Metaproteomic Identification of Diazotrophic Methanotrophs and Their Localization in Root Tissues of Field-Grown Rice Plants

Abstract

ABSTRACT In a previous study by our group, CH 4 oxidation and N 2 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 CH 4 oxidation and N 2 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 CH 4 oxidation and N 2 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 CH 4 oxidation and N 2 fixation are performed mainly by type II methanotrophs of the Methylocystaceae , including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots.