Characterization of the Arabidopsis nitrate transporter NRT1.6 reveals a role of nitrate in early embryo development

Abstract

This study of the Arabidopsis thaliana nitrate transporter NRT1.6 indicated that nitrate is important for early embryo development. Functional analysis of cDNA-injected Xenopus laevis oocytes showed that NRT1.6 is a low-affinity nitrate transporter and does not transport dipeptides. RT-PCR, in situ hybridization, and beta-glucuronidase reporter gene analysis showed that expression of NRT1.6 is only detectable in reproductive tissue (the vascular tissue of the silique and funiculus) and that expression increases immediately after pollination, suggesting that NRT1.6 is involved in delivering nitrate from maternal tissue to the developing embryo. In nrt1.6 mutants, the amount of nitrate accumulated in mature seeds was reduced and the seed abortion rate increased. In the mutants, abnormalities (i.e., excessive cell division and loss of turgidity), were found mainly in the suspensor cells at the one- or two-cell stages of embryo development. The phenotype of the nrt1.6 mutants revealed a novel role of nitrate in early embryo development. Interestingly, the seed abortion rate of the mutant was reduced when grown under N-deficient conditions, suggesting that nitrate requirements in early embryo development can be modulated in response to external nitrogen changes.

Figure 1.

Nitrate Uptake Activity of NRT1.6 cRNA-Injected Oocytes. (A) Substrate specificity of NRT1.6. Oocytes were voltage-clamped at −60 mV and stepped to a test voltage between 0 mV and −140 mV for 300 ms, in −20-mV increments. The currents (I) shown here are the difference between the currents flowing at +300 ms in the presence and in the absence of 10 mM nitrate or dipeptides at pH 5.5. Similar results were obtained from three other oocytes from different frogs. (B) and (C) Nitrate uptake activity of NRT1.6- or CHL1-injected oocytes. High-affinity (B) or low-affinity (C) nitrate uptake activity of NRT1.6- or CHL1 cRNA-injected oocytes determined using 0.25 or 10 mM ¹⁵N-labeled nitrate as described in Methods. Values are means ± sd (n = 14, 15, and 13 for the high-affinity assay of water-, CHL1-, and NRT1.6-injected oocytes, respectively, and n = 16, 11, and 12 for the low-affinity assay of water-, CHL1-, and NRT1.6-injected oocytes, respectively). Similar results were obtained using two other frogs. * Significant at P < 0.001 compared with the water-injected control. (D) Kinetics of nitrate-elicited currents in a single NRT1.6 cRNA-injected oocyte. The oocyte was voltage clamped at −60 mV. The inward current elicited by different concentration of nitrate at pH 5.5 is plotted as a function of the external nitrate concentration. The inset shows a Lineweaver-Burk plot. The example shown is representative of the results from nine oocytes from three different frogs (three oocytes/frog).

Figure 2.

NRT1.6 Expression in the Siliques and Seeds of Arabidopsis. (A) RT-PCR products of NRT1.6 (30 cycle-amplified) and UBQ10 (20 cycle-amplified). RNA was isolated from different plant tissues. Shoot (S) and root (R) tissues of plants grown for 14 d in NH₄NO₃. Inflorescence stem (St), cauline leaves (Cl), flower and flower buds (Fl), and silique (Sl) from 4-week-old plants grown in soil. Et, 5-d etiolated seedling. (B) Quantitative PCR. RNA isolated from siliques in different stages of development was used for quantitative PCR. Stage A, closed flower; stage B, opening flower; stage C, siliques ∼3 mm long; and stage D, siliques 4 to 5 mm long. Plants were grown under normal (circles) or nitrogen-limited conditions (diamonds). The relative expression level is the expression normalized to the expression of UBQ10. Values are means ± sd of three biological repeats. * Significant difference at P < 0.05 compared with the level in stage B. # Significant difference (P < 0.05) between normal and nitrogen-limited conditions.

Figure 3.

NRT1.6 Is Expressed in Funiculus, and the Protein Is Located in the Plasma Membrane. (A) and (B) In situ hybridization of the antisense NRT1.6 probe to a section of Arabidopsis silique. (C) and (D) In situ hybridization of the sense NRT1.6 probe to a section of Arabidopsis silique. Green arrows indicate the signals in funiculus (F). White triangles indicate the nonspecific signals in embryo sac wall and the epidermis of silique, which were found in sections hybridized with either antisense or sense probe. (E) and (F) Localization of GUS activity (blue) in the mutant line nrt1.6-1. (E) shows a longitudinal section of the silique. The area labeled with white box was enlarged and is shown in (F). The seed coat appears orange in this section. F, funiculus; VB, vascular bundle. (G) Subcellular localization of NRT1.6-GFP fusion protein in Arabidopsis protoplasts. Confocal laser scanning microscope pictures (left) and corresponding bright-field images (right) of Arabidopsis protoplasts transiently expressing NRT1.6-GFP (top panels) or GFP alone (bottom panels).

Figure 4.

Seed Abortion Rate Is Increased in nrt1.6 Mutants. (A) T-DNA insertion sites in the NRT1.6 coding region in the three different mutant lines used in this study. Mutants nrt1.6-1 and nrt1.6-2 had a single T-DNA inserted in the first or third introns, respectively, while two copies of T-DNA were inserted in the second and fourth exons of nrt1.6-3. The black boxes represent exons and the arrowheads indicate T-DNA left border primers. F, forward primer used for RT-PCR; R, reverse primer used for RT-PCR. (B) NRT1.6 expression in mutant lines. Total RNA from siliques of the wild-type (1) or the nrt1.6-2 (2) or nrt1.6-3 (3) mutant was used for RT-PCR analysis. Control reactions (UBQ10) were amplified for 18 cycles, while NRT1.6 samples were amplified for 30 cycles. (C) Silique from an nrt1.6-2 plant containing normal green seeds (black arrowheads) and aborted seeds (white arrowheads). (D) Quantification of seed abortion in the nrt1.6 mutants and the wild type. Plants were grown under normal (Regular) or nitrogen-deficient (Starved) conditions and the percentage of aborted seeds calculated. Values are means ± se of 18 to 30 siliques from three independent plants. The possible seeds (normal seeds plus aborted seed) per siliques were ∼45 to 55, which was not significantly different between the wild type and mutant. *, P < 0.05 compared with the wild type.

Figure 5.

Embryo Abnormalities in the nrt1.6 Mutants. (A) Percentage of abnormal embryos in siliques from the wild type and nrt1.6 mutants grown in the same pot. The siliques with embryos at the one- to eight-cell stages of development were examined. The frequency of abnormal embryos in the nrt1.6-2 mutant and wild type was counted in 16 siliques collected from four plants, and the frequency of abnormal embryos in the nrt1.6-3 mutant and the wild type was counted in eight siliques collected from three plants. Values are means ± se. Compared with the wild type, the percentage of abnormal embryos in nrt1.6 mutants was significantly higher (P < 0.05 based on Student's t test). (B) to (G) Embryos from the wild type ([B] and [C]) and nrt1.6-2 mutant ([D] to [G]) were compared. Photographs of one-cell embryos ([B], [D], and [F]), and four-cell embryos ([C] and [F]) are shown. Bars= 10 μm.

Figure 6.

Seed Nitrate Content Is Reduced in the nrt1.6-2 Mutant. The mother plants were grown under normal conditions or nitrogen-deficient conditions. Each point represents the nitrate content in seeds from a single plant. The error bar represents the sd of three sample measurements on the same batch of seeds. Five hundred seeds were used for each measurement. * P < 0.05 compared with the wild-type values.