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Methods in
Molecular Biology 2526

Amna Mhamdi Editor

Reactive Oxygen
Species in Plants
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

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

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
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advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
Reactive Oxygen Species in Plants

Methods and Protocols

Edited by

Amna Mhamdi
VIB Center for Plant Systems Biology, Ghent University, Zwijnaarde, Belgium
Editor
Amna Mhamdi
VIB Center for Plant Systems Biology
Ghent University
Zwijnaarde, Belgium

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-2468-5 ISBN 978-1-0716-2469-2 (eBook)
https://doi.org/10.1007/978-1-0716-2469-2

© Springer Science+Business Media, LLC, part of Springer Nature 2022

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
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Preface

Reactive oxygen species (ROS) refer to any molecule that is more reactive than oxygen itself.
ROS are produced spontaneously by metabolism at different subcellular locations such as
chloroplasts, mitochondria, peroxisomes, and the apoplast. In planta, ROS regulate a
myriad of processes including development, stress signaling, systemic responses, and cell
death. Literature analysis continues to document the tremendous ever-growing interest in
research areas related to ROS and redox-related metabolism certainly due to their impor-
tance in plant responses to fluctuating environments. I believe that these areas will continue
to be relevant to numerous aspects of plant biology and will continue to be of interest to
many researchers, even more with the future climate-change context. Advances in these
areas would not be possible without a reliable methodology; therefore, this guide is timely
and convenient.
This book includes detailed information on protocols and methods that can be used to
study reactive oxygen species (ROS) in plants and to characterize their roles in development
and stress responses and is written by recognized leaders in this field. The aim is to provide a
useful collection of protocols that any researcher, and in particular young researchers, can
use and reproduce with ease. The book contains 20 chapters and is divided into 4 parts. Part
I covers the strategies to induce ROS production. Part II focuses on methods to visualize
ROS and detect changes in redox homeostasis. Part III is devoted to small-scale and
targeted analyses that allow for investigating the effects of ROS accumulation during stress
on plant physiology and metabolism. The final part, Part IV, explores the benefit of using
systems biology approaches to understand ROS functions. While the methods described
here have been used mostly on our favorite model plant, Arabidopsis thaliana, they can be
easily adapted to study ROS in crops and other plant species.
This book covers complementary approaches to capture the ROS field from different
angles. The Notes section provides the experts’ views on how to handle pitfalls and guides
the users to get the best of their data, and for that, I would like to acknowledge all the
authors for their valuable contributions.

Zwijnaarde, Belgium Amna Mhamdi

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I STRATEGIES TO INDUCE ROS PRODUCTION

1 Modification of Chloroplast Antioxidant Capacity
by Plastid Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Shengchun Li, Pan Shen, Bipeng Wang, Xiujie Mu,
Mimi Tian, Tao Chen, and Yi Han
2 Analysis of Ascorbate Metabolism in Arabidopsis
Under High-Light Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Takanori Maruta and Takahiro Ishikawa
3 Genetic Manipulation of Reactive Oxygen Species (ROS)
Homeostasis Utilizing CRISPR/Cas9-Based Gene Editing in Rice. . . . . . . . . . . . 25
Sheng Xu, Tao Chen, Mimi Tian, Marie-Sylviane Rahantaniaina,
Linlin Zhang, Ren Wang, Wei Xuan, and Yi Han
4 Studying Plant Stress Reactions In Vivo by PAM Chlorophyll
Fluorescence Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Alexey Shapiguzov and Jaakko Kangasj€ a rvi

PART II METHODS TO VISUALIZE ROS AND TO DETECT

CHANGES IN REDOX HOMEOSTASIS

5 Live Monitoring of ROS-Induced Cytosolic Redox Changes

with roGFP2-Based Sensors in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
José Manuel Ugalde, Lara Fecker, Markus Schwarzl€ a nder,
Stefanie J. Müller-Schüssele, and Andreas J. Meyer
6 Quantitative Measurement of Ascorbate and Glutathione
by Spectrophotometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Graham Noctor and Amna Mhamdi
7 Measurement of NAD(P)H and NADPH-Generating Enzymes . . . . . . . . . . . . . . 97
Amna Mhamdi, Frank Van Breusegem, and Graham Noctor
8 Quantitative Analysis for ROS-Producing Activity and Regulation
of Plant NADPH Oxidases in HEK293T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Sachie Kimura, Hidetaka Kaya, Kenji Hashimoto,
Michael Wrzaczek, and Kazuyuki Kuchitsu

