Dissection of the AtNRT2.1:AtNRT2.2 Inducible High-Affinity Nitrate Transporter Gene Cluster

Abstract

Using a new Arabidopsis (Arabidopsis thaliana) mutant (Atnrt2.1-nrt2.2) we confirm that concomitant disruption of NRT2.1 and NRT2.2 reduces inducible high-affinity transport system (IHATS) by up to 80%, whereas the constitutive high-affinity transport system (CHATS) was reduced by 30%. Nitrate influx via the low-affinity transport system (LATS) was unaffected. Shoot-to-root ratios were significantly reduced compared to wild-type plants, the major effect being upon shoot growth. In another mutant uniquely disrupted in NRT2.1 (Atnrt2.1), IHATS was reduced by up to 72%, whereas neither the CHATS nor the LATS fluxes were significantly reduced. Disruption of NRT2.1 in Atnrt2.1 caused a consistent and significant reduction of shoot-to-root ratios. IHATS influx and shoot-to-root ratios were restored to wild-type values when Atnrt2.1-nrt2.2 was transformed with a NRT2.1 cDNA isolated from Arabidopsis. Disruption of NRT2.2 in Atnrt2.2 reduced IHATS by 19% and this reduction was statistically significant only at 6 h after resupply of nitrate to nitrogen-deprived plants. Atnrt2.2 showed no significant reduction of CHATS, LATS, or shoot-to-root ratios. These results define NRT2.1 as the major contributor to IHATS. Nevertheless, when maintained on agar containing 0.25 mM KNO 3 as the sole nitrogen source, Atnrt2.1-nrt2.2 consistently exhibited greater stress and growth reduction than Atnrt2.1. Evidence from real-time PCR revealed that NRT2.2 transcript abundance was increased almost 3-fold in Atnrt2.1. These findings suggest that NRT2.2 normally makes only a small contribution to IHATS, but when NRT2.1 is lost, this contribution increases, resulting in a partial compensation.Physiological evidence based upon nitrate influx versus nitrate concentration plots has demonstrated that nitrate uptake by roots of higher plants occurs via both high-affinity transport systems (HATS) and lowaffinity transport systems (LATS; for review, see Glass and Siddiqi, 1995;Crawford and Glass, 1998;Forde, 2000). In barley (Hordeum vulgare), for example, this physiological evidence suggested the existence of a constitutive high-affinity transport system (CHATS) and a nitrate-inducible high-affinity transport system (IHATS), whose V max was 30 times that of the CHATS (Siddiqi et al., 1990). In addition, a constitutive LATS showed no saturation even at 50 mM external nitrate. This appears to be the case for nitrate uptake by other species, including white spruce (Picea glauca), aspen (Populus tremuloides), and lodgepole pine (Pinus contorta; Min et al., 2000). In Arabidopsis (Arabidopsis thaliana), there are also CHATS and IHATS, but LATS is mediated by both constitutive and nitrate-inducible transport systems (Crawford and Glass, 1998).Physiological evidence based upon biphasic Lineweaver-Burk and Hofstee plots (Aslam et al., 1992;Kronzucker et al., 1995) indicates that, in nitrateinduced plants, both CHATS and IHATS operate independently and additively, suggesting the existence of discrete transporters for CHATS and IHATS. Likewise, total influx a...

Environmental nitrate signals through abscisic acid in the root tip

“…6 In the Arabidopsis root, 6 different transporters have been shown to transport nitrate from the environment into root cells. 6,20 Under conditions of low external nitrate, uptake is mediated primarily by the high-affinity transporter, AtNRT2.1, and to a lesser degree by AtNRT2.2 and AtNRT2.4 [21][22][23] ; under nitrate starvation conditions uptake is mediated mainly by AtNRT2.5, with some contribution by AtNRT2.1. 24,25 At high concentrations of environmental nitrate, the dual-affinity transporter AtNPF6.3 (AtNRT1.1) and the low-affinity transporter AtNPF4.6 (AtNRT1.2; AtAIT1) come into play.…”

TheArabidopsisNitrate Transporter NRT1.8 Functions in Nitrate Removal from the Xylem Sap and Mediates Cadmium Tolerance

“…The NRT2 family consists of seven members in the Arabidopsis genome. NRT2.1 and NRT2.2 are involved in inducible high-affinity nitrate uptake (Cerezo et al, 2001;Li et al, 2007). Though functionally and phylogenetically distinct, the nitrate transport functions of both NRT1 and NRT2 are believed to be proton dependent (Paulsen and Skurray, 1994;Orsel et al, 2002a).…”

The genomic organization and transcriptional pattern of genes encoding nitrate transporters 1 (NRT1) in cucumber

“…NRT2 are inducible (NRT2.1, NRT2.2) or constitutively (NRT2.4-NRT2.6) expressed high-affinity nitrate transporters, among which only a few members have been characterized in detail: NRT2.1 and NRT2.2 were shown to be responsible for nitrate uptake from the soil whereas NRT1.7 determines NO 3 − storage in seeds (Orsel et al 2002;Okamoto et al 2003;Tsay et al 2007;Wirth et al 2007;Li et al 2007;Chopin et al 2007). Three tonoplast-localized members of CLC family in Arabidopsis operate as nitrate transporters: CLCa, which functions as a 2NO 3 -/1H + antiporter critical for NO 3 − accumulation within vacuoles (Geelen et al 2000; de Angeli et al 2006) and CLC-b as well as CLC-c, which regulate the level of vacuolar nitrate in plant cells (Harada et al 2004;von der Fecht-Bartenbach et al 2010).…”

“…Although the Arabidopsis genome contains 53 NRT1 (PTR) genes, the function in NO 3 -transport was confirmed only for 8 NRT1s (AtNRT1.1-1.2 and AtNRT1.4-1.9). Arabidopsis NRT1.1 (CHL1) and NRT1.2 nitrate transporters were shown to be involved in nitrate uptake from the soil solution into root cells (Tsay et al 1993;Wang et al 1998;Huang et al 1999;Liu et al 1999;Orsel et al 2006;Li et al 2007). Root-to-shoot nitrate transport is probably governed by three members of AtNRT1 family: two closely related transporters NRT1.5 and NRT1.8, are responsible for loading or retrieving nitrate from xylem sap, respectively, whereas root stele-expressed AtNRT1.9 is involved in the loading of nitrate into the root phloem to enhance downward nitrate transport in roots (Wang and Tsay 2011).…”