Plant Molecular Biology 51: 99–108, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Molecular and cellular characterisation of LjAMT2;1, an ammonium
transporter from the model legume Lotus japonicus
Ulrike Simon-Rosin, Craig Wood and Michael K. Udvardi
Molecular Plant Nutrition Group, Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476
Golm, Germany (
author for correspondence; e-mail: email@example.com)
Received 26 October 2001; accepted in revised form 28 April 2002
Key words: ammonium transport, legume root nodule, Lotus japonicus,mRNAin situ hybridisation, nitrogen
ﬁxation, sub-cellular localisation
Two related families of ammonium transporters have been identiﬁed and partially characterised in plants in the
past; the AMT1 and AMT2 families. Most attention has focused on the larger of the two families, the AMT1
family, which contains members that are likely to fulﬁl different, possibly overlapping physiological roles in plants,
including uptake of ammonium from the soil. The possible physiological functions of AMT2 proteins are less clear.
Lack of data on cellular and tissue location of gene expression, and the intracellular location of proteins limit our
understanding of the physiological role of all AMT proteins. We have cloned the ﬁrst AMT2 family member
from a legume, LjAMT2;1 of Lotus japonicus, and demonstrated that it functions as an ammonium transporter
by complementing a yeast mutant defective in ammonium uptake. However, like AtAMT2 from Arabidopsis,
and unlike AMT1 transporters from several plant species, LjAMT2;1 was unable to transport methylammonium.
The LjAMT2;1 gene was found to be expressed constitutively throughout Lotus plants. In situ RNA hybridisation
revealed LjAMT2;1 expression in all major tissues of nodules. Transient expression of LjAMT2;1-GFP fusion
protein in plant cells indicated that the transporter is located on the plasma membrane. In view of the fact that
nodules derive ammonium internally, rather than from the soil, the results implicate LjAMT2;1 in the recovery
of ammonium lost from nodule cells by efﬂux. A similar role may be fulﬁlled in other organs, especially leaves,
which liberate ammonium during normal metabolism.
Abbreviations: CaMV, cauliﬂower mosaic virus; DIG, digoxygenin; ER, endoplasmic reticulum; GFP, green
ﬂuorescent protein; GS, glutamine synthetase; MA, methyammonium; NCBI, National Center of Biotechnological
Information; PBM, peribacteroid membrane; PBS, peribacteroid space.
Ammonium is a primary source of nitrogen for plants.
It is taken up from the soil by ammonium transporters
in the plasma membrane of root cells and incorporated
into glutamine via glutamine synthetases (GS) present
in the cytoplasm and plastids (for recent reviews see
Glass et al., 1997; Forde and Clarkson, 1999; Schjoer-
ring et al., 1999; Howitt and Udvardi, 2000; von Wiren
The nucleotide sequence data reported will appear in the
EMBL, GenBank and DDBJ nucleotide sequence databases under
the accession number AF187962 (LjAMT2;1).
et al., 2000a). Ammonium is also produced within
plants either by reduction of nitrate and nitrite ob-
tained from the soil, or by catabolism of endogenous
amino compounds. For instance, the photorespiratory
nitrogen cycle generates a large amount of ammonium
in leaf mitochondria that is subsequently transported
to chloroplasts for re-assimilation by GS (Lam et al.,
1996). Symbiotic nitrogen ﬁxation is another impor-
tant source of ammonium for some plant cells. In fact,
legumes and the few non-legumes that establish sym-
bioses with nitrogen-ﬁxing bacteria can obtain most,