Methods in molecular biology vol 1573 ethylene signaling methods and protocols

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 quanti

Ethylene Signaling

Brad M Binder

G Eric Schaller Editors

Methods and Protocols

Methods in

Molecular Biology 1573

Series Editor

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

For further volumes:

http://www.springer.com/series/7651

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

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

This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction

on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed.

The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to

be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 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)

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Hanover, NH, USA

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 senes-cence 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 regula-tor 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 contin-ues to be a focus for worldwide research

This volume in the Methods in Molecular Biology series provides a collection of

proto-cols 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 bio-chemical 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 cover-ing 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 begin-ning researchers, for those with prior experience in the study of ethylene signaling, and for those just entering this exciting research area

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.

Contents

Preface v Contributors ix

Part I analysIs of EthylEnE BIosynthEsIs

1 Gas Chromatography-Based Ethylene Measurement

of Arabidopsis Seedlings 3

Gyeong Mee Yoon and Yi-Chun Chen

2 Plant Ethylene Detection Using Laser-Based Photo-Acoustic Spectroscopy 11

Bram Van de Poel and Dominique Van Der Straeten

3 Treatment of Plants with Gaseous Ethylene and Gaseous Inhibitors

of Ethylene Action 27

Mark L Tucker, Joonyup Kim, and Chi-Kuang Wen

4 Analysis of 1-Aminocyclopropane-1-Carboxylic Acid Uptake

Using a Protoplast System 41

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 47

Shigeru Satoh and Yusuke Kosugi

6 Assay Methods for ACS Activity and ACS Phosphorylation

by MAP Kinases In Vitro and In Vivo 59

Xiaomin Han, Guojing Li, and Shuqun Zhang

Part II analysIs of thE EthylEnE sIgnalIng Pathway

7 Analysis of Ethylene Receptors: Ethylene-Binding Assays 75

Brad M Binder and G Eric Schaller

8 Analysis of Ethylene Receptors: Assay for Histidine Kinase Activity 87

G Eric Schaller and Brad M Binder

9 Analysis of Ethylene Receptor Interactions

by Co-immunoprecipitation Assays 101

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 113

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 163

Catharina Merchante and Anna N Stepanova

14 Time-Lapse Imaging to Examine the Growth Kinetics

of Arabidopsis Seedlings in Response to Ethylene 211

Brad M Binder

15 Inhibitors of Ethylene Biosynthesis and Signaling 223

G Eric Schaller and Brad M Binder

16 Analysis of Growth and Molecular Responses to Ethylene

in Etiolated Rice Seedlings 237

Biao Ma and Jin-Song Zhang

17 Love Me Not Meter: A Sensor Device for Detecting Petal Detachment

Forces in Arabidopsis thaliana 245

Andrew Maule, Graham Henning, and Sara Patterson

18 Effects of Ethylene on Seed Germination of Halophyte Plants

Under Salt Stress 253

Weiqiang Li and Lam-Son Phan Tran

19 Assessing Attraction of Nematodes to Host Roots Using Pluronic

Gel Medium 261

Valerie M Williamson and Rasa Čepulytė

Index 269

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

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

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

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 pro- duced 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 pro-cesses, including germination, fruit ripening, nodulation, cell elon-gation, and response to a wide range of stresses [2] Due to its broad and dynamic roles, the precise regulation of ethylene biosyn-thesis is crucial for maintaining the optimal levels of ethylene pro-duction 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- 1-carboxylic 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 regula-tion of the ethylene production in plant [9 10] Recent studies, however, suggest that protein stability of ACS also plays a role in

under-20], alter the levels of ethylene produced in plants, which leads to adaptation of the plant to given environmental conditions The rip-ening 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 ylene varies from nearly zero at the mature green stage to a maxi-mum of over 3 nL/g/h at the red ripening stage [24]

eth-Gas chromatography is a common analytical technique for lyzing 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 mea-suring ethylene produced by plants Automated sampling via a headspace unit connected to a gas chromatograph (GC) makes the

ana-GC an attractive tool for ethylene measurement as headspace pling enhances the sensitivity, reproducibility, and optimum injec-tion of ethylene from the headspace vials containing plant materials [26] Here, we describe a procedure for measuring ethylene pro-duced from dark-grown Arabidopsis seedlings using a GC equipped with a headspace unit As an example, we measured ethylene pro-duction of wild-type Arabidopsis seedlings in response to the phy-tohormone cytokinin, which increases ethylene biosynthesis by stabilizing ACS protein [27]

sam-2 Materials

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

1 Headspace vials and preassembled caps with septa

6 A pipette filler.

7 An automatic 20 mm headspace crimper (or manual crimper)

8 A laminar flow hood

1 A gas chromatograph with a headspace unit and column able 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

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

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 tions 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