vii
viii Contents

PART III SMALL-SCALE TARGETED ANALYSIS OF ROS ACCUMULATION

DURING STRESS AND EFFECTS ON PLANT PHYSIOLOGY

9 Estimation of the Level of Abasic Sites in Plant mRNA

Using Aldehyde Reactive Probe. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Jagna Chmielowska-Ba˛k, Karolina Izbiańska-Jankowska,
and Joanna Deckert
10 A Simplified Method to Assay Protein Carbonylation
by Spectrophotometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Corentin Moreau and Emmanuelle Issakidis-Bourguet
11 In Vitro Biochemical Analysis of Recombinant Plant Proteins
Under Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Zeya Chen and Jingjing Huang
12 Methods to Analyze the Redox Reactivity of Plant Proteins . . . . . . . . . . . . . . . . . . 161
Thualfeqar Al-Mohanna, George V. Popescu, and Sorina C. Popescu
13 Determination of ROS-Induced Lipid Peroxidation by HPLC-Based
Quantification of Hydroxy Polyunsaturated Fatty Acids . . . . . . . . . . . . . . . . . . . . . 181
Brigitte Ksas and Michel Havaux
14 Detection of Lipid Peroxidation-Derived Free Azelaic Acid, a Biotic
Stress Marker and Other Dicarboxylic Acids in Tobacco by Reversed-Phase
HPLC-MS Under Non-derivatized Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Attila L. Ádám, György Kátay, András Künstler,
and Lo ránt Király
15 Determination of Reactive Carbonyl Species, Which Mediate Reactive
Oxygen Species Signals in Plant Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Jun’ichi Mano, Md. Sanaullah Biswas, Koichi Sugimoto,
and Yoshiyuki Murata
16 Measuring Stress-Induced Changes in Defense Phytohormones
and Related Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Caroline Lelarge-Trouverie, Amna Mhamdi,
Florence Guérard, and Graham Noctor

PART IV SYSTEMS BIOLOGY APPROACHES TO UNDERSTAND ROS FUNCTIONS

17 Targeted Mass Spectrometry Analysis of Protein Phosphorylation

by Selected Ion Monitoring Coupled to Parallel Reaction
Monitoring (tSIM/PRM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Jesús Pascual and Saijaliisa Kangasj€ a rvi
18 Quantitative Analysis of Posttranslational Modifications
of Plant Histones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Hana Kuchařı́ková, Zuzana Plšková, Zbyněk Zdráhal,
Miloslava Fojtová, Pavel Kerchev, and Gabriela Lochmanová
 Contents ix

19 Characterization of RBPome in Oxidative Stress Conditions . . . . . . . . . . . . . . . . . 259

Zhicheng Zhang, Evy Timmerman, Francis Impens,
and Frank Van Breusegem
20 Analysis of ROS-Triggered Changes in the Transcriptome . . . . . . . . . . . . . . . . . . . 277
Patrick Willems

