title: High-resolution spatial and temporal analysis of phytoalexin production in oats.
abstract: The production of oat (Avena sativa L.) phytoalexins, avenanthramides, occurs in response to elicitor treatment with oligo-N-acetylchitooligosaccharides. In this study, avenanthramides production was investigated by techniques that provide high spatial and temporal resolution in order to clarify the process of phytoalexin production at the cellular level. The amount of avenanthramides accumulation in a single mesophyll cell was quantified by a combination of laser micro-sampling and low-diffuse nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) techniques. Avenanthramides, NAD(P)H and chlorophyll were also visualized in elicitor-treated mesophyll cells using line-scanning fluorescence microscopy. We found that elicitor-treated mesophyll cells could be categorized into three characteristic cell phases, which occurred serially over time. Phase 0 indicated the normal cell state before metabolic or morphological change in response to elicitor, in which the cells contained abundant NAD(P)H. In phase 1, rapid NAD(P)H oxidation and marked movement of chloroplasts occurred, and this phase was the early stage of avenanthramides biosynthesis. In phase 2, avenanthramides accumulation was maximized, and chloroplasts were degraded. Avenanthramides appear to be synthesized in the chloroplast, because a fluorescence signal originating from avenanthramides was localized to the chloroplasts. Moreover, our results indicated that avenanthramides biosynthesis and the hypersensitive response (HR) occurred in identical cells. Thus, the avenanthramides production may be one of sequential events programmed in HR leading to cell death. Furthermore, the phase of the defense response was different among mesophyll cells simultaneously treated with elicitor. These results suggest that individual cells may have different susceptibility to the elicitor.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan. izumi_yoshihiro@bio.eng.osaka-u.ac.jp
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Izumi
authors.forename: Shin'ichiro
authors.initials: S
authors.lastname: Kajiyama
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Nakamura
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ishihara
authors.forename: Atsushi
authors.initials: A
authors.lastname: Okazawa
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Fukusaki
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Kanematsu
authors.forename: Akio
authors.initials: A
authors.lastname: Kobayashi
mesh_terms: D017209:Apoptosis; D018554:Avena / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002853:Chromatography, Liquid; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009844:Oligosaccharides / Q000494:pharmacology; D018515:Plant Leaves / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012717:Sesquiterpenes; D021241:Spectrometry, Mass, Electrospray Ionization; D013729:Terpenes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D062367:ortho-Aminobenzoates / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000094162:Phytoalexins
chemical_list: C520160:N-acetylchitooligosaccharide; D009844:Oligosaccharides; D017382:Reactive Oxygen Species; D012717:Sesquiterpenes; D013729:Terpenes; D062367:ortho-Aminobenzoates; C514463:avenanthramide-2C; D000094162:Phytoalexins