The authors thank Dr. Dario Bonetta for providing Arabidopsis thaliana seeds and for technical assistance in regards to the triple response assay, as well as Simone Quaranta for help with FT-IR. This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (NSERC-DG) to JLS, an Ontario Graduate Scholarship (OGS) to RVA, and a Queen Elizabeth II Graduate Scholarship in Science and Technology (QEII-GSST) to AJV.

1. Chemicals
<ol>
	<li>Prepare a solution of 500 mM CEPA (144.49 g/mol), and a solution consisting of both 500 mM NaCl (58.44 g/mol) and 500 mM NaH2PO4&#183;H2O (137.99 g/mol) in acidified (pH 2.5) ultra-pure water or 0.1 N HCl. Mix using a vortex until the solutions are clear.</li>
	<li>Serially dilute (10x) the 500 mM solutions in the same solvent to obtain 5 mM and 50 mM stocks.</li>
	<li>Prepare a 10 mM solution of 1-aminocyclopropane carboxylic acid (ACC; 101.1 g/mol) in ultra-pure...

<ol>
	<li>Guzm&#225;n, P., Ecker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+triple+response+of+Arabidopsis+to+identify+ethylene-related+mutants.">Exploiting the triple response of Arabidopsis to identify ethylene-related mutants.</a> Plant Cell. 2 (6), 513-523 (1990).</li><li>Bakshi, A., Shemansky, J. M., Chang, C., Binder, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+research+on+the+plant+hormone+ethylene.">History of research on the plant hormone ethylene.</a> J. Plant Growth Regul. 34 (4), 809-827 (2015).</li><li>Schaller, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethylene+and+the+regulation+of+plant+development.">Ethylene and the regulation of plant development.</a> BMC Biol. 10 (1), (2012).</li><li>Hua, J., Sakai, 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=EIN4+and+ERS2+are+members+of+the+putative+ethylene+receptor+gene+family+in+Arabidopsis.">EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis.</a> Plant Cell. 10 (8), 1321-1332 (1998).</li><li>Bleecker, A. B., Estelle, M. A., Somerville, C., Kende, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insensitivity+to+ethylene+conferred+by+a+dominant+Mutation+in+Arabidopsis+thaliana.">Insensitivity to ethylene conferred by a dominant Mutation in Arabidopsis thaliana.</a> Science. 241 (4869), 1086-1089 (1988).</li><li>Hamilton, A. J., Bouzayen, M., Grierson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+tomato+gene+for+the+ethylene-forming+enzyme+by+expression+in+yeast.">Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast.</a> Proc. Natl. Acad. Sci. 88 (16), 7434-7437 (1991).</li><li>Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+ethylene+removal+with+Pseudomonas+strains.">Assessment of ethylene removal with Pseudomonas strains.</a> J. Hazard. Mater. 131 (3), 131-136 (2006).</li><li>Kim, H. E., Shitashiro, M., Kuroda, A., Takiguchi, N., Kato, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethylene+chemotaxis+in+Pseudomonas+aeruginosa+and+other+Pseudomonas+species.">Ethylene chemotaxis in Pseudomonas aeruginosa and other Pseudomonas species.</a> Microbes Environ. 22 (2), 186-189 (2007).</li><li>Augimeri, R. V., Strap, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phytohormone+ethylene+enhances+bacterial+cellulose+production,+regulates+CRP/FNRKx+transcription+and+causes+differential+gene+expression+within+the+cellulose+synthesis+operon+of+Komagataeibacter+(Gluconacetobacter)+xylinus+ATCC+53582.">The phytohormone ethylene enhances bacterial cellulose production, regulates CRP/FNRKx transcription and causes differential gene expression within the cellulose synthesis operon of Komagataeibacter (Gluconacetobacter) xylinus ATCC 53582.</a> Front. Microbiol. 6, 1459 (2015).</li><li>Zhang, W., Wen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+ethylene+gas+and+comparison+of+ethylene+responses+induced+by+ethylene,+ACC,+and+ethephon.">Preparation of ethylene gas and comparison of ethylene responses induced by ethylene, ACC, and ethephon.</a> Plant Physiol. Biochem. 48 (1), 45-53 (2010).</li><li>Zhang, W., Hu, W., Wen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethylene+preparation+and+its+application+to+physiological+experiments.">Ethylene preparation and its application to physiological experiments.</a> Plant Signal. Behav. 5 (4), 453-457 (2010).</li><li>Warner, H. L., Leopold, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethylene+evolution+from+2-chloroethylphosphonic+acid.">Ethylene evolution from 2-chloroethylphosphonic acid.</a> Plant Physiol. 44 (1), 156-158 (1969).</li><li>Biddle, E., Kerfoot, D. G. S., Kho, Y. H., Russell, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+studies+of+the+thermal+decomposition+of+2-chloroethylphosphonic+acid+in+aqueous+solution.">Kinetic studies of the thermal decomposition of 2-chloroethylphosphonic acid in aqueous solution.</a> Plant Physiol. 58 (5), 700-702 (1976).</li><li>Klein, I., Lavee, S., Ben-Tal, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+water+vapor+pressure+on+the+thermal+decomposition+of+2-chloroethylphosphonic+acid.">Effect of water vapor pressure on the thermal decomposition of 2-chloroethylphosphonic acid.</a> Plant Physiol. 63 (3), 474-477 (1979).</li><li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bio+assays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bio assays with tobacco tissue cultures.</a> Physiol. Plant. 15 (3), 473-497 (1962).</li><li>Schramm, M., Hestrin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+production+of+cellulose+at+the+air/liquid+interface+of+a+culture+of+Acetobacter+xylinum.">Factors affecting production of cellulose at the air/liquid interface of a culture of Acetobacter xylinum.</a> J. Gen. Microbiol. 11 (1), 123-129 (1954).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9 (7), 671-675 (2012).</li><li>Ciolacu, D., Ciolacu, F., Popa, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amorphous+cellulose-structure+and+characterization.">Amorphous cellulose-structure and characterization.</a> Cellul. Chem. Technol. 45 (1), 13-21 (2011).</li></ol>

