title: Two h-Type Thioredoxins Interact with the E2 Ubiquitin Conjugase PHO2 to Fine-Tune Phosphate Homeostasis in Rice.
abstract: Phosphate overaccumulator2 (PHO2) encodes a ubiquitin-conjugating E2 enzyme that is a major negative regulator of the inorganic phosphate (Pi)-starvation response-signaling pathway. A yeast two-hybrid (Y2H) screen in rice (Oryza sativa; Os) using OsPHO2 as bait revealed an interaction between OsPHO2 and two h-type thioredoxins, OsTrxh1 and OsTrxh4. These interactions were confirmed in vivo using bimolecular fluorescence complementation (BiFC) of OsPHO2 and OsTrxh1/h4 in rice protoplasts and by in vitro pull-down assays with 6His-tagged OsTrxh1/h4 and GST-tagged OsPHO2. Y2H assays revealed that amino acid Cys-445 of OsPHO2 and an N-terminal Cys in the "WCGPC" motif of Trxhs were required for the interaction. Split-ubiquitin Y2H analyses and BiFC assays in rice protoplasts confirmed the interaction of OsPHO2 with PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (OsPHF1), and PHOSPHATE1;2 (OsPHO1;2) in the endoplasmic reticulum and Golgi membrane system, where OsPHO2 mediates the degradation of OsPHF1 in both tobacco (Nicotiana benthamiana) leaves and rice seedlings. Characterization of rice pho2 complemented lines, transformed with an endogenous genomic OsPHO2 or OsPHO2C445S(a constitutively reduced form) fragment, indicated that OsPHO2C445Srestored Pi concentration in rice to statistically significant lower levels compared to native OsPHO2 Moreover, the suppression of OsTrxh1 (knockdown and knockout) resulted in slightly higher Pi concentration than that of wild-type Nipponbare in leaves. These results demonstrate that OsPHO2 is under redox control by thioredoxins, which fine-tune its activity and link Pi homeostasis with redox balance in rice.
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China (Y.Y., W.Y., S.W., S.L., M.W., Y.Z., C.W., C.M., H.S.); and.|Australian Research Council Centre of Excellence in Plant Energy Biology, Department of Animal, Plant, and Soil Science, School of Life Science, La Trobe University, Bundoora, Victoria 3086, Australia (J.W.).
authors.forename: Yinghui
authors.initials: Y
authors.lastname: Ying
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China (Y.Y., W.Y., S.W., S.L., M.W., Y.Z., C.W., C.M., H.S.); and.|Australian Research Council Centre of Excellence in Plant Energy Biology, Department of Animal, Plant, and Soil Science, School of Life Science, La Trobe University, Bundoora, Victoria 3086, Australia (J.W.).
authors.forename: Wenhao
authors.identifier: 0000-0003-0587-8958
authors.initials: W
authors.lastname: Yue
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China (Y.Y., W.Y., S.W., S.L., M.W., Y.Z., C.W., C.M., H.S.); and.|Australian Research Council Centre of Excellence in Plant Energy Biology, Department of Animal, Plant, and Soil Science, School of Life Science, La Trobe University, Bundoora, Victoria 3086, Australia (J.W.).
authors.forename: Shoudong
authors.initials: S
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China (Y.Y., W.Y., S.W., S.L., M.W., Y.Z., C.W., C.M., H.S.); and.|Australian Research Council Centre of Excellence in Plant Energy Biology, Department of Animal, Plant, and Soil Science, School of Life Science, La Trobe University, Bundoora, Victoria 3086, Australia (J.W.).
authors.forename: Shuai
authors.identifier: 0000-0002-8317-6321
authors.initials: S
authors.lastname: Li
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China (Y.Y., W.Y., S.W., S.L., M.W., Y.Z., C.W., C.M., H.S.); and.|Australian Research Council Centre of Excellence in Plant Energy Biology, Department of Animal, Plant, and Soil Science, School of Life Science, La Trobe University, Bundoora, Victoria 3086, Australia (J.W.).
authors.forename: Min
authors.identifier: 0000-0001-8077-0168
authors.initials: M
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China (Y.Y., W.Y., S.W., S.L., M.W., Y.Z., C.W., C.M., H.S.); and.|Australian Research Council Centre of Excellence in Plant Energy Biology, Department of Animal, Plant, and Soil Science, School of Life Science, La Trobe University, Bundoora, Victoria 3086, Australia (J.W.).
authors.forename: Yang
authors.identifier: 0000-0002-1635-9144
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China (Y.Y., W.Y., S.W., S.L., M.W., Y.Z., C.W., C.M., H.S.); and.|Australian Research Council Centre of Excellence in Plant Energy Biology, Department of Animal, Plant, and Soil Science, School of Life Science, La Trobe University, Bundoora, Victoria 3086, Australia (J.W.).
authors.forename: Chuang
authors.initials: C
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China (Y.Y., W.Y., S.W., S.L., M.W., Y.Z., C.W., C.M., H.S.); and.|Australian Research Council Centre of Excellence in Plant Energy Biology, Department of Animal, Plant, and Soil Science, School of Life Science, La Trobe University, Bundoora, Victoria 3086, Australia (J.W.).
authors.forename: Chuanzao
authors.identifier: 0000-0001-5126-2180
authors.initials: C
authors.lastname: Mao
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China (Y.Y., W.Y., S.W., S.L., M.W., Y.Z., C.W., C.M., H.S.); and.|Australian Research Council Centre of Excellence in Plant Energy Biology, Department of Animal, Plant, and Soil Science, School of Life Science, La Trobe University, Bundoora, Victoria 3086, Australia (J.W.).
authors.forename: James
authors.initials: J
authors.lastname: Whelan
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China (Y.Y., W.Y., S.W., S.L., M.W., Y.Z., C.W., C.M., H.S.); and huixia@zju.edu.cn.|Australian Research Council Centre of Excellence in Plant Energy Biology, Department of Animal, Plant, and Soil Science, School of Life Science, La Trobe University, Bundoora, Victoria 3086, Australia (J.W.) huixia@zju.edu.cn.
authors.forename: Huixia
authors.identifier: 0000-0001-6890-5672
authors.initials: H
authors.lastname: Shou
mesh_terms: D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D006706:Homeostasis; D012275:Oryza / Q000235:genetics / Q000378:metabolism*; D028061:Phosphate Transport Proteins / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010802:Phylogeny; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D054480:Thioredoxin h / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D028061:Phosphate Transport Proteins; D010710:Phosphates; D010940:Plant Proteins; D054480:Thioredoxin h; C514696:UBC24 protein, Arabidopsis; D044763:Ubiquitin-Conjugating Enzymes; D003545:Cysteine