Knockdown of a rice stelar nitrate transporter alters long-distance translocation but not root influx.

Zhong Tang,Qing Li,Anthony J Miller,Xiaorong Fan,Huimin Feng,Guohua Xu,Qirong Shen

doi:10.1104/pp.112.204461

Abstract

Root nitrate uptake is well known to adjust to the plant's nitrogen demand for growth. Long-distance transport and/or root storage pools are thought to provide negative feedback signals regulating root uptake. We have characterized a vascular specific nitrate transporter belonging to the high-affinity Nitrate Transporter2 (NRT2) family, OsNRT2.3a, in rice (Oryza sativa ssp. japonica 'Nipponbare'). Localization analyses using protoplast expression, in planta promoter-β-glucuronidase assay, and in situ hybridization showed that OsNRT2.3a was located in the plasma membrane and mainly expressed in xylem parenchyma cells of the stele of nitrate-supplied roots. Knockdown expression of OsNRT2.3a by RNA interference (RNAi) had impaired xylem loading of nitrate and decreased plant growth at low (0.5 mm) nitrate supply. In comparison with the wild type, the RNAi lines contained both nitrate and total nitrogen significantly higher in the roots and lower in the shoots. The short-term [(15)N]NO(3)(-) influx (5 min) in entire roots and NO(3)(-) fluxes in root surfaces showed that the knockdown of OsNRT2.3a in comparison with the wild type did not affect nitrate uptake by roots. The RNAi plants showed no significant changes in the expression of some root nitrate transporters (OsNRT2.3b, OsNRT2.4, and OsNAR2.1), but transcripts for nia1 (nitrate reductase) had increased and OsNRT2.1 and OsNRT2.2 had decreased when the plants were supplied with nitrate. Taken together, the data demonstrate that OsNRT2.3a plays a key role in long-distance nitrate transport from root to shoot at low nitrate supply level in rice.

Highlights

Root nitrate uptake is well known to adjust to the plant’s nitrogen demand for growth
We confirmed that OsNRT2.3a is a plasma membrane-localized nitrate transporter
We have shown previously that the transcripts of OsNRT2.3a are abundantly represented in nitratesupplied roots and are suppressed by ammonium N, while the transcripts of OsNRT2.3b are mainly in shoots and very faint in the roots irrespective of the supplied N form and concentration (Feng et al, 2011)

Summary

Introduction

Root nitrate uptake is well known to adjust to the plant’s nitrogen demand for growth. Knockdown expression of OsNRT2.3a by RNA interference (RNAi) had impaired xylem loading of nitrate and decreased plant growth at low (0.5 mM) nitrate supply. The data demonstrate that OsNRT2.3a plays a key role in long-distance nitrate transport from root to shoot at low nitrate supply level in rice. Several Arabidopsis NRT1 and NRT2 family members have been characterized for their functions in nitrate uptake and long-distance transport. AtNRT1.5 is expressed in the root pericycle cells close to the xylem and is responsible for the loading of nitrate into the xylem for root-to-shoot nitrate transport (Lin et al, 2008). AtNRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature and plays the role in the retrieval of nitrate from the xylem sap (Li et al, 2010). AtNRT1.9 facilitates the loading of nitrate into the root phloem, enhancing downward transport in roots, and its knockout increases root-toshoot xylem transport of nitrate (Wang and Tsay, 2011)

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Conclusion