Abstract
BackgroundNitrate plays an important role in grapevines vegetative and reproductive development. However, how grapevines uptake, translocate and utilize nitrate and the molecular mechanism still remains to be investigated.ResultsIn this study, we report the functional characterization of VvNPF6.5, a member of nitrate transporter 1/peptide transporter family (NRT1/PTR/NPF) in Vitis vinifera. Subcellular localization in Arabidopsis protoplasts indicated that VvNPF6.5 is plasma membrane localized. Quantitative RT-PCR analysis indicated that VvNPF6.5 is expressed predominantly in roots and stems and its expression is rapidly induced by nitrate. Functional characterization using cRNA-injected Xenopus laevis oocytes showed that VvNPF6.5 uptake nitrate in a pH dependent way and function as a dual-affinity nitrate transporter involved in both high- and low-affinity nitrate uptake. Further ectopic expression of VvNPF6.5 in Arabidopsis resulted in more 15NO3− accumulation in shoots and roots and significantly improved nitrogen use efficiency (NUE). Moreover, VvNPF6.5 might participate in the nitrate signaling by positively regulating the expression of primary nitrate response genes.ConclusionOur results suggested that VvNPF6.5 encodes a pH-dependent, dual-affinity nitrate transporter. VvNPF6.5 regulates nitrate uptake and allocation in grapevines and is involved in primary nitrate response.
Highlights
Nitrate plays an important role in grapevines vegetative and reproductive development
Cloning and sequence analysis of VvNPF6.5 To explore how grapevines uptake and translocate nitrate, we identified a highly homologous protein with CHL1 in Vitis vinifera and named it VvNPF6.5 (Fig. S1)
VvNPF6.5 is localized to the plasma membrane To investigate the subcellular localization of VvNPF6.5, VvNPF6.5 was fused in frame with the EYFP under the control of CaMV Cauliflower mosaic virus 35S promoter (35S) and the fusion protein was transiently expressed in Arabidopsis mesophyll protoplasts
Summary
Nitrate plays an important role in grapevines vegetative and reproductive development. As one of the most important nutrients for grapevines, nitrogen has a great impact on vine vegetative and reproductive development as well as grape composition [2, 3]. Nitrate is the major source of nitrogen for most terrestrial plants, especially those grown in aerobic soil conditions [8]. Nitrate concentrations in soil solutions range from very low levels of a few hundred micromolar to around 20 mM, even up to 70 mM [9] To cope with this wide range of concentrations, plants have evolved two nitrate uptake systems, the high affinity transport system (HATS) and the low-affinity transport system (LATS) [10]. During the past two decades, at least four gene families including nitrate transporter 1/peptide transporter family (NRT1/PTR/NPF), nitrate transporter 2 family (NRT2), chloride channel family (CLC) and slow anion channel-associated homologues (SLAC/SLAH), have been identified to play roles in nitrate transport in higher plants [11]
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