Name: forward genetic screen using transgenic calcium reporter aequorin to identify novel targets in calcium signaling
Uploaded: 2020-08-01T13:00:00
Description: Watch this Scientific Journal Video about Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling at JoVE.com

We thank National Institute of Plant Genome Research - Phytotron Facility for plant growth, Bombay Locale for the video shoot, and the Department of Biotechnology- eLibrary Consortium for providing access to e-resources. This work was supported by the Department of Biotechnology, India through the National Institute of Plant Genome Research Core Grant, Max Planck Gesellschaft-India Partner Group program; and CSIR-Junior Research Fellowship (to D.M and S.M) and Department of Biotechnology-Junior Research Fellowship (to R.P).

1. EMS mutagenesis and single pedigree-based seed collection (1-3 months)
<ol>
	<li>Weigh 150 mg of seeds (~7500) of aequorin for EMS mutagenesis (M0 seeds). Weigh another 150 mg of seeds to be used as a control.</li>
	<li>Transfer the seeds to a 50 mL tube and add 25 mL of 0.2% EMS (v/v) (CAUTION) (for treatment) or 25 mL of autoclaved water (for control). 
	NOTE: Ethyl-methane sulfonate is a chemical agent for mutagenizing plant material.</li>
	<li>Seal the tube with parafilm and wrap it in aluminum f...

