The nitrate transporter MtNPF6.8 (MtNRT1.3) transports abscisic acid and mediates nitrate regulation of primary root growth in Medicago truncatula

Abstract

Elongation of the primary root during postgermination of Medicago truncatula seedlings is a multigenic trait that is responsive to exogenous nitrate. A quantitative genetic approach suggested the involvement of the nitrate transporter MtNPF6.8 (for Medicago truncatula NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER Family6.8) in the inhibition of primary root elongation by high exogenous nitrate. In this study, the inhibitory effect of nitrate on primary root elongation, via inhibition of elongation of root cortical cells, was abolished in npf6.8 knockdown lines. Accordingly, we propose that MtNPF6.8 mediates nitrate inhibitory effects on primary root growth in M. truncatula. pMtNPF6.8:GUS promoter-reporter gene fusion in Agrobacterium rhizogenes-generated transgenic roots showed the expression of MtNPF6.8 in the pericycle region of primary roots and lateral roots, and in lateral root primordia and tips. MtNPF6.8 expression was insensitive to auxin and was stimulated by abscisic acid (ABA), which restored the inhibitory effect of nitrate in npf6.8 knockdown lines. It is then proposed that ABA acts downstream of MtNPF6.8 in this nitrate signaling pathway. Furthermore, MtNPF6.8 was shown to transport ABA in Xenopus spp. oocytes, suggesting an additional role of MtNPF6.8 in ABA root-to-shoot translocation. (15)NO3(-)-influx experiments showed that only the inducible component of the low-affinity transport system was affected in npf6.8 knockdown lines. This indicates that MtNPF6.8 is a major contributor to the inducible component of the low-affinity transport system. The short-term induction by nitrate of the expression of Nitrate Reductase1 (NR1) and NR2 (genes that encode two nitrate reductase isoforms) was greatly reduced in the npf6.8 knockdown lines, supporting a role of MtNPF6.8 in the primary nitrate response in M. truncatula.

Figure 1.

A and B, Subcellular localization of MtNPF6.8. MtNPF6.8-GFP fluorescence in leaf epidermal cells of M. truncatula (A) or N. benthamiana (B). C to F, Coexpression in N. benthamiana plasmolyzed leaf cells of MtNPF6.8-RFP (C) and the plasma membrane marker TM23:GFP (D). Near-perfect colocalization of coexpressed proteins is observed (E). A differential interference contrast image of the plasmolyzed cell highlighting the outline of the cell wall is shown (F). G, Overlay of C to F.

Figure 2.

MtNPF6.8 expression determined by quantitative RT-PCR in 10-d-old seedlings of M. truncatula. A, MtNPF6.8 relative expression in roots or shoots of seedlings grown on 5 mm nitrate. B and C, MtNPF6.8 relative expression in the entire root system (B) or in the primary root and the LR (C) in seedlings grown on either an N-free or an NO₃⁻-supplied (250 μm or 5 mm) medium. D, MtNPF6.8 relative expression from four sequential sections of the primary root starting with the tip: R1, R2, and R3 (0.5 cm long) and the root remainder R4. The R3 section was used as the calibrator. Seedlings were grown on 5 mm nitrate. Error bars represent the sd of three independent biological replicates.

Figure 3.

Histochemical localization of GUS activity in transgenic M. truncatula roots expressing the GUS reporter gene under the control of the MtNPF6.8 promoter. A, M. truncatula root expressing pMtNPF6.8:GUS stained histologically for GUS activity. B to E, Enlarged views of the primary root and of different developmental stages of LR: primary root near the shoot-root section (B), LR prior to emergence (C), LR immediately after emergence (D), and mature LR (E).

Figure 4.

Nitrate uptake by the M. truncatula wild type and npf6.8 knockdown lines. Seedlings were grown in hydroponic solution containing N-free or 5 mm nitrate for 10 d. Root ¹⁵NO₃⁻ influx was assayed by 5-min labeling in complete nutrient solutions containing ¹⁵NO₃⁻ (99 atom percentage ¹⁵N) at the concentration indicated. Values are the mean of four biological replicates ± se. P < 0.05 compared with the wild type for each condition (ANOVA). WT, Wild type.

Figure 5.

Effect of nitrate treatment on root architecture. A to D, Primary root length (A and B) and LR density (C and D) in the wild type or npf6.8 knockdown lines. Seedlings were grown on N-free medium for 24 h and then transferred on vertical plates for 10 d on medium containing nitrate at the indicated concentration. Primary root length was determined by image analysis (A and B), and LR density was calculated as the number of LRs per centimeter of primary root length (C and D). Means represent average values ± se of 32 seedlings of four biological replicates (A and C) and 16 seedlings of two biological replicates (B and D). Asterisks indicate significant differences compared with the untreated control (N-free) according to a one-way ANOVA (one asterisk indicates P < 0.05, whereas three asterisks indicate P < 0.001). WT, Wild type.

