Functional assessment of the Medicago truncatula NIP/LATD protein demonstrates that it is a high-affinity nitrate transporter

Abstract

The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 μm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function.

Figure 1.

Nitrate uptake in M. truncatula. Wild-type A17, Mtnip-1, and Mtnip-3 plants were placed in solutions containing 250 μm (A) or 5 mm (B) nitrate. Nitrate uptake was monitored by its depletion from the medium at 2-h intervals. Data are plotted for one biological replicate ± se.

Figure 2.

LR lengths of M. truncatula plants grown in different conditions. Wild-type A17 (black bars), Mtnip-1 (gray bars), and Mtnip-3 (horizontally striped bars) were grown in liquid buffered nodulation medium with no added NO₃⁻ (A), with 250 μm KNO₃ (B), or with 5 mm KNO₃ (C), with the medium changed every other day. LR lengths were measured after 2 weeks. Data are shown for one biological replicate ± se (n = 5). Replicates gave similar results. Asterisks mark LR lengths from plants grown at 250 μm KNO₃ and 5 mm KNO₃ that are significantly different from the same genotype grown at 0 mm KNO₃, using Student’s t test at P < 0.05.

Figure 3.

Nitrate uptake in X. laevis oocytes expressing MtNIP/LATD. Oocytes were microinjected with MtNIP/LATD mRNA (black bars, +) or water as a negative control (gray bars, −), incubated for 3 d, and then placed for the indicated times in medium containing 250 μm or 5 mm NO₃⁻ at pH 5.5 or 7.4. The oocytes were rinsed, lysed, and assayed for NO₃⁻ content. A and C, Treatment with 250 μm NO₃⁻. B and D, Treatment with 5 mm NO₃⁻. A and B, pH = 5.5. C and D, pH = 7.4. Data are shown for one biological replicate ± sd (n = 3–5 batches of 4–6 oocytes per batch). Asterisks mark NO₃⁻ uptake significantly different from the negative control, using Student’s t test at P < 0.05. Similar results were obtained in more than five repetitions of the experiment. E, Michaelis-Menten plot of oocyte NO₃⁻ uptake. MtNIP/LATD-injected oocytes (squares) or water-injected oocytes (circles) as control were incubated for 3 h in 50 μm to 10 mm NO₃⁻ in batches of five and assayed for NO₃⁻ uptake. Results for two biological replicates are indicated by the black and gray symbols, with error bars showing sd. All NO₃⁻ uptake was significantly different from the negative control, using Student’s t test at P < 0.05, except for that at 50 μm. F, Hanes-Woolf plot of averaged NO₃⁻ uptake data, in MtNIP/LATD-injected oocytes minus water-injected oocytes, presented in E. These data were used to calculate the K_m of 160 μm.

Figure 4.

Nitrate uptake in X. laevis oocytes expressing MtNIP/LATD or mutant Mtnip/latd mRNAs. Oocytes were microinjected with MtNIP/LATD mRNA, mutant mRNA, or water as a negative control, incubated for 3 d, and then placed for the indicated times in medium containing 5 mm or 250 μm nitrate, at pH 5.5, and assayed for nitrate uptake. A, Treatment with 5 mm nitrate. B, Treatment with 250 μm nitrate. C, Treatment with 5 mm nitrate. Oocytes expressing wild-type (WT) MtNIP/LATD (black bars), Mtnip-1 (dark gray bars), Mtnip-3 (horizontally striped bars), Mtlatd (hatched bars), or water as a negative control (gray bars) are shown. Data are shown for one biological replicate ± sd (n = 3–5 batches of 4–6 oocytes per batch). Asterisks mark nitrate uptake that is significantly different from the negative control, using Student’s t test at P < 0.05. Similar results were obtained in more than three repetitions of the experiment.

Figure 5.

MtNIP/LATD complements the chlorate-insensitivity phenotype of the Arabidopsis chl1-5 mutant. Arabidopsis chl1-5 plants were transformed with a construct containing MtNIP/LATD cDNA under the control of the Arabidopsis EF1α promoter, pAtEF1α-MtNIP/LATD, or a positive control construct containing the Arabidopsis AtNRT1.1 gene under the control of the same promoter, pAtEF1α-AtNRT1.1. The plants were treated with chlorate, a NO₃⁻ analog that can be converted to toxic chlorite after uptake, as described by Tsay et al. (1993). A, Arabidopsis Col-0 plant. B, Arabidopsis chl1-5 plant. C, Arabidopsis chl1-5/pAtEF1α-AtNRT1.1 plant. D, Arabidopsis chl1-5/pAtEF1α-MtNIP/LATD plant. Bars = one-quarter inch. The MtNIP/LATD gene was able to confer chlorate sensitivity on Arabidopsis chl1-5 plants, similar to the AtNRT1.1 gene.

Figure 6.

Arabidopsis NRT1.1 partially complements the M. truncatula nip-1 mutant for its root architecture phenotype. M. truncatula nip-1 and control wild-type composite plants transformed with pAtEF1α-AtNRT1.1 or empty pCAMBIA2301 vector, as a control, were grown in aeroponic chambers, inoculated with S. meliloti containing a constitutive lacZ gene, and grown in a 16/8-h light/dark cycle at 22°C. At 15 dpi, root architecture characteristics were evaluated. A to D, Appearance of the roots. Bars = 10 mm. A, A17, empty vector. B, A17, pAtEF1α-AtNRT1.1. C, Mtnip-1, empty vector. D, Mtnip-1, pAtEF1α-AtNRT1.1. E to G, Quantitation of primary root length (E), LR number (F), and LR length (G). Averaged values with sd are plotted (n = 5). Asterisks mark root attributes that are significantly different from the negative control, using Student’s t test at P < 0.01. Black bars, A17, empty vector; vertically striped bars, A17, pAtEF1α-AtNRT1.1; dark gray bars, Mtnip-1, empty vector; light gray bars, Mtnip-1, pAtEF1α-AtNRT1.1.

Figure 7.

Arabidopsis NRT1.1 does not complement the M. truncatula nip-1 nodule phenotype. M. truncatula nip-1 and control wild-type composite plants transformed with pAtEF1α-AtNRT1.1, empty vector pCAMBIA2301 as a negative control, or pAtEF1α-MtNIP/LATD were grown as in Figure 5 with S. meliloti containing a constitutive lacZ gene. At 15 dpi, nodule characteristics were evaluated after staining with X-Gal for localization of rhizobia, which stain blue. A, A17 transformed with empty vector. B, A17 transformed with pAtEF1α-AtNRT1.1. C, A17 transformed with pAtEF1α-MtNIP/LATD. D, Mtnip-1 transformed with empty vector. E, Mtnip-1 transformed with pAtEF1α-AtNRT1.1. F, Mtnip-1 transformed with pAtEF1α-MtNIP/LATD. Bars = 200 μm.