<ol>
	<li>Kudla, J., Batistic, O., Hashimoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signals:+The+lead+currency+of+plant+information+processing.">Calcium signals: The lead currency of plant information processing.</a> The Plant Cell. 22 (3), 541-563 (2010).</li><li>Wasternack, C., Hause, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jasmonates:+biosynthesis,+perception,+signal+transduction+and+action+in+plant+stress+response,+growth+and+development.+An+update+to+the+2007+review+in+Annals+of+Botany.">Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany.</a> Annals of Botany. 111 (6), 1021-1058 (2013).</li><li>Kiep, V., 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=Systemic+cytosolic+Ca2++elevation+is+activated+upon+wounding+and+herbivory+in+Arabidopsis.">Systemic cytosolic Ca2+ elevation is activated upon wounding and herbivory in Arabidopsis.</a> New Phytologist. 207 (4), 996-1004 (2015).</li><li>Zhu, X., 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=Aequorin-based+Luminescence+imaging+reveals+stimulus-+and+tissue-specific+Ca2++dynamics+in+Arabidopsis+plants.">Aequorin-based Luminescence imaging reveals stimulus- and tissue-specific Ca2+ dynamics in Arabidopsis plants.</a> Molecular Plant. 6 (2), 444-455 (2013).</li><li>White, P. J., Broadley, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+in+plants.">Calcium in plants.</a> Annals of Botany. 92, 487-511 (2003).</li><li>Johnson, J. M., Reichelt, M., Vadassery, J., Gershenzon, J., Oelmueller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+mutant+impaired+in+intracellular+calcium+elevation+is+sensitive+to+biotic+and+abiotic+stress.">An Arabidopsis mutant impaired in intracellular calcium elevation is sensitive to biotic and abiotic stress.</a> BMC Plant Biology. 14 (1), 162 (2014).</li><li>Dodd, A. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signals+in+plant+immunity:+a+spiky+issue.">Calcium signals in plant immunity: a spiky issue.</a> New Phytologist. 204 (4), 733-735 (2014).</li><li>Marcec, M. J., Gliroy, S., Poovaiah, B. W., Tanaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutual+interplay+of+Ca2++and+ROS+signaling+in+plant+immune+response.">Mutual interplay of Ca2+ and ROS signaling in plant immune response.</a> Plant Science. 283, 343-354 (2019).</li><li>McAnish, M. R., Pittman, J. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+plant+aequorin+reports+the+effects+of+touch+and+cold-shock+and+elicitors+on+cytoplasmic+calcium.">Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium.</a> Nature. 352 (6335), 524-526 (1991).</li><li>Tanaka, K., Choi, J., Stacey, G., Running, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aequorin+Luminescence-Based+Functional+Calcium+Assay+for+Heterotrimeric+G-Proteins+in+Arabidopsis.">Aequorin Luminescence-Based Functional Calcium Assay for Heterotrimeric G-Proteins in Arabidopsis.</a> G Protein-Coupled Receptor Signaling in Plants. Methods in Molecular Biology (Methods and Protocols). , 45-54 (2013).</li><li>Mith&#246;fer, A., Ebel, J., Bhagwat, A. 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H., 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=Ethyl+methane+sulfonate+induced+mutations+in+M2+generation+and+physiological+variations+in+M1+generation+of+peppers+(Capsicum+annuum+L.).">Ethyl methane sulfonate induced mutations in M2 generation and physiological variations in M1 generation of peppers (Capsicum annuum L.).</a> Frontiers in Plant Science. 6, 399 (2015).</li><li>Ranf, 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=Defense-related+calcium+signaling+mutants+uncovered+via+a+quantitative+high-throughput+screen+in+Arabidopsis+thaliana.">Defense-related calcium signaling mutants uncovered via a quantitative high-throughput screen in Arabidopsis thaliana.</a> Molecular Plant. 5 (1), 115-130 (2012).</li><li>Rentel, M. C., Knight, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress-induced+calcium+signalling+in+Arabidopsis.">Oxidative stress-induced calcium signalling in Arabidopsis.</a> Plant Physiology. 135 (3), 1471-1479 (2004).</li><li>Jankowicz-Cieslak, J., Till, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+mutagenesis+of+seed+and+vegetatively+propagated+plants+using+EMS.">Chemical mutagenesis of seed and vegetatively propagated plants using EMS.</a> Current Protocols in Plant Biology. 1 (4), 617-635 (2016).</li><li>Espina, M. J., 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=Development+and+Phenotypic+Screening+of+an+Ethyl+Methane+Sulfonate+Mutant+Population+in+Soybean.">Development and Phenotypic Screening of an Ethyl Methane Sulfonate Mutant Population in Soybean.</a> Frontiers in Plant Science. 9, 394 (2018).</li><li>Weigel, D., Glazebrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+Genetics+in+Arabidopsis:+Finding+Mutations+that+Cause+Particular+Phenotypes.">Forward Genetics in Arabidopsis: Finding Mutations that Cause Particular Phenotypes.</a> Cold Spring Harbor Protocols. 5, (2006).</li><li>Maple, J., Moeller, S. 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J., Flanders, D., Dean, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMS+Mutagenesis+of+Arabidopsis.">EMS Mutagenesis of Arabidopsis.</a> Arabidopsis: the Complete Guide (Electronic v. 1.4). , 9-10 (1993).</li><li>Pauly, 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=The+nucleus+together+with+the+cytosol+generates+patterns+of+specific+cellular+calcium+signatures+in+tobacco+suspension+culture+cells.">The nucleus together with the cytosol generates patterns of specific cellular calcium signatures in tobacco suspension culture cells.</a> Cell Calcium. 30 (6), 413-421 (2001).</li><li>Mith&#246;fer, A., Mazars, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aequorin-based+measurements+of+intracellular+Ca2+-signatures+in+plant+cells.">Aequorin-based measurements of intracellular Ca2+-signatures in plant cells.</a> Biological Procedures Online. 4 (1), 105-118 (2002).</li><li>Mehlmer, 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=A+toolset+of+aequorin+expression+vectors+for+in+planta+studies+of+subcellular+calcium+concentrations+in+Arabidopsis+thaliana.">A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana.</a> Journal of Experimental Botany. 63 (4), 1751-1761 (2012).</li><li>Knight, H., Trewavas, A. 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EMS mutagenesis is a powerful tool to generate mutations in population. The classical forward genetic screens using EMS has been an effective tool to identify novel genes for two major reasons: firstly, they do not require any prior assumptions on gene identity and secondly, they do not introduce any bias. There are several methods to generate a screening populations like EMS, T-DNA insertions, radiations etc. Out of all the methods, EMS-based mutagenesis has few advantages over the other methods. First, it is easier to ...

