Proceedings of the National Academy of Sciences Acta Medica Okayama 0027-8424 123 17 2026 A magnesium efflux transporter required for seed development and eating quality in rice e2536813123 EN Sheng Huang Institute of Plant Science and Resources, Okayama University Kiyosumi Hori National Institute of Crop Science, National Agriculture Research Organization Naoki Yamaji Institute of Plant Science and Resources, Okayama University Yuma Yoshioka Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Min Ning Institute of Plant Science and Resources, Okayama University Yu Nagaya Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takaaki Miyaji Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Namiki Mitani-Ueno Institute of Plant Science and Resources, Okayama University Shin-ichiro Inoue Department of Regulatory Biology, Saitama University June-Sik Kim Institute of Plant Science and Resources, Okayama University Miho Kashino Institute of Plant Science and Resources, Okayama University Jian Feng Ma Institute of Plant Science and Resources, Okayama University As a staple food for half the world’s population, rice is an important dietary source of magnesium (Mg), an essential mineral for human health. Enhanced Mg accumulation in rice grains has also been linked to eating quality. However, the mechanisms underlying Mg transport to the grains remains poorly understood. Here, we report that OsMGR2, a member belonging to Magnesium Release (MGR) family, is required for Mg accumulation in rice grains. OsMGR2 encodes a plasma membrane-localized transporter that mediates Mg efflux. OsMGR2 is constitutively and highly expressed in the stele tissues of roots, the phloem region of both enlarged and diffused vascular bundles in nodes, and the ovular vascular trace of caryopses. Knockout of this gene results in decreased root-to-shoot translocation and altered distribution of Mg to different organs; less Mg is allocated to the second newest leaf with high Mg requirement for active photosynthesis. The osmgr2 mutants exhibit decreased Mg accumulation in the grain, which are smaller, lighter, and shriveled, but show increased accumulation in the husk. The eating quality of the mutant grains is significantly decreased compared with the wild-type rice. These results indicate that OsMGR2 plays multiple roles within the rice; facilitating the root-to-shoot Mg translocation, mediating phloem-to-xylem Mg transfer at nodes for preferential distribution to the most active leaf, and exporting Mg from maternal vascular tissues of the caryopsis to the grains, processes essential for grain development and eating quality in rice. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0960-7412 120 1 2024 OsHAK4 functions in retrieving sodium from the phloem at the reproductive stage of rice 76 90 EN Jing Che Institute of Plant Science and Resources, Okayama University Naoki Yamaji Institute of Plant Science and Resources, Okayama University Shao Fei Wang Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences Yue Xia Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences Shun Ying Yang Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences Yan Hua Su Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences Ren Fang Shen Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences Jian Feng Ma Institute of Plant Science and Resources, Okayama University Soil salinity significantly limits rice productivity, but it is poorly understood how excess sodium (Na+) is delivered to the grains at the reproductive stage. Here, we functionally characterized OsHAK4, a member of the clade IV HAK/KUP/KT transporter subfamily in rice. OsHAK4 was localized to the plasma membrane and exhibited influx transport activity for Na+, but not for K+. Analysis of organ- and growth stage-dependent expression patterns showed that very low expression levels of OsHAK4 were detected at the vegetative growth stage, but its high expression in uppermost node I, peduncle, and rachis was found at the reproductive stage. Immunostaining indicated OsHAK4 localization in the phloem region of node I, peduncle, and rachis. Knockout of OsHAK4 did not affect the growth and Na+ accumulation at the vegetative stage. However, at the reproductive stage, the hak4 mutants accumulated higher Na+ in the peduncle, rachis, husk, and brown rice compared to the wild-type rice. Element imaging revealed higher Na+ accumulation at the phloem region of the peduncle in the mutants. These results indicate that OsHAK4 plays a crucial role in retrieving Na+ from the phloem in the upper nodes, peduncle, and rachis, thereby preventing Na+ distribution to the grains at the reproductive stage of rice. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0031-9317 177 4 2025 Comparative Transcriptome Reveals ART1-Dependent Regulatory Pathways for Fe Toxicity Response in Rice Roots e70398 EN Yoshiaki Ueda Crop, Livestock and Environment Division, Japan International Research Center for Agricultural Sciences Naoki Yamaji Institute of Plant Science and Resources, Okayama University Matthias Wissuwa Crop, Livestock and Environment Division, Japan International Research Center for Agricultural Sciences Iron (Fe) is an essential element for plants, but an excess supply can have detrimental effects. Fe toxicity induces complex physiological and genetic responses, and due to this complexity, the knowledge of transcriptional regulatory mechanisms under Fe toxicity is very limited. Previous studies suggested that plant responses to excess Fe involve oxidative stress caused by reactive oxygen species (ROS), which itself causes transcriptional changes. We hypothesized that dissecting these complex responses could lead to the identification