A vacuolar phosphate transporter essential for phosphate homeostasis in Arabidopsis

Abstract

Inorganic phosphate (Pi) is stored in the vacuole, allowing plants to adapt to variable Pi availability in the soil. The transporters that mediate Pi sequestration into vacuole remain unknown, however. Here we report the functional characterization of Vacuolar Phosphate Transporter 1 (VPT1), an SPX domain protein that transports Pi into the vacuole in Arabidopsis. The vpt1 mutant plants were stunted and consistently retained less Pi than wild type plants, especially when grown in medium containing high levels of Pi. In seedlings, VPT1 was expressed primarily in younger tissues under normal conditions, but was strongly induced by high-Pi conditions in older tissues, suggesting that VPT1 functions in Pi storage in young tissues and in detoxification of high Pi in older tissues. As a result, disruption of VPT1 rendered plants hypersensitive to both low-Pi and high-Pi conditions, reducing the adaptability of plants to changing Pi availability. Patch-clamp analysis of isolated vacuoles showed that the Pi influx current was severely reduced in vpt1 compared with wild type plants. When ectopically expressed in Nicotiana benthamiana mesophyll cells, VPT1 mediates vacuolar influx of anions, including Pi, SO4(2-), NO3(-), Cl(-), and malate with Pi as that preferred anion. The VPT1-mediated Pi current amplitude was dependent on cytosolic phosphate concentration. Single-channel analysis showed that the open probability of VPT1 was increased with the increase in transtonoplast potential. We conclude that VPT1 is a transporter responsible for vacuolar Pi storage and is essential for Pi adaptation in Arabidopsis.

Keywords: anion channel; patch clamp; phosphorus nutrition; vacuolar phosphate sequestration.

Fig. 1.

Genetic characterization and phenotypic analysis of vpt1 mutant plants. (A) Scheme of the Arabidopsis VPT1 gene structure and localization of the T-DNA insertion site of SALK_006647. Solid boxes and lines indicate exons and introns, respectively. The position of the T-DNA insertions in vpt1 is indicated by a solid triangle. (B) qRT-PCR analysis of VPT1 and ACTIN2 mRNA levels in wild type (Col-0), water, vpt1 mutant plants (vpt1), and two complementation lines transformed with At1g63010 genomic DNA (COM1 and COM2). (C) Phenotype of wild type (Col-0), vpt1, COM1, and COM2 plants grown in soil for 3 wk. (D) Comparative analysis of the aerial biomass in 3-wk-old seedlings of various genotypes as in C. Three independent experiments were performed. Values are mean ± SD. n = 12 for each genotype. (E and F) Growth phenotype (E) and primary root length (F) of 2-wk-old wild type (Col-0), vpt1, COM1, and COM2 plants grown in hydroponic culture containing 6.5 mM Pi. Three independent experiments were performed. Data are mean ± SD. n = 16 for each genotype. The plants shown in C and E were planted at a light intensity of 150 μmol/m²/s with a long-day cycle (16 h light/8 h dark).

Fig. 2.

Growth phenotype of vpt1 mutants under variable Pi conditions. (A) Growth phenotype of 2-wk-old wild type (Col-0) and vpt1 mutants in hydroponic medium containing 1.3 μM, 13 μM, 130 μM, 1.3 mM, 3.9 mM, or 6.5 mM Pi. (B and C) Quantitative analyses of primary root length (B) and whole-plant biomass (C) of wild type (Col-0) and vpt1 seedlings under the various Pi concentrations as described in A. Sixteen 2-wk-old seedlings of wild type (Col-0) and vpt1 mutants were gathered for root length measurements with Image J software and biomass analysis. Three independent experiments were performed. Data are mean ± SD. (D) The phenotype of wild type (Col-0) and vpt1 plants transplanted from various Pi concentrations to the Pi-deficient condition. The wild type (Col-0) and vpt1 mutants were grown in hydroponic medium containing 1.3 μM, 13 μM, 130 μM, 1.3 mM, 3.9 mM, or 6.5 mM Pi for 14 d (a), and then transferred to medium containing 1.3 μM Pi and grown for another 7 d (b). (E) Anthocyanin content in the leaves of plants described in D. Four independent experiments were performed. Data are mean ± SD.

Fig. 3.