A change in cytosolic calcium (Ca2+) concentration upon perception of biotic or abiotic stimulus is a well-studied early signaling event that activates many signaling pathways1,2,3,4. A cell in its basal resting state maintains a lower Ca2+ concentration in the cytosol and sequesters excess Ca2+ in various intracellular organelles and extracellular apoplast leading to a steep Ca2+ gradient5,6. Upon signal perception, Ca2+ levels rise in the cytosol due to an influx of Ca2+ from extracellular and/or intracellular sources and generate a stimuli specific calcium signature7,8,9. Ca2+ elevations in the cytosol are activated by many stimuli, but specificity is maintained by distinct stores releasing Ca2+, a unique Ca2+ signature and appropriate sensor proteins10,11.
The use of alkylating agent, ethyl-methane sulfonate (EMS) for mutagenesis is a powerful tool in classical forward genetic screens to identify multiple independent genes involved in a process. EMS is a chemical mutagen predominantly inducing C to T and G to A transitions randomly throughout the genome and produces a 1 bp change in every 125 kb of the genome. EMS mutagenesis will induce &#8776;1000 single base pair changes, either insertion/deletions (InDel) or single nucleotide polymorphism (SNP) per genome12. EMS-induced mutations are multiple point mutations with a mutation frequency ranging from 1/300 to 1/30000 per locus. This reduces the number of M1 plants needed to find a mutation in a given gene. A M1 seed population range of 2000-3000 is typically used to obtain mutations of interest in Arabidopsis thaliana13,14.
Aequorin transgenics are Arabidopsis Columbia-0 (Col-0) ecotype plants expressing p35S-apoaequorin (pMAQ2) in the cytosol15. Aequorin is a Ca2+ binding protein composed of apoprotein and a prosthetic group consisting of luciferin molecule, coelenterazine.&#160;The binding of Ca2+ to aequorin, which has three Ca2+ binding EF-hands sites, results in coelenterazine being oxidized and cyclized to give the dioxetanone intermediate, followed by a conformational change of the protein accompanied by the release of carbon dioxide and singlet-excited coelenteramide16. The coelenteramide so produced emits a blue light (&#955;max, 470 nm) that can be detected by the luminometer17. The extremely fast Ca2+ elevations can thus be measured in real time, and exploited for rapid forward genetic screens. This protocol aims to use the specificity of calcium response to identify novel key players that are involved in the Ca2+ signature. To achieve this task, we use EMS mutagenesis in transgenic aequorin and identify the SNPs associated with altered Ca2+ signaling. The protocol identifies mutants that show no or reduced Ca2+ elevations upon stimuli addition. These mutants can then be mapped to identify the genes responsible for the Ca2+ response. The method is applicable to any kind of liquid stimuli in plants that results in a Ca2+ elevation. Since Ca2+ elevation is one of the first responses in the plant defense signaling pathway, the identification of upstream response components can provide candidates for genetic engineering to develop resilient plants.

