The Nitrate Transporter Family Protein LjNPF8.6 Controls the N-Fixing Nodule Activity

Abstract

N-fixing nodules are new organs formed on legume roots as a result of the beneficial interaction with soil bacteria, rhizobia. The nodule functioning is still a poorly characterized step of the symbiotic interaction, as only a few of the genes induced in N-fixing nodules have been functionally characterized. We present here the characterization of a member of the Lotus japonicus nitrate transporter1/peptide transporter family, LjNPF8.6 The phenotypic characterization carried out in independent L. japonicus LORE1 insertion lines indicates a positive role of LjNPF8.6 on nodule functioning, as knockout mutants display N-fixation deficiency (25%) and increased nodular superoxide content. The partially compromised nodule functioning induces two striking phenotypes: anthocyanin accumulation already displayed 4 weeks after inoculation and shoot biomass deficiency, which is detected by long-term phenotyping. LjNPF8.6 achieves nitrate uptake in Xenopus laevis oocytes at both 0.5 and 30 mm external concentrations, and a possible role as a nitrate transporter in the control of N-fixing nodule activity is discussed.

Figure 1.

LjNPF8.6 transcriptional regulation. A, Expression in different organs. RNAs were extracted from wild-type plants grown on Gamborg B5 derivative medium without N source at 4 weeks post inoculation (wpi). Mature flowers were obtained from L. japonicus plants propagated in the growth chamber. B, Time course of expression in root and nodule tissues after M. loti inoculation. RNAs were extracted from roots of wild-type seedlings grown in N starvation conditions at different times after inoculation (R0, 24 h, and 72 h) and from young (10 d post inoculation) and mature (28 d post inoculation) nodules. Expression levels are normalized with respect to the internal control ubiquitin (UBI) gene and plotted relative to the expression of flowers (A) and R0 (B). White bars, LjNPF8.6; gray bars, LjNIN. Data bars represent means and sd of data obtained with RNA extracted from three different sets of plants and three real-time PCR experiments.

Figure 2.

Exon/intron organization of the LjNPF8.6 gene. Insertion sites and relative orientations of the LORE1 retrotransposon element in the 53155, 19899, and 49638 lines are indicated.

Figure 3.

Quantitative analysis of shoot biomass and nodulation capacity of L. japonicus wild-type (wt) and LjNPF8.6 null mutant plants, grown in the presence of different KNO₃ concentrations, in symbiotic and nonsymbiotic conditions. A, Shoot length per plant. B, Fresh shoot weight per plant. C, Nodule numbers per plant. The different KNO₃ concentrations and, when determined, M. loti inoculations are indicated. Bars corresponding to wild-type and different LORE1 plants are indicated. Data bars represent means and se of measures from three experiments (12 plants per experiment per condition). Data in A and B were scored 25 d after sowing (21 d after transferring the plants from water agar). Data in C were scored 28 d after inoculation.

Figure 4.

Analysis of anthocyanin content. A, Three representative wild-type and 49638 plants are shown on the right and left sides of the petri dish, respectively. B, Higher magnification showing intense purple colors in the stems of mutant plants (on the left). C, Anthocyanin content in stems of wild-type (wt) and LORE1 lines. The different KNO₃ concentrations and, when determined, M. loti inoculations are indicated. Bars corresponding to wild-type and different LORE1 plants are indicated. Data bars represent means and se of measures from three experiments (12 plants per experiment per condition). Data in C were scored 28 d after inoculation. Asterisks indicate significant differences (P < 0.001) from wild-type levels.

Figure 5.

A, ARA per nodule weight of wild-type and 49638 plants. The different KNO₃ conditions are indicated. Data bars indicate means and se of three independent experiments (n = 8 plants per experiment). Asterisks indicate significant differences (P < 0.005) between wild-type and 49638 nodules in no-N and 1 mm KNO₃ conditions. The asterisk over the bracket across the 10 mm condition indicates a significant reduction of ARA activity in nodules of both plant genotypes shifted onto high-nitrate conditions compared with nodules of plants maintained on low permissive conditions (P < 0.001). B and C, Representative shoot phenotypes of 8-wpi wild-type and 49638 plants transferred onto hydroponic conditions in the presence of 0.5 mm KNO₃ at 4 wpi. Wild-type and mutant plants were maintained in the same vessels (four vessels, 16 plants).

Figure 6.

Phenotypic symbiotic characterization of Ljnpf8.6 mutants. A to D, Histochemical detection of β-galactosidase activity to test M. loti (carrying the hemA::lacZ-expressing plasmid) density in young and mature nodules of wild-type (A and C) and 49638 (B and D) plants. E to H, Whole-mount NBT staining for O₂⁻ anion detection of wild-type (E and G) and 49638 (F and H) nodule primordia and mature nodules. I and J, Sections (100 μm) of wild-type (I) and 49638 (J) mature nodules stained with NBT. Arrows indicate staining in the parenchyma (p) and nodular vascular bundles (vb). K, Quantification of NBT staining in wild-type (wt) and 49638 mutant lines. Data bars represent means and se of nodules from three independent samples (eight plants per experiment). Asterisks indicate significant differences from wild-type values (P < 0.05). FW, Fresh weight.

Figure 7.

Functional expression of LjNPF8.6 in X. laevis oocytes in low (0.5 mm) and high (30 mm) external nitrate concentrations. Nitrate uptake is shown in control oocytes (black bars), injected with cRNAs, expressing LjNPF8.6 (white bars) or AtNPF6.3 (gray bars; n = 5–8). Values are means ± se.

Figure 8.

A, Nitrate content of shoots and roots from wild-type and 49638 plants grown on 1 mm KNO₃ and inoculated with M. loti. Data bars represent means and se from three independent samples (10 plants per sample). B, Nitrate content in wild-type and 49638 4-week-old nodules. Data bars represent means and se of nodules from three independent samples (10 plants per sample). Bars corresponding to wild-type and 49638 plants are indicated. FW, Fresh weight.