Vacuolar nitrate efflux requires multiple functional redundant nitrate transporter in Arabidopsis thaliana.

Nitrate in plants is preferentially stored in vacuoles; however, how vacuolar nitrate is reallocated and to which biological process(es) it might contribute remain largely elusive. In this study, we functionally characterized three nitrate transporters NPF5.10, NPF5.14, and NPF8.5 that are tonoplast-localized. Ectopic expression in Xenopus laevis oocytes revealed that they mediate low-affinity nitrate transport. Histochemical analysis showed that these transporters were expressed preferentially in pericycle and xylem parenchyma cells. NPF5.10, NPF5.14, and NPF8.5 overexpression significantly decreased vacuolar nitrate contents and nitrate accumulation in Arabidopsis shoots. Further analysis showed that the sextuple mutant (npf5.10 npf5.14 npf8.5 npf5.11 npf5.12 npf5.16) had a higher 15NO3-uptake rate than the wild-type Col-0, but no significant difference was observed for nitrate accumulation between them. The septuple mutant (npf5.11 npf5.12 npf5.16 npf5.10 npf5.14 npf8.5 clca) generated by using CRISPR/Cas9 showed essentially decreased nitrate reallocation compared to wild type when exposed to nitrate starvation, though no further decrease was observed when compared to clca. Notably, NPF5.10, NPF5.14, and NPF8.5 as well as NPF5.11, NPF5.12, and NPF5.16 were consistently induced by mannitol, and more nitrate was detected in the sextuple mutant than in the wild type after mannitol treatment. These observations suggest that vacuolar nitrate efflux is regulated by several functional redundant nitrate transporters, and the reallocation might contribute to osmotic stress response other than mineral nutrition.

The nitrate transporter OsNPF7.9 mediates nitrate allocation and the divergent nitrate use efficiency between indica and japonica rice.

Nitrate allocation in Arabidopsis (Arabidopsis thaliana) represents an important mechanism for mediating plant environmental adaptation. However, whether this mechanism occurs or has any physiological/agronomic importance in the ammoniphilic plant rice (Oriza sativa L.) remains unknown. Here, we address this question through functional characterization of the Nitrate transporter 1/Peptide transporter Family (NPF) transporter gene OsNPF7.9. Ectopic expression of OsNPF7.9 in Xenopus oocytes revealed that the gene encodes a low-affinity nitrate transporter. Histochemical and in-situ hybridization assays showed that OsNPF7.9 expresses preferentially in xylem parenchyma cells of vasculature tissues. Transient expression assays indicated that OsNPF7.9 localizes to the plasma membrane. Nitrate allocation from roots to shoots was essentially decreased in osnpf7.9 mutants. Biomass, grain yield, and nitrogen use efficiency (NUE) decreased in the mutant dependent on nitrate availability. Further analysis demonstrated that nitrate allocation mediated by OsNPF7.9 is essential for balancing rice growth and stress tolerance. Moreover, our research identified an indica-japonica divergent single-nucleotide polymorphism occurring in the coding region of OsNPF7.9, which correlates with enhanced nitrate allocation to shoots of indica rice, revealing that divergent nitrate allocation might represent an important component contributing to the divergent NUE between indica and japonica subspecies and was likely selected as a favorable trait during rice breeding.

Tonoplast-localized nitrate uptake transporters involved in vacuolar nitrate efflux and reallocation in Arabidopsis.

A great proportion of nitrate taken up by plants is stored in vacuoles. Vacuolar nitrate accumulation and release is of great importance to nitrate reallocation and efficient utilization. However, how plants mediate nitrate efflux from vacuoles to cytoplasm is largely unknown. The current study identified NPF5.11, NPF5.12 and NPF5.16 as vacuolar nitrate efflux transporters in Arabidopsis. Histochemical analysis showed that NPF5.11, NPF5.12 and NPF5.16 were expressed preferentially in root pericycle cells and xylem parenchyma cells, and further analysis showed that these proteins were tonoplast-localized. Functional characterization using cRNA-injected Xenopus laevis oocytes showed that NPF5.11, NPF5.12 and NPF5.16 were low-affinity, pH-dependent nitrate uptake transporters. In npf5.11 npf5.12 npf5.16 triple mutant lines, more root-fed 15NO3- was translocated to shoots compared to the wild type control. In the NPF5.12 overexpression lines, proportionally less nitrate was maintained in roots. These data together suggested that NPF5.11, NPF5.12 and NPF5.16 might function to uptake nitrate from vacuoles into cytosol, thus serving as important players to modulate nitrate allocation between roots and shoots.