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

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 ating system

4 Make an ethylene standard curve (Fig 1) using at least five ferent calibration points by running various dilutions of a known ethylene standard (e.g., 10 μL/L) (see Note 6).

dif-3.3 Measurement

of Ethylene with a Gas

Chromatograph

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)Gyeong Mee Yoon and Yi-Chun Chen

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

correspond-ing 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

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 tration 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 sis 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

4 Not more than 50 seeds per vial are recommended for ethylene measurement of dark-grown Arabidopsis seedlings The bio-synthesis 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 ples The ethylene standard curve has to be recalibrated when-ever the GC running method changes (e.g., changes of oven temperature, duration at specific temperature, and split ratio of gas flow through the column)

sam-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 mined by comparison to the predetermined ethylene standard curve Error bars

deter-represent standard deviation (n = 3)

Gyeong Mee Yoon and Yi-Chun Chen

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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19 Orihuel-Iranzo B, Miranda M, Zacarias L, Lafuente MT (2010) Temperature and ultra low oxygen effects and involvement of ethyl- ene in chilling injury of ‘Rojo Brillante’ per- simmon fruit Food Sci Technol Int 16(2):159–167

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Gyeong Mee Yoon and Yi-Chun Chen

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

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 devel-opment 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 produc-tion of apple fruit using GC The versatility and (relative) afford-ability 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 sys-tem 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

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 con-tent without the necessity of long-term headspace accumulation, preventing any possible feedback effects of the accumulated ethyl-ene [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

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

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 bon-free air is transferred to the control box (VC) containing a valve controller (to switch between channels)

hydrocar-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 detec-

tor 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]

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

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 pro-

cedure how the snapshot sample was made (see Note 12).

3.1 Preparation

of Measurements

3.2 Calibration

of the ETD

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

Bram Van de Poel and Dominique Van Der Straeten

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 brate 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

1 Before the start of a new set of measurements or a new ment, 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

1 Flush the ETD until a stable baseline is reached for each nel using empty vials (as described in Subheading 3.3)

2 Start a new continuous flow experiment in the ETD software

3.3 Flushing the ETD

3.4 Measurements

in Continuous Flow

Mode

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 ple (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

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 ene measured (nL) during this averaging period

ethyl-3.5 Data Analysis

in Continuous Flow

Mode

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 state 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 pro-

steady-duction 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)

Bram Van de Poel and Dominique Van Der Straeten

4 Check the tick box “concentration x flow” to calculate the exact ethylene production rate of each sample (nL/h).

5 Subtract the values of the reference cuvette by selecting the correct channel in the drop-box “Reference cuvette” in the experimental settings panel (channel number 6 in Fig 3)

6 The calculated ethylene production values are saved in a rate Excel file, which can be viewed or analyzed at a later stage

1 Flush the ETD until a stable baseline is reached for each nel using empty vials (as described in Subheading 3.3)

2 Start a new stop-and-flow experiment in the ETD software

3 Set the flow rate between 2 and 3 L/h (see Note 16).

4 Set the measuring time for each sample to 10 min (see Note 16).

5 Program the schedule for each sample that needs to be

ana-lyzed (see Note 16).

6 Press start

7 Attach the tubing of each channel to its corresponding cuvette (as described in Subheading 3.1) after starting the measurement Make sure not to waste too much time in between the start of the measurement and the attachment of the first cuvette

1 Figure 4 shows an example of the raw ethylene recordings in the stop-and-flow mode Each sample was measured for 10 min

at a flow rate of 2 L/h, allowing sufficient time to purge out all ethylene gas from the cuvette, resulting in a typical bell-shaped

curve (peak) (see Note 17).

2 In the experimental settings panel, set the start and end point that corresponds to the time period that completely incorpo-

rates the peak (see Note 18).

3 The software will automatically calculate the amount of ene produced (nL)

4 Check the tick box “integral/accumulation time” to calculate the ethylene production rate per unit of time (nL/h) for each sample

5 Subtract the values of the reference cuvette by selecting the correct channel in the drop-box “Reference cuvette” in the experimental settings panel The reference cuvette is an empty cuvette, or an untreated control, representing background eth-ylene values

6 The calculated ethylene production values are saved in a rate Excel file, which can be viewed or analyzed at a later stage

1 Prepare a snapshot sample by taking 1–2 mL from the cuvettes containing the plant samples, and injecting this volume in

4 Perform an experiment-specific calibration using the same

sampling procedure as for the snapshot samples (see Note 12).

