Methods in molecular biology vol 1573 ethylene signaling methods and protocols

Methods in
Molecular Biology 1573

Brad M. Binder
G. Eric Schaller Editors

Ethylene
Signaling
Methods and Protocols


Methods

in

Molecular Biology

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:
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Ethylene Signaling
Methods and Protocols

Edited by

Brad M. Binder
Department of Biochemistry & Cellular and Molecular Biology,
University of Tennessee, Knoxville, TN, USA

G. Eric Schaller
Department of Biological Sciences, Dartmouth College, Hanover, NH, USA


Editors
Brad M. Binder
Department of Biochemistry & Cellular and
Molecular Biology
University of Tennessee
Knoxville, TN, USA

G. Eric Schaller
Department of Biological Sciences
Dartmouth College
Hanover, NH, USA

ISSN 1064-3745    ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6852-7    ISBN 978-1-4939-6854-1 (eBook)
DOI 10.1007/978-1-4939-6854-1
Library of Congress Control Number: 2017931962
© Springer Science+Business Media LLC 2017
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Cover illustration: Apical hook of a dark-grown Arabidopsis seedling grown in the presence of ethylene. The seedling
was visualized by collapsing multiple Z-stack images from a confocal, with red fluorescence arising from propidium
iodide staining of the cell wall. Green fluorescence arises from an EIN3-GFP reporter, this appearing predominantly
yellow due to overlap with red fluorescence. (photograph by Yan Zubo)
Printed on acid-free paper
This Humana Press imprint is published by Springer Nature
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The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.


Preface
Ethylene was the first gaseous hormone discovered, and its discovery was prompted by the
pronounced effects of “illuminating gas” on plant growth and development. Illuminating
gas, a coal by-product, was piped throughout cities during the Victorian era as a fuel source
for the lamps lighting streets and houses. Gas leaking from the pipes induced early senescence as well as leaf and petal abscission in nearby plants, which prompted a search for its
active component. In 1901, Dimitry Neljubow demonstrated that this active component
was the simple hydrocarbon ethylene. In the 1930s, Richard Gane established that plants
produced their own ethylene, establishing ethylene as an endogenous plant growth regulator. Ethylene is now most popularly known for its role in controlling fruit ripening, but
ethylene also regulates many other traits of agricultural significance including senescence,
abscission, biomass, and responses to biotic and abiotic stresses. As such, ethylene continues to be a focus for worldwide research.
This volume in the Methods in Molecular Biology series provides a collection of protocols for the research scientist appropriate to the study of ethylene signaling in plants. Topics
covered relate to ethylene biosynthesis, the signal transduction pathway, and the diverse
ethylene responses of dicots and monocots. The section on ethylene biosynthesis includes

six chapters, with techniques for the measurement of activities related to the biosynthetic
enzymes ACC synthase and ACC oxidase, for quantifying the levels of ethylene synthesized
by plants, as well as for the treatment of plants with exogenous ethylene. The section on the
signal transduction pathway includes six chapters and focuses on the analysis of the novel
membrane-associated proteins involved in the initial perception and transduction of the
ethylene signal, including the ethylene receptors, CTR1 and EIN2. Many of these biochemical techniques were derived from work in Arabidopsis where these signaling elements
were first discovered, but the approaches are readily transferable to the study of similar
proteins in other species. The section on ethylene responses includes seven chapters covering assays applicable to dicots and monocots, including methods related to the roles of
ethylene in germination, growth, abscission, abiotic stress, and defense. This section also
includes information on Arabidopsis mutants and the variety of chemical inhibitors that
affect ethylene responses.
The chapters follow the established format used throughout the Methods in Molecular
Biology™ series. They include an Abstract, an Introduction, a detailed Materials section
with lists of chemicals, buffers, and equipment, a step-by-step Methods section, as well as
Notes and References. The Notes are often of particular use to investigators as these give
additional background, provide alternative approaches, and describe potential difficulties
and how these can be resolved. The protocols are intended for both experienced and beginning researchers, for those with prior experience in the study of ethylene signaling, and for
those just entering this exciting research area.

v


vi

Preface

The editors thank their “scientific parents”: Michael Sussman who pushed them over the
edge and down that slippery slope of plant membrane biochemistry and Tony Bleecker who
enthusiastically introduced them to that deceptively simple hydrocarbon ethylene and the
myriad effects it has on plants. The editors also thank all those colleagues who so willingly

shared their protocols for this Methods in Molecular Biology volume on Ethylene Signaling.
Knoxville, TN, USA
Hanover, NH, USA

