High-resolution spatial and temporal analysis of phytoalexin production in oats
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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.
KeywordsPhytoalexin production Oats High-resolution spatial and temporal analysis Laser-assisted single-cell sampling Low-diffuse nanoflow liquid chromatography–electrospray ionization-ion trap mass spectrometry (LC–ESI-MS/MS) Line-scanning fluorescence microscopy
Liquid chromatography–electrospray ionization-ion trap mass spectrometry
Multiple reaction monitoring
Reactive oxygen species
Programmed cell death
5-(and 6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester
The hollow optical fiber used in this experiment was supplied by Dr. Yuji Matsuura (Department of Electrical Communications, Graduate School of Engineering, Tohoku University, Japan). This work was supported by grants from the Promotion of Basic Research Activities for Innovative Biosciences program of BRAIN (Bio-oriented Technology Research Advancement Institution, Japan) and from a Grant-in-Aid for Scientific Research (KAKENHI) in Priority Area “Molecular Nano Dynamics” from the Ministry of Education, Culture, Sports, Science and Technology of Japan to A. Kobayashi.
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