338.99 µmoles h^-1 g^-1 root DW. Altogether, our results suggest that ScAMT2;1 is a functional high-affinity ammonium transporter that might contribute to ammonium uptake and presumably to root-to-shoot translocation under high NH₄ ⁺ conditions.

Keywords: AMT2 subfamily; ammonium uptake; nitrogen use efficiency; quadruple mutant; transport kinetics; xylem loading.

Figure 1

Physical map of genomic sequences (100 Kb) containing AMT2;1 from sugarcane (Saccharum spp. ‘R570’) BAC clones (032_A12, 038_G02, 118_C18, 216_D16, and 235_F05) and S. bicolor (chromosome 9; NC_012878).

Figure 2

Expression of ScAMT2;1 in sugarcane organs after subjecting plants to distinct inorganic N sources or no N. (A) RT–qPCR analysis of ScAMT2;1 expression in roots, culms, and young (+1) or mature (+3) leaves of sugarcane grown under 2 mM NH₄NO₃ for 2 d. Bars indicate ± SE (n = 3). ScUBQ2 was adopted as a reference gene. Gene expression levels were normalized to expression levels in culms. Asterisks represent significant differences in relation to culms according to Student’s t test (p < 0.01). (B) ScAMT2;1 relative expression levels in sugarcane roots, culm, young, and mature leaves of plants under +N: 2 mM NH₄NO₃, -N: no N; ${\text{NO}}_3^{-}$: 4 mM KNO₃, or NH₄ ⁺: 4 mM NH₄Cl for 14 d. ScUBQ2 was used as a reference gene. The gene expression level was normalized to the +N treatment. Bars indicate ± SE (n = 3). Asterisks represent significant differences between treatments and +N according to Student’s t test (p < 0.01).

Figure 3

Functional complementation of the yeast mutant defective for ammonium uptake. Growth of the ammonium transporter-deficient yeast strain triple mepΔ (31019b) expressing AtAMT1;1 (positive control), the empty pDR196 vector (negative control), or ScAMT2;1 on media supplied with 0.5 to 5 mM ammonium chloride (NH₄ ⁺), 100 mM methylammonium (MeA), or 1 mM arginine (Arg; positive control) as the sole N source. Culture media pH was adjusted to 6.0 when not indicated otherwise. Each transformant line was grown to OD_600nm = 1 and plated in concentrated and sequential four 10-fold dilutions.

Figure 4

Functional evaluation of complemented Arabidopsis qko mutant. (A) Total biomass (DW, dry weight) of homozygous lines of Arabidopsis overexpressing ScAMT2;1 (qko+p35S::ScAMT2;1) subjected to either 0.5 mM KNO₃ (${\text{NO}}_3^{-}$) or 2 mM NH₄Cl (NH₄ ⁺) for 14 d. Bars indicate means ± SE (n = 6). Different letters indicate significant differences among means according to Tukey’s test (p ≤ 0.05). (B) Phenotype of qko and transgenic events grown in vitro under different N sources. (C) Influx of ¹⁵N-labeled ammonium into the roots of qko and a transgenic line (#1) overexpressing ScAMT2;1 subjected to either N-free (-N) or 0.2 mM NH₄ ⁺ (+N) nutrient solution for 3 d. Bars indicate means ± SE (n = 4). Asterisks indicate significant differences between qko and the transgenic line according to Student’s t test (p < 0.05). (D) Concentration-dependent influx of ¹⁵NH₄ ⁺ into roots of qko or qko+p35S::ScAMT2;1 (#1). Symbols indicate six biological replicates (n = 6).

Figure 5

Endogenous ScAMT2;1 promoter (p2) driving GUS expression in Col-0 Arabidopsis plants subjected to 1 mM NH₄NO₃, 2 mM NH₄ ⁺, or no N for 1, 3, 5, and 10 d in (A) shoots; and (B) roots. The blue color shows GUS activity. Bars = 200 μm.

Figure 6

Biomass accumulation in Arabidopsis mutant plants and lines complemented with ScAMT2;1 driven by the sugarcane endogenous promoter. (A) Fresh weight of homozygous lines of qko+p2ScAMT2;1::ScAMT2;1 subjected to 2 mM KNO₃ (${\text{NO}}_3^{-}$) or 0.2, 2, and 4 mM NH₄Cl (NH₄ ⁺) for 14 d. Bars indicate means ± SE (n = 30). Different letters indicate significant differences among means according to Tukey’s test (p ≤ 0.05). (B) Influx of ¹⁵N-labeled ammonium (NH₄ ⁺) into roots of qko and qko+p2ScAMT2;1::ScAMT2;1 lines upon 3-d exposure to N-free (-N), 2 mM ${\text{NO}}_3^{-}$, or 2 mM NH₄ ⁺ nutrient solution. Bars indicate means ± SE (n = 4). Asterisks indicate significant differences between qko and transgenic plants according to Student’s t test (*p < 0.10 and **p < 0.05).

Figure 7

¹⁵N accumulation in roots and shoots of qko and complemented qko transgenic lines expressing ScAMT2;1 under the regulation of its sugarcane endogenous promoter (p2). The assay was performed in plants after 3 d of exposure to an N-free nutrient solution, followed by 1 h of treatment in either a 0.2 mM or 4.0 mM ¹⁵NH₄ ⁺ solution. Bars indicate means ± SE (n = 5). Asterisks indicate significant differences in root and shoot ¹⁵NH₄ ⁺ accumulation between qko and transgenic plants according to Student’s t test (*p < 0.1 and **p < 0.05). (B) Root-to-shoot translocation of ¹⁵N in percent of ¹⁵N accumulation in shoots in relation to the whole plant. Asterisks indicate significant differences between qko and transgenic plants according to Student’s t test (** p < 0.05).