Léran S et al. (2015), AtNPF5.5, a nitrate transporter affecting nitro...

XB-ART-50398

Sci Rep 2015 Jan 22;5:7962. doi: 10.1038/srep07962.

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AtNPF5.5, a nitrate transporter affecting nitrogen accumulation in Arabidopsis embryo.

Léran S , Garg B , Boursiac Y , Corratgé-Faillie C , Brachet C , Tillard P , Gojon A , Lacombe B .

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Dipeptide (Leu-Leu) and nitrate transport activities of 26 Arabidopsis NPF (NRT1/PTR Family) proteins were screened in Saccharomyces cerevisiae and Xenopus laevis oocytes, respectively. Dipeptide transport activity has been confirmed for 2 already known dipeptide transporters (AtNPF8.1 and AtNPF8.3) but none of the other tested NPFs displays dipeptide transport. The nitrate transport screen resulted in the identification of two new nitrate transporters, AtNPF5.5 and AtNPF5.10. The localization of the mRNA coding for NPF5.5 demonstrates that it is the first NPF transporter reported to be expressed in Arabidopsis embryo. Two independent homozygous npf5.5 KO lines display reduced total nitrogen content in the embryo as compared to WT plants, demonstrating an effect of NPF5.5 function on the embryo nitrogen content. Finally, NPF5.5 gene produces two different transcripts (AtNPF5.5a and AtNPF5.5b) encoding proteins with different N-terminal ends. Both proteins are able to transport nitrate in xenopus oocytes.

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Species referenced: Xenopus laevis

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	Figure 1. Screen for dipeptide transport complementation by NPFs in S. cerevisiae.Control (C, non-transformed) and NPF-transformed ΔPTR2 yeast mutants were grown for 20 hours on SC minimal media (complemented with uracil for control yeast) containing His, Leu and Met (black bars), SC minimal media containing Met (red bars) and SC minimal media containing His, Met and Leu-Leu (green bars). (inset) Growth kinetics in the 3 different media for yeasts expressing NPF8.1. Results from one experiment representative of 2–3 independent repeats.
	Figure 2. Screen for nitrate transport activity of NPFs in xenopus oocytes.Control (non-injected) and NPF-injected oocytes were bathed in 30 mM K15NO3 for 2 hours.15N accumulation is expressed as % of accumulation in NPF6.3/NRT1.1-expressing oocytes. Values are mean +/− SEM from 3 experiments. *** indicate significant difference with control oocytes at p < 0.001 (t-test).
	Figure 3. Characterization of npf5.5 KO lines.(a) Schematic representation of NPF5.5 insertion lines. Blue arrows represent the sens and antisens oligonucleotides used for Q-PCR analysis. (b) Q-RT-PCR analysis were performed on mRNA isolated from embryo at the bent cotyledon stage. Values are mean +/− SEM from 3 experiments. *** indicate significant difference with control oocytes at p < 0.001 (t-test). (c) Nitrogen content in embryos of two NPF5.5 KO lines. The total nitrogen content was determined in embryos of two NPF5.5 KO lines (npf5.5-1 and npf5.5-2). Values are expressed in percent of N content decrease compared to total N content in WT embryos. The average was made on 5 batches of 75 embryos, n = 5.
	Figure 4. Alternative NPF5.5 transcripts.(a) Schematic representation of NPF5.5 5′ genomic and RNA sequences. Blue arrows represent the sens oligonucleotides used for identification of NPF5.5a and b. (b) Amino-acid alignment of the N-terminal region of the 3 different NPF5.5 proteins. (c) Influx in control (non-injected) oocytes (white bar), in NPF6.3-expresssing oocytes (black bar) and in the 3 forms NPF5.5-expressing oocytes (grey bars) after 2 hours of incubation in 30 mM15NO3 solution. P: predicted sequence, a and b: 2 NPF5.5 forms. Influx in control oocytes (white bar), in NPF6.3-expresssing oocytes (black bar) and in NPF5.5-expressing oocytes (grey bars) after 2 hours of incubation in 30 mM15NO3 solution. * and indicate significant difference with control oocytes at p < 0.001 and p < 0.005 respectively (t-test).

References [+] :

Almagro, Characterization of the Arabidopsis nitrate transporter NRT1.6 reveals a role of nitrate in early embryo development. 2008, Pubmed, Xenbase