Molecular Cloning, In Silico Analysis and Expression of plasma membrane-associated NAR2 Protein, SaNAR2.2, from Euhalophyte Suaeda altissima

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Abstract
Plasma membrane-associated proteins of NAR2 family are involved in the uptake of nitrate by plant roots as partners of NRT2 family proteins, which carry out high- affinity nitrate transport across membranes of plant cells. In this study, the coding sequence of NAR2 family gene, SaNAR2.2, was cloned from the euhalophyte Suaeda altissima . This species is able to withstand high concentrations of NaCl, up to 750 mM, in the nutrient medium. The protein SaNAR2.2, of 196 amino acids in size, with a calculated molecular weight of 21.7 kDa, has been characterized in terms of structure and phylogeny. According to the prediction of cellular localization and protein topology, SaNAR2.2 is a resident of plasma membrane. It has one transmembrane segment at the C-terminal part and a small C-end located in cytoplasm. The large N-terminal part of SaNAR2.2 protein is located in the extracellular space. On the phylogenetic tree of NAR2 family proteins, the protein from S. altissima lies in a clade with the proteins of NAR2.2 subfamily from other plants of Amaranthaceae family. We detected the interaction between SaNAR2.2 and putative partner proteins SaNRT2.1 and SaNRT2.5, which are the members of high-affinity nitrate transporter family NRT2 from S. altissima , using the bimolecular fluorescence complementation (BiFC) assay in Nicotiana benthamiana leaves. The expression of SaNAR2.2 gene in organs of S. altissima plants grown at low (0.5 mM) or high (15 mM) nitrate concentrations in the nutrient medium, without NaCl or in the presence of increasing salt concentrations, was analyzed using RT-qPCR method. It has been found that SaNAR2.2 is expressed nearly exclusively in roots of S. altissima . As the concentrations of NaCl in the nutrient medium increased (up to 500 mM), the expression of SaNAR2.2 decreased at high external nitrate concentrations and significantly increased, up to ten-fold, at low nitrate concentrations; this points to the significant role of SaNAR2.2 protein under salinity conditions as a participant of high-affinity nitrate transport system. SaNAR2.2 is the first components of a high affinity nitrate transport system characterized in a terrestrial halophytic plant.

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