A 150 kDa plasma membrane complex of AtNRT2.5 and AtNAR2.1 is the major contributor to constitutive high-affinity nitrate influx in Arabidopsis thaliana.

In plants that have been deprived of nitrate for a significant length of time, a constitutive high-affinity nitrate transport system (cHATS) is responsible for initial nitrate uptake. This absorbed nitrate leads to the induction of the major nitrate transporters and enzymes involved in nitrate assimilation. By use of (13) NO3 (-) influx measurements and Blue Native polyacrylamide gel electrophoresis we examined the role of AtNRT2.5 in cHATS in wild type (WT) and various T-DNA mutants of Arabidopsis thaliana. We demonstrate that AtNRT2.5 is predominantly expressed in roots of nitrate-deprived WT plants as a 150 kDa molecular complex with AtNAR2.1. This complex represents the major contributor to cHATS influx, which is reduced by 63% compared with WT in roots of Atnrt2.5 mutants. The remaining cHATS nitrate influx in these mutants is due to a residual contribution by the inducible high-affinity transporter encoded by AtNRT2.1/AtNAR2.1. Estimates of the kinetic properties of the NRT2.5 transporter reveal that its low Km for nitrate makes this transporter ideally suited to detect and respond to trace quantities of nitrate in the root environment.

Characterization of nitrite uptake in Arabidopsis thaliana: evidence for a nitrite-specific transporter.

Nitrite-specific plasma membrane transporters have been described in bacteria, algae and fungi, but there is no evidence of a nitrite-specific plasma membrane transporter in higher plants. We have used 13NO2(-) to characterize nitrite influx into roots of Arabidopsis thaliana. Hydroponically grown Arabidopsis mutants, defective in high-affinity nitrate transport, were used to distinguish between nitrate and nitrite uptake by means of the short-lived tracers 13NO2(-) and 13NO3(-). This approach allowed us to characterize a nitrite-specific transporter. The Atnar2.1-2 mutant, lacking a functional high-affinity nitrate transport system, is capable of nitrite influx that is constitutive and thermodynamically active. The corresponding fluxes conform to a rectangular hyperbola, exhibiting saturation at concentrations above 200 µM (Km = 185 µM and Vmax = 1.89 µmol g(-1) FW h(-1)). Nitrite influx via the putative nitrite transporter is not subject to competitive inhibition by nitrate but is downregulated after 6 h exposure to ammonium. These results signify the existence of a nitrite-specific transporter in Arabidopsis. This transporter enables Atnar2.1-2 mutants, which are incapable of sustained growth on low nitrate, to maintain significant growth on low nitrite. In wild-type plants, this nitrite flux may increase nitrogen acquisition and also participate in the induction of genes specifically induced by nitrite.

Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2.1.

• Interactions between the Arabidopsis NitRate Transporter (AtNRT2.1) and Nitrate Assimilation Related protein (AtNAR2.1, also known as AtNRT3.1) have been well documented, and confirmed by the demonstration that AtNRT2.1 and AtNAR2.1 form a 150-kDa plasma membrane complex, thought to constitute the high-affinity nitrate transporter of Arabidopsis thaliana roots. Here, we have investigated interactions between the remaining AtNRT2 family members (AtNRT2.2 to AtNRT2.7) and AtNAR2.1, and their capacity for nitrate transport. • Three different systems were used to examine possible interactions with AtNAR2.1: membrane yeast split-ubiquitin, bimolecular fluorescence complementation in A. thaliana protoplasts and nitrate uptake in Xenopus oocytes. • All NRT2s, except for AtNRT2.7, restored growth and ß-galactosidase activity in the yeast split-ubiquitin system, and split-YFP fluorescence in A. thaliana protoplasts only when co-expressed with AtNAR2.1. Thus, except for AtNRT2.7, all other NRT2 transporters interact strongly with AtNAR2.1. • Co-injection into Xenopus oocytes of cRNA of all NRT2 genes together with cRNA of AtNAR2.1 resulted in statistically significant increases of uptake over and above that resulting from single cRNA injections.

