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Ammonium is a primary source of nitrogen (N) for both perennial and annual species. It is taken up from the soil by ammonium transporters through the plasma membrane of root cells (Kaiser et al., 2002) and incorporated into glutamine via glutamine synthetase (GS) present in the cytoplasm and plastids, the biochemistry of which has also been described in perennial species (Suarez et al., 2002). Ammonium is also produced within plants either by reduction of nitrate and nitrite obtained from the soil, or by catabolism of endogenous amino compounds. For example, the photorespiratory nitrogen cycle generates a large amount of ammonium in leaf mitochondrias that is subsequently transported to chloroplasts for reassimilation by GS, although this statement has now to be interpreted with caution, given the recent finding that the nuclear encoded GS GLN2 may also be addressed to the mitochondria (Taira et al., 2004).
The biochemistry and molecular biology of ammonium transport in plants has been extensively studied and recent comprehensive reviews are available (Schjoerring et al., 2002; Loque & von Wiren, 2004). Physiological studies on ammonium uptake by plant roots has provided evidence for the existence of a low affinity nonsaturable transport system (LATS), which operates in the millimolar concentration range and a high-affinity transport system (HATS), which operates in the submillimolar concentration range. The HATS exhibits saturation kinetics, energy dependence, and leads to depolarization of the plasma membrane electrical potential (Ludewig et al., 2002).
The first ammonium transporter (AMT) genes were identified in yeast (Marini et al., 1994) and Arabidopsis (Ninnemann et al., 1994) by functional complementation of a yeast mutant deficient in high-affinity ammonium uptake. In plants, the AMT family can be subdivided into two subfamilies (Loque & von Wiren, 2004). Members of the AMT1 subfamily are intron-free, except LjAMT1;1 (Salvemini et al., 2001); whereas members of AMT2 subfamily contain some introns in their gene sequences. The AMT2 family has been further divided into three subclades in rice (Suenaga et al., 2003). Several plant AMT1 subfamily members, including homologs from Arabidopsis thaliana (AtAMT1;1, AtAMT1;2, AtAMT1;3: Ninnemann et al., 1994; Gazzarrini et al., 1999), Brassica napus (BnAMT1;2: Pearson et al., 2002), Lotus japonicus (LjAMT1;1, LjAMT1;2, LjAMT1;3: Salvemini et al., 2001; D’Apuzzo et al., 2004), Lycopersicon esculentum (LeAMT1;1, LeAMT1;2 and LeAMT1;3: Lauter et al., 1996; von Wiren et al., 2000b; Becker et al., 2002; Ludewig et al., 2002) or Oryza sativa (OsAMT1;1, OsAMT1;2, OsAMT1;3; Sonoda et al., 2003) have been characterized in yeast or in Xenopus oocytes. AtAMT1;1 encodes a high-affinity transporter with a Km value of < 0.5 µm and is expressed in roots and leaves, while AtAMT1;2 and AtAMT1;3 encode transporters of lower affinity (Km values of 25–40 µm) and are mainly expressed in roots (Gazzarrini et al., 1999). AtAMT1;2 expressed in yeast mutant displays biphasic kinetics (Km values of 36 µm and 3.0 mm) for methylammonium uptake (Shelden et al., 2001). AMT expression has been studied in detail in Arabidopsis and rice, and the data demonstrated the close relationship between AMT1;1 and AMT1;3 expression with glutamine (Rawat et al., 1999) and sugar (Gazzarrini et al., 1999; Rawat et al., 1999) concentrations, respectively.
More recently, a second subfamily of ammonium transporters (AMT2) with distinct biochemical features, has been identified in several plants such as A. thaliana (Sohlenkamp et al., 2002), L. japonicus (Simon-Rosin et al., 2003) and O. sativa (Suenaga et al., 2003). Plant AMT2 family members are more closely related to ammonium transporters from prokaryotes than they are to plant AMT1. While plant members of the AMT1 subfamily are preferentially expressed in roots (with the exception of LeAMT1;3 (von Wiren et al., 2000b), LjAMT1;1 and LjAMT1;2 (D’Apuzzo et al., 2004), AtAMT2 showed a higher level of gene expression in shoots compared with roots (Sohlenkamp et al., 2002) and OsAMT2;1 presented a constitutive expression in shoots and roots (Suenaga et al., 2003). These distinct expression patterns may support the fact that individual members of the AMT family would function not only in ammonium uptake in roots, but also in ammonium recycling during leaf senescence or photorespiration (Howitt & Udvardi, 2000; von Wiren et al., 2000a). Multiple forms of ammonium transporters in higher plants allow a greater regulatory flexibility and organelle-, cell-, tissue- or organ-specialization, and enable cells to take up ammonium over a wide range of concentrations (D’Apuzzo et al., 2004).
As exemplified above, most studies have focused on a few annual species (A. thaliana, L. esculentum, L. japonicus and O. sativa), thus available information concerning N transport in perennial woody models is much more limited, particularly at the molecular level. The perennial life style of trees implies specific physiological traits that have been recently discussed (Suarez et al., 2002; Bhalerao et al., 2003; Andersson et al., 2004; Sterky et al., 2004). Related to N nutrition, one of these traits is the mobilization and storage in perennial tissues of the N present in leaves during autumn, which is remobilized at the beginning of the next growing season (Suarez et al., 2002). Thus, transport mechanisms for nitrogenous compounds are likely to be different from those used by annual species, and probably rely on perennial-specific transport functions.
Taking advantage of the shotgun sequenced Populus trichocarpa (Nisqually 1) genome (Tuskan et al., 2006), we present here the expression analysis of AMT members from the perennial tree species poplar and extend the analysis of five AMT members to include the functional characterization of three AMT1 and two AMT2 members by heterologous expression in yeast.