The methods described here outline the in situ production of ethylene from CEPA for the study of bacterial ethylene response using the model organism, K. xylinus. This method is very useful as ethylene can be produced by supplementing any aqueous medium that has a pH greater than 3.510,11 with CEPA negating the need for pure ethylene gas or specialized laboratory equipment. This method is not limited to studying the effects of CEPA-derived ethylene on bacteria but can be also be adapted to st...

The olefin ethylene (C2H4) was first discovered as a plant hormone in 1901 when it was observed that pea seedlings, grown in a laboratory that used coal gas lamps, exhibited an abnormal morphology in which stems (hypocotyls) were shorter, thicker and bent sideways compared to normal pea seedlings; a phenotype later termed the triple response1,2. Subsequent studies demonstrated that ethylene is a vital phytohormone that regulates numerous developmental processes such as growth, stress response, fruit ripening and senescence3. Arabidopsis thaliana, a model organism for plant biology research, has been well studied in regards to its response to ethylene. Several ethylene response mutants have been isolated by exploiting the triple response phenotype observed in dark-grown A. thaliana seedlings in the presence of ethylene1,4,5. The biosynthetic precursor for ethylene production in plants is 1-aminocyclopropane carboxylic acid (ACC)6 and is commonly used during the triple response assay to increase endogenous ethylene production that leads to the triple response phenotype1,4,5.
Although the ethylene response is widely studied in plants, the effect of exogenous ethylene on bacteria is vastly understudied despite the close association of bacteria with plants. One study reported that certain Pseudomonas strains can survive using ethylene as a sole source of carbon and energy7. However, only two studies have demonstrated that bacteria respond to ethylene. The first study showed that strains of Pseudomonas aeruginosa, P. fluorescens, P. putida, and P. syringae were chemotactic toward ethylene using an agarose plug assay in which molten agarose was mixed with a chemotaxis buffer equilibrated with pure ethylene gas8. However, to our knowledge, there have been no further reports using pure ethylene gas to characterize bacterial ethylene response, likely due to the difficultly of handling gases in the laboratory without specialized equipment. The second report of bacterial ethylene response demonstrated that ethylene increased bacterial cellulose production and influenced gene expression in the fruit-associated bacterium, Komagataeibacter (formerly Gluconacetobacter) xylinus9. In this case, the ethylene-releasing compound, 2-chloroethylphosphonic acid (CEPA) was used to produce ethylene in situ within the bacterial growth medium, bypassing the need for pure ethylene gas or specialized equipment.
CEPA produces ethylene at a 1:1 molar ratio above pH 3.510,11 through a base-catalyzed, first-order reaction12-14. The degradation of CEPA is positively correlated with pH and temperature13,14 and results in the production of ethylene, chloride and phosphate. CEPA provides researchers interested in studying bacterial responses to ethylene with a convenient alternative to gaseous ethylene.
The overall goal of the following protocols is to provide a simple and efficient method to study bacterial ethylene response and includes validation of physiologically relevant levels of ethylene production from CEPA decomposition in bacterial growth medium, analysis of culture pH to ensure CEPA decomposition is not impaired during bacterial growth, and assessment of the effect of ethylene on bacterial morphology and phenotype. We demonstrate these protocols using K. xylinus, however, these protocols can be adapted to study ethylene response in other bacteria by using the appropriate growth medium and phenotype analyses.

<table border="0" cellpadding="0" cellspacing="0" width="1018">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">1-aminocyclopropane carboxylic acid (ACC)</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">A3903</td>
			<td style="width:432px;">Biosynthetic precursor of ethylene in plants</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">4-sector Petri dish</td>
