Characterization of Arabidopsis AtAMT2, a High-Affinity Ammonium Transporter of the Plasma Membrane

Abstract

AtAMT2 is an ammonium transporter that is only distantly related to the five members of the AtAMT1 family of high-affinity ammonium transporters in Arabidopsis. The short-lived radioactive ion 13 NH 4 ϩ was used to show that AtAMT2, expressed in yeast (Saccharomyces cerevisiae), is a high-affinity transporter with a K m for ammonium of about 20 m. Changes in external pH between 5.0 and 7.5 had little effect on the K m for ammonium, indicating that NH 4 ϩ , not NH 3 , is the substrate for AtAMT2. The AtAMT2 gene was expressed in all organs of Arabidopsis and was subject to nitrogen (N) regulation, at least in roots where expression was partially repressed by high concentrations of ammonium nitrate and derepressed in the absence of external N. Although expression of AtAMT2 in shoots responded little to changes in root N status, transcript levels in leaves declined under high CO 2 conditions. Transient expression of an AtAMT2-green fluorescent protein fusion protein in Arabidopsis leaf epidermal cells indicated a plasma membrane location for the AtAMT2 protein.Thus, AtAMT2 is likely to play a significant role in moving ammonium between the apoplast and symplast of cells throughout the plant. However, a dramatic reduction in the level of AtAMT2 transcript brought about by dsRNA interference with gene expression had no obvious effect on plant growth or development, under the conditions tested.Ammonium and nitrate are thought to be the primary sources of nitrogen (N) for most plants growing in agricultural soils. Acquisition of these inorganic nutrients from the soil solution involves a variety of different transporters, which transport the ions from the apoplast of root epidermal and cortical cells into the symplast. Although ammonium concentrations are often 10 to 1,000 times lower than those of nitrate in well-aerated soil, ammonium nutrition plays an essential role in waterlogged and acid soils (Marschner, 1995). Furthermore, ammonium seems to be a preferred source of N and is taken up more rapidly than nitrate when both ions are presented simultaneously to plants (Gazzarrini et al., 1999).Physiological studies of ammonium transport into roots have revealed biphasic kinetics in several species (Fried et al., 1965;Vale et al., 1988;Wang et al., 1993). The so-called high-affinity ammonium transport system is predominant at low (submillimolar) concentrations of substrate (NH 4 ϩ ) and exhibits saturation kinetics. A second component of ammonium uptake is the low-affinity transport system, which becomes significant at higher external ammonium concentrations (above 1 mm) and exhibits nonsaturation kinetics (Wang et al., 1993;Kronzucker et al., 1996). Although the molecular basis of low-affinity transport system activity remains unknown, there is growing evidence that members of the AMT1 family of transporters are responsible for high-affinity ammonium transport system activity in plants. The first AMT1 gene to be discovered in plants was AtAMT1;1 from Arabidopsis, which was cloned by complementation of a yeast (Sacchar...

Molecular mechanisms of ammonium transport and accumulation in plants

“…The transcripts of four root expressed AtAMTs are upregulated by nitrogen deficiency, by photosynthetic products such as sugars and are diurnally regulated, albeit to a different extent [6,8,10]. The AtAMT1;1 transcript expression and ammonium influx were down-regulated after ammonium re-supply to N-starved plants and these effects negatively correlated with glutamine levels, suggesting that glutamine may be a feedback signal for inhibition of ammonium influx after re-supply [7].…”

“…The transcript was expressed in the vasculature, cortical and root tip cells and was de-repressed by nitrogen deprivation in roots, but not in shoots. The loss of AtAMT2;1 by RNAi did not affect plant growth [10]. In addition to the AMT-dependent high-affinity ammonium transport systems in plants, a lower affinity, non-saturable at least partially protein-mediated influx exists in plants, including Arabidopsis [7].…”

Tissue-specific down-regulation of LjAMT1;1 compromises nodule function and enhances nodulation in Lotus japonicus

“…Members of the AMT1 and AMT2 subfamilies differ in their biochemical and sequence characteristics with the latter being more closely related to bacterial AMT transporters (Sohlenkamp et al 2000). Genome sequencing has revealed three AMT1 genes in Oriza sativa , five in Arabidopsis thaliana and eight in Populus trichocarpa (Couturier et al 2007); whereas one, six and seven AMT2 genes were identified in A. thaliana (Sohlenkamp et al 2000) P. trichocarpa (Couturier et al 2007) and O. sativa (Suenaga et al 2003), respectively.…”

Feedback Inhibition of Ammonium Uptake by a Phospho-Dependent Allosteric Mechanism inArabidopsis

“…The Arabidopsis thaliana genome encodes six members of the family, which fall into two clades, AMT1 and AMT2. In addition to their function in ammonium uptake from soil, AMTs are most likely involved in ammonium transport through the plant, ammonia retrieval in roots and leaves, and in the supply of nitrogen to pollen (Sohlenkamp et al, 2002;Yuan et al, 2007bYuan et al, , 2009). …”