5 Set the flow rate between 2 and 3 L/h (see Note 19).

6 Set the measuring time for each sample to 10 min (see Note 19).

7 Program the schedule for each sample that needs to be lyzed and name each sample accordingly

8 Select the “Pause after each cycle” tick box if you wish to pause the measurements after each loop as defined in the schedule

9 Press start

10 Attach the tubing of each channel to its corresponding cuvette (as described in Subheading 3.1) after starting the measurement Make sure not to waste too much time in between the start of the measurement and the attachment of the first cuvette

Fig 4 Overview of a typical “stop-and-flow” output of the ETD software Each sample is represented by a bell-

shaped curve in a different color, which can be integrated (in this case a numerical integration was chosen) to quantify the amount of ethylene measured Therefore, the start and end time point of the integration period should match the beginning and end of the peak The area underneath the curve represents the total amount

of ethylene (nL) multiplied by the flow rate The total amount of ethylene can also be adjusted for the

accumu-lation time to calculate the ethylene production rate (nL/h) The x-axis represents the measurement time (s) and the double y-axis represents the concentration of ethylene (raw data points in ppb) and the calculated

ethylene production rate (diamonds in nL/h)

Bram Van de Poel and Dominique Van Der Straeten

11 After each loop of six samples, a new loop of six different ples can be initiated by pressing the continue button.

1 The data analysis of the samples mode (Fig 5) is similar to the

stop-and-flow mode (see Subheading 3.7) The only difference

is that it is not possible to correct the peak area (ethylene tent in nL) with the accumulation time This should be done manually by calculating the time each original sample was sealed up to the moment when the snapshot sample was made

con-4 Notes

1 The ETD-300 (Sensor Sense, Nijmegen, NL) has a limit of detection of 300 ppt ethylene This machine can be equipped with a control box (valve and flow controllers) and comes with

an optional catalyzer that removes residual hydrocarbons from the carrier gas (in principle, air) The ETD is best operated in

3.9 Data Analysis

in Samples Mode

Fig 5 Overview of a typical “samples mode” output of the ETD software Many different samples can be

pro-grammed to be analyzed in series of six, with a pause in between each loop of six (by checking the tick box

“Pause after each cycle.” Each sample is represented by a bell-shaped curve that can be integrated (in this

case a “fit then integrate” integration was chosen using the Levenberg-Marquardt Algorithm for curve fitting with the slope parameters set to 1) The start and end time point of the integration period should match the beginning and end of the peak The area underneath the curve represents the total amount of ethylene (nL) multiplied by the flow rate The data cannot be corrected for the accumulation time in the samples mode In this example, the first four samples are used for making a new calibration curve (the calibration tick boxes are marked and the concentration of ethylene is given) The calibration graph can be displayed by pressing the

button “calibration graph.” The x-axis represents the measurement time (s) and the double y-axis the

concen-tration of ethylene (raw data points in ppb) and the total amount of ethylene (diamonds in nL)

a temperature-controlled environment between 10 and 26 °C, avoiding strong temperature fluctuations Ideally, the equip-ment is placed in an air-conditioned room For most plant sci-ence applications this temperature range is workable In case different temperature conditions are required, tubes should be diverted from the machine into a different room/space where the samples are stored It is important to switch on the ETD detector 60–90 min before the start of an experiment to ensure proper warm-up of the system

2 All gas handling is best done with PFA (perfluoroalkoxy alkane) tubing with an outer diameter of 1/8” Connections can be made with Swagelok connectors, quick-lock connectors or flexible rubbers Syringe needles attached to tubing ends using flexible rubbers can also be used if septa need to be punctured

3 Carrier gas, mostly air, can be supplied by a compressor, or by bottles of compressed air Bottles with compressed air will con-tain trace amounts of CO2 (originating from ambient air), although the exact concentration is variable depending on time and method of fabrication (consult with the air supplier for more details) Compressed air is well suited for most exper-iments with plants, but sometimes it can be desired to treat plants with a known concentration of CO2 or other gasses, and then a gas mixture with a predefined composition should be used

4 The outlet of the gas bottle can be equipped with a two-stage pressure regulator that allows a precise control of the outlet pres-sure Typically, a final output pressure of 1 atm is used for all experiments with the ETD In order to ensure optimal valve and flow controller operation, the maximum outlet pressure cannot exceed 6 atm The carrier gas from the bottle is directed to a cata-lyzer (Sensor Sense supplies the CAT1) to remove residual hydrocarbons and particulate matter The catalyzer ensures that

no external ethylene gets in the tubing of the experimental setup The catalyzer only requires 5 min to warm up