Brad M. Binder
G. Eric Schaller


Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Part I Analysis of Ethylene Biosynthesis
  1 Gas Chromatography-Based Ethylene Measurement
of Arabidopsis Seedlings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gyeong Mee Yoon and Yi-Chun Chen
  2 Plant Ethylene Detection Using Laser-Based Photo-Acoustic Spectroscopy . . .
Bram Van de Poel and Dominique Van Der Straeten
  3 Treatment of Plants with Gaseous Ethylene and Gaseous Inhibitors
of Ethylene Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mark L. Tucker, Joonyup Kim, and Chi-Kuang Wen
  4 Analysis of 1-Aminocyclopropane-1-Carboxylic Acid Uptake
Using a Protoplast System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Won-Yong Song, Sumin Lee, and Moon-Soo Soh
 5 Escherichia coli-Based Expression and In Vitro Activity Assay
of 1-Aminocyclopropane-1-Carboxylate (ACC) Synthase
and ACC Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Shigeru Satoh and Yusuke Kosugi
  6 Assay Methods for ACS Activity and ACS Phosphorylation

by MAP Kinases In Vitro and In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Xiaomin Han, Guojing Li, and Shuqun Zhang

3
11

27

41

47

59

Part II Analysis of the Ethylene Signaling Pathway
  7 Analysis of Ethylene Receptors: Ethylene-Binding Assays . . . . . . . . . . . . . . . . .
Brad M. Binder and G. Eric Schaller
  8 Analysis of Ethylene Receptors: Assay for Histidine Kinase Activity . . . . . . . . . .
G. Eric Schaller and Brad M. Binder
  9 Analysis of Ethylene Receptor Interactions
by Co-immunoprecipitation Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Zhiyong Gao and G. Eric Schaller
10 Localization of the Ethylene-Receptor Signaling Complex
to the Endoplasmic Reticulum: Analysis by Two-Phase Partitioning
and Density-Gradient Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G. Eric Schaller
11 Kinase Assay for CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1)
in Arabidopsis thaliana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Han Yong Lee and Gyeong Mee Yoon

vii

75
87

101

113

133


viii

Contents

12 Circular Dichroism and Fluorescence Spectroscopy to Study Protein
Structure and Protein–Protein Interactions in Ethylene Signaling . . . . . . . . . . . 141
Mareike Kessenbrock and Georg Groth

Part III Analysis of Ethylene Responses
13 The Triple Response Assay and Its Use to Characterize Ethylene
Mutants in Arabidopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Catharina Merchante and Anna N. Stepanova
14 Time-Lapse Imaging to Examine the Growth Kinetics
of Arabidopsis Seedlings in Response to Ethylene . . . . . . . . . . . . . . . . . . . . . . .
Brad M. Binder
15 Inhibitors of Ethylene Biosynthesis and Signaling . . . . . . . . . . . . . . . . . . . . . . .
G. Eric Schaller and Brad M. Binder
16 Analysis of Growth and Molecular Responses to Ethylene

in Etiolated Rice Seedlings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biao Ma and Jin-Song Zhang
17 Love Me Not Meter: A Sensor Device for Detecting Petal Detachment
Forces in Arabidopsis thaliana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Andrew Maule, Graham Henning, and Sara Patterson
18 Effects of Ethylene on Seed Germination of Halophyte Plants
Under Salt Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Weiqiang Li and Lam-Son Phan Tran
19 Assessing Attraction of Nematodes to Host Roots Using Pluronic
Gel Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

163

211
223

237

245

253

261

Valerie M. Williamson and Rasa Čepulytė
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269


Contributors
Brad M. Binder  •  Department of Biochemistry & Cellular and Molecular Biology,