			<td style="width:136px;">Phoenix Biomedical</td>
			<td style="width:119px;">CA73370-022</td>
			<td>For testing triple response</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Agar</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">AGR001.1</td>
			<td>To solidify medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Canon Rebel T1i DLSR camera</td>
			<td style="width:136px;">Canon</td>
			<td style="width:119px;">3818B004</td>
			<td>For pictures of pellicles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Cellulase from Trichoderma reesei ATCC 26921&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">C2730</td>
			<td>Aqueous solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Citric acid</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">CIT002.500</td>
			<td>For SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Commercial bleach</td>
			<td style="width:136px;">Life Brand</td>
			<td style="width:119px;">57800861874</td>
			<td>Bleach for seed sterilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Concentrated HCl</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">HCL666.500</td>
			<td>Hydrochloric acid for pH adjustment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Digital USB microscope</td>
			<td style="width:136px;">Plugable</td>
			<td style="width:119px;">N/A</td>
			<td>For pictures of colonies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ethephon (&#8805; 96%; 2-chloroethylphosphonic acid)</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">C0143</td>
			<td>Ethylene-releasing compound</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Glucose</td>
			<td style="width:136px;">BioBasic</td>
			<td style="width:119px;">GB0219</td>
			<td>For SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Komagataeibacter xylinus ATCC 53582</td>
			<td style="width:136px;">ATCC</td>
			<td style="width:119px;">53582</td>
			<td>Bacterial cellulose-producing alphaproteobacterium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Microcentrifuge tube</td>
			<td style="width:136px;">LifeGene</td>
			<td style="width:119px;">LMCT1.7B</td>
			<td>1.7 mL microcentrifuge tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Murashige and Skoog (MS) basal medium&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">M5519</td>
			<td>Arabidopsis thaliana growth medium</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Na2HPO4&#183;7H2O&#160;</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SPD579.500</td>
			<td>Sodium phosphate, dibasic heptahydrate for SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">NaCl</td>
			<td style="width:136px;">BioBasic</td>
			<td style="width:119px;">SOD001.1</td>
			<td>Sodium chloride for saline and control solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">NaH2PO4&#183;H2O&#160;</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SPM306.500</td>
			<td>Sodium phosphate, monobasic monohydrate for control solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">NaOH</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SHY700.500</td>
			<td>Sodium hydroxide for pH adjustment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Paraffin film</td>
			<td style="width:136px;">Parafilm</td>
			<td style="width:119px;">PM996</td>
			<td>For sealing plates and flasks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Peptone (bacteriological)</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">PEP403.1</td>
			<td>For SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Petroff-Hausser counting chamber</td>
			<td style="width:136px;">Hausser scientific</td>
			<td style="width:119px;">3900</td>
			<td>Bacterial cell counting chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Polyethersulfone sterilization filter 0.2 &#181;m</td>
			<td style="width:136px;">VWR</td>
			<td style="width:119px;">28145-501</td>
			<td>For sterilizing cellulase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sucrose</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SUC600.1</td>
			<td>Sucrose for MS medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Yeast extract</td>
			<td style="width:136px;">BioBasic</td>
			<td style="width:119px;">G0961</td>
			<td>For SH medium</td>
		</tr>
	</tbody>
</table>

