An improved microtiter plate assay to monitor the oxidative burst in monocot and dicot plant cell suspension cultures

Abstract

Background A screening method for elicitor and priming agents does not only allow detecting new bioactive substances, it can also be used to understand structure–function relationships of known agents by testing different derivatives of them. This can not only provide new lead compounds for the development of novel, more environment-benign, bio-based agro-chemicals, it may eventually also lead to a better understanding of defense mechanisms in plants. Reactive oxygen species (ROS) are sensitive indicators of these mechanisms but current assay formats are not suitable for multiplex screening, in particularly not in the case of monocot systems. Results Here we describe continuous monitoring of ROS in 96-well microtiter plates using the chemiluminescent probe L012, a luminol derivative producing chemiluminescence when oxidised by ROS like hydrogen peroxide, superoxide, or hydroxyl radical that can thus be used as an indicator for these ROS. We were able to measure ROS in both monocot ( Oryza sativa ) and dicot ( Medicago truncatula ) cell suspension cultures and record dose dependencies for the carbohydrate elicitors and priming agents ulvan and chitosan at low substrate concentrations (0.3–2.5 µg/ml). The method was optimized in terms of cell density, L012 concentration, and pre-incubation time. In contrast to the single peak observed using a cuvette luminometer, the improved method revealed a double burst in both cell systems during the 90-min measuring period, probably due to the detection of multiple ROS rather than only H 2 O 2 . Conclusion We provide a medium throughput screening method for monocot and dicot suspension-cultured cells that enables direct comparison of monocot and dicot plant systems regarding their reaction to different signaling molecules. Electronic supplementary material The online version of this article (doi:10.1186/s13007-016-0110-1) contains supplementary material, which is available to authorized users.

Plant phenotyping relevance

植物細胞培養における酸化バーストを測定するアッセイの改良が主題であり、植物の生理状態を取得する測定法の開発に該当する。

Code and data availability

The article describes a microtiter plate ROS assay but contains no public phenotype datasets, images, analysis code, models, or deposited supplements. Supplementary files (Additional files 1–3) are referenced but no public repository, URL, or availability statement is provided; the only URLs present are license and f f

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