University of Tennessee, Knoxville, TN, USA
Rasa Čepulytė  •  Department of Plant Pathology, University of California, Davis, CA, USA
Yi-Chun Chen  •  Department of Botany and Plant Pathology, Purdue University, West
Lafayette, IN, USA
Zhiyong Gao  •  State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan
University, Wuhan, China
Georg Groth  •  Institute of Biochemical Plant Physiology, Heinrich-Heine University
Düsseldorf, Düsseldorf, Germany
Xiaomin Han  •  College of Life Sciences, Inner Mongolia Agricultural University, Hohhot,
Inner Mongolia, P. R. China
Graham Henning  •  Department of Horticulture, University of Wisconsin, Madison,
WI, USA
Mareike Kessenbrock  •  Institute of Biochemical Plant Physiology, Heinrich-Heine
University Düsseldorf, Düsseldorf, Germany
Joonyup Kim  •  Department of Cell Biology and Molecular Genetics, University
of Maryland, Biosciences Research Bldg., College Park, MD, USA
Yusuke Kosugi  •  Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, Japan
Han Yong Lee  •  Department of Botany and Plant Pathology, Purdue University, West
Lafayette, IN, USA
Sumin Lee  •  Department of Integrative Bioscience and Biotechnology, College of Life
Science, Sejong University, Seoul, Republic of Korea
Guojing Li  •  College of Life Sciences, Inner Mongolia Agricultural University, Hohhot,
Inner Mongolia, P. R. China
Weiqiang Li  •  Signaling Pathway Research Unit, RIKEN Center for Sustainable Resource
Science, Yokohama, Japan
Biao Ma  •  State Key Lab of Plant Genomics, Institute of Genetics and Developmental
Biology, Chinese Academy of Sciences, Beijing, China
Andrew Maule  •  Department of Horticulture, University of Wisconsin, Madison, WI, USA
Catharina Merchante  •  Departamento de Biología Molecular y Bioquímica, Instituto de
Hortofruticultura Subtropical y Mediterranea (IHSM)-UMA-CSIC, Universidad de

Málaga, Málaga, Spain
Sara Patterson  •  Department of Horticulture, University of Wisconsin, Madison, WI, USA
Bram Van de Poel  •  Faculty of Sciences, Laboratory of Functional Plant Biology,
Department of Physiology, Ghent University, Gent, Belgium
Shigeru Satoh  •  Faculty of Agriculture, Ryukoku University, Otsu, Japan
G. Eric Schaller  •  Department of Biological Sciences, Dartmouth College, Hanover,
NH, USA

ix


x

Contributors

Moon-Soo Soh  •  Department of Integrative Bioscience and Biotechnology, College of Life
Science, Sejong University, Seoul, Republic of Korea
Won-Yong Song  •  Department of Life Science, Pohang University of Science and
Technology, Pohang, Republic of Korea
Anna N. Stepanova  •  Department of Plant and Microbial Biology, North Carolina State
University, Raleigh, NC, USA; Genetics Graduate Program, North Carolina State
University, Raleigh, NC, USA
Dominique Van Der Straeten  •  Faculty of Sciences, Laboratory of Functional Plant
Biology, Department of Physiology, Ghent University, Gent, Belgium
Lam-Son Phan Tran  •  Signaling Pathway Research Unit, RIKEN Center for Sustainable
Resource Science, Yokohama, Japan
Mark L. Tucker  •  Soybean Genomics and Improvement Lab, USDA/ARS, BARC-­West,
Beltsville, MD, USA
Chi-Kuang Wen  •  National Key Laboratory of Plant Molecular Genetics, CAS Center for
Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology,

Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Valerie M. Williamson  •  Department of Plant Pathology, University of California, Davis,
CA, USA
Gyeong Mee Yoon  •  Department of Botany and Plant Pathology, Purdue University,
West Lafayette, IN, USA
Jin-Song Zhang  •  State Key Lab of Plant Genomics, Institute of Genetics and
Developmental Biology, Chinese Academy of Sciences, Beijing, China
Shuqun Zhang  •  Division of Biochemistry, Interdisciplinary Plant Group, and Bond Life
Sciences Center, University of Missouri, Columbia, MO, USA


Part I
Analysis of Ethylene Biosynthesis


Chapter 1
Gas Chromatography-Based Ethylene Measurement
of Arabidopsis Seedlings
Gyeong Mee Yoon and Yi-Chun Chen
Abstract
Plants tightly regulate the biosynthesis of ethylene to control growth and development and respond to a
wide range of biotic and abiotic stresses. To understand the molecular mechanism by which plants regulate
ethylene biosynthesis as well as to identify stimuli triggering the alteration of ethylene production in plants,
it is essential to have a reliable tool with which one can directly measure in vivo ethylene concentration.
Gas chromatography is a routine detection technique for separation and analysis of volatile compounds
with relatively high sensitivity. Gas chromatography has been widely used to measure the ethylene produced by plants, and has in turn become a valuable tool for ethylene research. Here, we describe a protocol
for measuring the ethylene produced by dark-grown Arabidopsis seedlings using a gas chromatograph.
Key words Ethylene, Gas chromatography, Arabidopsis, Dark-grown seedlings, ACC synthase

