化学发光检测植物叶片中的氧化迸发

Markus Albert; Melinka A. Butenko; Reidunn B. Aalen; Georg Felix; Mari Wildhagen

doi:10.21769/BioProtoc.1423

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Peer-reviewed

Chemiluminescence Detection of the Oxidative Burst in Plant Leaf Pieces

化学发光检测植物叶片中的氧化迸发

MA Markus Albert email

MB Melinka A. Butenko

RA Reidunn B. Aalen

GF Georg Felix

MW Mari Wildhagen

发布: 2015年03月20日第5卷第6期 DOI: 10.21769/BioProtoc.1423 浏览次数: 19832

评审: Tie LiuMasahiro Morita

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Cover of The Plant Cell, featuring study using the protocol.

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Abstract

The production of 'reactive oxygen species' (ROS), also termed oxidative burst, is a typical cellular response of animals and plants to diverse biotic and abiotic stresses. Here, we outline the detection of the ROS-burst in plant leaf pieces using a luminol-based bioassay which allows for the detection of chemiluminescence. The assay was originally described by Keppler et al. (1989) and subsequently adapted for other plant cells and tissues (Felix et al., 1999) and also used in recent publications (Albert et al., 2013; Albert et al., 2010; Butenko et al., 2014; Halter et al., 2014). In this protocol we outline a standardized version of this assay including remarks and recommendations for data evaluation and interpretation of results.

Materials and Reagents

Plant leaf pieces (any plant of interest, e.g. Arabidopsis or N. benthamiana transiently expressing a construct of interest)
Water
200 µM luminol L-012 (Wako Chemicals USA, catalog number: 075-05111 ; http://www.wako-chem.co.jp/english/labchem/journals/wpu_bio1/10.htm)
10 µg/ml peroxidase, horseradish peroxidase (AppliChem, catalog number: A3791 )
MAMPs or any elicitors of interest
Known MAMP as positive control (e.g. flg22, chitin-oligomers, etc.)
10x luminol master mix (see Recipes)

Equipment

Scissors
Petridishes
Small spatula
96 well plates, white, flat-bottomed (e.g., LIA-plate, whit, 96-well, flat-bottom, Greiner Bio-One GmbH) or suitable cuvettes for single cell instruments
Luminometer, either a 96 well plate reader (e.g., Centro LB 960, Microplate Luminometer, BERTHOLD TECHNOLOGIES) or a single cell instrument

Procedure

English

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Albert, M., Butenko, M. A., Aalen, R. B., Felix, G. and Wildhagen, M. (2015). Chemiluminescence Detection of the Oxidative Burst in Plant Leaf Pieces. Bio-protocol 5(6): e1423. DOI: 10.21769/BioProtoc.1423.