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Figure 1. OsNPF2.2 is a low-affinity nitrate transporter.(a) Nitrate uptake activity of OsNPF2.2-injected oocytes at pH 5.5. High-affinity or low-affinity nitrate uptake activity was examined by incubating oocytes with 0.25 or 10 mM K 15NO3 for 1.5 h and 3 h and then measuring the levels of 15N in the oocytes (n = 8–13 oocytes for the water- and OsNPF2.2-injected oocytes, respectively). (b) The pH dependence of the nitrate uptake activity of OsNPF2.2. The OsNPF2.2-injected oocytes were incubated with 10 mM K 15NO3 buffered at pH 5.5 or 7.4 for 2 h, and then the levels of 15N in the oocytes were measured (n = 10–13 oocytes for the water- and OsNPF2.2-injected oocytes, respectively). (c) Uptake kinetics of OsNPF2.2. OsNPF2.2-injected oocytes were incubated with different concentrations of K 15NO3 at pH 5.5 for 1.5 h, and then their 15N contents were determined. The Km, calculated from one experiment by fitting the data to the Michaelis–Menten equation using a nonlinear least squares methods in the ORIGIN 5.0 program (Microcal Software; GE Healthcare), was ~16.6 ± 12.9 mM. The values are mean ± SE (n = 4–9 oocytes for each concentration). *P < 0.05 when compared to the water-injected control; **P < 0.01 when compared to the water-injected control.
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Figure 2. OsNPF2.2 is located in the rice plasma membrane.(a–e) Fluorescence of OsNPF2.2-GFP coexpressed with the plasma membrane marker pm-rk-mCherry in transiently transformed rice protoplasts (bar = 5 μm). Transformed rice protoplasts were first identified by their green fluorescent protein (GFP) fluorescence from OsNPF2.2-GFP (a); then, these cells were checked for mCherry fluorescence (b) and finally for chlorophyll autofluorescence (c). The corresponding bright-field image is shown in (e). (d) Merged images from (a), (b), and (c). (f) GFP fluorescence from free GFP expressed under the 35S promoter as a control (bar = 5 μm).
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Figure 3. OsNPF2.2 expression in leaf blades is nitrate inducible.(a) Time-course analysis of OsNPF2.2 expression following NO3− and NH4+ induction by quantitative real-time–PCR. Rice seedlings were grown on 1/2 Murashige and Skoog solid medium for 10 d; next, seedlings were washed and deprived of NO3− for 1 d and then subcultured in International Rice Research Institute (IRRI) solution with 10 mM KNO3 or 10 mM NH4Cl as the N source, respectively, or mock-treated with KCl. The plants were collected for RNA extraction at the indicated times. Relative expression was normalized to the expression level of eEF-1a. The values are mean ± SE for triplicate samples, with each replicate containing seven seedlings. (b–g) β-Glucuronidase activity analysis in leaf blades of the transgenic rice plants harboring the uidA gene driven by the cauliflower mosaic virus 35S promoter (b–d) or the 2.5 kb OsNPF2.2 promoter (e–g) after nitrate induction. The transgenic plants were first grown in 1.4 mM NH4NO3 solution (b, e) and then deprived of NO3− for 1 d (c, f); finally, the plants were transferred back into IRRI solution with 10 mM KNO3 as the N source (d, g) for 2 h. Bar = 0.25 cm.
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Figure 4. OsNPF2.2 is strongly expressed in parenchyma cells around xylem vessels.(a–f) OsNPF2.2 expression patterns in various organs by β-glucuronidase staining analysis, bar = 1000 μm. The transgenic plants harboring the uidA gene driven by the 2.5 kb OsNPF2.2 promoter were grown in a paddy with normal nitrogen fertilizer. GUS activity was detectable in the roots and leaves of germinating seeds (a), in old roots (b), in filling seeds (c), in young inflorescences and stems (d), in flag leaves (e), and in panicles (f). (g–l) Longitudinal (g, j) and transverse (h, i, k, and l) section analysis of GUS-stained plants transgenic for pOsNPF2.2-uidA and grown under normal nitrate conditions. GUS staining revealed that OsNPF2.2 is strongly expressed in vascular parenchyma cells in leaves (g, h), stems (i, j, k), and roots (l). X, xylem; P, phloem; PC, parenchyma cell. Bar = 5 µm in (g), 100 µm in (h), 50 µm in (i, j and l), and 10 µm in (k).