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Contributors

ATTILA L. ÁDÁM • Plant Protection Institute, Centre for Agricultural Research, Eötvös
Loránd Research Network (ELKH), Budapest, Hungary
THUALFEQAR AL-MOHANNA • Department of Biochemistry, Molecular Biology, Entomology,
and Plant Pathology, Mississippi State University, Mississippi State, MS, USA
MD. SANAULLAH BISWAS • Department of Horticulture, Bangabandhu Sheikh Mujibur
Rahman Agricultural University, Gazipur, Bangladesh
FRANK VAN BREUSEGEM • Department of Plant Biotechnology and Bioinformatics, Ghent
University, Ghent, Belgium; VIB-UGent Center for Plant Systems Biology, Ghent, Belgium
TAO CHEN • School of Food and Biological Engineering, Hefei University of Technology,
Hefei, Anhui, China
ZEYA CHEN • Department of Plant Biotechnology and Bioinformatics, Ghent University,
Ghent, Belgium; Center for Plant Systems Biology, VIB, Ghent, Belgium
JAGNA CHMIELOWSKA-BA˛K • Department of Plant Ecophysiology, Institute of Experimental
Biology, Faculty of Biology, School of Natural Sciences, Adam Mickiewicz University,
Poznan, Poland
JOANNA DECKERT • Department of Plant Ecophysiology, Institute of Experimental Biology,
Faculty of Biology, School of Natural Sciences, Adam Mickiewicz University, Poznan,
Poland
LARA FECKER • Institute of Crop Science and Resource Conservation (INRES), Rheinische
Friedrich-Wilhelms-Universit€ at Bonn, Bonn, Germany
MILOSLAVA FOJTOVÁ • Mendel Centre for Plant Genomics and Proteomics, Central European
Institute of Technology, Masaryk University, Brno, Czech Republic
FLORENCE GUÉRARD • Plateforme Métabolisme-Métabolome, Institut des Sciences des Plantes
de Paris-Saclay, Unité Mixte de Recherche 8618 Centre National de la Recherche
Scientifique, Orsay Cedex, France
YI HAN • School of Food and Biological Engineering, Hefei University of Technology, Hefei,
Anhui, China; National Engineering Laboratory of Crop Stress Resistance Breeding, School
of Life Sciences, Anhui Agricultural University, Hefei, China
KENJI HASHIMOTO • Department of Applied Biological Science, Tokyo University of Science,
Noda, Chiba, Japan
MICHEL HAVAUX • Aix-Marseille University, CNRS, CEA, UMR7265, Biosciences and
Biotechnologies Institute of Aix-Marseille, CEA/Cadarache, Saint-Paul-lez-Durance,
France
JINGJING HUANG • Department of Plant Biotechnology and Bioinformatics, Ghent
University, Ghent, Belgium; Center for Plant Systems Biology, VIB, Ghent, Belgium
FRANCIS IMPENS • VIB Center for Medical Biotechnology, Ghent, Belgium; UGent
Department of Biomolecular Medicine, Ghent, Belgium; VIB Proteomics Core, Ghent,
Belgium
TAKAHIRO ISHIKAWA • Department of Life Sciences, Faculty of Life and Environmental
Science, Shimane University, Matsue, Shimane, Japan; Institute of Agricultural and Life
Sciences, Academic Assembly, Shimane University, Matsue, Shimane, Japan
EMMANUELLE ISSAKIDIS-BOURGUET • Université Paris-Saclay, CNRS, INRAE, Univ Evry,
Institute of Plant Sciences Paris-Saclay (IPS2), Orsay, France

