Name: real-time detection of reactive oxygen species production in immune response in rice with a chemiluminescence assay
Uploaded: 2022-11-25T15:00:00
Description: Watch this Scientific Journal Video about Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay at JoVE.com

This work was supported by grants from Shanghai Natural Science Foundation (Grant Number: 21ZR1429300/BS1500016),&#160;Shanghai Jiao Tong University (Agri-X program, Grant Number:AF1500088/002), Shanghai Collaborative Innovation Center of Agri-Seeds (Grant Number: ZXWH2150201/001)&#160;to Jiangbo Fan,&#160;and by the Medical-Engineering Collaboration Project of Shanghai Jiao Tong Univesity (grant number: 21X010301734) to Can Li.

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

<ol>
	<li>Gechev, T. S., Van Breusegem, F., Stone, J. M., Denev, I., Laloi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+as+signals+that+modulate+plant+stress+responses+and+programmed+cell+death.">Reactive oxygen species as signals that modulate plant stress responses and programmed cell death.</a> Bioessays. 28 (11), 1091-1101 (2006).</li><li>Mittler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+are+good.">ROS are good.</a> Trends in Plant Science. 22 (1), 11-19 (2017).</li><li>Gilroy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS,+calcium,+and+electric+signals:+key+mediators+of+rapid+systemic+signaling+in+plants.">ROS, calcium, and electric signals: key mediators of rapid systemic signaling in plants.</a> Plant Physiology. 171 (3), 1606-1615 (2016).</li><li>Mittler, R., Vanderauwera, S., Gollery, M., Van Breusegem, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+gene+network+of+plants.">Reactive oxygen gene network of plants.</a> Trends in Plant Science. 9 (10), 490-498 (2004).</li><li>Marino, D., Dunand, C., Puppo, A., Pauly, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+burst+of+plant+NADPH+oxidases.">A burst of plant NADPH oxidases.</a> Trends in Plant Science. 17 (1), 9-15 (2012).</li><li>Mittler, R., Zandalinas, S. I., Fichman, Y., Van Breusegem, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+signalling+in+plant+stress+responses.">Reactive oxygen species signalling in plant stress responses.</a> Nature Reviews Molecular Cell Biology. 23 (10), 663-679 (2022).</li><li>Suzuki, N., Koussevitzky, S., Mittler, R., Miller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+and+redox+signalling+in+the+response+of+plants+to+abiotic+stress.">ROS and redox signalling in the response of plants to abiotic stress.</a> Plant, Cell &amp; Environment. 35 (2), 259-270 (2012).</li><li>Suzuki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+burst+oxidases:+the+engines+of+ROS+signaling.">Respiratory burst oxidases: the engines of ROS signaling.</a> Current Opinion in Plant Biology. 14 (6), 691-699 (2011).</li><li>Kadota, Y., Shirasu, K., Zipfel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+NADPH+oxidase+RBOHD+during+plant+immunity.">Regulation of the NADPH oxidase RBOHD during plant immunity.</a> Plant and Cell Physiology. 56 (8), 1472-1480 (2015).</li><li>Segonzac, C., Zipfel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+plant+pattern-recognition+receptors+by+bacteria.">Activation of plant pattern-recognition receptors by bacteria.</a> Current Opinion in Microbiology. 14 (1), 54-61 (2011).</li><li>Roda, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+chemical+luminescence-based+biosensors:+A+critical+review.">Progress in chemical luminescence-based biosensors: A critical review.</a> Biosensors and Bioelectronics. 76, 164-179 (2016).</li><li>Hong, D., Joung, H. -. A., Lee, D. Y., Kim, S., Kim, M. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attomolar+detection+of+cytokines+using+a+chemiluminescence+immunoassay+based+on+an+antibody-arrayed+CMOS+image+sensor.">Attomolar detection of cytokines using a chemiluminescence immunoassay based on an antibody-arrayed CMOS image sensor.</a> Sensors and Actuators B: Chemical. 221, 1248-1255 (2015).</li><li>Nishinaka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.+new+sensitive+chemiluminescence+probe,+L-012,+for+measuring+the+production+of+superoxide+anion+by+cells.">et al. new sensitive chemiluminescence probe, L-012, for measuring the production of superoxide anion by cells.</a> Biochemical and Biophysical Research Communications. 193 (2), 554-559 (1993).</li><li>Grundy, J., Stoker, C., Carre, I. 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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.