<table border="0" cellpadding="0" cellspacing="0" style="width:695px;" width="694">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">24 well tissue culture plate</td>
			<td style="width:125px;">Jetbiofil</td>
			<td style="width:116px;">11024</td>
			<td style="width:241px;">for growing seedlings</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well white cliniplate</td>
			<td>Thermo Scientific</td>
			<td>9502887</td>
			<td>for luminometer measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aequorin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agropet</td>
			<td>Lab Chem India</td>
			<td></td>
			<td>for plant growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>12135</td>
			<td>for discharge solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coelenterazine</td>
			<td>PJK</td>
			<td>55779-48-1</td>
			<td>prosthetic group for aequorin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ehtylmethane sulfonate</td>
			<td>Sigma Aldrich</td>
			<td>M0880-5G</td>
			<td>for seed mutagenesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Analytical reagent</td>
			<td>1170</td>
			<td>for discharge solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td>Merck Life Sciences</td>
			<td>1.93001.0521</td>
			<td>sterlization solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrogen peroxide</td>
			<td>Fisher Scientific</td>
			<td>15465</td>
			<td>as stimulus for Calcium elevation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luminoskan ascent</td>
			<td>Thermo Scientific</td>
			<td>5300172</td>
			<td>aequorin luminescence measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MES buffer</td>
			<td>Himedia</td>
			<td>RM1128-100G</td>
			<td>plant growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Murashige and skoog media</td>
			<td>Himedia</td>
			<td>PT021-25L</td>
			<td>plant growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>27805</td>
			<td>for neutralizing EMS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hypochlorite</td>
			<td>Merck Life Sciences</td>
			<td>1.93607.5021</td>
			<td>sterlization solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium thiosulfate</td>
			<td>Fisher Scientific</td>
			<td>28005</td>
			<td>for seed washing in step 1.6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">soilrite</td>
			<td>Lab Chem India</td>
			<td></td>
			<td>for plant growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Square pots</td>
			<td>Lab Chem India</td>
			<td></td>
			<td>for plant growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389</td>
			<td>plant growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taxim</td>
			<td>Alkem</td>
			<td>7180720</td>
			<td>for seedling rescue</td>
		</tr>
	</tbody>
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forward genetic screen using transgenic calcium reporter aequorin to identify novel targets in calcium signaling

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets.

The EMS population was screened for H2O2 induced Ca2+ elevation. As discussed earlier, 12 individual M2 seedlings were screened from each M1 line. In Figure 3, one such M1 line is plotted with each panel showing 12 individual M2 seedlings. A wild-type aequorin is used as control for comparing and evaluating the mutant response. A recessive mutant segregates in the ratio of 1:7 (mutant: non mutant). When screening 12...

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Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

A forward genetic screen based on Ca2+ elevation as a read-out leads to identification of genetic components involved in calcium dependent signaling pathways in plants.

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis ...

