title: Development of a colorimetric assay for determining the amount of H2O2 generated in tobacco cells in response to elicitors and its application to study of the structure-activity relationship of flagellin-derived peptides.
abstract: Plants exhibit various defense responses after recognizing elicitor molecules derived from pathogenic microorganisms and host plants. In this study, we developed an improved colorimetric assay for quantifying the generation of H2O2 in plant cells, one of the defense responses, to evaluate elicitor activity quantitatively. H2O2 is detected using a dye, N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine sodium salt (DA-64), which can be measured by conventional spectrometers in a highly sensitive and quantitative manner. Using this method, we successfully measured the elicitor activity of flagellin-derived peptides in cultured tobacco cells, and identified several structural features of the peptides important for the elicitor activity. The results suggest that the structural factors required for expression of the elicitor activity differ slightly among plant species. The efficient and sensitive assay developed in this study should be useful not only for studying structure-activity relationships, but also for the screening of novel compounds that can induce defense responses.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Miyata
authors.forename: Masahiro
authors.initials: M
authors.lastname: Miyashita
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Nose
authors.forename: Yukari
authors.initials: Y
authors.lastname: Otake
authors.forename: Hisashi
authors.initials: H
authors.lastname: Miyagawa
mesh_terms: D000595:Amino Acid Sequence; D003124:Colorimetry / Q000379:methods*; D004396:Coloring Agents / Q000737:chemistry; D005408:Flagellin / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014026:Nicotiana / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
chemical_list: D004396:Coloring Agents; D010446:Peptide Fragments; D005408:Flagellin; D006861:Hydrogen Peroxide