1  Introduction

Ethylene has been considered a plant hormone for over a century
[1–3]. It influences many plant growth and developmental processes, including germination, fruit ripening, nodulation, cell elongation, and response to a wide range of stresses [2]. Due to its
broad and dynamic roles, the precise regulation of ethylene biosynthesis is crucial for maintaining the optimal levels of ethylene production throughout the plant life cycle. The biosynthesis of
ethylene is simple and straightforward [4–6]; it requires only three
enzymatic reactions. The initial ethylene precursor, amino acid
methionine, is converted to S-Adenosyl methionine (SAM) by
SAM synthase. SAM is then converted to 1-aminocyclopropane-­1carboxylic acid (ACC) by ACC synthase (ACS). This step is the
first committed and generally rate-limiting step of the pathway [7,
8]. ACC is finally converted to ethylene by ACC oxidase (ACO).
Transcriptional regulation of ACS plays a central role in the regulation of the ethylene production in plant [9, 10]. Recent studies,
however, suggest that protein stability of ACS also plays a role in
Brad M. Binder and G. Eric Schaller (eds.), Ethylene Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1573,
DOI 10.1007/978-1-4939-6854-1_1, © Springer Science+Business Media LLC 2017

3


4

Gyeong Mee Yoon and Yi-Chun Chen

regulating the production of ethylene in response to various input
stimuli [9, 11, 12].
Measurement of ethylene in plants is a critical step in understanding the underlying mechanisms by which ethylene regulates
the physiological and developmental processes of plants. Many biotic
and abiotic stimuli, such as pathogen attack [13], herbivorous predation [14], flooding [15, 16], drought [17], and temperature [18–
20], alter the levels of ethylene produced in plants, which leads to
adaptation of the plant to given environmental c­ onditions. The ripening of climacteric fruit also depends on ethylene action [21, 22].
Ethylene can stimulate the ripening of fruit at concentration as low
as tens of nL/L [23]. In tomato fruits, the biosynthesis rate of ethylene varies from nearly zero at the mature green stage to a maximum of over 3 nL/g/h at the red ripening stage [24].

Gas chromatography is a common analytical technique for analyzing compounds that are in vapor form or can be vaporized at an
appropriate temperature [25]. Due to its versatility, efficiency, and
sensitivity, gas chromatography has become instrumental for measuring ethylene produced by plants. Automated sampling via a
headspace unit connected to a gas chromatograph (GC) makes the
GC an attractive tool for ethylene measurement as headspace sampling enhances the sensitivity, reproducibility, and optimum injection of ethylene from the headspace vials containing plant materials
[26]. Here, we describe a procedure for measuring ethylene produced from dark-grown Arabidopsis seedlings using a GC equipped
with a headspace unit. As an example, we measured ethylene production of wild-type Arabidopsis seedlings in response to the phytohormone cytokinin, which increases ethylene biosynthesis by
stabilizing ACS protein [27].

2  Materials
2.1  Sterilize
Arabidopsis Seeds

1.Wild-type Arabidopsis Col-0 seeds.
2.Sterilized double-distilled water (ddH2O).
3.Bleach solution: 30% (v/v) bleach, 0.05% (v/v), and
Tween-20.
4.95% (v/v) ethanol.
5.Sterilized microcentrifuge tubes.

2.2  Preparation
of Headspace GC Vials
with Arabidopsis
Seeds

1.Headspace vials and preassembled caps with septa.
2.Aluminum foil.
3. Murashige and Skoog (MS) media: MS salts, 1% (w/v) sucrose,
and 0.6% (w/v) agar.
4.0.6% (w/v) top agarose.

5.Racks to hold headspace vials.


Ethylene Measurement Using GC

5

6.A pipette filler.
7.An automatic 20 mm headspace crimper (or manual crimper).
8.A laminar flow hood.
2.3  Measurement
of Ethylene with a Gas
Chromatograph

1.A gas chromatograph with a headspace unit and column suitable for resolving mixtures of organic and inorganic gases (e.g.,
resolving air, CO, methane, CO2, ethylene, and ethane).
2.Carrier gases (e.g., hydrogen or helium) with high purity and
air with zero grade (see Note 1).
3.A decapper.
4.White weighing dishes.

3  Methods
3.1  Surface Sterilize
Arabidopsis Seeds

1.Add Arabidopsis Col-0 seeds into a microcentrifuge tube.
2.Add 900 μL of 95% (v/v) ethanol into the tube and incubate
for 1 min at room temperature.
3.Discard the ethanol using a 1 mL pipette.
4.Add 30% (v/v) bleach solution and gently shake for 20 min at

room temperature.
5.Discard the bleach solution and add 900 μL ddH2O to wash
the seeds by gently inverting the tube several times.
6.Repeat ddH2O wash at least five times.
7.Discard the ddH2O and add 1 mL of ddH2O into the tube.

