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Tonoplast-localized nitrate uptake transporters involved in vacuolar nitrate efflux and reallocation in Arabidopsis.
He YN
,
Peng JS
,
Cai Y
,
Liu DF
,
Guan Y
,
Yi HY
,
Gong JM
.
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A great proportion of nitrate taken up by plants is stored in vacuoles. Vacuolar nitrate accumulation and release is of great importance to nitrate reallocation and efficient utilization. However, how plants mediate nitrate efflux from vacuoles to cytoplasm is largely unknown. The current study identified NPF5.11, NPF5.12 and NPF5.16 as vacuolar nitrate efflux transporters in Arabidopsis. Histochemical analysis showed that NPF5.11, NPF5.12 and NPF5.16 were expressed preferentially in root pericycle cells and xylem parenchyma cells, and further analysis showed that these proteins were tonoplast-localized. Functional characterization using cRNA-injected Xenopus laevis oocytes showed that NPF5.11, NPF5.12 and NPF5.16 were low-affinity, pH-dependent nitrate uptake transporters. In npf5.11 npf5.12 npf5.16 triple mutant lines, more root-fed 15NO3- was translocated to shoots compared to the wild type control. In the NPF5.12 overexpression lines, proportionally less nitrate was maintained in roots. These data together suggested that NPF5.11, NPF5.12 and NPF5.16 might function to uptake nitrate from vacuoles into cytosol, thus serving as important players to modulate nitrate allocation between roots and shoots.
Figure 1. Subcellular localization of NPF5.11, NPF5.12 and NPF5.16. NPF5.11-EYFP (aâc), NPF5.12-EYFP (dâf), NPF5.16-EYFP (gâi) or EYFP (jâl) was driven by the cauliflower mosaic virus 35âS promoter and transiently expressed in Arabidopsis mesophyll protoplasts. Overlap images of EYFP (green) and chlorophyll (red) fluorescence (a,d,g,j), EYFP fluorescence (b,e,h,k), and bright-field (c,f,i,l) images are shown. Barsâ=â20âμm.
Figure 2. Functional characterization of NPF5.11, NPF5.12 and NPF5.16 in oocytes. (aâe) Currents elicited in oocytes injected with H2O (a), CHL1 cRNA (b), NPF5.11 cRNA (c), NPF5.12 cRNA (d) or NPF5.16 cRNA (e). Oocytes were voltage clamped at â60 mV and representative inward currents elicited by 10âmM NO3
â at pH 5.5 were recorded. (fâh) Nitrate uptake activity in oocytes injected with H2O, NPF5.11 cRNA, NPF5.12 cRNA, NPF5.16 cRNA, NRT1.8 cRNA or CHL1 cRNA. Oocytes were incubated with 10âmM 15NO3
â at pH 5.5 (f), 0.25âmM 15NO3
â at pH 5.5 (g) or 10âmM 15NO3
â at pH 7.4 (h) for 12âh. Values are meansâ±âSD (nâ=â8â12). Asterisks indicate difference at Pâ<â0.01 (**) compared with the H2O-injected oocytes by Studentâs t-test. (iâk) Uptake kinetics of NPF5.11 (i), NPF5.12 (j) and NPF5.16 (k). Oocytes injected with NPF5.11 cRNA (i), NPF5.12 cRNA (j) or NPF5.16 cRNA (k) were incubated with indicated concentrations of 15NO3
â at pH 5.5 for 1.5âh, and the 15N contents were determined. Values are meansâ±âSD (nâ=â6â12). The Km was calculated by fitting to the Michaelis-Menten equation using a nonlinear least squares method in the SigmaPlot program. The Km was 2.57âmM, 4.84âmM, or 2.91âmM for NPF5.11, NPF5.12 or NPF5.16, respectively.
Figure 3.
NPF5.11, NPF5.12 and NPF5.16 are preferentially expressed in vascular tissues. Histochemical localization of GUS activity in NPF5.11
pro::GUS transgenic plants (aâd), NPF5.12
pro::GUS transgenic plants (eâh) and NPF5.16
pro::GUS transgenic plants (iâl). The expression patterns of NPF5.11, NPF5.12 and NPF5.16 were determined in whole-mount seedlings (a,e,i), seedling roots (b,f,j) or cross-sectioned seedling roots (c,d,g,h,k,l). (m,n,o) Transcript expression of NPF5.11 (m), NPF5.12 (n) and NPF5.16 (o) in 28 d old plants. 1â8 indicated leaf positions arranged according to leaf ages (old to young); R, root; F, flower; S, stem. Data were normalized to that of SAND. Values are meansâ±âSD, nâ=â3. Barsâ=â10âμm.
Figure 4. Root-to-shoot nitrate transport enhanced in the triple mutant plants npf5.11 npf5.12 npf5.16. Plants were grown in hydroponics for 28 days and treated with 2.25âmM K15NO3 for 30âmin. 15N contents in shoots and roots were analyzed. 15N concentration ratio between shoots and roots (S/R ratio, a) and root uptake activity (b) were determined. Values are meansâ±âSD, nâ=â3. Asterisks indicate difference between wild type and triple mutant lines at Pâ<â0.05 (*) by Studentâs t-test.
Figure 5. Decreased nitrate accumulation in roots of NPF5.12 overexpression lines. (a,b) Identification of NPF5.12 overexpression lines by RT-PCR (a) and quantitative PCR analysis (b). (c) 24 days old hydroponically grown plants were subjected to nitrogen-starvation for 30âh, then roots were sampled and nitrate contents were determined by HPLC. 12OE1 and 12OE2 were two independent NPF5.12 overexpression lines. Values are meansâ±âSD, nâ=â5â7. Asterisks indicate difference between wild type and overexpression lines at Pâ<â0.05 (*) and Pâ<â0.01 (**) by Studentâs t-test.
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