Figure 6.

Effect of nitrate treatment on the cortical cell length of primary root. Seedlings of the wild type and the npf6.8-3 knockdown line were germinated on N-free medium for 24 h and then transferred on vertical plates on N-free or 5 mm nitrate for 6 d. A, Roots were stained with propidium iodide and observed under a confocal microscope. B, Cortical cell length was then measured on the 1-mm part indicated by vertical black lines (primary meristem [PM] region) of three sequential sections of the primary root starting with the tip: root sections R1 (0.7 cm long), R2 (0.7 cm long), and R3 (the root remainder) by image analysis. Results are the means ± se of 6 to 12 replicates. The mean was modeled in a linear model (two asterisks indicate P < 0.01) with genotype and condition as quantitative factors and taking genotype and condition interactions into account. c, Cortex; e, endoderm; m, medulla; r, rhizoderm; WT, wild type; x, xylem.

Figure 7.

Effect of nitrate treatment on the expression of cell division marker genes. Seedlings were grown as described in the legend for Figure 6. Expression of cell division marker genes (Aurora, Cyclin A2, Cyclin B1.1, and Cyclin B1.2) was determined by quantitative RT-PCR in response to nitrate in the primary root tip. Each value represents the mean ± se of three independent biological experiments. Gene numbers are as follows: Aurora (TC901033), Cyclin A2 (Medtr2g102520), Cyclin B1.1 (Medtr5g088980), and Cyclin A2 (Medtr2g102520). WT, Wild type.

Figure 8.

Combined effect of nitrate and ABA or IAA on MtNPF6.8 expression and on primary root length. A and B, MtNPF6.8 expression in root determined by quantitative RT-PCR. Ten-day-old seedlings of M. truncatula were grown on N-free medium and exposed to 10 µm IAA or ABA during 5 h without (A) and with (B) 5 mm nitrate. Error bars represent the sd of three independent biological replicates. C and D, Combined effect of nitrate and hormonal treatment on primary root length in the wild type or npf6.8 knockdown lines. C and D, Seedlings were grown on medium for 24 h and then transferred on vertical plates for 10 d on medium containing N-free or 5 mm nitrate with 10 µm IAA (C) or ABA (D). Primary root length was determined by image analysis. Means represent average values ± se of 16 seedlings of two biological replicates. Asterisks indicate significant differences compared with the untreated control (N-free) according to a one-way ANOVA (asterisk indicates P < 0.05). WT, Wild type.

Figure 9.

ABA uptake activities in MtNPF6.8-cRNA- or AtNPF4.6-cRNA-injected Xenopus spp. oocytes. Injected or control oocytes were incubated with 1 µm [³H]ABA at pH 5.5. Each data point is a mean ± se for eight oocytes. Asterisks indicate significant differences compared with the control according to a one-way ANOVA (two asterisks indicate P < 0.01, whereas three asterisks indicate P < 0.001). Similar results were obtained with oocytes isolated from three different frogs.

Figure 10.

Nitrate induction of the expression of nitrate reductase genes (NR1 and NR2) and Gln synthetase gene (GS2) in roots. A and B, Seedlings of the wild type and the npf6.8 knockdown line were grown on vertical plates on N-free for 6 d and then transferred to N-free or 250 µm nitrate (A) and to N-free or 5 mm nitrate (B) for 30 min. The root mRNA level was determined by quantitative RT-PCR. Nitrate induction was calculated using N-free medium as the control condition. Error bars represent ± sd of biological replicates (n = 3). Gene numbers are as follows: NR1 (Medtr3g073180), NR2 (Medtr5g059820), and GS2 (Medtr2g021250). WT, Wild type.

Figure 11.

Schematic representation of a model of ABA-dependent nitrate signaling pathway for the regulation of primary root growth. This model includes two nitrate transporters: Nitrate-induced inhibition of primary root elongation was abolished in mutants knocked down in either MtNPF6.8 (this work) or MtNPF1.7 (Yendrek et al., 2010). In this work, the rescue of nitrate-induced inhibition of primary root growth by exogenous ABA in the npf6.8 mutant shows that MtNPF6.8 acts upstream of ABA, whereas MtNPF1.7 (LATD) was shown to act downstream of ABA (Liang et al., 2007). The rescue of nitrate inhibitory effect by exogenous ABA in the npf6.8 mutant suggests that nitrate is sensed at another site than MtNPF6.8. MtNPF1.7, a high-affinity nitrate transporter, is a good candidate for the integration of both nitrate and ABA signals.