title: A low temperature-inducible protein AtSRC2 enhances the ROS-producing activity of NADPH oxidase AtRbohF.
abstract: Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in plant environmental responses. Arabidopsis thaliana NADPH oxidase AtRbohF-mediated ROS-production is involved in abiotic stress responses. Because overproduction of ROS is highly toxic to cells, the activity of AtRbohF needs to be tightly regulated in response to diverse stimuli. The ROS-producing activity of AtRbohF is activated by Ca(2+) and protein phosphorylation, but other regulatory factors for AtRbohF are mostly unknown. In this study, we screened for proteins that interact with the N-terminal cytosolic region of AtRbohF by a yeast two-hybrid screen, and isolated AtSRC2, an A. thaliana homolog of SRC2 (soybean gene regulated by cold-2). A co-immunoprecipitation assay revealed that AtSRC2 interacts with the N-terminal region of AtRbohF in plant cells. Intracellular localization of GFP-tagged AtSRC2 was partially overlapped with that of GFP-tagged AtRbohF at the cell periphery. Co-expression of AtSRC2 enhanced the Ca(2+)-dependent ROS-producing activity of AtRbohF in HEK293T cells, but did not affect its phosphorylation-dependent activation. Low-temperature treatment induced expression of the AtSRC2 gene in Arabidopsis roots in proportion to levels of ROS production that was partially dependent on AtRbohF. Our findings suggest that AtSRC2 is a novel activator of Ca(2+)-dependent AtRbohF-mediated ROS production and may play a role in cold responses.
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kawarazaki
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Sachie
authors.initials: S
authors.lastname: Kimura
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Iizuka
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Hanamata
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Nibori
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Michikawa
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Imai
authors.affiliation: Department of Biological Science, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Mitsutomo
authors.initials: M
authors.lastname: Abe
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan; Department of Biological Science, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address: hidetaka@biol.s.u-tokyo.ac.jp.
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Kaya
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan. Electronic address: kuchitsu@rs.noda.tus.ac.jp.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Kuchitsu
mesh_terms: D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D003080:Cold Temperature; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D019255:NADPH Oxidases / Q000737:chemistry / Q000378:metabolism*; D059828:Plant Cells / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects / Q000235:genetics; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; D002118:Calcium; D049452:Green Fluorescent Proteins; D019255:NADPH Oxidases; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; C510500:SRC2 protein, Arabidopsis; C000589475:RBOH F protein, Arabidopsis
keywords: ABA; Arabidopsis thaliana; Arabidopsis thaliana respiratory burst oxidase homologue; AtRboh; AtRbohF; Cold stress; NADPH oxidase; NADPH oxidase (NOX); NOX; ROS; RT-PCR; Reactive oxygen species (ROS); Respiratory burst oxidase homologue (Rboh); SRC2; TM; abscisic acid; co-IP; co-immunoprecipitation; reactive oxygen species; reverse transcription-polymerase chain reaction; soybean gene regulated by cold-2; transmembrane