37. Characterization of rice ammonium transporters, OsAMT1;1 and OsAMT1;2

BioScience Center and Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan

Ammonium and nitrate ions are important nitrogen sources for plants. In aerobic agricultural soil, because nitrate is the major nitrogen source, many crops prefer to take up nitrate. On the other hand in anaerobic agricultural soil such as paddy field, ammonium is the major form of available nitrogen, and rice prefers the uptake of ammonium rather than nitrate. Ammonium is taken up from the soil into plant cells by ammonium transporters. Ammonium transporters have been characterized in several plant species, including Arabidopsis thaliana (Gazzarrini et al. 1999) and Lycopersicon esculentum (Lauter et al. 1996).

In this study, we have cloned and characterized ammonium transporter cDNAs of rice (Oryza sativa L.), OsAMT1;1 and OsAMT1;2. These clones were identified by searching the rice database of expressed sequence tags for homologs of AtAMT1;1. The OsAMT1;1 clone is identical to that reported previously (von Wiren et al. 1997). The open reading frames of OsAMT1;1 and OsAMT1;2 encode 499- and 497-amino acid residues with predicted molecular masses of 52.6 and 52.2 kD, respectively. Hydropathy analysis of the deduced amino acid sequence suggests that OsAMT1;1 and OsAMT1;2 contain 10-11 putative transmembrane domains. Sequence comparison shows that OsAMT1;1 and OsAMT1;2 are closely related to Arabidopsis and tomato ammonium transporters (67-63%, Fig. 1).

Following nitrogen starvation, hydroponically grown 3-week-old rice seedlings were transferred to nutrient solutions containing no nitrogen, 0.3 mM ammonium or 0.3 mM nitrate as nitrogen sources. After 2 hr of treatment, the roots and shoots were sampled, and mRNA accumulation of OsAMT1;1 and OsAMT1;2 was analyzed. OsAMT1;1 transcripts were detectable in both roots and shoots irrespective of the nitrogen source (Fig. 2), while negligible amount of OsAMT1;2 transcripts were detectable only in the root. The expression was largely enhanced by the ammonium but not by the nitrate treatment (Fig. 2).

To study ammonium-inducibility of OsAMT1;1 and OsAMT1;2 in roots, hydroponically grown rice was exposed to nitrogen deficiency for two weeks, and then transferred to nutrient solution containing 0.3 mM ammonium as the nitrogen source. After 0, 15, 30, 60, 90 and 120 min of the treatment, mRNA accumulation of OsAMT1;1 and OsAMT1;2 in roots was analyzed. The expression of OsAMT1;1 was observed in all the treatments, while an increased level of OsAMT1;2 transcripts was detectable at 60 min after the ammonium treatment (data not shown).

To study ammonium-dependent expression of OsAMT1;1 and OsAMT1;2 in roots, following nitrogen starvation, hydroponically grown rice was exposed to 0.3 mM ammonium

as the nitrogen source for 2 hr, and transferred to a nitrogen-deficient solution for 0.5, 1, 2 and 4 hr. The expression of OsAMT1;1 was enhanced by the ammonium treatment. However, even when transferred to the nitrogen-deficient solution, expression of OsAMT1;1 was constantly detectable (Fig. 3). OsAMT1;2 transcripts disappeared in the nitrogendeficient condition, indicating that the expression of OsAMT1;2 is ammonium-dependent.

Consequently, while OsAMT1;1 was constitutively expressed (Figs. 2 and 3), accumulation of OsAMT1;2 transcripts was roots-specific and ammonium-inducible (Figs. 2 and 3). We complemented a yeast mutant to determine whether the protein encoded by the cDNA is a functional ammonium transporter. Both cDNAs were able to functionally complement the mutant which is deficient in ammonium uptake, indicating that OsAMT1;1 and OsAMT1;2 encodes functional ammonium transporters (data not shown). Thus, OsAMT1;1 and OsAMT1;2 may have different physiological roles in ammonium uptake in rice. There results imply that OsAMT1;1 is involved in uptake and/or salvage of the ammonium in vascular tissues, and OsAMT1;2 in the uptake in root epidermis. Further experiments will be needed to evaluate this hypothesis.


Gazzarrini S., L. Lejay, A. Gohon, O. Ninnemann, W.B. Frommer and N. von Wiren, 1999. Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots. Plant Cell 11: 937-947.

Lauter F.R., O. Ninnemann, M. Bucher, J.W. Riesmeier and W.B. Frommer, 1996. Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato. Proc. Natl. Acad. Sci. USA 93: 8139-8144.

von Wiren N., A. Bergfeld, O, Ninnemann and W.B. Frommer, 1997. OsAMT1;1: a high-affinity ammonium transporter from rice (Oryza sativa cv. Nipponbare). Plant Mol. Biol. 3: 681.