Two-component high-affinity nitrate transport system in barley: Membrane localization, protein expression in roots and a direct protein-protein interaction

Abstract

Nitrate is one of the major sources of nitrogen for higher plants and is taken up from the soil by active transporters coupled with H ϩ across the plasma membrane (PM) of root cells. Nitrate uptake systems have been classified into two groups: low-affinity transport systems (LATS) and high-affinity transport systems (HATS). The LATS contribute to nitrate uptake at high nitrate concentrations above 1 mM whereas the HATS operate at micromolar concentrations of external nitrate and display MichaelisMenten kinetics saturating at 0.2-0.5 mM nitrate. The HATS are further divided into two categories: constitutive HATS (cHATS) and inducible HATS (iHATS), which are significantly affected by the supply of external nitrate. Many studies of the molecular basis of nitrate uptake reveal the existence of two gene families, namely the NRT1 and NRT2 families, which potentially encode for LATS and HATS respectively. NRT2 genes are identified in a variety of organisms including fungi, certain yeasts, green algae, and higher plants ( Unkles et al. 1991;Quesada et al. 1994;Trueman et al. 1996;Pérez et al. 1997;Quesada et al. 1997;Amarasinghe et al. 1998;Zhuo et al., 1999;Araki and Hasegawa 2006;Tsujimoto et al. 2007). In most species, NRT2 genes are members of a multigene family: for example, seven Arabidopsis genes (AtNRT2.1-AtNRT2.7) and four rice genes (OsNRT2.1-OsNRT2.4) have been found in their genomes (Orsel et al. 2002; Araki and Hasegawa 2007), and at least four NRT2 genes (HvNRT2.1-HvNRT2.4) exist in barley (Vidmar 2000a). Amino acid sequences deduced from these genes indicate that the NRT2 proteins are typically 480-510 amino acids in length and predicted to be integral to membranes with 12 transmembrane helices (Forde 2000).It has been well documented that iHATS activity is strongly induced by nitrate supply, and is down-regulated by the accumulation of nitrate assimilation products, especially ammonium and glutamine (Crawford and Glass 1998). In several plant species, it has been shown that a particular member of the NRT2 gene family (e.g., NpNRT2.1 for Nicotiana plumbaginifolia, AtNRT2.1 for Arabidopsis, HvNRT2.1 for barley) contribute to iHATS, because those transcript levels are highly correlated with changes in iHATS activity in such species (Krapp et al. 1998;Lejay et al. 1999;Zhuo et al. 1999;Vidmar et al. 2000a Abstract A high affinity transport system (HATS) for nitrate in plants is operated by a two-component NRT2/NAR2 transport system. However, the regulation and localization of NRT2 and NAR2 at protein level are largely unknown and especially so in crop plant species. In this study with barley (Hordeum vulgare), membrane localization, protein expression in the roots, and a direct protein-protein interaction of HvNRT2 and HvNAR2 proteins were investigated. Immunochemical analysis showed that both HvNRT2 and HvNAR2 proteins were co-localized in the plasma membrane of the roots. Expression of HvNRT2 and HvNAR2 proteins was more strongly induced by treatment with higher concentrations of external nitrate, while H...

Regulation of High-Affinity Nitrate Uptake in Roots of Arabidopsis Depends Predominantly on Posttranscriptional Control of the NRT2.1/NAR2.1 Transport System

“…gene expression and HATS activity, on the other hand Ishikawa et al, 2009). These data support the previous proposal that posttranscriptional regulatory mechanisms participate in the control of the HATS, because expression of a 35S::NpNRT2.1 or RolD:: NpNRT2.1 transgene in N. plumbaginifolia did not prevent down-regulation of the HATS activity by high NH 4 NO 3 supply (Fraisier et al, 2000).…”

“…More recently, the abundance of the Arabidopsis NRT2.1 protein in the root plasma membrane was reported to be little affected by treatments that strongly regulate both NRT2.1 transcript accumulation and NO 3 2 HATS activity . In barley too, changes in HvNRT2.1 protein were not correlated with those of HvNRT2.1 transcript accumulation during induction by NO 3 2 (Ishikawa et al, 2009). Finally, studies on NRT2 homologs in lower eukaryotes, e.g.…”

Nitrogen acquisition by roots: physiological and developmental mechanisms ensuring plant adaptation to a fluctuating resource

“…Nevertheless, the importance of such a regulation has been supported by data obtained in tobacco (Fraisier et al 2000), barley (Ishikawa et al 2009) and A. thaliana Laugier et al 2012). In the later species, ectopic expression of NRT2.1 actually resulted in a high and almost constant mRNA level in the roots, which did not prevent downregulation of NO 3 − HATS activity by high N supply.…”

Multiple roles of nitrate transport accessory protein NAR2 in plants

“…8 The NAR2 protein plays a role in establishing HATS activity by targeting NRT2 to the plasma membrane. 12,13 In the rnc1 mutant of Arabidopsis, it was suggested that an aspartate residue in the central loop of NAR2 was important for HATS activity. 14 Immunological results demonstrated an oligomer, proposed to contain a tetramer consisting of two subunits each of AtNRT2.1 and AtNAR2.1, was active in HATS.…”

“…13 In barley, a direct protein-protein interaction was shown to occur between the HvNRT2.1 C-terminal, S463A, and the HvNAR2.3 central region. 12 However, it is difficult to determine if this serine is a key residue because it is not conserved in AtNRT2.1 and HvNRT2.1 C-terminal. 12 In rice roots we detected some differences in the tissue specific expression of OsNAR2.1 and its interacting NRT2 members.…”