Scientific Correspondence A Reevaluation of the Role of Arabidopsis NRT1.1 in High-Affinity Nitrate Transport

In papers by Wang et al. (1998), Liu et al. (1999), and Liu and Tsay (2003), it was proposed that Arabidopsis thaliana Nitrate Transporter1.1 (AtNRT1.1; CHL1) encodes a dual-affinity nitrate transporter that “plays a major role in high-affinity nitrate uptake.” Here, we evaluate this concept by reexamining the uptake kinetics of Arabidopsis (Arabidopsis thaliana) mutant lines defective in NRT1.1 or other nitrate transporters. The uptake of inorganic ions by plant roots conforms to a pattern of biphasic kinetics. At low external ion concentration, ions are absorbed by saturable highaffinity transport systems (HATS), while at high concentrations, nonsaturating low-affinity transport systems (LATS) operate. Such is the case for K, NH4 , NO3 , and ClO3 2 (a NO3 2 analog; Kochian and Lucas, 1982; Ullrich et al., 1984; Pace and McClure, 1986; Guy et al., 1988; Siddiqi et al., 1990; Aslam et al., 1992). The LATS for ClO3 2 uptake was linear at [ClO3 ] down to 200 mM in tobacco (Nicotiana tabacum; Guy et al., 1988) and for nitrate uptake by barley (Hordeum vulgare) down to 100 mM NO3 2 (Aslam et al., 1992). These concentrations were the lowest examined by the latter authors. In the studies by Pace and McClure (1986), Guy et al. (1988), Siddiqi et al. (1990), and Aslam et al. (1992), LATS fluxes were extremely small at low external [NO3 ] and linear at both low and high [NO3 ]. In barley, both constitutive HATS (CHATS) and inducible HATS (IHATS) were demonstrated at low [NO3 ], while a constitutive LATS (CLATS) failed to saturate even at 50 mM NO3 2 (Siddiqi et al., 1990). Likewise, CHATS and IHATS for nitrate have been demonstrated in Arabidopsis, as well as CLATS and inducible LATS (ILATS; Tsay et al., 1993; Huang et al., 1999). Doddema and Telkamp (1979) isolated an Arabidopsis B1 mutant that was defective in the LATS for nitrate (but not the HATS) by screening for survival on ClO3 . Tsay et al. (1993) isolated the nitrate-inducible AtNRT1.1 gene that encodes the ILATS. Interestingly, Touraine and Glass (1997) were unable to detect reduced LATS or HATS influxes in AtNRT1.1 mutants grown on KNO3, while Muños et al. (2004) reported increased HATS influx in AtNRT1.1 mutants. Likewise, Remans et al. (2006) failed to detect reduced uptake rates at low (0.5 mM) or high (10 mM) nitrate in AtNRT1.1 mutants. Among eukaryotes, genes encoding IHATS for nitrate were first isolated from Aspergillus nidulans (Unkles et al., 1991) and subsequently from Chlamydomonas reinhardtii (Quesada et al., 1994) and several higher plants (Glass, 2009), and based on the correlations between AtNRT2.1 expression and IHATS influx, it became accepted that IHATS was encoded by AtNRT2.1. This conclusion was supported by the demonstration that transfer DNA mutants disrupted in both AtNRT2.1 and AtNRT2.2 exhibited 67% reduction of HATS but no reduction in LATS function (Filleur et al., 2001). A gene encoding CHATS has not yet been identified, although a mutant with defective CHATS has been isolated (Wang and Crawford, 1996). In summary, it was held that in Arabidopsis, AtNRT2.1 was responsible for IHATS, while AtNRT1.1 and AtNRT1.2 encoded ILATS and CLATS, respectively (Forde, 2000; Li et al., 2007). In papers by Wang et al. (1998), Liu et al. (1999), and Liu and Tsay (2003), it was demonstrated thatAtNRT1.1 mutants of Arabidopsis exhibited reduced nitrate uptake even at 10 mM nitrate. The authors concluded that AtNRT1.1 fluxes exhibited saturation kinetics in planta and in Xenopus laevis oocytes and proposed that NRT1.1 encodes a dual-affinity nitrate transporter that “plays a major role in high-affinity nitrate uptake” (Wang et al., 1998). Liu and Tsay (2003) demonstrated that the AtNRT1.1 protein was capable of switching between highand low-affinity states by phosphorylation of Thr residue 101; under low-nitrogen (N) conditions, phosphorylation mediated via the activation of protein kinase CIPK23 generated a high-affinity transporter (Ho et al., 2009), whereas high-N favored the dephosphorylated low-affinity configuration.