Distinct roles of nitrate and nitrite in regulation of expression of the nitrate transport genes in the moss Physcomitrella patens.

Five NRT2 genes and three Nar2 genes, encoding putative high-affinity nitrate transporters, and the respective cDNAs were identified and characterized in Physcomitrella patens. The deduced moss NRT2 and NAR2 proteins were more similar to the corresponding proteins of higher plants than to those of the green alga Chlamydomonas reinhardtii. Expression of all the genes was inhibited by ammonium added to the medium. The regulation by ammonium was abolished by an inhibitor of glutamine synthetase, but the effect of this inhibitor was counteracted by an inhibitor of glutamate synthase. Negative correlation was observed between the glutamine content of protonemata and the transcript levels of PpNRT2 and PpNar2. These results indicated that glutamine is the signal for repression of the genes. All the genes except PpNRT2;5 showed transient expression stimulated by nitrate but not by nitrite, peaking at 2-4 h after the medium was deprived of ammonium. When the glutamine synthetase inhibitor was used to inhibit assimilation of the ammonium generated intracellularly from nitrate or nitrite, the second phase of activation of genes became manifest at approximately 8 h after the medium was deprived of ammonium. Surprisingly, both nitrate and nitrite stimulated gene expression at this stage. PpNRT2;5 was distinct from the other genes in that its expression is sharply induced by nitrite, is strictly dependent on nitrite or nitrate, and is much less susceptible to the feedback regulation, retaining a constant level in nitrate-containing medium. These results indicated that P. patens has multiple mechanisms for sensing nitrate and nitrite.