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Front Plant Sci
2016 Jan 01;7:2013. doi: 10.3389/fpls.2016.02013.
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AtNPF2.5 Modulates Chloride (Cl-) Efflux from Roots of Arabidopsis thaliana.
Li B
,
Qiu J
,
Jayakannan M
,
Xu B
,
Li Y
,
Mayo GM
,
Tester M
,
Gilliham M
,
Roy SJ
.
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The accumulation of high concentrations of chloride (Cl-) in leaves can adversely affect plant growth. When comparing different varieties of the same Cl- sensitive plant species those that exclude relatively more Cl- from their shoots tend to perform better under saline conditions; however, the molecular mechanisms involved in maintaining low shoot Cl- remain largely undefined. Recently, it was shown that the NRT1/PTR Family 2.4 protein (NPF2.4) loads Cl- into the root xylem, which affects the accumulation of Cl- in Arabidopsis shoots. Here we characterize NPF2.5, which is the closest homolog to NPF2.4 sharing 83.2% identity at the amino acid level. NPF2.5 is predominantly expressed in root cortical cells and its transcription is induced by salt. Functional characterisation of NPF2.5 via its heterologous expression in yeast (Saccharomyces cerevisiae) and Xenopus laevis oocytes indicated that NPF2.5 is likely to encode a Cl- permeable transporter. Arabidopsis npf2.5 T-DNA knockout mutant plants exhibited a significantly lower Cl- efflux from roots, and a greater Cl- accumulation in shoots compared to salt-treated Col-0 wild-type plants. At the same time, [Formula: see text] content in the shoot remained unaffected. Accumulation of Cl- in the shoot increased following (1) amiRNA-induced knockdown of NPF2.5 transcript abundance in the root, and (2) constitutive over-expression of NPF2.5. We suggest that both these findings are consistent with a role for NPF2.5 in modulating Cl- transport. Based on these results, we propose that NPF2.5 functions as a pathway for Cl- efflux from the root, contributing to exclusion of Cl- from the shoot of Arabidopsis.
Figure 1. In silico analysis of NPF2.5. (A) Protein sequence alignment of NPF2.5 to NAXT family members with known functions (NPF2.4, NPF2.3, and NPF2.7/NAXT1). Sequence alignment was performed using ClustalW2 with a gap open of 10 and a gap extension of 0.1. Black and shaded regions represent identical residues and conservative substitutions respectively. (B) NPF2.5 protein was predicted to have 12 trans-membrane domains and a hydrophobic loop. Trans-membrane domain prediction of NPF2.5 was performed using TMHMM 2.0 with default settings.
Figure 2. NPF2.5 is predominantly expressed in the root and is salt-inducible. (A)
NPF2.5 transcript abundance in the root and the shoot. Col-0 wild type Arabidopsis plants were grown hydroponically for 4 weeks before whole root and shoot were harvested separately for quantitative RT-PCR. (B) Transcript abundance of NPF2.5 was normalized relative to control group (2 mM NaCl) to show expression of NPF2.5 in response to salt stress. Four-week old Col-0 Arabidopsis plants were grown hydroponically before application of 2 mM, 50 mM or 100 mM NaCl for 72 h. Whole root was harvested for quantitative RT-PCR. Results are presented as mean ± SEM (n = 4); significance is indicated by the asterisks (one way ANOVA and Tukey test, **P ≤ 0.01, ***P ≤ 0.001).
Figure 3. NPF2.5 is preferentially expressed in the cortical cells in the root. T2
proNPF2.5:uidA plants were grown on ½ MS media for 2 weeks. Plants were harvested and stained for GUS activity for 1 h. (A) No GUS activity in a wild type Col-0 plant. (B) GUS activity predominantly in the root of proNPF2.5:uidA plants. (C) GUS activity in the root of proNPF2.5:uidA plants. (D) GUS activity in the root-to-shoot junction of proNPF2.5:uidA plants. (E) Root cross-section of non-transformed Col-0 plant. (F) Root cross-section of proNPF2.5:uidA plant. Scale bars = 4 mm in (A,B), Scale bars = 0.3 mm in (C,D), Scale bars = 0.05 mm in (E,F).