5 The control box contains a valve controller that directs the flow toward the six cuvettes that are connected to the control box Each channel has a flow controller that precisely regulates the flow of the cuvette to which it is attached The valve con-troller can be programmed to flush all six cuvettes at the same time (continuous flow mode) or only one cuvette at the time (stop-and-flow and samples mode) The flow rate of the sup-plied air can be adjusted between 0.25 and 5 L/h using channel- specific flow controllers

6 Each cuvette (sample) has two tubes connected with the control box One is the inlet tube that directs the carrier gas from the Bram Van de Poel and Dominique Van Der Straeten

control box to the cuvette, and the other one is the outlet tube that directs the carrier gas from the cuvette back to the control box The control box is also connected with the ETD detector This setup creates a loop that allows to purge out the headspace above the plants or plant tissues in the cuvette and directs it via the control box to the ETD detector for analysis Different types

of airtight cuvettes can be used, depending on the size and growth conditions of the samples It is essential to try to use cuvettes that have similar dimensions as the plants/plant parts that are sampled, minimizing the free headspace This avoids dilution of ethylene in the headspace and ensures that the ethyl-ene concentration remains above the limit of detection Sometimes cuvettes are vials that need to be sealed with a rubber septa, which can be punctured with a syringe needle to purge out the headspace Make sure that these septa are sealed airtight, preferentially by capping them with a metal ring that is firmly attached using a crimper Examples of different type of cuvettes are given by [9 10] and are sold by several companies (e.g., Qubit Systems, Chromacol, Waters, Agilent, and others)

7 When the carrier gas, together with the headspace gas, is rected from the cuvette via the control box to the ETD detec-tor, it passes a scrubber to filter out water vapor and CO2 to avoid interference with the ethylene signal The scrubbers are placed in series, with first a CO2 scrubber, containing KOH (or NaOH or soda lime) and second a water vapor scrubber, con-taining CaCl2 (or CaSO4, also called Drierite) It is important

redi-to place the CO2 scrubber before the water vapor scrubber because the CO2 scrubber generates moisture when CO2 is removed from the gas stream

8 Whole plant samples can be germinated in the cuvette or ferred from a different growth medium into an airtight cuvette for analysis When plants are germinated and grown in the cuvette it is best to use MS medium or an inert substrate in sterile conditions to prevent interference from unwanted eth-ylene production from microorganisms or decaying organic matter When plants are transferred from a growth medium to

trans-an airtight cuvette, they ctrans-an be placed on a sterile pre-wetted filter paper or miracloth tissue to avoid desiccation Again, it is advised to work in sterile conditions to prevent unwanted eth-ylene production from microorganisms It is also possible to measure ethylene levels in the headspace above liquid cultures (hydroponics, aquatic species, or algal cultures) in cuvettes, but it is important that a sufficiently large headspace is present

to prevent water leakage into the control box When working

in liquid conditions one should take into account the solubility

of ethylene in liquid (in principle water) using the law of Henry under atmospheric equilibrium conditions:

c H cc c

a = ´ g

with ca being the concentration (in molarity) of ethylene in

the aqueous phase, cg being the concentration (in molarity) of

ethylene in the gas phase, and Hcc the Henry’s law solubility

constant [11] More details on how to calculate Henry’s law constant under different environmental conditions including different solutes, as well as examples of Henry’s law constants for ethylene dissolved in water, are given in [11]

9 When using detached plant parts it is important to take into account the release of wound-induced ethylene [12] Wound-ethylene is produced rapidly after wounding; hence, in princi-ple, it is observed as a first peak Plant parts can also be exposed

to the surrounding air for 5–15 min before the start of the analysis to eliminate the first burst in wound ethylene

10 Connections between two tubes can be made by using flexible rubbers or quick-lock connectors This type of connections can also be made to attach tubing to the sample cuvette or the control box of the ETD When samples are sealed in a cuvette using an airtight septum, the connection with the ETD can be made by puncturing through the septum with a syringe needle that is attached to the tubing by a flexible rubber

11 The ETD software records the raw data separately from the analyzed data in different Excel files This means that any soft-ware manipulations during data analysis do not affect the raw data recordings, but only the analyzed data

