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AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis.

Miyaji T, Kuromori T, Takeuchi Y, Yamaji N, Yokosho K, Shimazawa A, Sugimoto E, Omote H, Ma JF, Shinozaki K, Moriyama Y - Nat Commun (2015)

Bottom Line: Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter.The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane.Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.

View Article: PubMed Central - PubMed

Affiliation: Advanced Science Research Center, Okayama University, Okayama 700-8530, Japan.

ABSTRACT
Ascorbate is an antioxidant and coenzyme for various metabolic reactions in vivo. In plant chloroplasts, high ascorbate levels are required to overcome photoinhibition caused by strong light. However, ascorbate is synthesized in the mitochondria and the molecular mechanisms underlying ascorbate transport into chloroplasts are unknown. Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter. In vitro analysis shows that proteoliposomes containing the purified AtPHT4;4 protein exhibit membrane potential- and Cl(-)-dependent ascorbate uptake. The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane. Knockout of AtPHT4;4 results in decreased levels of the reduced form of ascorbate in the leaves and the heat dissipation process of excessive energy during photosynthesis is compromised. Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.

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Phylogenetic tree of the plant SLC17 transporter family and ascorbate transporter of Arabidopsis SLC17 transporter family.(a) Phylogenetic tree of the plant SLC17 transporter family. Arabidopsis SLC17 transporters are indicated in red boxes. (b) Purification of Arabidopsis SLC17 transporter family. (Left) The purified fraction (10 μg) was analysed by 10% SDS-PAGE and visualized by CBB staining. (Right) A duplicate gel was analysed by immunoblotting with anti-6 × His antibody. The positions of marker proteins are indicated on the left. The positions of recombinant proteins are indicated by arrowheads. (c) Na+/Pi uptake by proteoliposomes containing purified AtPHT4 proteins at 2 min. ΔNa+-driven Pi uptake by proteoliposomes was assayed in the presence (closed bars) or absence (open bars) of Na+. (d) Ascorbate uptake by the proteoliposomes at 2 min. Δψ-driven ascorbate uptake by proteoliposomes was assayed in the presence (closed bars) or absence (open bars) of 2 μM valinomycin. Data are means±s.e., n=3–6.
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f1: Phylogenetic tree of the plant SLC17 transporter family and ascorbate transporter of Arabidopsis SLC17 transporter family.(a) Phylogenetic tree of the plant SLC17 transporter family. Arabidopsis SLC17 transporters are indicated in red boxes. (b) Purification of Arabidopsis SLC17 transporter family. (Left) The purified fraction (10 μg) was analysed by 10% SDS-PAGE and visualized by CBB staining. (Right) A duplicate gel was analysed by immunoblotting with anti-6 × His antibody. The positions of marker proteins are indicated on the left. The positions of recombinant proteins are indicated by arrowheads. (c) Na+/Pi uptake by proteoliposomes containing purified AtPHT4 proteins at 2 min. ΔNa+-driven Pi uptake by proteoliposomes was assayed in the presence (closed bars) or absence (open bars) of Na+. (d) Ascorbate uptake by the proteoliposomes at 2 min. Δψ-driven ascorbate uptake by proteoliposomes was assayed in the presence (closed bars) or absence (open bars) of 2 μM valinomycin. Data are means±s.e., n=3–6.

Mentions: The PHT4 family can be classified into four groups according to amino-acid sequence homology (Fig. 1a). To identify the ascorbate transporter from the PHT4 family, we selected one gene from each subgroup of the Arabidopsis PHT4 family (subgroup 1: AtPHT4;3, subgroup 2: AtPHT4;5, subgroup 3: AtPHT4;6, and subgroup 4: AtPHT4;4), and their cDNAs were cloned into Escherichia coli expression vectors with a His-tag and soluble α-helix protein (β) coupled to both ends16. Each transporter was overexpressed in E. coli, solubilized and purified using Ni-NTA affinity column chromatography. The purified proteins were then electrophoresed and stained with Coomassie Brilliant Blue (Fig. 1b left), and their immunological properties were confirmed by immunoblotting with anti-6 × His antibodies (Fig. 1b right). The final fractions contained the major protein bands of the expected apparent molecular masses and immunological properties (Fig. 1b). These purified proteins were incorporated into proteoliposomes. By analogy to mammalian SLC17 family transporters, we investigated whether the transporters possess Na+-dependent transport activity for inorganic phosphate (Pi). The Na+/Pi transport activity was detected in proteoliposomes containing all of these transporters, supporting the suggestion that all of the purified recombinant transporters were active in nature (Fig. 1c). Using the same batch of proteoliposomes, we employed Δψ (positive-inside) by addition of valinomycin in the presence of K+. The proteoliposomes established an inside-positive Δψ of ~90 mV through K+ diffusion, as reported previously17. Under these conditions, only proteoliposomes containing purified AtPHT4;4 exhibited significant ascorbate uptake activity, while those containing AtPHT4;3 or AtPHT4;6 did not (Fig. 1d). Proteoliposomes containing purified AtPHT4;5 exhibited slightly Δψ-dependent ascorbate uptake activity.


AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis.