utilizing the ethylene-releasing compound, 2-chloroethylphosphonic acid, as a tool to study ethylene response in bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

A schematic plate setup for verification of ethylene liberation from CEPA in SH medium (pH 7) by the triple response assay is shown in Figure 1A-C. A flow-chart illustrating the pellicle protocol is shown in Figure 2. Dark-grown A. thaliana seedlings exhibit the triple response phenotype (shorter and thicker hypocotyl with an exaggerated apical hook) in the presence of ACC and in the presence of ethylene produced through the dec...

Watch this Scientific Journal Video about Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria at JoVE.com

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

The overall goal of this procedure is to provide a simple and safe method for the study of bacterial response to ethylene. This method will answer key questions regarding plant-microbe interactions, such as the role that ethylene plays in bacterial adhesion and colonization of plants. The main advantage of this technique is that it does not require any specialized equipment and it's much safer than handling gaseous ethylene in the lab.Although this method can provide insight into how bacterial cellulose production is affected by ethylene, it can also be applied to other plant-associated microorganisms for the in vitro study of how ethylene-mediated processes are involved in plant colonization. In preparation, make 100 milliliters of growth medium for the Arabidopsis seedlings and make 100 milliliters of bacterial growth medium for the CEPA decomposition. Once the arabidopsis medium has tempered, divide 40 milliliters out to a sterile flask and supplement the aliquot with 40 microliters of 10 millimolar ACC.Now, load the plate quadrants with five milliliters of medium and allow the agar to solidify. Opposing quadrants of all plates have bacterial growth medium and arabidopsis medium. Use three different medium configurations.The CEPA treatment will occur later in the protocol. Prepare each plate configuration in triplicate. Once the agar has cooled, evenly distribute approximately 50 seeds onto each quadrant of arabidopsis medium.Then, incubate the plates in the dark at four degrees Celsius for three to four days. Later, after incubating the seeds, expose them to fluorescent light for two hours. Then, spread 10 microliters of 500 millimolar CEPA stock solution onto the bacterial agar of one plate configuration.The final CEPA concentration in the agar will be one millimolar. Now, seal all the plates with laboratory film and wrap them in foil. Then, place the plates in a dark environment at 23 degrees Celsius with the agar side down and let them incubate for three days to germinate the seeds.After the seeds germinate, use flame sterilized forceps to remove seedlings corresponding to each treatment. Select 30 seedlings from each quadrant for a total of 180 per treatment. Image the seedlings under a dissecting microscope or with a high resolution digital camera against a black background next to a ruler for scale.Then, measure their hypocotyl length using ImageJ software. Using the straight tool, measure 10 millimeters and select Set Scale under the Analyze tab to set the scale. Then, use the Segmented tool under the Straight Line tool to measure each hypocotyl and press M to calculate their lengths.The pellicle assay is a standard analysis tool for bacterial cellulose production. PH and morphological analysis are detailed in sex protocol. In preparation, harvest cells from starter cultures in triplicate and quantify the cells using a Petroff-Hauser Counting Chamber.Then, prepare four medium master mixes with different CEPA concentrations. For each master mix, eliquate 60 milliliters of pH 7 SH medium and add 120 microliters of either zero, five, 50 or 500 millimolar CEPA stock solution to obtain final CEPA concentrations of zero, 01, 0.1, or 1.0 millimolar. Mix these media by swirling the flasks.One set of media is needed for each of the triplicates and a fourth set is needed to function as a sterile control. So, divide each 60 milliliter medium preparation into four 14 