xi
xii Contributors

KAROLINA IZBIAŃSKA-JANKOWSKA • Department of Plant Ecophysiology, Institute of

Experimental Biology, Faculty of Biology, School of Natural Sciences, Adam Mickiewicz
University, Poznan, Poland
JAAKKO KANGASJA€ RVI • Organismal and Evolutionary Biology Research Programme, Faculty
of Biological and Environmental Sciences, and Viikki Plant Science Center, University of
Helsinki, Helsinki, Finland
SAIJALIISA KANGASJA€ RVI • Organismal and Evolutionary Biology Research Programme,
Faculty of Biological and Environmental Sciences, FIN-00014 University of Helsinki,
Helsinki, Finland; Department of Agricultural Sciences, Faculty of Agriculture and
Forestry, FIN-00014 University of Helsinki, Helsinki, Finland
GYÖRGY KÁTAY • Plant Protection Institute, Centre for Agricultural Research, Eötvös Loránd
Research Network (ELKH), Budapest, Hungary
HIDETAKA KAYA • Department of Food Production Science, Ehime University, Matsuyama,
Ehime, Japan
PAVEL KERCHEV • Phytophthora Research Centre, Department of Molecular Biology and
Radiobiology, Faculty of AgriSciences, Mendel University in Brno, Brno, Czech Republic
SACHIE KIMURA • Ritsumeikan Global Innovation Research Organization, Ritsumeikan
University, Kusatsu, Shiga, Japan
LÓRÁNT KIRÁLY • Plant Protection Institute, Centre for Agricultural Research, Eötvös
Loránd Research Network (ELKH), Budapest, Hungary
BRIGITTE KSAS • Aix-Marseille University, CNRS, CEA, UMR7265, Biosciences and
Biotechnologies Institute of Aix-Marseille, CEA/Cadarache, Saint-Paul-lez-Durance,
France
HANA KUCHAŘÍKOVÁ • Mendel Centre for Plant Genomics and Proteomics, Central European
Institute of Technology, Masaryk University, Brno, Czech Republic
KAZUYUKI KUCHITSU • Department of Applied Biological Science, Tokyo University of Science,
Noda, Chiba, Japan
ANDRÁS KÜNSTLER • Plant Protection Institute, Centre for Agricultural Research, Eötvös
Loránd Research Network (ELKH), Budapest, Hungary
CAROLINE LELARGE-TROUVERIE • Institut des Sciences des Plantes de Paris-Saclay, Unité
Mixte de Recherche 8618 Centre National de la Recherche Scientifique, Orsay Cedex,
France
SHENGCHUN LI • State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life
Sciences, Hubei University, Wuhan, China
GABRIELA LOCHMANOVÁ • Mendel Centre for Plant Genomics and Proteomics, Central
European Institute of Technology, Masaryk University, Brno, Czech Republic
JUN’ICHI MANO • Science Research Center, Organization for Research Initiatives,
Yamaguchi University, Yamaguchi, Japan
TAKANORI MARUTA • Department of Life Sciences, Faculty of Life and Environmental
Science, Shimane University, Matsue, Shimane, Japan; Institute of Agricultural and Life
Sciences, Academic Assembly, Shimane University, Matsue, Shimane, Japan
ANDREAS J. MEYER • Institute of Crop Science and Resource Conservation (INRES),
Rheinische Friedrich-Wilhelms-Universit€ at Bonn, Bonn, Germany
AMNA MHAMDI • VIB Center for Plant Systems Biology, Ghent University, Zwijnaarde,
Belgium
CORENTIN MOREAU • Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of
Plant Sciences Paris-Saclay (IPS2), Orsay, France
 Contributors xiii

STEFANIE J. MÜLLER-SCHÜSSELE • Department of Biology, Molecular Botany, Technische

Universit€at Kaiserslautern, Kaiserslautern, Germany
YOSHIYUKI MURATA • Graduate School of Environmental and Life Science, Okayama
University, Okayama, Japan
XIUJIE MU • School of Food and Biological Engineering, Hefei University of Technology,
Hefei, Anhui, China
GRAHAM NOCTOR • Institut des Sciences des Plantes de Paris-Saclay, Unité Mixte de
Recherche 8618 Centre National de la Recherche Scientifique, Université de Paris-Sud,
Orsay, France; Institut Universitaire de France (IUF), Paris, France
JESÚS PASCUAL • Molecular Plant Biology, Department of Biochemistry, University of Turku,
Turku, Finland
ZUZANA PLŠKOVÁ • Phytophthora Research Centre, Department of Molecular Biology and
Radiobiology, Faculty of AgriSciences, Mendel University in Brno, Brno, Czech Republic
GEORGE V. POPESCU • Department of Biochemistry, Molecular Biology, Entomology, and
Plant Pathology, Mississippi State University, Mississippi State, MS, USA; Institute for
Genomics, Biocomputing, and Biotechnology, Mississippi State University, Mississippi State,
MS, USA
SORINA C. POPESCU • Department of Biochemistry, Molecular Biology, Entomology, and
Plant Pathology, Mississippi State University, Mississippi State, MS, USA
MARIE-SYLVIANE RAHANTANIAINA • Institute of Ecology and Environmental Sciences of Paris,
Molecular Ecophysiology of Stressed plant, Faculté des Sciences et Technologie, Université
Paris Est Créteil, Créteil, France
MARKUS SCHWARZLA€ NDER • Institute of Plant Biology and Biotechnology, Westf€ alische
Wilhelms-Universit€ at Münster, Münster, Germany
ALEXEY SHAPIGUZOV • Organismal and Evolutionary Biology Research Programme, Faculty
of Biological and Environmental Sciences, and Viikki Plant Science Center, University of
Helsinki, Helsinki, Finland; Natural Resources Institute Finland (Luke), Piikkiö, Finland
PAN SHEN • State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life
Sciences, Hubei University, Wuhan, China
KOICHI SUGIMOTO • Science Research Center, Organization for Research Initiatives,
Yamaguchi University, Yamaguchi, Japan; Tsukuba Plant Innovation Research Center,
University of Tsukuba, Ibaraki, Japan
MIMI TIAN • School of Food and Biological Engineering, Hefei University of Technology,
Hefei, Anhui, China
EVY TIMMERMAN • VIB Center for Medical Biotechnology, Ghent, Belgium; UGent
Department of Biomolecular Medicine, Ghent, Belgium; VIB Proteomics Core, Ghent,
Belgium
JOSÉ MANUEL UGALDE • Institute of Crop Science and Resource Conservation (INRES),
Rheinische Friedrich-Wilhelms-Universit€ at Bonn, Bonn, Germany
BIPENG WANG • State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life
Sciences, Hubei University, Wuhan, China
REN WANG • Institute of Botany, Jiangsu Province and Chinese Academy of Sciences,
Nanjing, Jiangsu, China
PATRICK WILLEMS • Department of Plant Biotechnology and Bioinformatics, Ghent
University, Ghent, Belgium; Center for Plant Systems Biology, VIB, Ghent, Belgium
MICHAEL WRZACZEK • Organismal and Evolutionary Biology Research Programme, Viikki
Plant Science Centre, University of Helsinki, Helsinki, Finland; Institute of Plant
xiv Contributors

