Date of Award
Doctor of Philosophy
Biochemistry and Cellular and Molecular Biology
Daniel M. Roberts
Beth Mullin, Rebecca Prosser, Jeffrey Becker, Cynthia Peterson
A cDNA was isolated from soybean (Glycine max) nodules that encodes a putative transporter (GmN70). GmN70 is expressed predominantly in mature nitrogenfixing root nodules. By western-blot and immunocytochemical analyses, GmN70 was localized to the symbiosome membrane of infected root nodule cells, suggesting a transport role in symbiosis. To investigate its transport function, cRNA encoding GmN70 was expressed in Xenopus laevis oocytes, and two-electrode voltage clamp analysis was performed. Ooctyes expressing GmN70 showed outward currents that are carried by anions with a selectivity of nitrate > nitrite >> chloride. These currents showed little sensitivity to pH or the nature of the counter cation in the oocyte bath solution. One-half maximal currents were induced by nitrate concentrations between 1 to 3 mM.
A global protein BLAST search for NLAT-related proteins revealed the presence of multiple homologs in all plant genomes sequenced with members segregating into 6 distinct monophyletic clades. Two genes from Arabidopsis clustering within the same clade 1 (chromosome nomenclature:At2g39210 and At2g28120) and shared a high amino acid sequence identity with GmN70 (respective 63.7% and 56.3%) and cluster in the same clade (Clade 1) as GmN70 and LjN70. Investigation of the transport properties of these two Arabidopsis NLAT-like genes (renamed AtNLAT1;1 [At2g39210] and AtNLAT1;2 [At2g28120] showed similar transport properties as GmN70 and LjN70 and transported the inorganic anions, nitrate, nitrite, and chloride. Expression analysis and sub-cellular localization of AtNLAT1;1 show it to be expressed predominately in the leaf on the plasma membrane. Its expression is regulated by a diverse set of biotic and abiotic stress signals including Pseudomonas syringae pv. tomato, salicylic acid, salinity, touch, and wounding suggesting a role in stress adaptation in plants. Consistent with these findings, transgenic Arabidopsis lines in which AtNLAT1;1 expression is knocked-down show an enhanced sensitivity to NaCl as well as a perturbed ionomic profile as revealed by inductively coupled plasma-mass spectroscopy (ICP-MS). Overall, the data presented in this body of work have provided insight into the biochemical and biophysical function and expression profiles of two members of the NLAT family, GmN70 and AtNLAT1;1.
Vincill, Eric Daniel, "The Identification, Functional Characterization and Phylogeny of the Nodulin-Like Anion Transporter (NLAT) Family in Plants. " PhD diss., University of Tennessee, 2008.