of a novel factor and conducted a comparative transcriptome analysis using roots of rice plants exposed to nutrient solutions containing 1 or 5 mM of hydrogen peroxide (a major form of ROS) or 300 mg L－1 of Fe (as FeSO4). Genes induced by hydrogen peroxide overlapped with 62%, 49%, and 30% of Fe toxicity-upregulated genes at 3 h, 1 day, and 3 days following treatment initiation. Subsequent gene co-expression analyses classified genes into 21 groups with varying responsiveness to ROS and Fe toxicity. Genes in group 15 were specifically upregulated by Fe toxicity and overlapped significantly with aluminum (Al)-inducible genes and target genes of the Zn-finger transcription factor, ART1, which regulates Al response in rice roots. Additional experiments using the art1 knock-out mutant demonstrated that ART1 is crucial for upregulating genes such as STAR2 and FRDL4 in response to Fe toxicity. This study reveals the contribution of ART1-dependent regulatory pathways in rice roots under Fe toxicity. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2041-1723 16 1 2025 A node-localized efflux transporter for loading iron to developing tissues in rice 9916 EN Jing Che Institute of Plant Science and Resources, Okayama University Sheng Huang Institute of Plant Science and Resources, Okayama University Yuting Qu State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences Yuma Yoshioka Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiyuri Tomita Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takaaki Miyaji Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Zhenyang Liu State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences Renfang Shen State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences Naoki Yamaji Institute of Plant Science and Resources, Okayama University Jian Feng Ma Institute of Plant Science and Resources, Okayama University Iron (Fe) is an essential micronutrient for plant growth and development. It plays crucial roles in various organs and tissues of plants, but the molecular mechanisms governing its distribution to the above-ground parts after root uptake remain unclear. In this study, we identify OsIET1 (Oryza sativa Iron Efflux Transporter 1), a rice gene highly expressed in the nodes. OsIET1 encodes a plasma membrane-localized protein, which shows efflux transport activity for ferrous iron. It is predominantly expressed in the xylem regions of diffuse vascular bundles, and its expression is upregulated under high Fe conditions. Disruption of OsIET1 impairs Fe allocation, reducing Fe transport to developing tissues (young leaves and grains), while increasing accumulation in nodes and older leaves. This misdistribution causes chlorosis in young leaves and decreases grain yield, especially under Fe-deficient conditions. Furthermore, we detect excessive Fe deposition around the xylem of diffuse vascular bundles in the nodes. Given the pivotal role of nodes in mineral distribution, our results indicate that OsIET1 mediates inter-vascular Fe transfer by facilitating Fe loading into the xylem of diffuse vascular bundles. This process ensures preferential Fe delivery to developing tissues, thereby promoting optimal plant growth and productivity. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0003-6072 118 10 2025 Duganella hordei sp. nov., Duganella caerulea sp. nov., and Duganella rhizosphaerae sp. nov., isolated from barley rhizosphere 146 EN Katsumoto Kishiro Institute of Plant Science and Resources, Okayama University Nurettin Sahin Egitim Fakultesi, Mugla Sitki Kocman University Daisuke Saisho Institute of Plant Science and Resources, Okayama University Naoki Yamaji Institute of Plant Science and Resources, Okayama University Jun Yamashita Institute of Plant Science and Resources, Okayama University Yuki Monden Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Tomoyuki Nakagawa Faculty of Applied Biological Sciences, Gifu University Keiichi Mochida RIKEN Center for Sustainable Resource Science Akio Tani Institute of Plant Science and Resources, Okayama University Duganella sp. strains R1T, R57T, and R64T, isolated from barley roots in Japan, are Gram-stain-negative, motile, rod-shaped bacteria. Duganella species abundantly colonized barley roots. Strains R1T, R57T, and R64T were capable of growth at 4 °C, suggesting adaptation to colonize winter barley roots. Strains R57T and R64T formed purple colonies, indicating violacein production, while strain R1T did not. Based on 16S rRNA gene sequence similarities, strains R1T, R57T, and R64T were most closely related to D. violaceipulchra HSC-15S17T (99.10%), D. vulcania FT81WT (99.45%), and D. violaceipulchra HSC-15S17T (99.86%), respectively. Their genome sizes ranged from 7.05 to 7.38 Mbp, and their genomic G+C contents were 64.2–64.7%. The average nucleotide identity and digital DNA–DNA hybridization values between R1T and D. violaceipulchra HSC-15S17T, R57T and D. vulcania FT81WT, R64T and D. violaceipulchra HSC-15S17T were 86.0% and 33.2%, 95.7% and 67.9%, and 92.7% and 52.6%, respectively. Their fatty acids were predominantly composed of C16:0, C17:0 cyclo, and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). Based on their distinct genetic and phenotypic characteristics, and supported by chemotaxonomic analyses, we propose that strains R1T, R57T, and R64T represent novel species within the Duganella genus, for which the names Duganella hordei (type strain R1T = NBRC 115982 T = DSM 115069 T), Duganella caerulea (type strain R57T = NBRC 115983 T = DSM 115070 T), and Duganella rhizosphaerae (type strain R64T = NBRC 115984 T = DSM 115071 T) are proposed. No potential conflict of interest relevant to this article was reported.