VPT1 contributes to Pi homeostasis in Arabidopsis plants. (A) Two-week-old seedlings of vpt1 mutants contain less Pi than wild type (Col-0) when grown in hydroponic medium with various Pi concentrations. Data are mean ± SD. n = 5. (B) Time course and dose course of Pi accumulation in the wild type and vpt1 mutant. Here 3-wk-old wild type (Col-0) and vpt1 mutants were transferred into the medium containing 0.13 mM, 1.3 mM, or 13 mM Pi, and rosette leaves were collected at the indicated time points for Pi content measurement. Data are mean ± SD. n = 4. (C) Expression pattern of pVPT1::GUS in transformed Arabidopsis plants. (Scale bar: 2 mm.) (D) VPT1 expression levels in leaves of different ages (young leaves: the upper two leaves that newly emerged; mature leaves: the middle leaves; old leaves: the two true leaves close to the base of plants) and roots of 3-wk-old wild type seedlings grown in the hydroponic solution containing 0.13 mM Pi. Data are mean ± SD. n = 4. (E) VPT1 expression levels in leaves and roots of the wild type (Col-0) seedlings after treatment with 1.3 mM Pi. Three-week-old wild type plants (Col-0) cultured in the hydroponic solution containing 0.13 mM Pi were transplanted into the solution containing 1.3 mM Pi, after which rosette leaves and roots were obtained at the indicated time points for mRNA measurement. Data are mean ± SD. n = 4. (F) VPT1 expression levels in leaves of different ages from wild type (Col-0) seedlings after treatment with 1.3 mM Pi for 72 h. Data are mean ± SD. n = 4. Values were normalized to ACTIN2, and the relative expression of VPT1 was calculated as the ratio of VPT1 mRNA level to the lowest level (as 1.0) in the group D–F. (G) Pi distribution in the wild-type plants, showing Pi content in roots and leaves of different ages under normal Pi conditions (0.13 mM). (H) Percentage of Pi increase in the wild type and vpt1 mutant plants after high-Pi treatment. Three-week-old seedlings of wild type (Col-0) and mutant vpt1 plants grown in hydroponic solution containing 0.13 mM Pi were transferred into the solution containing 0.13 mM or 1.3 mM Pi and grown for another 5 d. Leaves of different ages without petioles and roots were collected, and Pi content was measured. Data are mean ± SD. n = 6.

Fig. 4.

Subcellular localization of VPT1-GFP in the vacuole membrane. (A) Confocal microscopy of GFP signals in the epidermal cells from a cotyledon of a 3-d-old transgenic vpt1 seedling expressing 35S:VPT1-GFP. The panels, from left to right, show the GFP signals (green), the plasma membrane fluorescence stained with FM4-64 (red), and an overlay (of green and red) from the same sample. (Scale bar: 10 μm.) (B) VPT1-GFP signal overlaid with a γ-TIP-mCherry signal in the epidermal cells from a cotyledon of a 5-d-old seedling expressing 35S:VPT1-GFP and 35S:γ-TIP-mCherry. The panels, from left to right, show the GFP signals (green), the mCherry signals (red), and an overlay (of green and red) from the same sample. TIP (a tonoplast intrinsic protein) is a tonoplast marker (21). (Inset) The intermembrane space and sag area pressed by nuclei or plastids (21). (C) A vacuole released from a mesophyll protoplast isolated from transgenic plants expressing VPT1-GFP. (Scale bar: 10 μm.)

Fig. 5.

VPT1 contributes to the Pi current amplitudes recorded in isolated Arabidopsis vacuoles. (A) The representative current traces were generated by the vacuoles isolated from the wild type (Col-0) (a), vpt1 mutant (b), or 35S:VPT1-GFP^vpt1 (c) leaves. (B) The current–voltage relationships were deduced from isolated vacuoles under each condition as in A. Data are mean ± SD. n = 16. A series of test voltages from 20 to −160 mV in steps of −20 mV with a prepulse holding potential of 0 mV was applied to record currents in the whole-vacuole mode with a symmetrical phosphate condition (100 mM phosphate in cytoplasm/100 mM phosphate in vacuole lumen).

Fig. 6.

The VPT1-mediated currents in N. benthamiana vacuoles are sensitive to phosphate and membrane potentials. (A and B) VPT1 expression enhances the currents generated from N. benthamiana vacuoles. Shown are typical whole-vacuole current traces (A) and the voltage–current relationship (B) of channel activities recorded from the mesophyll vacuoles isolated from empty vector-transformed and VPT1-GFP–overexpressing N. benthamiana leaves. The isolated vacuoles were bathed in a solution containing 100 mM phosphate. The pipette solution contained 100 mM phosphate. The data in B are mean ± SD. n = 17. (C–E) The whole-vacuole currents generated by VPT1-GFP–overexpressing vacuoles were sensitive to the phosphate concentration in bath solution. (C) The typical whole-vacuole current traces generated by a VPT1-GFP–expressing vacuole bathed in solution containing 1 mM, 10 mM, 50 mM, or 100 mM phosphate. (D) The current–voltage relationship was deduced from recordings of VPT1-GFP–expressing vacuoles as in C. The data are mean ± SD. n = 17. (E) Reversal potentials of phosphate currents at various cytosolic phosphate concentrations. Data points are mean ± SD. The solid line represents the calculated phosphate equilibrium potentials. (F) Anion selectivity reflected by the current ratio of VPT1 overexpression (OE) vs. empty vector control. The channel currents at −160 mV were recorded from VPT1-GFP–expressing vacuoles or empty vector-transformed vacuoles bathed in solution containing 100 mM phosphate, sulfate, nitrate, chloride, or malate. The current ratio of VPT1-GFP–tagged vacuoles vs. empty vector-transformed ones was calculated. Data are mean ± SD. n = 15. (G) The open probability (P_o) of channels in excised outside-out tonoplast patches at different voltages. Representative current traces (Left) and corresponding amplitude histograms (Right) in an excised outside-out patch recorded at membrane potentials of −40, −60, −80, or −100 mV. (Left) The horizontal lines represent the channel open state. (Right) The conductance (γ) was calculated as the current-to-voltage ratio. (H) Current–voltage relationship of recordings from eight excised outside-out patches under various membrane potentials. Data are mean ± SD.