3.2  Preparation
of Headspace GC Vials
with Arabidopsis
Seeds

1.Sterilize 22 mL headspace GC vials, preassembled caps with
septa, and a pack of 2 × 2 inches precut aluminum foil (see
Note 2) using an autoclave on dry cycle.
2.Let the headspace vials and caps cool in a sterile laminar flow
cabinet.
3. Prepare MS media and 0.6% (w/v) top agarose and keep them
in a 65 °C water bath (see Note 3).
4.Place the sterilized headspace GC vials on a rack that can
securely hold the vials.
5.Prepare MS media with different cytokinin concentrations (0,
0.1, 0.5, and 1 μM).
6. Aliquot 3 mL of the prepared MS media with different concentrations of cytokinin into the headspace vials using a pipette filler.
7.Let the media in the vials solidify for at least 20 min in the
laminar hood.
8.Discard the ddH2O from the tube of surface sterilized seeds.
9.Add 0.6% (w/v) top agarose to the tube and mix well using a
1 mL pipette.

6

Gyeong Mee Yoon and Yi-Chun Chen

10.Withdraw 30–50 seeds (see Note 4) mixed with 0.6% (w/v)
agarose from the microcentrifuge tube and place in the middle
of the headspace vials.
11.Let the vials with seeds solidify in the sterile hood for 10 min.
12.Seal the headspace vials with the sterilized aluminum foil
securely after confirming the seeds are settled in the middle of
the vials.
13. Maintain the headspace vials at 4 °C for 2–4 days in the dark to
stratify the seeds.
14. After stratification, bring vials to room temperature and remove
the foil.
15.Using an electronic 20 mm automatic crimper (or manual
crimper), securely crimp the headspace vials with the sterilized
caps (see Note 5).
16. Place the vials in a plant growth chamber with dark conditions
for 3 days.
3.3  Measurement
of Ethylene with a Gas
Chromatograph

1.Open gas valves to let gas flow into a GC before turning the
GC on.
2.Turn on the GC and headspace unit.
3. Turn on the software program for the headspace and GC operating system.
4. Make an ethylene standard curve (Fig. 1) using at least five different calibration points by running various dilutions of a
known ethylene standard (e.g., 10 μL/L) (see Note 6).

Fig. 1 Ethylene standard curve. The standard curve was generated using five
concentrations (0.1, 0.25, 0.5, 2.5, and 5 μL/L) of ethylene diluted from a known
ethylene standard (10 μL/L) and displays excellent linearity over a wide dynamic
range (R2 = 1)


Ethylene Measurement Using GC

7

5.Set up the parameters for running the software program and
designate a folder for saving the data file.
6. Number the headspace vials and place them in the corresponding headspace unit (see Note 7).
7.Run the GC according to the manufacturer’s instruction.
8.Open the real-time running screen to monitor the peaks.
Identification of ethylene peaks can be done by finding the peak
in the samples that has the same retention time as the ethylene
peaks obtained when determining the ethylene standard curve.
3.4  Calculation
of Ethylene
Concentration
from the Samples

1.Collect the sample headspace vials from the headspace auto
sampler after the GC run is finished.
2.Open the vials using a decapper.
3.Place 2–3 vials at a time in a microwave and run them for 10 s
or until the MS agar in the vials is melted (see Note 8).
4.Pour the seedlings from the headspace vials into the white

weighing dish.
5.Count the number of seedlings per vial and record.
6. Open the data files from the GC and retrieve the total concentration of ethylene that has been automatically determined by
comparison to the predetermined ethylene standard curve.
7.Divide the total concentration of ethylene with the number of
seedlings and incubation days (or time), which will give the unit
of ethylene concentration (e.g., 10 μL/L per seedling per day).
8.Graph the data to determine the dose-response characteristics
(Fig. 2).

4  Notes
1. The purity of carrier gases is critical for obtaining the best analysis result and normally ultra-pure gases are required (e.g.,
99.995–99.999%). The use of high-purity gases results in higher
sensitivity and longer life-time of a column. Installation of a gas
trap can be an alternative to increase the purity of carrier gases.
2.As an alternate way to sterilize caps and aluminum foil, place
them in a hood and spray with 90% (v/v) ethanol and let them
dry for 20–30 min before use. Caps should be upside down
when the ethanol is applied. Sterilization of preassembled caps
with septa by autoclave may cause the septa to melt resulting in
the blockage of a needle hole in a headspace unit.
3.0.6% (v/v) agarose helps seeds to set in the middle of the MS
agar in the headspace vials.