Figure 4. NPF2.5 is localized to the plasma membrane in Arabidopsis. (A) Green fluorescence detected in root cells of 2-week old GFP::NPF2.5 plants. (B) Transmitted light image of root cells of 2-week old GFP::NPF2.5 plants. (C–E) Confocal image of plasmolysis performed on root cells of 2-week GFP::NPF2.5 plants. (F) Yellow florescence of YFP::NPF2.5 fusion protein observed in an Arabidopsis mesophyll protoplast. (G) Cyan fluorescence of plasma membrane marker eCFP::ROP11 observed in the protoplast. (H) Transmitted light image of the protoplast. White arrow points to the separation of the tonoplast from the plasma membrane. (I) Merged image showing co-localisation of yellow fluorescence of YFP::NPF2.5 fusion protein and cyan fluorescence of plasma membrane marker. Scale bars = 50 μm in (A,B); Scale bars = 10 μm in (C–E); Scale bars = 20 μm in (F–I).
Figure 5. Growth of NPF2.5 transformed yeast was inhibited by high levels of external Br−. Yeast was transformed with pYES2-DEST52/NPF2.5, or empty vector. Five 10 × serial dilutions of liquid culture were spotted and incubated at 28°C for 2 days on SD medium (-uracil) containing 2% (w/v) galactose, 1.67% (w/v) agar and salts as indicated. (A) 500 mM KCl; (B) 400 mM NaCl; (C) 400 mM KBr; (D) 300 mM NaBr.
Figure 6. NPF2.5 encodes a protein that is able to facilitate Cl− efflux. MIFE and TEVC were performed to study putative Cl− transporter NPF2.5 both in planta and in heterologous system. (A) Inward currents of oocytes expressing NPF2.5 when membrane potential was clamped at −120 mV. Oocytes were incubated in a solution of ND50 (50 mM Cl−). Results are presented as mean ± SEM (n = 7); significance is indicated by asterisks (Two-tailed t-test, **P ≤ 0.005) (derived from Supplementary Figure 2A). (B) MIFE measurement of X. laevis oocytes for Cl− efflux. Oocytes were incubated in ND50 solution. Flux measurements were made for 15 min. Net fluxes of 5 or 7 individual oocytes were averaged for each treatment. Results are presented as mean ± SEM (n = 7 for NPF2.5 injected ones; n = 5 for controls; Two-tailed t-test, ***P ≤ 0.001). (C) MIFE measurement of plants for Cl− efflux. 2-week old npf2.5 and Col-0 plants were transferred to 48 mM NaCl solution for 30 min before a 15-min long measurement. Net fluxes of 6 or 9 individual seedlings were averaged for each genotype. Results are presented as mean ± SEM (n = 9 for npf2.5; n = 6 for Col-0; Two-tailed t-test, ***P ≤ 0.001). (D)
NPF2.5 knockout mutant plants accumulated higher Cl− in the shoot. Four-week old T4
npf2.5 and Col-0 plants were grown hydroponically before being treated with 75 mM NaCl for 5 days. Cl− accumulation in the shoot of npf2.5 after salt treatment is presented as mean ± SEM (n = 9 for npf2.5; n = 4 for Col-0); significance is indicated by asterisks (Two-tailed t-test, *P ≤ 0.05).
Figure 7. NPF2.5 amiRNA lines treated with 75 mM NaCl. Four-week old hydroponically grown T2
NPF2.5 amiRNA lines were treated with 75 mM NaCl for 5 days before harvest. (A)
NPF2.5 transcript abundance detected in roots of knockdown lines and null segregate controls. (B) Shoot Cl− accumulation in knockdown lines and null segregate controls. (C) Shoot NO3− accumulation in knockdown lines and null segregate controls. (D) Shoot Na+ accumulation in knockdown lines and null segregate controls. (E) Shoot K+ accumulation in knockdown lines and null segregate controls. Results are presented as mean ± SEM (n = 4); significance is indicated by asterisks (one way ANOVA and Tukey test, *P ≤ 0.05; **P ≤ 0.01).
Figure 8. NPF2.5 over-expression lines treated with low/high salt. Four-week old hydroponically grown T3
NPF2.5 over-expression lines were treated with 2 mM (low salt) or 75 mM NaCl (high salt) for 5 days before harvest. (A)
NPF2.5 transcript abundance detected in roots of mutant lines and null segregant control plants. Columns with different letters indicate statistically significant difference (P ≤ 0.05). (B) Shoot Cl− accumulation in over-expressing lines and null segregate controls after low salt treatment. (C) Shoot NO3− accumulation in over-expressing lines and null segregate controls after low salt treatment. (D) Shoot Cl− accumulation in over-expressing lines and null segregate controls after high salt treatment. (E) Shoot NO3− accumulation in over-expressing lines and null segregate controls after high salt treatment. Results are presented as mean ± SEM (n = 4); significance is indicated by asterisks (one way ANOVA and Tukey test, *P ≤ 0.05).
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