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

Ya-Ni He,Hong-Ying Yi,Jia-Shi Peng,Yao Cai,Yuan Guan,Ji-Ming Gong,De-Fen Liu

doi:10.1038/s41598-017-06744-5

Abstract

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.

Highlights

Nitrate is the major nitrogen source for most plants, especially those grown in aerobic soil conditions[1]
Regarding nitrate efflux from vacuoles, indirect evidences imply that AtCLCb and OsNPF7.2 might get involved, as they both were tonoplast-localized, and heterologous expression in Xenopus laevis oocytes indicated that they mediated nitrate uptake, but no evidence showed that functional disruption of these genes led to nitrate accumulation in vacuoles[24, 25]
Significant progresses have been made in clarifying the nitrate uptake and transport in Arabidopsis by the characterization of the transporters in NRT1/NPF, NRT2, CLC and SLAC/SLAH families[34]

Summary

Introduction

Nitrate is the major nitrogen source for most plants, especially those grown in aerobic soil conditions[1]. AtNRT2.7, was a tonoplast localized transporter expressed exclusively in seeds, which regulated the kinetics of seed germination by affecting nitrate storage in seed vacuoles[21]. AtCLCc was supposed to be involved in vacuolar nitrate accumulation, because www.nature.com/scientificreports/ It was tonoplast localized and the related mutants showed lower nitrate contents[22, 23]. Regarding nitrate efflux from vacuoles, indirect evidences imply that AtCLCb and OsNPF7.2 might get involved, as they both were tonoplast-localized, and heterologous expression in Xenopus laevis oocytes indicated that they mediated nitrate uptake, but no evidence showed that functional disruption of these genes led to nitrate accumulation in vacuoles[24, 25]. Our data suggested that these three transporters were all tonoplast localized, and mediated nitrate uptake in a pH-dependent low-affinity manner when heterologously expressed in oocytes. Further analysis indicated that they possibly modulated nitrate allocation between roots and shoots via vacuolar nitrate release

Methods

Results

Conclusion