Functional characterization of nitrate transporters in maize.

Abstract

Nitrate is an essential nutrient for plant growth. Nitrate acquisition by roots and its intercellular translocation is mediated by nitrate permeable transport proteins. Nitrate transporters have been extensively studied in the model plant, Arabidopsis thaliana. Nitrate transporters belong to three protein families: NPF (Nitrate Transporter 1/Peptide Transporter), NRT2 (Nitrate Transporter 2) and CLC (Chloride Channel) (Miller et al., 2007; Wang et al., 2012). However, there is little known about how these proteins orchestrate nitrate transport in maize. Four putative nitrate transporter genes (ZmNPF6.4, ZmNPF6.5, ZmNPF6.6, and ZmNPF7.10) were cloned from a maize root cDNA population. Preliminary localization studies using C-terminal YFP-fusions showed maize NPF proteins targeting to the plasma membrane, with the exception of ZmNPF7.10, where targeting could not be resolved. Gene expression studies indicated ZmNPF6.6 was induced strongly in roots by nitrate. Its shoot expression was mostly absent. In contrast, ZmNPF6.4 exhibited a constitutive expression pattern in both root and shoot tissues and was not sensitive to nitrate. Both ZmNPF6.5 and ZmNPF7.10 showed little expression in either root or shoot tissues. Functional characterization studies were conducted on ZmNPF6.4 and ZmNPF6.6 as there was no nitrate transport activity measured with ZmNPF6.5 and ZmNPF7.10 using a preliminary screening experiment in Xenopus laevis oocytes. Combining electrophysiology and chemical flux analysis, ZmNPF6.4 was characterized as a pH-dependent, low-affinity, non-selective nitrate and chloride transporter. On the other hand, ZmNPF6.6 encoded a pH-dependent, dual-affinity, nitrate specific transporter, which was also permeable to chloride in the absence of nitrate. The functional differences between ZmNPF6.4 and ZmNPF6.6 were explored using site-directed mutagenesis experiments. The “affinity switch” Thr101 within the nitrate transceptor, AtNPF6.3, is conserved in ZmNPF6.6 (Thr104) (Liu, 2003). However, mutating ZmNPF6.6:Thr104 to alanine or aspartate (dephosphorylation and phosphorylation mimics, respectively), did not transform the dual-affinity transporter into either a high- or low-affinity monophasic transporter. Instead, both HATS and, predominantly, the LATS activities of ZmNPF6.6 were repressed by both T104A and T104D mutations. The equivalent of the predicted nitrate-binding residue in AtNPF6.3 (His356) was investigated in ZmNPF6.4 and ZmNPF6.6. In ZmNPF6.4, a tyrosine residue (Tyr370) is present instead of a histidine. Replacement of Y370 with histidine (ZmNPF6.4:Y370H) conferred dual-affinity nitrate transport and enhanced nitrate specificity over chloride. However, replacing His362 in ZmNPF6.6 with Tyr362 made the transporter non-functional. A preliminary analysis of the high-affinity nitrate transport system was conducted by functionally characterizing ZmNRT2.1 and ZmNRT3.1A. The plasma membrane targeting of ZmNRT2.1 required the presence of ZmNRT3.1A. This was confirmed using a C-terminal fusion of NRT2.1 with YFP. Signal was only detected in onion epidermal cells that were co-transformed with both ZmNRT2.1 and ZmNRT3.1A. Gene expression analysis identified both a N-starvation induced expression and a nitrate induced expression pattern for ZmNRT2.1. In contrast, ZmNRT3.1A exhibited a constitutive expression in both roots and shoots. When ZmNRT2.1 and ZmNRT3.1A were co-injected into Xenopus laevis oocytes, high-affinity nitrate transport activity was measured. Single injections of either cRNA failed to elicit a nitrate transport phenotype.