12 The ETD can be calibrated separately in the samples mode, ing into account the procedure how the snapshot sample is pre-pared This is done by first injecting a certain volume (e.g., 1–2 mL) of a calibration gas into an empty airtight vial (snap-shot sample), by the same sampling procedure used for the analysis of unknown samples Make sure to use at least two dif-ferent concentrations for the calibration and prepare at least three technical replicates for each concentration Subsequently, tick the “calibration (ppv)” box in the ETD software (in the experimental settings panel) for each sample that will be analyzed to generate a calibration curve Define the concentra-tion of each calibration sample in the ETD software (in the experimental settings panel) and measure each calibration sam-ple It is best to repeat the calibration procedure each time new experimental settings (flow rate and measuring time) or a new sampling procedure (volume of the snapshot sample) is used

13 The flow rate in the continuous flow mode should be chosen wisely to ensure that an equilibrium is established between the amount of ethylene that is produced by the sample and the Bram Van de Poel and Dominique Van Der Straeten

amount of ethylene flushed out of the cuvette A typical flow rate for these types of experiments is between 0.5 and 2 L/h, although this is best determined experimentally.

14 In the continuous flow mode, each sample is measured during

a 10–30 min time period (depending on the programmed flow rate and the rate of ethylene emanation from the samples) The exact measuring time is best chosen experimentally to ensure sufficient time to reach the equilibrium state when switching from one channel to another Especially if large differences in ethylene production are expected between different samples, it

is important to increase the measuring time Due to the very low level of detection and the fast response time (5 s) of the ETD, continuous real-time measurements of ethylene produc-tion of most plants or plant tissues are possible, in contrast to conventional GC setups that are not sensitive nor fast enough

In addition, the continuous flow mode prevents ethylene mulation in the headspace of the cuvettes, eliminating possible unwanted effects of ethylene on the plant metabolism

15 In the continuous flow mode, the raw data points have a moidal shape for each sample, reaching a plateau level after a

sig-certain time (see Fig 3) The initial lag phase of the curve responds to the rest air of the previous sample that needs to be flushed out from the tubing Next, the signal increases (or decreases) because the first ethylene molecules of the current sample are detected After a while the amount of ethylene pro-duced by the sample is equal to the amount of ethylene that is flushed out the cuvette, resulting in a stable signal (equilib-rium) It is important to maintain this equilibrium for several minutes (3–10 min) to have a good estimate of the average ethylene production rate

16 In the stop-and-flow mode it is common to use a flow rate around 2–3 L/h, unless very high concentrations of ethylene are expected (then a higher flow rate should be used) The total time of ethylene accumulation in the headspace should be chosen so that all the ethylene present in the headspace of the cuvette is purged out, meaning that the raw data recordings are peak-shaped and return to the baseline at the end of the peak If large differences in ethylene production are expected,

it is advisable to program the time of analysis and the flow rate

of each cuvette individually, allowing a longer accumulation time for samples with a lower ethylene production rate When six samples are attached to the control box, it is common to analyze each sample for 10 min, so that each sample is analyzed once every hour It is also possible that one of the six cuvettes

is used as a blanc control, which can be analyzed for a longer time period, to create a longer accumulation period for the other five cuvettes For example, cuvettes 1–5 contain samples

and are measured for 10 min, while cuvette 6 is empty and is measured for 2 h and 10 min This way cuvettes 1–5 are only analyzed once every 3 h, ensuring a longer accumulation period, which can be convenient for samples that produce little ethylene

17 During the stop-and-flow mode the ethylene concentration in the headspace of one cuvette is measured after a certain accu-mulation time, resulting in a bell-shaped curve (peak) that rep-resents the total amount of ethylene flowing through the detector The initial lag phase of the peak corresponds to the rest air of the previous sample that needs to be flushed out from the tubing Next, the accumulated ethylene passes through the detector and will result in the bell-shaped output

At the end of the measurement, the signal drops again and reaches the equilibrium state (or the baseline) It is important that the analysis time is long enough so that the peak has reached the baseline or equilibrium state at the end of each measurement This is achieved by setting an optimal flow rate and measurement time A typical ethylene measurement of plant material would last 10 min when using a flow rate of

2 L/h A rule of thumb is that the flow rate and analysis time should be set so that the volume of the headspace (including the volume of tubing) is flushed at least five times

18 In the stop-and-flow mode, the area underneath a peak sents the total amount of ethylene of the corresponding sample, and is calculated by integrating this peak over a certain time period The start and end point of the integration period should

repre-be chosen by the user These time points are selected so that the peak is completely incorporated in the integration period There are two integration methods available in the ETD software: the Levenberg-Marquardt Algorithm and a numerical integration The Levenberg-Marquadt Algorithm will first calculate the best fit through the raw data points, facilitating the integration of the peak surface (=fit then integrate) It is important to adjust the shape of the “parabolic” fit to match the shape of the peak

of the raw data points by adjusting the slope values (the higher the value, the steeper the slope) The numerical integration method is faster and does not require manual adjustment of the slopes In practice, the results of the numerical integration method are not much different compared to the integration results of the Levenberg- Marquardt Algorithm curve (less than 1% based on an experimental comparison of 385 individual integration events) Therefore, the numerical integration method is more practical in use