Molecular Biology, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech
Republic
WEI XUAN • MOA Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle
Reaches of the Yangtze River and State Key Laboratory of Crop Genetics and Germplasm
Enhancement, Nanjing Agricultural University, Nanjing, Jiangsu, China
SHENG XU • Institute of Botany, Jiangsu Province and Chinese Academy of Sciences,
Nanjing, Jiangsu, China
ZBYNĚK ZDRÁHAL • Mendel Centre for Plant Genomics and Proteomics, Central European
Institute of Technology, Masaryk University, Brno, Czech Republic
LINLIN ZHANG • School of Food and Biological Engineering, Hefei University of Technology,
Hefei, Anhui, China
ZHICHENG ZHANG • Department of Plant Biotechnology and Bioinformatics, Ghent,
Belgium; VIB-UGent Center for Plant Systems Biology, Ghent, Belgium
 Part I

Strategies to Induce ROS Production

 Chapter 1

Modification of Chloroplast Antioxidant Capacity by Plastid

Transformation
Shengchun Li, Pan Shen, Bipeng Wang, Xiujie Mu, Mimi Tian, Tao Chen,
and Yi Han

Abstract
As immobile organisms, green plants must be frequently challenged by a broad range of environmental
stresses. During these constantly adverse conditions, reactive oxygen species (ROS) levels can rise extremely
in plants, leading to cellular dysfunction and cell death presumably due to irreversible protein overoxida-
tion. Once considered merely as deleterious molecules, cells seek to remove them as efficiently as possible.
To enhance ROS scavenging capacity, genes encoding antioxidative enzymes can be directly expressed from
the genome of plastid (chloroplast), a major compartment for ROS production in photosynthetic organ-
isms. Thus, overexpression of antioxidant enzymes by plastid engineering may provide an alternative to
enhance plant’s tolerance to stressful conditions specifically related with chloroplast-derived ROS. Here, we
describe basic procedures for expressing glutathione reductase, a vital component of ascorbate-glutathione
pathway, in tobacco via plastid transformation technology.

