Molecular cloning and expression analysis of cDNAs encoding a putative Nrt2 nitrate transporter from peach

Abstract

Two cDNA clones encoding a putative high-affinity nitrate transporter (Nrt2) were isolated from a cDNA library of peach (Prunus persica (L.) Batsch) roots. The nucleotide sequences of the clones, named PpNrt2.1 and PpNrt2.2, showed 99% identity in their coding regions, but their 3'-untranslated sequences showed only 40% identity. The deduced amino acid sequences of the clones showed 44-90% homology to NRT2 proteins from other plants. Both clones contained open reading frames encoding polypeptides of 530 amino acid residues, each with a predicted molecular mass of about 57 kDa. Both polypeptides had 12 putative transmembrane domains and a long hydrophilic C-terminal domain like other members of this protein family. Analyses of the genomic structures and of Southern blots suggested that PpNrt2.1 and PpNrt2.2 originated from different loci in the peach genome. In Northern blots, transcripts of PpNrt2.1 and PpNrt2.2 were detected in roots but not shoots of seedlings after nitrate application. They were not detected in leaves, stems, flowers, fruits or seeds of mature trees in the field, indicating root-specific expression of PpNrt2.1 and PpNrt2.2. Application of nitrate to roots of nitrogen-deprived seedlings strongly induced expression of PpNrt2.1 within 0.5 h, and gradually induced expression of PpNrt2.2. Application of ammonium to roots of nitrogen-deprived seedlings repressed expression of PpNrt2.1 and PpNrt2.2. The relationship of PpNrt2.1 and PpNrt2.2 to the inducible high-affinity transport system (iHATS) involved in the uptake of nitrate in peach roots is discussed.