19 The samples mode is used when multiple (more than six) samples, originating from snapshot samples, need to be analyzed These snapshot samples are made by taking a 1–2 mL gas specimen from Bram Van de Poel and Dominique Van Der Straeten

the sample vials that contain plants or plant parts, and injecting this gas specimen into another empty airtight vial This way, a snapshot of the headspace of the sample is transferred to another vial These secondary vials can be stored and subsequently ana-lyzed with the ETD in the samples mode It is thus possible to analyze more than six samples, although only six different samples can be attached to the control box in one loop of analysis There

is an option to pause the analysis after each loop, allowing the operator to change the cuvettes without having to worry that a new series of measurements has already started The sample mode

is typically programmed with a flow rate of 2 L/h for 10 min, but these settings can be adjusted according to the volume size of the cuvettes and the amount of ethylene present in the headspace The data analysis procedure for the samples mode is the same as for the stop-and- flow mode and will result in the typical bell-

shaped curves (see Fig 5) There is no option available to matically correct the amount of ethylene for its accumulation time The calculated ethylene production values (nL) should be manually corrected for the accumulation time (time before the snapshot sample was made) by the operator using a separate soft-ware (e.g., Excel)

auto-Acknowledgments

BVdP is a postdoctoral fellow of the Research Foundation Flanders (FWO Vlaanderen) This work was supported by projects from Ghent University (Bijzonder Onderzoeksfonds, 01B02112) and the Research Foundation Flanders (FWO Vlaanderen, G.0656.13N) to DVDS

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katharometer and its application to the

mea-surement of ethylene and other gases of

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1:245–259

2 Huelin FE, Kennett BH (1959) Nature of the

olefines produced by apples Nature 184:996

3 Meigh DF (1960) Ethylene production of

stored apples—use of gas chromatography in

measuring the ethylene production of stored

apples J Sci Food Agr 11:381–385

4 Cristescu SM, Mandon J, Arslanov D, De

Pessemier J, Hermans C, Harren FJM (2013)

Current methods for detecting ethylene in

plants Ann Bot 111:347–360

5 Reid MS, Wu M-J (1992) Ethylene and flower

senescence Plant Growth Regul 11:37–43

6 Harren FJM, Cotti G, Oomens J, te Lintel Hekkert S (2000) Photoacoustic spectroscopy

in trace gas monitoring In: Meyer RA (ed) Encyclopedia of analytical chemistry John Wiley & Sons, Chichinster, pp 2203–2226

7 te Lintel Hekkert S, Stall MJ, Nabben RHM, Zuckermann H, Persijn S, Stal LJ, Voesenek LACJ, Harren FK, Reuss J, Parker DH (1997) Laster photoacoustic trace gas detection, an extremely sensitive technique applied in biologi- cal research Instrum Sci Technol 26:157–175

8 Vandenbussche F, Vaseva I, Vissenberg K, Van Der Straeten D (2012) Ethylene in vegetative development: a tale with a riddle New Phytol 194:895–909

9 Cristescu SM, Woltering E, Hermans C, Harren FJM, te Lintel Hekkert S (2015) Research tools:

ethylene detection In: Wen C-K (ed) Ethylene

in plants Springer, Dordrecht, pp 263–286

10 Tucker M, Wen C-K (2015) Research tools:

ethylene production: treatment with ethylene

and its replacements In: Wen C-K (ed) Ethylene

in plants Springer, Dordrecht, pp 245–261

11 Sander R (2015) Compilation of Henry’s law constants (version 4.0) for water as solvent Atmos Chem Phys 15:4399–4981

12 Boller T, Kende H (1980) Regulation of wound ethylene synthesis in plants Nature 286:256–260

Bram Van de Poel and Dominique Van Der Straeten

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_3, © Springer Science+Business Media LLC 2017

