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...

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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. -. 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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 ...

This protocol describes an optimized L-012-based chemiluminescence method to detect ROS production in real time upon PAMP elicitation in rice tissues. This method is easy, standardized, and highly reproducible under firmly controlled conditions. Demonstrating this procedure will be On Ying, a PhD student from my lab.To begin, sterilize the dehusked rice seeds with 70%ethanol for one minute, then with 40%sodium hypochlorite for one hour. Rinse the seeds five times with sterile water to remove residual chlorine. In the rice sheath method, directly plate the seeds in the sterile glass vessel with Murashige and Skoog or MS medium.To plate the seeds in the leaf disc method, Plate them on MS plates for five to seven days and then transplant them to the growth matrix or soil. Grow the seedlings in a growth room with a 12 hour light, 12 hour dark photo period. Cut the sheath from 10 day old rice seedlings into three millimeter segments with a sharp razor blade or surgical blade for pre-treatment one day before the ROS assay.Place five sheath segments in an individual well of a 96 well microtiter plate containing 100 microliters of double distilled water for 10 to 12 hours. Cut the leaf discs from four to six week old rice plants using a biopsy punch with a plunger. Always cut the leaf discs from the middle third of the second leaf of the main tiller to reduce data variation.Next, place one leaf disc in an individual well of a 96 well microtiter plate containing 100 microliters of double distilled water for 10 to 12 hours for pre-treatment. Keep all the leaf discs floating and facing up in the wells of a microtiter plate for water pre-treatment to avoid leaf side associated variation. Prepare elicitation solution by combining 9.4 milliliters of 50 micromolar Tris Hcl, 400 microliters of L012 solution, 100 microliters of horse radish peroxidase, and 100 microliters of Flg22.Add 200 microliters of elicitation solution to the wells. Start the software and click on the experiments button to create a new protocol or use an existing protocol. Click procedure in the popup to set up the plate and select the wells from the plate to be monitored.Click start kinetic and set the runtime to 30 minutes or longer, depending on the experimental requirements. Select minimum interval to obtain the readings as frequently as possible. And for integration time, choose one second or longer depending on the signal intensity.Click on validate, followed by Okay to confirm the settings, then click on detect the new plate in the popup and wait for the software to prompt the load plate dialogue box. Carefully remove the double distilled water from the wells containing the pre-treated tissues, avoiding any tissue damage or desiccation. Use a multi-channel pipette to add 200 microliters of the elicitation solution to the wells containing the tissues.Place the plate to be tested on the carrier and begin detection. To induce ROS by Flg22, leaf discs and three millimeter long sheaths were used. ROS generation is monitored for 35 minutes.The bars indicate the means of standard deviations calculated from five technical repeats. In rice, the increase in ROS production was first detected in one to two minute, peaked at 10 to 12 minute and returned to the baseline in around 30 to 35 minute. The total amount of ROS was calculated from the curve.To obtain the total amount of ROS values, apply the formula to the corresponding data sets to calculate the ROS generated at each time interval, which can be combined by applying the formula sum to calculate the total amount generated. A single hole or two halves of a leaf disc were placed into the wells of a 96 well microtiter plate pretreated with 100 microliters of double distilled water for 10 to 12 hours and then treated with Flg22 for ROS induction. The reading values from the two half disc samples are much higher than that from the whole leaf disc.On average, the total values from the two half disc samples are almost 1.6 times that from the whole leaf disc, which is proportional to the edge length, not to the area of the samples. This result supports that ROS are mainly generated in cells at the wound site. When attempting with this procedure, operate with tissues gently and do not make extra cuts out which could be a source of a data variation.This method can be applied to any other ROS-producing physiological processes in plant tissues.

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3.1K Views.  Shanghai Jiao Tong University. Questo protocollo descrive un metodo di chemiluminescenza ottimizzato basato su L-012 per rilevare la produzione di ROS in tempo reale dopo l'elicitazione di PAMP nei tessuti di riso. Questo metodo è semplice, standardizzato e altamente riproducibile in condizioni saldamente controllate. A dimostrare questa procedura sarà On Ying, uno studente di dottorato del mio laboratorio.Per iniziare, sterilizzare i semi di riso decorticati con etanolo al 70% per un minuto, quindi con ipoclorito...