8

Gyeong Mee Yoon and Yi-Chun Chen

Fig. 2 Cytokinin-induced ethylene production in dark-grown wild-type Arabidopsis

seedlings. Arabidopsis seedlings were grown in sealed vials in the presence of
an indicated concentration of cytokinin. The concentration of ethylene was determined by comparison to the predetermined ethylene standard curve. Error bars
represent standard deviation (n = 3)

4. Not more than 50 seeds per vial are recommended for ethylene
measurement of dark-grown Arabidopsis seedlings. The biosynthesis of ethylene may be hampered when there are too
many seeds due to a negative feedback regulation. 30–50 seeds
per vial are optimum for measuring ethylene produced by
dark-­
grown Arabidopsis seedlings. The optimal number of
seeds for measuring ethylene from light-grown Arabidopsis
seedlings should be experimentally determined.
5.Proper crimper handling is crucial for obtaining reproducible
results. Low levels of ethylene or irreproducible results may be
due to leaks at the headspace vial seal. This is likely due to an
improperly adjusted vial crimper. An inadequately crimped vial
seal will leak ethylene during thermal equilibrium and/or the
pressurization step, which increases an internal pressure in the
headspace vials.
6. A proper standard curve is essential. The standard curve is used
to calculate the peak area of ethylene from experimental samples. The ethylene standard curve has to be recalibrated whenever the GC running method changes (e.g., changes of oven
temperature, duration at specific temperature, and split ratio of
gas flow through the column).


Ethylene Measurement Using GC

9

7.At least the first vial should be without a sample to avoid

potential contamination from the carryover from the previous
GC run.
8. 10–15 s of microwaving should be enough for a vial containing
3 mL MS media to melt MS agar. Longer microwaving makes
it difficult to accurately count the number of seedlings.

Acknowledgments
This work was supported by a startup fund from Purdue University
to GMY.
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606–614


Chapter 2
Plant Ethylene Detection Using Laser-Based

Photo-­Acoustic Spectroscopy
Bram Van de Poel and Dominique Van Der Straeten
Abstract
Analytical detection of the plant hormone ethylene is an important prerequisite in physiological studies.
Real-time and super sensitive detection of trace amounts of ethylene gas is possible using laser-based
photo-acoustic spectroscopy. This Chapter will provide some background on the technique, compare it
with conventional gas chromatography, and provide a detailed user-friendly hand-out on how to operate
the machine and the software. In addition, this Chapter provides some tips and tricks for designing and
performing physiological experiments suited for ethylene detection with laser-based photo-acoustic
spectroscopy.
Key words Ethylene, Laser-based photo-acoustic spectroscopy, Real-time measurements, ETD-300

1  Introduction
The plant hormone ethylene (C2H4) is a unique growth regulator
due to its volatile nature and its pleiotropic effects on plant development and stress responses. Accurate detection of ethylene
requires sensitive equipment that is suited to detect trace amounts
of the gas. Ethylene (ethene according to IUPAC nomenclature) is
the smallest unsaturated hydrocarbon with a double bond. Since
the introduction of gas chromatography (GC) in plant science,
ethylene became a detectable molecule opening new opportunities
for research [1–3]. These first reports quantified ethylene production of apple fruit using GC. The versatility and (relative) affordability of GCs has made it the most used analytical technique for
scientific and commercial detection of ethylene [4]. One of the
major drawbacks of using GC for ethylene detection is that the
analysis time can be quite long (2–10 min depending on the system used), eliminating the ability to monitor ethylene production
in real time. Furthermore, the level of detection (around
1–0.1 ppm) is sometimes insufficient to detect trace amounts of
ethylene, which can be physiologically relevant [5]. An alternative
Brad M. Binder and G. Eric Schaller (eds.), Ethylene Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1573,
DOI 10.1007/978-1-4939-6854-1_2, © Springer Science+Business Media LLC 2017

11


12

Bram Van de Poel and Dominique Van Der Straeten

technique that overcomes these last two limitations is laser-based
photo-acoustic spectroscopy [4, 6]. This technique uses a CO2
laser that excites ethylene molecules in the mid infrared region
(absorption range 2–12 μm) resulting in heat production of the
excited ethylene molecules. By continuously switching the light
source on and off (by means of a chopper), the ethylene molecules
are periodically excited, a process that generates heat pulses, that
gives rise to periodic pressure changes (sound waves), which in
turn can be detected by a sensitive acoustic microphone [4, 6].
The magnitude of the acoustic signal is proportional to the ethylene concentration in the sample. Unprecedented detection limits
as low as 6 pL/L (6 ppt) have been reported [7]. Besides a low
detection limit, a short response time of laser-based photo-acoustic
spectroscopy facilitates real-time measurements of ethylene content without the necessity of long-term headspace accumulation,
preventing any possible feedback effects of the accumulated ethylene [8].