Proceedings of the National Academy of Sciences Acta Medica Okayama 0027-8424 122 32 2025 Structural insights into a citrate transporter that mediates aluminum tolerance in barley e2501933122 EN Tran Nguyen Thao Degree Program in Interdisciplinary Sciences, Graduate School of Environmental, Life, Natural Science, and Technology, Okayama University Namiki Mitani-Ueno Research Core for Plant Stress Science, Institute of Plant Science and Resources, Okayama University Ryo Urano Division of Superconducting and Functional Materials, Research Institute for Interdisciplinary Science, Okayama University Yasunori Saitoh Degree Program in Interdisciplinary Sciences, Graduate School of Environmental, Life, Natural Science, and Technology, Okayama University Peitong Wang Research Core for Plant Stress Science, Institute of Plant Science and Resources, Okayama University Naoki Yamaji Research Core for Plant Stress Science, Institute of Plant Science and Resources, Okayama University Jian-Ren Shen Degree Program in Interdisciplinary Sciences, Graduate School of Environmental, Life, Natural Science, and Technology, Okayama University Wataru Shinoda Degree Program in Interdisciplinary Sciences, Graduate School of Environmental, Life, Natural Science, and Technology, Okayama University Jian Feng Ma Research Core for Plant Stress Science, Institute of Plant Science and Resources, Okayama University Michihiro Suga Degree Program in Interdisciplinary Sciences, Graduate School of Environmental, Life, Natural Science, and Technology, Okayama University HvAACT1 is a major aluminum (Al)-tolerance gene in barley, encoding a citrate transporter that belongs to the multidrug and toxic compound extrusion (MATE) family. This transporter facilitates citrate secretion from the roots, thereby detoxifying external Al ions―a major constraint of crop production on acidic soils. In this study, we present the outward-facing crystal structure of HvAACT1, providing insights into a citrate transport mechanism. The putative citrate binding site consists of three basic residues―K126 in transmembrane helix 2 (TM2), R358 in TM7, and R535 in TM12―creating substantial positive charges in the C-lobe cavity. Proton coupling for substrate transport may involve two pairs of aspartate residues in the N-lobe cavity, one of which corresponds to the essential Asp pair found in prokaryotic H+-coupled MATE transporters belonging to the DinF subfamily. Structural coupling between proton uptake in the N-lobe and citrate extrusion in the C-lobe can be enabled by an extensive, unique hydrogen-bonding network at the extracellular half of the N-lobe. Mutation-based functional analysis, structural comparisons, molecular dynamics simulation, and phylogenic analysis suggest an evolutionary link between citrate MATE transporters and the DinF MATE subfamily. Our findings provide a solid structural basis for citrate transport by HvAACT1 in barley and contribute to a broader understanding of citrate transporter structures in other plant species. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 0022-0957 2025 Dual roles of suberin deposition at the endodermal Casparian strip in manganese uptake of rice 1 11 EN Toshiki Fujii Institute of Plant Science and Resources, Okayama University Naoki Yamaji Institute of Plant Science and Resources, Okayama University Jian Feng Ma Institute of Plant Science and Resources, Okayama University Rice roots are characterized by having two Casparian strips (CSs) at the exodermis and endodermis, where transporters for mineral nutrients are expressed. However, the exact role of the CS in expression of the transporters and subsequent nutrient uptake is poorly understood. Here, we first investigated the role of the CS in manganese (Mn) uptake by using a rice mutant (oscasp1) defective in formation of the endodermal CS. Knockout of OsCASP1 resulted in decreased Mn uptake under limited Mn conditions, but increased Mn uptake at high Mn concentration. Immunostaining revealed that knockout of OsCASP1 did not affect the cell specificity of localization of two transporters (OsNramp5 and OsMTP9) required for Mn uptake, but decreased the protein abundance of these transporters at the endodermis regardless of Mn concentrations tested. Furthermore, we found that overaccumulation of suberin at the endodermis of the mutants suppressed the expression of two transporters; the expression of the two transporters was only observed in the endodermal cells without suberin deposition, but not in the cells with suberin deposition. Taken together, our results indicate that there are two roles for the CS in Mn uptake; maintaining normal expression of the transporters at limited Mn concentration and preventing Mn diffusion to the stele at high Mn concentration. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2041-1723 15 1 2024 Shoot-Silicon-Signal protein to regulate root silicon uptake in rice 10712 EN Naoki Yamaji Institute of Plant Science and Resources, Okayama University Namiki Mitani-Ueno Institute of Plant Science and Resources, Okayama University Toshiki Fujii Institute of Plant Science and Resources, Okayama University Tomonori Shinya Institute of Plant Science and Resources, Okayama University Ji Feng Shao State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture & Forestry University Shota Watanuki Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yasunori Saitoh Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Jian Feng Ma Institute of Plant Science and Resources, Okayama University Plants accumulate silicon to protect them from biotic and abiotic stresses. Especially in rice (Oryza sativa), a typical Si-accumulator, tremendous Si accumulation is indispensable for healthy growth and productivity. Here, we report a shoot-expressed signaling protein, Shoot-Silicon-Signal (SSS), an exceptional homolog of the flowering hormone “florigen” differentiated in Poaceae. SSS transcript is only detected in the shoot, whereas the SSS protein is also detected in the root and phloem sap. When Si is supplied from the root, the SSS transcript rapidly decreases, and then the SSS protein disappears. In sss mutants, root Si uptake and expression of Si transporters are decreased to a basal level regardless of the Si supply. The grain yield of the mutants is decreased to 1/3 due to insufficient Si accumulation. Thus, SSS is a key phloem-mobile protein for integrating root Si uptake and shoot Si accumulation underlying the terrestrial adaptation strategy of grasses. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0140-7791 45 11 2022 FE UPTAKE‐INDUCING PEPTIDE1 maintains Fe translocation by controlling Fe deficiency response genes in the vascular tissue of Arabidopsis 3322 3337 EN Satoshi Okada Group of Environmental Stress Response Systems, Institute of Plant Science and Resources, Okayama University Gui J. Lei Group of Plant Stress Physiology, Institute of Plant Science and Resources, Okayama University Naoki Yamaji Group of Plant Stress Physiology, Institute of Plant Science and Resources, Okayama University Sheng Huang Group of Plant Stress Physiology, Institute of Plant Science and Resources, Okayama University Jian F. Ma Group of Plant Stress Physiology, Institute of Plant Science and Resources, Okayama University Keiichi Mochida Crop Design Research Team, Institute of Plant Science and Resources, Okayama University Takashi Hirayama Group of Environmental Stress Response Systems, Institute of Plant Science and Resources, Okayama University FE UPTAKE-INDUCING PEPTIDE1 (FEP1), also named IRON MAN3 (IMA3) is a short peptide involved in the iron deficiency response in Arabidopsis thaliana. Recent studies uncovered its molecular function, but its physiological function in the systemic Fe response is not fully understood. To explore the physiological function of FEP1 in iron homoeostasis, we performed a transcriptome analysis using the FEP1 loss-of-function mutant fep1-1 and a transgenic line with oestrogen-inducible expression of FEP1. We determined that FEP1 specifically regulates several iron deficiency-responsive genes, indicating that FEP1 participates in iron translocation rather than iron uptake in roots. The iron concentration in xylem sap under iron-deficient conditions was lower in the fep1-1 mutant and higher in FEP1-induced transgenic plants compared with the wild type (WT). Perls staining revealed a greater accumulation of iron in the cortex of fep1-1 roots than in the WT root cortex, although total iron levels in roots were comparable in the two genotypes. Moreover, the fep1-1 mutation partially suppressed the iron overaccumulation phenotype in the leaves of the oligopeptide transporter3-2 (opt3-2) mutant. These data suggest that FEP1 plays a pivotal role in iron movement and in maintaining the iron quota in vascular tissues in Arabidopsis. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2041-1723 12 1 2021 Structural basis for high selectivity of a rice silicon channel Lsi1 6236 EN Yasunori Saitoh Research Institute for Interdisciplinary Science, Okayama University Namiki Mitani-Ueno Institute of Plant Science and Resources, Okayama University Keisuke Saito Research Center for Advanced Science and Technology, The University of Tokyo Kengo Matsuki Graduate School of Natural Science and Technology, Okayama University Sheng Huang Institute of Plant Science and Resources, Okayama University Lingli Yang Research Institute for Interdisciplinary Science, Okayama University Naoki Yamaji Institute of Plant Science and Resources, Okayama University Hiroshi Ishikita Research Center for Advanced Science and Technology, The University of Tokyo Jian-Ren Shen Research Institute for Interdisciplinary Science, Okayama University Jian Feng Ma Institute of Plant Science and Resources, Okayama University Michihiro Suga Research Institute for Interdisciplinary Science, Okayama University Silicon (Si), the most abundant mineral element in the earth’s crust, is taken up by plant roots in the form of silicic acid through Low silicon rice 1 (Lsi1). Lsi1 belongs to the Nodulin 26-like intrinsic protein subfamily in aquaporin and shows high selectivity for silicic acid. To uncover the structural basis for this high selectivity, here we show the crystal structure of the rice Lsi1 at a resolution of 1.8 Å. The structure reveals transmembrane helical orientations different from other aquaporins, characterized by a unique, widely opened, and hydrophilic selectivity filter (SF) composed of five residues. Our structural, functional, and theoretical investigations provide a solid structural basis for the Si uptake mechanism in plants, which will contribute to secure and sustainable rice production by manipulating Lsi1 selectivity for different metalloids. No potential conflict of interest relevant to this article was reported.

