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Front Plant Sci
2016 Jan 01;7:1529. doi: 10.3389/fpls.2016.01529.
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Knock-Down of a Tonoplast Localized Low-Affinity Nitrate Transporter OsNPF7.2 Affects Rice Growth under High Nitrate Supply.
Hu R
,
Qiu D
,
Chen Y
,
Miller AJ
,
Fan X
,
Pan X
,
Zhang M
.
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The large nitrate transporter 1/peptide transporter family (NPF) has been shown to transport diverse substrates, including nitrate, amino acids, peptides, phytohormones, and glucosinolates. However, the rice (Oryza sativa) root-specific family member OsNPF7.2 has not been functionally characterized. Here, our data show that OsNPF7.2 is a tonoplast localized low-affinity nitrate transporter, that affects rice growth under high nitrate supply. Expression analysis showed that OsNPF7.2 was mainly expressed in the elongation and maturation zones of roots, especially in the root sclerenchyma, cortex and stele. It was also induced by high concentrations of nitrate. Subcellular localization analysis showed that OsNPF7.2 was localized on the tonoplast of large and small vacuoles. Heterologous expression in Xenopus laevis oocytes suggested that OsNPF7.2 was a low-affinity nitrate transporter. Knock-down of OsNPF7.2 retarded rice growth under high concentrations of nitrate. Therefore, we deduce that OsNPF7.2 plays a role in intracellular allocation of nitrate in roots, and thus influences rice growth under high nitrate supply.
FIGURE 1. OsNPF7.2 is mainly expressed in the elongation and maturation zones of roots.
(A) qPCR analysis of OsNPF7.2 expression in 5-day-old rice seedlings. UBC was used as reference gene. Data represent mean ± SD from one experiment of six seedlings, three independent experiments showed the same result. (B–E) GUS staining of POsNPF7.2: GUS seedlings. GUS staining of whole plant (B), roots (C), and cross section of roots (D,E). Bar = 1 mm in (B), 3 mm in (C), 20 μm in (D) and 10 μm in (E). CO, coleoptile; RA, radical root; CR, crown root; LR, lateral root; EP, epidermis; X, exodermis; S, sclerenchyma; C, cortex; E, endodermis; R, pericycle; XY, xylem; PL, phloem; L, late metaxylem.
FIGURE 2. High nitrate induces OsNPF7.2 expression in roots.
(A,B) qPCR analysis of short-term induction of OsNPF7.2 in rice roots under 10 mM (A) and 0.5 mM (B) nitrate solution, respectively. ZH11 seedlings were grown in IRRI solution for 2 weeks, and then transferred for 3-day nitrate starvation, finally shifted in IRRI solution with 10 mM or 0.5 mM KNO3 or KCl instead of original N sources. In the solution, equal molar of KCl instead of KNO3 was used as control. (C) Long-term induction of OsNPF7.2 in rice roots. ZH11 seedlings were grown on 1/2 MS medium with various concentrations of KNO3 for 2 weeks. Ammonium was used to maintain total N concentrations equal in these medium. Data in (A–C) represent mean ± SD from one experiment of 10 seedlings, two independent experiments showed same result.
FIGURE 3. OsNPF7.2 is mainly localized on tonoplast.
(A,B) Rice protoplasts expressing 35S: EGFP as control. (C–F) Rice protoplasts expressing 35S: OsNPF7.2: EGFP and a tonoplast localized marker vac-rk. (G–J) Rice protoplasts expressing 35S: EGFP: OsNPF7.2 and a tonoplast localized marker vac-rk. BF, bright field; GFP, green fluorescent protein. Merged shows the signal of GFP merged with corresponding mCherry. Arrowhead indicates the small vacuoles can be merged with vac-rk. Arrows indicate the small vacuoles cannot be merged with vac-rk. Bar = 5 μm.
FIGURE 4. OsNPF7.2 is a low-affinity NRT.
(A) Nitrate uptake assay by Xenopus oocytes expression system. (B) Nitrate efflux assay by Xenopus oocytes expression system. AtNPF6.3 was used as positive control. Water-injected Xenopus oocytes were used as negative control. Data represent means of five oocytes and SD. Two independent experiments showed same result. Asterisks upon the bars indicate statistically significant differences (P < 0.05) between the cRNA-injected Xenopus oocytes and water-injected Xenopus oocytes by t-test.
FIGURE 5. osnpf7.2 mutants and OsNPF7.2-RNAi lines are knock-down lines of OsNPF7.2.
(A) Diagram of the insertional positions of retrotransposon Tos17 and T-DNA in two osnpf7.2 mutants. osnpf7.2-1 is inserted by Tos17, and osnpf7.2-2 is inserted by T-DNA. (B,C) Detection of OsNPF7.2 expression level in two osnpf7.2 mutants with semi-quantitative-PCR (B) and qPCR (C). (D) Detection of OsNPF7.2 expression level in OsNPF7.2-RNAi lines with qPCR. Data of (C,D) represent mean ± SD from one experiment of 10 seedlings, three independent experiments showed same result.
FIGURE 6. osnpf7.2-1 shows retardant growth under high concentrations of nitrate.
(A,B) Root and shoot length of osnpf7.2-1 and wild type plants (ZH11). Wild type and mutant plants grew on 1/2 MS medium containing various concentration of KNO3 for 7 d. Ammonium was used to maintain the total N concentrations in the medium. (C–E) Root and shoot length of osnpf7.2-1 and wild type plants in 10 mM NH4NO3 and (NH4)2SO4. Plants were grown on 1/2 MS medium containing NH4NO3 or (NH4)2SO4 as N source for 7 days. Bar = 1 cm in (C). For all the subfigures, data represent means of 16 samples and SE. Two independent experiments showed same result. Asterisks indicate significant differences (P < 0.01) between osnpf7.2-1 and wild type plants (ZH11) by t-test.
FIGURE 7. OsNPF7.2-RNAi plants show retardant growth compared with the wild type plants in hydroponic solution.
(A) Fresh weight per plant. (B) Length of the whole plants. Plants grew in IRRI solution for 5 weeks, then grew in IRRI solution containing 5 mM Ca(NO3)2 (10 NO3−), 0.25 mM Ca(NO3)2 (0.5 NO3−) or 5 mM NH4NO3 (5 NH4NO3) as N source for 3 weeks. The wild type of the RNAi lines (R29-5 and R30-1) and osnpf7.2-1 is ZH11. The wild type of osnpf7.2-2 is Hwayoung. Data represent means of 10 samples and SE. Two independent experiments showed same result. Asterisks upon the bars indicate significant differences (P < 0.05) between the transgenic line with their corresponding wild type plants in the LSD-test following one-way ANOVA.
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