Physiological and biochemical characterization of AnNitA, the Aspergillus nidulans high-affinity nitrite transporter.

High-affinity nitrite influx into mycelia of Aspergillus nidulans has been characterized by use of (13)NO(2)(-), giving average K(m) and V(max) values of 48 ± 8 µM and 228 ± 49 nmol mg(-1) dry weight (DW) h(-1), respectively. Kinetic analysis of a plot that included an additional large number of low-concentration fluxes gave an excellent monophasic fit (r(2) = 0.96), with no indication of sigmoidal kinetics. Two-dimensional (2D) and three-dimensional (3D) models of AnNitA are presented, and the possible roles of conserved asparagine residues N122 (transmembrane domain 3 ]Tm 3]), N173 (Tm 4), N214 (Tm 5), and N246 (Tm 6) are discussed.

Characterization of an intact two-component high-affinity nitrate transporter from Arabidopsis roots.

AtNRT2.1, a polypeptide of the Arabidopsis thaliana two-component inducible high-affinity nitrate transport system (IHATS), is located within the plasma membrane. The monomeric form of AtNRT2.1 has been reported to be the most abundant form, and was suggested to be the form that is active in nitrate transport. Here we have used immunological and transient protoplast expression methods to demonstrate that an intact two-component complex of AtNRT2.1 and AtNAR2.1 (AtNRT3.1) is localized in the plasma membrane. A. thaliana mutants lacking AtNAR2.1 have virtually no IHATS capacity and exhibit extremely poor growth on low nitrate as the sole source of nitrogen. Near-normal growth and nitrate transport in the mutant were restored by transformation with myc-tagged AtNAR2.1 cDNA. Membrane fractions from roots of the restored myc-tagged line were solubilized in 1.5% dodecyl-ß-maltoside and partially purified in the first dimension by blue native gel electrophoresis. Using anti-NRT2.1 antibodies, an oligomeric polypeptide (approximate molecular mass 150 kDa) was identified, but monomeric AtNRT2.1 was absent. This oligomer was also observed in the wild-type, and was resolved, using SDS-PAGE for the second dimension, into two polypeptides with molecular masses of approximately 48 and 26 kDa, corresponding to AtNRT2.1 and myc-tagged AtNAR2.1, respectively. This result, together with the finding that the oligomer is absent from NRT2.1 or NAR2.1 mutants, suggests that this complex, rather than monomeric AtNRT2.1, is the form that is active in IHATS nitrate transport. The molecular mass of the intact oligomer suggests that the functional unit for high-affinity nitrate influx may be a tetramer consisting of two subunits each of AtNRT2.1 and AtNAR2.1.

Effect of boron on the expression of aluminium toxicity in Phaseolus vulgaris.

The interaction of boron (B) and aluminium (Al) was investigated in 5-day-old seedlings of soybean cv. Maple Arrow. Al treatment inhibited root elongation and callose formation in root tips particularly after 4-h Al treatment. After 10 and 24 h, both parameters indicated increasing recovery from Al stress. B deficiency aggravated Al toxicity compared with B sufficiency. B deficiency did lead to an increase in unmethylated pectin in the first 3 mm of the root tip. This increase in potential binding sites is reflected in generally higher Al contents in root tips of B-deficient plants. A fractionated extraction of Al from the root tips showed that citrate-exchangeable and non-exchangeable Al steeply increased up to 4 h, but then decreased after 10- and 24-h Al treatment faster in B-sufficient than in B-deficient plants. This decrease of Al contents can be explained by an Al-enhanced release of citrate from the root tips after 10-h Al treatment. However, the citrate exudation rate was the same (after 10 h) or even lower (after 24 h) in B-sufficient plants and thus cannot explain the faster decrease in Al contents of the root tips compared with the B-deficient plants. We, therefore, propose that under B deficiency, Al is more strongly bound by the pectic network of the cell wall of the root tips, which delays or prevents the recovery from initial Al stress through exudation of citrate, and thus explains the greater Al sensitivity of B-deficient common bean roots.