UBC Theses and Dissertations
Characterization of high-affinity nitrate and nitrite transporters in Arabidopsis thaliana Kotur, Zorica
Nitrite is a potential nitrogen source in the environment. Algae and fungi posses nitrite-specific transporters, whereas plant counterparts have not been identified. Because nitrate transporters can take up nitrite, I used nitrate uptake-defective Arabidopsis mutants to eliminate the masking effect of nitrate transporters and measured ¹³NO₂- influx to characterize nitrite uptake. The Atnar2.1-2 mutant, lacking a functional high-affinity nitrate transport system, is capable of nitrite influx that is constitutive and thermodynamically active. This provides strong evidence for a nitrite-specific transporter that enables Atnar2.1-2 mutants, which are incapable of sustained growth on low nitrate, to maintain significant growth on low nitrite. To accommodate the variable nitrate concentration in soil solution, plants have high- and low-affinity uptake systems (HATS and LATS, respectively). AtNRT2.1, the major inducible HATS transporter, requires expression of a second polypeptide AtNAR2.1 to be functional. Immunological and transient protoplast expression methods revealed that an intact two-component complex of AtNRT2.1 and AtNAR2.1 is localized in the plasma membrane, has a size of ~150kDa, and half-life of 35h. Based on the absence of monomeric AtNRT2.1 in protein isolates, and lack of the oligomer in mutants of NRT2.1 or NAR2.1, I propose that this complex, rather than monomeric AtNRT2.1, is the form active in inducible HATS nitrate transport. After the large central cytosolic loop from the Aspergillus nidulans iHATS (NRTA) was introduced into AtNRT2.1, instead of its smaller loop, interaction with AtNAR2.1 was abolished. This observation shows that the central loop of AtNRT2.1 is required for interaction with AtNAR2.1. The Arabidopsis NRT2 family has 6 other members in addition to AtNRT2.1. By using heterologous expression in the yeast-two-hybrid system, Xenopus oocytes and leaf protoplasts, I have shown that, with the exception of AtNRT2.7, all NRT2s interact with AtNAR2.1 and are capable of nitrate transport.Plants also have a constitutive high-affinity transport system (cHATS) with proposed role of upregulation of many nitrate-inducible genes, including AtNRT2.1. For the first time in plants, a gene required for cHATS was isolated. Atnrt2.5 mutants exhibit ~60% reduction of the cHATS activity compared to WT plants at low nitrate concentrations, suggesting that AtNRT2.5 encodes the saturable cHATS transporter.
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