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

Abstract

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.

Overexpressing NRT2.7 induces nitrate export from the vacuole and increases growth of Arabidopsis

“…These data suggest that the ectopic expression of NRT2.7 drives nitrate use for biomass production by increasing nitrate assimilation. This difference may probably due to a lower vacuolar storage capacity and/or an increased nitrate flux out of the vacuole as it was previously showed for other vacuolar nitrate transporters (He et al ., 2017; Lu et al ., 2022; Hodin et al ., 2023; Shi et al ., 2023) and no difference in nitrate uptake though the HATS was previously observed (Fig. 3).…”

“…Next to CLC transporters, some members of NRT2 and NPF transporters family are localized in the vacuolar membrane (Hu et al, 2016;He et al, 2017;Wang et al, 2018b;Lu et al, 2022). Among them, NRT2.7 belongs to the seven members of the NRT2 gene family in Arabidopsis that encodes major high affinity nitrate transporters.…”

Revealing the relationship between nitrogen use efficiency‐related QTLs and carbon and nitrogen metabolism regulation in poplar

“…Once a comprehensive functional linkage network is generated, genes whose functions are unknown can be easily annotated based on their linkages to genes with known functions. AtNPF5.10 mediates nitrate efflux from vacuoles (Lu et al, 2022), and AtNPF8.1 transports dipeptides in planta (Komarova et al, 2008). The coexpression network revealed that the seven candidate genes had important regulatory roles with the nitrogen transporter family genes NPF5.10 and NPF8.1 .…”

Host organelles and transporters in underground plant-pathogen interactions

“…Nitrate, one of the main sources of nitrogen, also acts as a signalling molecule for plant growth by influencing meristem dynamics and root/shoot architecture (Fredes et al 2019). A web of functionally redundant nitrate sensors and transporters is responsible for uptake and transport locally and systemically (Hsu & Tsay 2013;Wang et al 2018;Lu et al 2022). Exposure to nitrogen causes a nitrogen response that affects more than one thousand genes (Contreras-López et al 2022).…”

“…Although the nitrate transport system is extensive and at least seven NPFs have nitrate efflux functions (Lu et al 2022), we speculated that transcriptional downregulation of NPF5.12 could be a metabolic starvation tactic to sequester nitrates from invading hyphae and limit the growth of V. longisporum in susceptible plants. Nitrate depletion experiments showed that V. longisporum is indeed dependent on host nitrate transport for proper establishment in the host (Paper II.…”