2  Materials
1. A laser-based photo-acoustic spectrophotometer for ethylene
detection. We use the ETD-300 (Sensor Sense, Nijmegen,
NL) hereafter referred to as ETD, which is equipped with
six channels (Fig. 1). This can also be a custom-built system
(see Note 1).
2.Computer with ETD software (Valve controller 1.4.2, Sensor
Sense, Nijmegen, NL), or similar.

3.Carrier gas tubing, connectors, and syringe needles (see Note 2).
4.Carrier gas supply and catalyzer (see Notes 3 and 4).
5.Control box (see Note 5).
6.Sample cuvettes, rubber septa, metal caps, and crimper (see
Note 6).
7.Scrubbers (see Note 7).

3  Methods
The ETD can be used in three different modes of operation: the
continuous flow mode, the stop-and-flow mode, and the samples
mode. During operation in continuous flow mode, all channels
attached to the control box (maximum six cuvettes) are flushed at
the same time, while only one channel is analyzed by the ETD
detector in real time. Thus, samples are analyzed one by one in a
consecutive order, while constantly being flushed. When the
amount of ethylene produced by the sample is too low to be


Laser-Based Photo-Acoustic Ethylene Detection

13

Fig. 1 Overview of the experimental setup of the laser-based photo-acoustic spectrophotometer (ETD). The
flow-through system requires a carrier gas (air or a gas mixture of choice) originating from a gas bottle (or
a compressor), which is passed through a catalyzer (Cat) to remove residual hydrocarbons. The hydrocarbon-free air is transferred to the control box (VC) containing a valve controller (to switch between channels)
and flow controllers (to regulate the flow rate of each channel) which has six different channels, in order to
consecutively measure ethylene in these six cuvettes. Built-in flow controllers regulate the flow rate
(0–5 L/h) and the valve controller selects the channel that will be analyzed and/or flushed. Each channel is
connected with a corresponding sample (cuvette) with an inlet and outlet tube. The VC selects the sample
for which the headspace is flushed and redirected to the ETD detector. Before the air enters the ETD detector it passes over a scrubber (Src) to remove both CO2 and water vapor by KOH and CaCl2 respectively. After

the gas sample has passed the ETD detector for analysis, it is exhausted into the room or can be redirected
outside. Image courtesy of [4]

detected in the continuous flow mode, it is possible to use the
stop-and-flow mode. In this mode, only one of the six cuvettes
attached to the control box will be flushed with the carrier gas and
simultaneously analyzed by the ETD detector. The other five
cuvettes remain sealed during this measurement, allowing them to
accumulate ethylene in their headspace. This will ensure an accrual
of ethylene gas beyond the limit of detection. The stop-and-flow
mode can be programmed so that up to six different samples are
measured sequentially and repeatedly. The samples mode is used
when a lot of samples need to be analyzed only once instead of
over a certain time period, or when the sampling time is shorter
than the analysis time of the ETD. When using the samples mode,
it is important to make a snapshot sample by drawing a 1–2 mL gas
specimen from the headspace of the vial that contains the plant
sample, and injecting this specimen in a different empty airtight
cuvette, which will be analyzed with the ETD at a later stage. The
samples mode is particularly useful if many samples need to be
analyzed shortly after each other.


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Bram Van de Poel and Dominique Van Der Straeten

3.1  Preparation
of Measurements

1. Place your samples in an airtight cuvette. Three different types
of samples can be analyzed: detached plant parts (e.g., a
detached leaf or fruit), attached plant parts (e.g., an attached
leaf), or whole plants (see Note 8).
2.Take into account the production of wound ethylene when
using detached plant parts (see Note 9).
3.Connect your sample with one inlet tube and one outlet tube
to the inlet and outlet connector, respectively, of the control
box of the ETD (see Note 10).
4.Open the ETD software to start an experiment and select the
desired settings (type of experiment, flow rate, measurement
time, and schedule). Figure 2 shows an overview of the most
important panels and configuration settings of the software
(see Note 11). More details about the different settings are
described below for each individual mode of operation.

3.2  Calibration
of the ETD

1. The ETD is calibrated in the same way for the continuous flow
mode and the stop-and-flow mode. For the samples mode, the
ETD can be calibrated separately, taking into account the procedure how the snapshot sample was made (see Note 12).

