Date of Award

8-2008

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Daniel M. Roberts

Committee Members

Beth Mullin, Rebecca Prosser, Jeffrey Becker, Cynthia Peterson

Abstract

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.

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