Chapter 3

Treatment of Plants with Gaseous Ethylene and Gaseous

Inhibitors of Ethylene Action

Mark L Tucker, Joonyup Kim, and Chi-Kuang Wen

Abstract

The gaseous nature of ethylene affects not only its role in plant biology but also how you treat plants with the hormone In many ways, it simplifies the treatment problem Other hormones have to be made up in solution and applied to some part of the plant hoping the hormone will be taken up into the plant and translocated throughout the plant at the desired concentration Because all plant cells are connected by an intercellular gas space the ethylene concentration you treat with is relatively quickly reached throughout the plant In some instances, like mature fruit, treatment with ethylene initiates autocatalytic synthesis of ethylene However, in most experiments, the exogenous ethylene concentration is saturating, usually

>1 μL L −1 , and the synthesis of additional ethylene is inconsequential Also facilitating ethylene research compared with other hormones is that there are inhibitors of ethylene action 1-MCP (1-methylcyclopro- pene) and 2,5-NBD (2,5-norbornadiene) that are also gases wherein you can achieve nearly 100% inhibi- tion of ethylene action quickly and with few side effects Inhibitors for other plant hormones are applied

as a solution and their transport and concentration at the desired site is not always known and difficult to measure Here, our focus is on how to treat plants and plant parts with the ethylene gas and the gaseous inhibitors of ethylene action.

Key words Ethylene, Ethephon, 1-methylcyclopropene, 1-MCP, Norbornadiene, Gaseous treatment

1 Introduction

The Merriam-Webster dictionary [1] defines a hormone as “a product of living cells that circulates in body fluids (as blood) or sap and produces a specific often stimulatory effect on the activity

of cells usually remote from its point of origin.” Ethylene, because

it is a gas, does not fit neatly into this definition of a hormone Nonetheless, the immediate biological precursor to ethylene, ACC (1-aminocyclopropane-1-carboxylic acid) has long been presumed

to be translocated within the plant and then converted to ethylene but it was only recently that a putative ACC transporter was identi-fied [2]; however, more often than not, where ACC is synthesized

is where ethylene is made [2] Because ethylene is a gas, the response to ethylene can be local In other words, the

concentration of the gas dissipates quickly as you move away from the source, e.g., site of injury; however, in an organ with a less permeable skin, e.g., waxy cuticle with few stomata or lenticels as might be the case in a fruit, ethylene can accumulate to high concentrations

Let us put this in terms that everybody can understand out sophisticated mathematics The first fact that is worth knowing

with-is that gas diffusion with-is 10,000 times faster in air than water [3] It

is intuitive that this is why each cell in a multicellular plant is connected by an intercellular gas space Most plant cells cannot withstand anaerobic conditions for very long If the intercellular gas space collapses due to injury or cellular leakage, it is likely those cells will die because of reduced gas exchange

inter-But just how fast is gas diffusion? We have all experienced being in a closed space with someone who had a bit of indigestion and passed some gas The smell spreads fast and, if there is no cir-culation, someone jumps up to open a window or door However,

if the room is very large or you are outside in the open air, the smell dissipates quickly Now, to transfer the above analogy to plants In a fruit like a tomato, which is like a room with moderate ventilation, you do not need a lot of ethylene synthesis inside the fruit to achieve an effective concentration throughout the fruit; however, in a leaf, with open stomata, ethylene synthesized by an injured cell will dissipate quickly and the ethylene response will be local because the concentration declines exponentially as you move away from the source

What happens when ethylene is provided from the outside of the plant? Ethylene diffuses through stomata and lenticels at the surface and then through the intercellular gas spaces fairly quickly Because the intercellular space is circuitous, diffusion is not quite

as fast as if it were an open room, but still reasonably quick Plant cells are generally small with a large surface area for gases to enter Ethylene is soluble in both lipids and water The concentration of ethylene in μmoles L−1 is approximately equal in air and lipids at

25 °C and approximately 1/10 as much in water, but still quite high [4] Ethylene must cross the water-filled cell wall and then a very short distance across a membrane bilayer into the cytoplasm, which streams and thereby, in addition to diffusion, actively moves ethylene throughout the cell Although ethylene diffuses 10,000 times more slowly in water and lipid, because the distances across the cell wall and membrane are relatively small, the change in media is probably not rate limiting Moreover, although most plants have the ability to metabolize ethylene, it is in most instances considered to be too slow to have an impact on the concentration

of ethylene inside the cell [5] Nevertheless, there are instances where ethylene metabolism, which is independent of ethylene binding, can influence the concentration of ethylene and therefore binding to a receptor [5] In most experiments, we treat with a Mark L Tucker et al.

concentration of ethylene that is >10-fold the concentration of ethylene needed to produce a maximal response, which is typically

1 μL L−1 [6] A high concentration of ethylene both shortens the time needed to initiate the ethylene response and greatly mini-mizes any effect due to its degradation