The aim of this protocol is to identify a method which can be used for identifying novel genes which are involved in calcium signaling. To do this, we use the classical forward genetic screen. Calcium signaling is an important defense response pathway in several plant systems.In order to identify the genes involved in this pathway, we have used the classical forward genetic screen. In this method, EMS would be used as an alkylating agent to introduce random mutations in the plant systems. The developed mutant generation can then be used for screening for a phenotype of interest.Once the phenotype of interest has been identified, the causal gene can be mapped. In this study, we use the model plant Arabidopsis which has the calcium reporter aequorin in its spectrum. Weigh 150 mg seeds of aequorin for EMS mutagenesis and another 150 mg seeds to be used as control.Transfer the seeds to a 50 ml Falcon tube and add 2%EMS or autoclaved water. While using EMS, be careful because it is carcinogenic. Protective gears should be worn while using EMS and any kind of spillage should be prevented.Seal the Falcon tubes with paraffin and wrap it aluminum foil. Rotate the tube end over end for 18 hours at room temperature. Allow the seeds to settle and remove the EMS solution carefully and discard in a waste container containing one molar NaOH.Wash the mutagenized seeds thoroughly with 40 ml of autoclaved water at least eight times. For the final wash, add 100 millimolar sodium thiosulfate and rinse for at least three times to remove traces of EMS. Soak the seeds in 40 ml of autoclaved water for one hour to diffuse EMS out of the seeds and then place them on Whatman paper until completely dry.Transfer the seeds to Eppendorf and incubate at four degrees Celsius for two to four days for stratification. Transfer both the mutagenized seeds and water-treated seeds onto soil and transfer them to growth rooms with 16-hour light and eight-hour dark photo period at 22 degrees Celsius and 70%relative humidity. In order to determine if mutagenesis was successful, look for chlorophyll sectoring.We have used the single pedigree-based seed collection method and each M1 plant is given a unique number. Upon maturation, seeds are harvested from these individual mutant plants and stored as individual M1 lines. A high throughput seed sterilization and hydroponic plant root protocol is used, adapted from Ranf et al.On day one, place nearly 12 to 15 M2 seeds per M1 one line in individual wells of a 24-well tissue culture plate and sterilize using sodium hypochlorite and hydrochloric acid solution at a ratio of three is to one. This releases chlorine gas that sterilizes the seeds. Leave the plate for overnight to evaporate remaining chlorine gas completely.After sterilization, add half strength liquid MS media to individual wells and stratify the seeds for two to four days at four degrees Celsius, and then move the seeds to a growth chamber with photo period of 10-hour light and 14-hour dark at 22 degrees Celsius, 70%relative humidity. On day 14, once the seedlings are eight to 12 days old, place 12 M2 seedlings from each line individually in a 96-well luminometer plate. After seedling transfer, add 150 microliters of five micromolar Coelenterazine solution in individual wells and store in dark at 21 degrees Celsius for eight hours.Coelenterazine is a prosthetic group which binds to apoaequorin to form functional aequorin. Coelenterazine is light sensitive and is hence stored in dark colored bottles protected from light. Next day, screen mutants using hydrogen peroxide as stimulus and measure subsequent calcium elevation.For simultaneous measurement of 24 wells, an out automated kinetic program is made, which includes measuring the background for one minute, followed by stimulus addition and measurement for 10 minutes, followed by discharge injection 150 microliter and measurement for three to five minutes. And any discharge for the daughter aequorin would be included in the reading to quantify the measured calcium and as additional control for functional aequorin. The readings provided from the above method is in relative light units.For calculating cytosolic calcium ion concentration, a previously described concentration equation given by Rental and Knight in 2004 is used, which is described in the protocol. The RLUs obtained from luminometer are added to the equation to calculate calcium ion concentration. The obtained cytosolic calcium ion values are then graphically plotted against a wild-type control for identifying putative hydrogen peroxide mutants to calcium response.This is a representative result of the protocol shown. We've shown one M1 line with 12 individual seedlings which have been measured for elevation of calcium ion concentration. Green line in the graph indicates wild-type aequorin which was measured along with mutants.Red is average of all the 12 seedlings, and black line is individual M2 seedlings measured for the calcium ion elevation. Seedlings showing a highly reduced response to hydrogen peroxide application are then rescued. Rescued mutants are then reconfirmed in the next generation, followed by mapping to identify causal genes.Caution should be given when using EMS as a mutagen since it's a carcinogen. Protective gearing should be worn at all times and any spillage done should be dealt with good laboratory practices. Additionally, when doing a pedigree-based collection of seeds, individual plants should be harvested with utmost care so that there is no mixing of any seeds at any stage.This will help one to go back and trace the mother plant, which is the homozygous mutant plant. Additionally, since this method does not introduce any bias or any prior assumptions into the screening method, it can be used for one or more conditions to identify a phenotype of interest. Additionally, multiple independent genes could also be identified using the same protocol.The protocol is easy to use since with the small dosage, a huge number of plants can be mutagenized. These huge number of mutant plants can be used for several populations and for several conditions and to identify several genes. A partial loss of function, an altered function, a complete loss of function, or also constitutive gene function can be identified through this method.Mapping should be an easy task given the advancement in mapping technologies in the current scenarios. A de novo-based mapping system, a SNP-based racial plotting and many others can be used to identify the causal gene causing the phenotype of interest. Given the applicability of this method, it can be used not just on the Arabidopsis model plants, but other model plants too provided that a phenotype of interest readout can be established.

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