Miyaji T, Kuromori T, Takeuchi Y, Yamaji N, Yokosho K, Shimazawa A, Sugimoto E, Omote H, Ma JF, Shinozaki K, Moriyama Y - Nat Commun (2015)

Phylogenetic tree of the plant SLC17 transporter family and ascorbate transporter of Arabidopsis SLC17 transporter family.(a) Phylogenetic tree of the plant SLC17 transporter family. Arabidopsis SLC17 transporters are indicated in red boxes. (b) Purification of Arabidopsis SLC17 transporter family. (Left) The purified fraction (10 μg) was analysed by 10% SDS-PAGE and visualized by CBB staining. (Right) A duplicate gel was analysed by immunoblotting with anti-6 × His antibody. The positions of marker proteins are indicated on the left. The positions of recombinant proteins are indicated by arrowheads. (c) Na+/Pi uptake by proteoliposomes containing purified AtPHT4 proteins at 2 min. ΔNa+-driven Pi uptake by proteoliposomes was assayed in the presence (closed bars) or absence (open bars) of Na+. (d) Ascorbate uptake by the proteoliposomes at 2 min. Δψ-driven ascorbate uptake by proteoliposomes was assayed in the presence (closed bars) or absence (open bars) of 2 μM valinomycin. Data are means±s.e., n=3–6.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4308718&req=5

f1: Phylogenetic tree of the plant SLC17 transporter family and ascorbate transporter of Arabidopsis SLC17 transporter family.(a) Phylogenetic tree of the plant SLC17 transporter family. Arabidopsis SLC17 transporters are indicated in red boxes. (b) Purification of Arabidopsis SLC17 transporter family. (Left) The purified fraction (10 μg) was analysed by 10% SDS-PAGE and visualized by CBB staining. (Right) A duplicate gel was analysed by immunoblotting with anti-6 × His antibody. The positions of marker proteins are indicated on the left. The positions of recombinant proteins are indicated by arrowheads. (c) Na+/Pi uptake by proteoliposomes containing purified AtPHT4 proteins at 2 min. ΔNa+-driven Pi uptake by proteoliposomes was assayed in the presence (closed bars) or absence (open bars) of Na+. (d) Ascorbate uptake by the proteoliposomes at 2 min. Δψ-driven ascorbate uptake by proteoliposomes was assayed in the presence (closed bars) or absence (open bars) of 2 μM valinomycin. Data are means±s.e., n=3–6.
Mentions: The PHT4 family can be classified into four groups according to amino-acid sequence homology (Fig. 1a). To identify the ascorbate transporter from the PHT4 family, we selected one gene from each subgroup of the Arabidopsis PHT4 family (subgroup 1: AtPHT4;3, subgroup 2: AtPHT4;5, subgroup 3: AtPHT4;6, and subgroup 4: AtPHT4;4), and their cDNAs were cloned into Escherichia coli expression vectors with a His-tag and soluble α-helix protein (β) coupled to both ends16. Each transporter was overexpressed in E. coli, solubilized and purified using Ni-NTA affinity column chromatography. The purified proteins were then electrophoresed and stained with Coomassie Brilliant Blue (Fig. 1b left), and their immunological properties were confirmed by immunoblotting with anti-6 × His antibodies (Fig. 1b right). The final fractions contained the major protein bands of the expected apparent molecular masses and immunological properties (Fig. 1b). These purified proteins were incorporated into proteoliposomes. By analogy to mammalian SLC17 family transporters, we investigated whether the transporters possess Na+-dependent transport activity for inorganic phosphate (Pi). The Na+/Pi transport activity was detected in proteoliposomes containing all of these transporters, supporting the suggestion that all of the purified recombinant transporters were active in nature (Fig. 1c). Using the same batch of proteoliposomes, we employed Δψ (positive-inside) by addition of valinomycin in the presence of K+. The proteoliposomes established an inside-positive Δψ of ~90 mV through K+ diffusion, as reported previously17. Under these conditions, only proteoliposomes containing purified AtPHT4;4 exhibited significant ascorbate uptake activity, while those containing AtPHT4;3 or AtPHT4;6 did not (Fig. 1d). Proteoliposomes containing purified AtPHT4;5 exhibited slightly Δψ-dependent ascorbate uptake activity.

Bottom Line: Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter.The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane.Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.

View Article: PubMed Central - PubMed

Affiliation: Advanced Science Research Center, Okayama University, Okayama 700-8530, Japan.

ABSTRACT
Ascorbate is an antioxidant and coenzyme for various metabolic reactions in vivo. In plant chloroplasts, high ascorbate levels are required to overcome photoinhibition caused by strong light. However, ascorbate is synthesized in the mitochondria and the molecular mechanisms underlying ascorbate transport into chloroplasts are unknown. Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter. In vitro analysis shows that proteoliposomes containing the purified AtPHT4;4 protein exhibit membrane potential- and Cl(-)-dependent ascorbate uptake. The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane. Knockout of AtPHT4;4 results in decreased levels of the reduced form of ascorbate in the leaves and the heat dissipation process of excessive energy during photosynthesis is compromised. Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.

Show MeSH
Related in: MedlinePlus