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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

Abstract

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

Introduction

Reactive oxygen species (ROS) comprise a series of chemically active oxygen derivatives, including superoxide anion radicals (O2-) and its derivatives, hydroxyl radicals (OH-), hydrogen peroxide, and products of singlet oxygen or oxidation-reduction reactions, which are constantly produced in plastids and chloroplasts, mitochondria, peroxisomes, and other subcellular locations1. ROS play important roles in many biological processes and are essential for all plants2,3,4. The broad spectrum of ROS fu....

Protocol

NOTE: The protocol is applicable to different plant tissues. Rice sheath and leaf discs were used in this protocol for ROS detection upon PAMP elicitation. As differences mainly arise due to the method of sampling, only the common procedures are described below, with specific steps being mentioned wherever necessary.

1. Plant culture

  1. Sterilize the dehusked rice seeds with 70% ethanol for 1 min, then with 40% sodium hypochlorite (NaClO) for 1 h. Then, rinse the seed.......

Representative Results

Here, we take rice material as an example to determine the ROS produced with flg22 treatment. The generation of ROS after elicitation is transient. In rice, the increase in ROS production was first detected in 1-2 min, peaked at 10-12 min, and returned to the baseline in ~30-35 min (Figure 3). Compared to the control test, in which PAMP was absent in the elicitation solution resulting in no obvious ROS induction, a specific ROS burst was induced only when the elicitation solution containing .......

Discussion

The purpose of this study was to establish a highly efficient method to quantify early ROS production in response to PAMP in rice tissues. This method provides a standardized procedure for the real-time determination of apoplast ROS produced from treated rice tissues. This method is simple in operation, low in cost, clear in composition, and independent of commercial kits. Using this method, researchers can study the real-time production of apoplast ROS when plants are subjected to biotic or abiotic stresses.

Acknowledgements

This work was supported by grants from Shanghai Natural Science Foundation (Grant Number: 21ZR1429300/BS1500016), Shanghai Jiao Tong University (Agri-X program, Grant Number:AF1500088/002), Shanghai Collaborative Innovation Center of Agri-Seeds (Grant Number: ZXWH2150201/001) to Jiangbo Fan, and by the Medical-Engineering Collaboration Project of Shanghai Jiao Tong Univesity (grant number: 21X010301734) to Can Li.

....

Materials

NameCompanyCatalog NumberComments
96-well microtiter plateWHBWHB-96-01
Ethanol absoluteInnochemA43543
flg22Sangon Biotechp20973PAMP
Gen5BioTeksoftware
L-012FUJIFILM120-048918-amino-5-chloro-7-phenyl-2,3-dihydropyrido [3,4-d] pyridazine-1,4-dione, CAS #:143556-24-5
Microplate readerBioTekSynergy 2
MS MediumSolarbioM8521
NaCLOAladdinS101636
Peroxidase from horseradish (HRP)SigmaP8375
PhytagelSigmaP8169
SamplerMiltex 15110-40
SucroseSangon BiotechA502792
TrisSangon BiotechA610195

References

  1. Gechev, T. S., Van Breusegem, F., Stone, J. M., Denev, I., Laloi, C. Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. Bioessays. 28 (11), 1091-1101 (2006).
  2. Mittler, R. ROS are good.

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