title: Metaproteomic identification of diazotrophic methanotrophs and their localization in root tissues of field-grown rice plants.
abstract: In a previous study by our group, CH4 oxidation and N2 fixation were simultaneously activated in the roots of wild-type rice plants in a paddy field with no N input; both processes are likely controlled by a rice gene for microbial symbiosis. The present study examined which microorganisms in rice roots were responsible for CH4 oxidation and N2 fixation under the field conditions. Metaproteomic analysis of root-associated bacteria from field-grown rice (Oryza sativa Nipponbare) revealed that nitrogenase complex-containing nitrogenase reductase (NifH) and the alpha subunit (NifD) and beta subunit (NifK) of dinitrogenase were mainly derived from type II methanotrophic bacteria of the family Methylocystaceae, including Methylosinus spp. Minor nitrogenase proteins such as Methylocella, Bradyrhizobium, Rhodopseudomonas, and Anaeromyxobacter were also detected. Methane monooxygenase proteins (PmoCBA and MmoXYZCBG) were detected in the same bacterial group of the Methylocystaceae. Because these results indicated that Methylocystaceae members mediate both CH4 oxidation and N2 fixation, we examined their localization in rice tissues by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The methanotrophs were localized around the epidermal cells and vascular cylinder in the root tissues of the field-grown rice plants. Our metaproteomics and CARD-FISH results suggest that CH4 oxidation and N2 fixation are performed mainly by type II methanotrophs of the Methylocystaceae, including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots.
authors.affiliation: Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Bao
authors.affiliation: Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Okubo
authors.affiliation: Department of Civil and Environmental Engineering, Tohoku University, Sendai, Miyagi, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Kubota
authors.affiliation: Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kasahara
authors.affiliation: Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan.
authors.forename: Hirohito
authors.initials: H
authors.lastname: Tsurumaru
authors.affiliation: Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan.
authors.forename: Mizue
authors.initials: M
authors.lastname: Anda
authors.affiliation: Memuro Research Station, National Agricultural Research Center for Hokkaido Region, Memuro-cho, Kasaigun, Hokkaido, Japan.
authors.forename: Seishi
authors.initials: S
authors.lastname: Ikeda
authors.affiliation: Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan kiwamu@ige.tohoku.ac.jp.
authors.forename: Kiwamu
authors.initials: K
authors.lastname: Minamisawa
mesh_terms: D052818:Autotrophic Processes; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D008697:Methane / Q000378:metabolism*; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation; D009591:Nitrogenase / Q000235:genetics / Q000378:metabolism; D012275:Oryza / Q000254:growth & development / Q000382:microbiology*; D010802:Phylogeny; D018517:Plant Roots / Q000254:growth & development / Q000382:microbiology; D040901:Proteomics; D012988:Soil Microbiology
chemical_list: D001426:Bacterial Proteins; D009591:Nitrogenase; D008697:Methane