Fig. 2 Overview of the main panels of the ETD software (Valve controller 1.4.2, Sensor Sense, Nijmegen, NL).
The dark blue panel (upper left, experimental settings) allows adjusting the settings of the experiment (mode
of operation, flow rate, measurement time). The red panel (lower left, view settings) allows adjusting the view
options presented in the raw data panel (center). The light blue panel (lower central panel, data recordings)
lists the measured data points. The green panel (upper right, instrument settings) shows the actual instrument
settings of the laser, the flow controller, and the detector

Laser-Based Photo-Acoustic Ethylene Detection

15

2. Attach a calibration bottle (e.g., 500 ppb ethylene) to the inlet
of the valve control box to supply a constant flow of ethylene
gas.
3. Do not connect the catalyzer in between the calibration bottle
and the valve control box of the ETD.
4. Attach an empty cuvette to one channel (e.g., channel 1) which
will be used for the calibration.
5.Analyze this channel (e.g., channel 1) in the continuous flow
mode with a flow rate of 5 L/h for at least 30 min.
6.Wait until a stable recording of the online raw data points is
reached (ethylene concentration in ppb).
7.Adjust the calibration factor (in the instrument settings panel)
so that the online recordings of the raw data points match the
concentration of the calibration gas. If the calibration factor is
increased, the raw ethylene reads will also increase, while if the
calibration factor is lowered, the raw ethylene reads will also
lower.
8.Allow sufficient time for the online raw data points to equilibrate every time the calibration factor is adjusted.
9.Repeat steps 7 and 8 until the raw online raw data recordings
match the concentration of the calibration gas. The ETD is
now calibrated.
10.Repeat the calibration procedure once every year, or more
often when the ETD is used frequently.
3.3  Flushing the ETD

1. Before the start of a new set of measurements or a new experiment, it is important to flush the system to ensure that a stable
baseline is reached, and any residual ethylene in the detector
and/or tubing is removed.
2.Attach the tubing of all six channels to six different empty
cuvettes.
3.Set the ETD software in the continuous flow mode.
4.Set the flow rate at 5 L/h.
5.Set the measuring time for each sample to 5 min.
6.Program the schedule of the samples so that each channel is
flushed (set: 1–6).
7.Press start and wait until the online recordings of the raw data
shows a stable baseline signal for each channel.
8.It is possible that the baseline is stable but not exactly zero.

3.4  Measurements
in Continuous Flow
Mode

1. Flush the ETD until a stable baseline is reached for each channel using empty vials (as described in Subheading 3.3).
2.Start a new continuous flow experiment in the ETD software.


16

Bram Van de Poel and Dominique Van Der Straeten

3.Set the flow rate between 0.5 and 2 L/h (see Note 13).
4. Set the measuring time for each sample to 30 min (see Note 14).
5.Program the schedule for each sample that needs to be
analyzed.

6.Incorporate one reference cuvette that does not contain a sample (empty cuvette or untreated control). This reference cuvette
represents the background ethylene or the baseline signal.
7. Attach the tubing of each channel to its corresponding cuvette (as
described in Subheading 3.1) after starting the measurement.
8.Press start.
3.5  Data Analysis
in Continuous Flow
Mode

1.Figure 3 shows an example of the raw ethylene recordings in
the continuous flow mode. Each sample was measured for
20 min at a flow rate of 2 L/h, allowing sufficient time to reach
and maintain an equilibrium state (see Note 15).
2.In the experimental settings panel, set the start and end point
that corresponds to the time period during which the raw data
recordings are stable for each sample, which corresponds to
the equilibrium phase (see Fig. 3).
3.The software will automatically calculate the amount of ethylene measured (nL) during this averaging period.

Fig. 3 Overview of a typical “continuous flow” output of the ETD software. Each sample is represented by
a sigmoidal-shaped curve in a different color. Each sample reaches a plateau level, reflecting the steadystate situation of the measurement. This indicates that an equilibrium is reached between the amount of
ethylene produced by the sample and the amount of ethylene that is flushed out of the headspace of the
cuvette. The average amount of ethylene (ppb or nL/L) produced is calculated from the raw data points
during which the equilibrium state is maintained (averaging period). The calculated rate of ethylene production can be adjusted for the flow rate (diamonds in nL/h). The x-axis represents the measurement time
(s) and the double y-axis represents the amount of ethylene (raw data points in ppb) and the calculated
ethylene production rate (diamonds in nL/h)