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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 functions varies from the regulation of growth and development to the perception of abiotic and biotic stresses5,6,7,8.
In the plant immune system, plant cell plasma membrane-localized receptors-so-called pattern recognition receptors (PRRs)-perceive pathogen-derived chemicals-pathogen-associated molecular patterns (PAMPs). This recognition triggers a series of fast immune responses, including calcium influx, ROS burst, and MAPK cascade; thus, this layer&#160;of immunity is named PAMP-triggered immunity (PTI). ROS burst is a hallmark PTI response, the determination of which is widely applied to PTI-related studies9,10. ROS production triggered by PAMPs is attributed to plasma membrane-resident NADPH oxidase, or respiratory burst oxidase homolog (RBOH) family proteins, which transfer electrons from cytosolic NADPH or NADH to extracellular oxygen to produce superoxide (O2-) which is spontaneously converted to hydrogen peroxide (H2O2) by superoxide dismutase8. PAMP-triggered ROS burst is quite rapid, appearing only a few minutes after PAMP treatment and peaking at ~10-12 min. The vast majority of the ROS molecules comprise hydrogen peroxide (H2O2), which can be easily and steadily detected with a chemiluminescence assay.
In chemiluminescence, the chemiluminescence reagent reacts with active oxygen, under the action of a catalyst, to produce the excited state intermediates. Then, the electrons in the product return to the ground state through non-radiative transition and emit photons. Common chemiluminescence reagents include luminol and L-012, with luminol dominating the application11,12,13. However, more researchers are choosing L-012 to detect ROS production, since L-012 has a much higher light emission efficiency under neutral or near neutral pH conditions compared to luminol.
This paper describes an optimized chemiluminescence method, based on L-012, for the real-time detection of ROS production after the elicitation of PAMPs in rice (Oryza sativa) tissues-leaf discs and sheath. The method provided here is simple, stable, and standardized, and is highly adaptable to meet different experimental needs. The data obtained with this method are highly reproducible under firmly controlled conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well microtiter plate</td>
			<td>WHB</td>
			<td>WHB-96-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Innochem</td>
			<td>A43543</td>
			<td></td>
		</tr>
		<tr>
			<td>flg22</td>
			<td>Sangon Biotech</td>
			<td>p20973</td>
			<td>PAMP</td>
		</tr>
		<tr>
			<td>Gen5</td>
			<td>BioTek</td>
			<td></td>
			<td>software</td>
		</tr>
		<tr>
			<td>L-012</td>
			<td>FUJIFILM</td>
			<td>120-04891</td>
			<td>8-amino-5-chloro-7-phenyl-2,3-dihydropyrido [3,4-d] pyridazine-1,4-dione, CAS #:143556-24-5</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>Synergy 2</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Medium</td>
			<td>Solarbio</td>
			<td>M8521</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCLO</td>
			<td>Aladdin</td>
			<td>S101636</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase from horseradish (HRP)</td>
			<td>Sigma</td>
			<td>P8375</td>
			<td></td>
		</tr>
		<tr>
			<td>Phytagel</td>
			<td>Sigma</td>
			<td>P8169</td>
			<td></td>
		</tr>
		<tr>
			<td>Sampler</td>
			<td>Miltex&#160;</td>
			<td>15110-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sangon Biotech</td>
			<td>A502792</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sangon Biotech</td>
			<td>A610195</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time detection of reactive oxygen species production in immune response in rice with a chemiluminescence assay

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.

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 ...

Watch this Scientific Journal Video about Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay at JoVE.com

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

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.

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...

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