Oxford University Press Acta Medica Okayama 0022-0957 71 6 2019 Decrosslinking enables visualization of RNA-guided endonuclease-in situ labeling signals for DNA sequences in plant tissues 1792 1800 EN K. Nagaki N. Yamaji Institute of Plant Science and Resources, Okayama University Information about the positioning of individual loci in the nucleus and the status of epigenetic modifications at these loci in each cell contained in plant tissue increases our understanding of how cells in a tissue coordinate gene expression. To obtain such information, a less damaging method of visualizing DNA in tissue that can be used with immunohistochemistry is required. Recently, a less damaging DNA visualization method using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/associated caspase 9) system, named RNA-guided endonuclease-in situ labeling (RGEN-ISL), was reported. This system made it possible to visualize a target DNA locus in a nucleus fixed on a glass slide with a set of simple operations, but it could not be applied to cells in plant tissues. In this work, we have developed a modified RGEN-ISL method with decrosslinking that made it possible to simultaneously detect the DNA loci and immunohistochemistry signals, including histone modification, in various types of plant tissues and species. No potential conflict of interest relevant to this article was reported.

Oxford University Press Acta Medica Okayama 0022-0957 70 10 2019 The tonoplast-localized transporter OsHMA3 plays an important role in maintaining Zn homeostasis in rice 2717 2725 EN Hongmei Cai Institute of Plant Science and Resources, Okayama University Sheng Huang Institute of Plant Science and Resources, Okayama University Jing Che Institute of Plant Science and Resources, Okayama University Naoki Yamaji Institute of Plant Science and Resources, Okayama University Jian Feng Ma Institute of Plant Science and Resources, Okayama University In order to respond to fluctuating zinc (Zn) in the environment, plants must have a system to control Zn homeostasis. However, how plants maintain an appropriate level of Zn during their growth and development is still poorly understood. In this study, we found that OsHMA3, a tonoplast-localized transporter for Zn/Cd, plays an important role in Zn homeostasis in rice. Accessions with the functional allele of OsHMA3 showed greater tolerance to high Zn than those with the non-functional allele based on root elongation test. A 67Zn-labeling experiment showed that accessions with loss of function of OsHMA3 had lower Zn accumulation in the roots but similar concentrations in the shoots compared with functional OsHMA3 accessions. When exposed to Zn-free growing medium, the concentration in the root cell sap was rapidly decreased in accessions with functional OsHMA3, but less dramatic changes were observed in non-functional accessions. A mobility experiment showed that more Zn in the roots was translocated to the shoots in accessions with functional OsHMA3. Higher expression levels of OsZIP4, OsZIP5, OsZIP8, and OsZIP10 were found in the roots of accessions with functional OsHMA3 in response to Zn deficiency. Taken together, our results indicate that OsHMA3 plays an important role in rice roots in both Zn detoxification and storage by sequestration into the vacuoles, depending on Zn concentration in the environment. No potential conflict of interest relevant to this article was reported.

Nature Publishing Group Acta Medica Okayama 2041-1723 6 2015 AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis EN Takaaki Miyaji Takashi Kuromori Yu Takeuchi Naoki Yamaji Kengo Yokosho Atsushi Shimazawa Eriko Sugimoto Hiroshi Omote Jian Feng Ma Kazuo Shinozaki Yoshinori Moriyama 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. No potential conflict of interest relevant to this article was reported.