Above we have discussed the physical properties of ethylene that affects its movement into a cell, but how fast is it in terms that

we can actually measure Probably, the best data to show this is work by Brad Binder et al [7] where they exposed Arabidopsis seedlings to 10 μL L−1 ethylene and measure a reduction in growth

in minutes More often researchers treat with ethylene and wait 3

or more hours before measuring changes in respiration, gene expression, or growth responses [8 9], which was without ques-tion enough time to see significant changes

At this point, we can probably agree that ethylene moves into the plant tissue quickly and is fairly uniformly distributed in the tissue, but how fast is the decline if you remove ethylene and how quickly should you see changes in gene expression If the outside concentration of ethylene is made close to zero by opening the chamber, diffusion out of the plant tissue has similar properties as its movement in; however, the problem here is that we usually treat with a high outside concentration of ethylene (e.g., 10 μL L−1) to quickly obtain a high effective concentration inside the cell Getting below an effective concentration of <0.1 μL L−1 may take a bit longer Moreover, endogenous ethylene synthesis may have been evoked by the ethylene treatment However, if required for the experiment, there is a way to get around this The container can be opened and then closed and inhibitors such as 1- methylcyclopropene (1-MCP) or 2,5-norbornadiene (2,5-NBD) added These inhibi-tors will bind to the ethylene receptors and block any new induc-tion of an ethylene response

1-MCP is a strong competitive inhibitor of ethylene action that binds to the receptor with a higher affinity than ethylene itself [10] The very strong binding makes recovery of ethylene respon-siveness after removal of 1-MCP slow because synthesis of new receptor may be required to replace the inhibited receptor [11] These properties have led to 1-MCP being used commercially to delay ripening of fruits or senescence (aging) of vegetables and ornamental flowers (http://www.agrofresh.com/technology) However, for research purposes, it is sometimes useful to be able to overcome the ethylene inhibition 2,5-NBD is also a competitive inhibitor of ethylene binding but with lower affinity than 1-MCP

In general, you need approximately 1500-fold more 2,5-NBD than ethylene to effectively inhibit ethylene action [12] The use of

a competitive inhibitor such as 2,5-NBD can provide some tages for experimental design where the ethylene response can be inhibited and then initiated by the addition of a high concentration

advan-of ethylene (e.g., 100 μL L−1) that will overcome the inhibition by

it is important to harvest tissues at approximately the same time of day, e.g., morning, afternoon, or evening If working with intact plants, it may be necessary to have a consistent light intensity and cycle In this case, the container used for the ethylene or inhibitor treatments must be transparent to light.

The maintenance of temperature is as important as is light To maintain a constant temperature, the chamber can be kept inside

an incubator or, in a flow-through system, the gas mixture may be passed through a sealed flask held in a temperature-regulated cir-culating water bath to bring all the gases to the desired tempera-ture before entering the sealed chamber However, if temperature cycling is required for the experiment, again, as with light, it is important to harvest the tissue at a similar time in the temperature cycle to assure reproducibility In this chapter, we discuss the use of

a closed system for ethylene treatment, the use of ethephon as an alternative to ethylene, the use of ethylene inhibitors 1-MCP and 2,5-NBD, and finally the use of a flow-through system for ethylene treatment

2 Materials

1 100% ethylene in a lecture bottle with appropriate single-stage regulator (Fig 1k) A small lecture bottle of 100% ethylene will provide enough ethylene for many experiments Tanks of com-pressed ethylene can also be purchased from several compa-nies, at larger volumes and various dilutions

2 Gas syringes (a 3 or 6 mL disposable syringe generally works

well, see Note 1).

3 Disposable needles (22 gauge generally works well)

4 Flask sealed with septum for ethylene dilutions (here we use a

500 mL Erlenmeyer flask with a single hole rubber stopper that has a tube inserted and sealed with a plug-type (sleeve-type) rubber seal stopper (Fig 1j, see Note 2)).

5 Chamber sealed with septum (here we use a 2.6 L glass desiccator)

2.1 Closed System

for Ethylene Treatment

Mark L Tucker et al.

tum glued onto lid with silicon sealer; (c) enlarged view of septums glued on with silicon sealer; (d, e, f) a

variety of containers with septums; (g) 2.6 L glass desiccator with rubber stopper and syringe needles that can

be used to inject ethylene and liquid into attached microcentrifuge tube; (h) modification to rubber stopper that

can be used for flow-through system; (i) gas syringe; (j) depiction of a variety of connecters with sleeve-type

rubber stoppers that can be used to seal a chamber; (k) lecture bottle of ethylene, regulator, and tubing