milliliter eliquates. Then, pre-cool the 14 milliliter eliquates before inoculating with their respective starter cultures for a final concentration of 100, 000 cells per milliliter.Keep all the inoculated tubes on ice to prevent cellulose production. Use the remaining 14 milliliter eliquates for sterile control wells. Now, from each 14 milliliter master mix, load two milliliter eliquates into six wells of a sterile 24 well plate for a total of four loaded plates per starter culture analyzed.Carefully seal the plates with paraffin film and incubate them statically for seven days at 30 degrees Celsius. During the incubation, incubate a 25 milliliter liquid culture in parallel to periodically measure the medium's acidity. If the acidity drops below a PH of five, then the organism's growth will inhibit the decomposition of CEPA to ethylene and is thus incompatible with this assay.A week later, harvest and measure pellicle wet weight, thickness, dry weight and crystallinity. To collect a pellicle from the plate, depress one side of the pellicle to elevate the opposing edge and pick it up with forceps. While retaining grip, place them on a fresh paper towel for three seconds to remove excess medium.Then, weigh each pellicle and record this as the wet weight. Next, align the pellicles along a ruler and photograph them from the side using a high resolution digital camera. Analyze these photos for the pellicles'thicknesses.Next, individually transfer the pellicles into the wells of a six well plate. Treat them with 12 milliliters of 0.1 normal sodium hydroxide at 80 degrees Celsius for 20 minutes to lice the cells. Then, remove the sodium hydroxide and neutralize the treated pellicles by thoroughly washing them with 12 milliliters of ultra pure water for 24 hours with agitation in a six well plate.During this incubation, change the water every six hours. When the incubation is over, the pellicles should be white. Then, place the pellicles on silicon mats and dry them at 50 degrees Celsius for 48 hours to prepare them for dry weight measurement.The pellicles'crystallinity can be analyzed using Fourier-transform infrared spectroscopy. Dark grown arabidopsis thaliana seedlings exhibited the triple response phenotype of a short, thick hypocotyl with an exaggerated apical hook in the presence of ACC and in the presence of ethylene produced through the decomposition CEPA on SH medium, but not under untreated conditions. Clearly, the hypocotyl length of the treated seeds was significantly less than the untreated controls.Thus, ethylene was released from CEPA on SH medium at a physiologically relevant concentration. All concentrations of CEPA derived ethylene significantly decreased pellicle wet weight, but did not affect pellicle thickness, and all concentrations of CEPA derived ethylene significantly increased pellicle dry weight. Furthermore, pellicle hydration was reduced by all concentrations of CEPA derived ethylene.While attempting this procedure, it's important to measure the PH of the growth medium to ensure that the PH does not drop below five so that CEPA degradation into ethylene is not inhibited. Remember that powdered CEPA is extremely hazardous to mucous membranes. Proper care should always be taken when performing this protocol.This technique will enable researchers who are interested in plant bacteria interactions to safely explore ethylene mediated responses in vitro. This will help shed light on how plant signals mediate bacterial colonization and adhesion. Currently, this area has been under studied.

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JoVE Journal

Biochemistry

7.8K vues.  University of Ontario Institute of Technology. The overall goal of this procedure is to provide a simple and safe method for the study of bacterial response to ethylene. This method will answer key questions regarding plant-microbe interactions, such as the role that ethylene plays in bacterial adhesion and colonization of plants. The main advantage of this technique is that it does not require any specialized equipment and it's much safer than handling gaseous ethylene in the lab.Although this method can provide insight into how b...