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Figure 5. Disruption of OsNPF2.2 hinders plant growth and seed filling.(a) Schematic diagram of OsNPF2.2 and insertion positions of transfer DNA (T-DNA) in the osnpf2.2-1 and osnpf2.2-2 mutants. The boxes indicate two exons, and the connecting black line represents the intron. Black boxes indicate the open reading frame, and the gray boxes indicate the 5′- and 3′-untranslated regions. The T-DNA is indicated by the triangle. The abbreviations LB and RB indicate the left and right borders, respectively, of the T-DNA. Arrows with letters show the locations of the primers used to amplify the flanking sequence tags and screen the homozygous mutants. (b) Semi-quantitative–PCR analyses of OsNPF2.2 expression in osnpf2.2 mutants. Total RNA was extracted from the leaves of homozygous osnpf2.2-1 and osnpf2.2-2 plants and their parental (WT) varieties. The gene eEF-1a was amplified as an internal control. (c–f) Seed germinating comparison between the osnpf2.2 mutants and WT plants. The surface-sterilized seeds were germinated on 1/2 Murashige and Skoog solid medium; bar = 2 cm. (g–n) Main phenotypes of the osnpf2.2 mutants grown in a paddy with normal nitrogen fertilizer. (g, j) Dwarf plants; bar = 5 cm; (h, k) Short panicles; bar = 1.5 cm; (i, l) Reduction in filling for seeds from the osnpf2.2 mutants as compared to WT seeds; bar = 2 mm. (m) Plant height statistics of the osnpf2.2 mutants and WT plants at the mature stage; **P < 0.01 when compared to WT plants. (n) Comparison analysis of the fully filled seeds (FFS), the unfilled seeds with expanded ovaries (UFS), and the empty spikelets (ES) per plant between the osnpf2.2 mutants and WT plants. The values are mean ± SE from 60 plants at two seasons.
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Figure 6. Disruption of OsNPF2.2 affects nitrate transport from root to shoot.(a–c) Nitrate concentrations in the xylem exudate (a), total nitrate concentration in the roots and shoots (b), and the shoot:root nitrate ratio (c) of the wild-type (WT) plants and the osnpf2.2 mutants. The plants were grown in International Rice Research Institute (IRRI) solution containing 1.4 mM NH4NO3 for 8 weeks for the steady-state nitrate treatment. To collect the xylem sap, the plants were cut 4 cm above the roots, and the roots were immediately transferred to 10 mM nitrate IRRI solution for 2 h. Xylem sap was collected over the 2 h period. To measure the total nitrate concentration in the roots and shoots, the samples were directly harvested after cultivation. The values represent mean ± SE for triplicate samples; *P < 0.05 when compared to WT plants; **P < 0.01 when compared to WT plants.
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Figure 7. Disruption of OsNPF2.2 affects nitrate unloading from the xylem.(a, b) Nitrate concentrations in the xylem exudates (a), and total nitrate concentration in the roots and shoots (b) of wild-type (WT) plants and the osnpf2.2 mutants under 2 h short-term nitrate feeding. The plants were grown in International Rice Research Institute (IRRI) solution containing 1.4 mM NH4NO3 for 8 weeks; next, they were transferred to IRRI solution containing 1.4 mM NH4SO4 for NO3-starvation for 1 week and then transferred back to IRRI solution with 10 mM KNO3 for 2 h for xylem sap collection (a) or for measuring the total nitrate concentration in the roots and shoots (b). The values are mean ± SE for triplicate samples; *P < 0.05 as compared to WT plants; **P < 0.01 as compared to WT plants.
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Figure 8. Disturbed vasculatures in the osnpf2.2-1 mutants, as shown by transmission electron microscopy and semisection analysis.(a, b) Anther cross section from a wild-type (WT) plant (a) and an osnpf2.2-1 mutant (b); the sieve tubes (St) and vessels (Ve) in the osnpf2.2-1 mutant are disrupted; bar = 2 µm. (c, d) Ultrastructure of leaf blades from a WT plant (c) and an osnpf2.2-1 mutant (d); a few vessels in the venation in the mutant plant showed delayed dying as compared with those in the WT plant; bar = 5 µm. (e, f) Semisection of primary branches from a WT plant (e) and an osnpf2.2-1 mutant (f), bar = 50 µm. There was no obvious vascular sheath around the vascular bundle (Vb) in the osnpf2.2-1 mutant, and its sieve tubes and vessels were irregularly distributed in the vascular bundles.
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