Key words ROS, Glutathione reductase, Chloroplast, Plastid transformation, Tobacco

1 Introduction

Reactive oxygen species (ROS) are partially reduced or excited

forms of atmospheric oxygen. The major forms of ROS in plants
are singlet oxygen, hydrogen peroxide, superoxide anion, and
hydroxyl radical [1]. ROS were initially recognized as mere toxic
by-products of metabolic processes and can result in cellular dys-
function and cell death. ROS are able to cause irreversible modifi-
cations to cysteine residues in proteins, leading to their degradation
and/or structural alterations [2, 3]. On the other hand, intensive
research has demonstrated that ROS play an integral role as signal-
ling molecules in the regulation of numerous biological processes
such as growth, development, and responses to a variety of biotic
and/or abiotic stimuli in plants [4–7]. To utilize ROS as signalling
molecules, non-toxic levels must be maintained in a delicate balance
between ROS production and elimination that is achieved via a

Amna Mhamdi (ed.), Reactive Oxygen Species in Plants: Methods and Protocols, Methods in Molecular Biology, vol. 2526,
https://doi.org/10.1007/978-1-0716-2469-2_1, © Springer Science+Business Media, LLC, part of Springer Nature 2022

3
4 Shengchun Li et al.

range of antioxidative systems, including superoxide dismutase,

catalase, the ascorbate-glutathione pathway, and numerous antiox-
idant metabolites [8, 9].
In plants, chloroplast is the key ROS-producing compartment,
which mainly produces superoxide anion, primarily by photosystem
I acceptors, and singlet oxygen, which is generated in photosystem
II [10]. Hence, overexpression of ROS scavenging enzymes in the
chloroplast can straightforward modify chloroplastic antioxidant
capacity, and improve plant performance under stresses. Asides
from targeting recombinant enzyme to the chloroplast with a tran-
sit sequence via nuclear transformation, expressing genes encoding
ROS-scavenging enzymes directly in the chloroplast via plastid
transformation is a promising alternative to protect plants against
various oxidative stresses [11–14]. Compared with nuclear genetic
engineering, plastid transformation offers several notable advan-
tages, such as highly precise transgene insertion by efficient homol-
ogous recombination, the potential for expressing foreign proteins
to extraordinarily high levels, the absence of epigenetic and position
effects, the possibility of stacking transgenes in synthetic operons,
and the excellent biosafety due to exclusion of plastids from pollen
transmission in most crops [15, 16]. Therefore, plastid engineering
has been extensively applied in biopharmaceuticals and industrial
enzyme production and in crop improvement against a variety of
environmental stimuli [17–19].
We propose that chloroplast transformants expressing antiox-
idative genes represent an excellent and challenging tool in which
to study the roles of various components of ROS-processing sys-
tems in the chloroplasts and their relationship with ROS in
response to adverse environmental conditions. The procedure
described here is based on the research of Wang et al. [13] with
slight modifications. The main steps of the procedure include vec-
tor construction, biolistic transformation, selection, regeneration
of plastid transformants, and molecular analysis of homoplasmic
plants, as well as glutathione reductase (GR) activity and glutathi-
one contents measurement.

2 Materials

2.1 Preparation of 1. Aseptically cultivated tobacco plants (Nicotiana tabacum L. cv

Plant Materials Petit Havana).
2. Growth chamber-grown plants of Arabidopsis thaliana
(Col-0).
3. Dilute sodium hypochlorite (2% active chlorine).
4. Sterile Milli-Q water.
5. Petri dishes (φ ¼ 90 mm), Magenta boxes.
 Antioxidant and Plastid Transformation 5

2.2 Culture Media 1. 20 Macro salts for MS medium (1 L): 38.0 g KNO3, 33.0 g
Components NH4 NO3, 8.8 g CaCl2·2 H2O, 7.4 g MgSO4·7H2O, 3.4 g
KH2PO4. Store at 4  C.
2. 200 Micro salts for MS medium (1 L): 4.46 g MnSO4·4H2O,
1.72 g ZnSO4·7H2O, 1.24 g H3BO3, 166.0 mg KI, 50.0 mg
Na2MoO4·2H2O, 5.0 mg CuSO4·5H2O, 5.0 mg CoCl2·6-
H2O. Store at 4  C.
3. 200 iron solution for MS (100 mL): 556 mg FeSO4·7H2O
and 746.0 mg Na2EDTA. Store at 4  C.
4. 200 vitamins/organic compounds for MS medium
(100 mL): 2 g inositol, 2.0 mg thiamine HCl, 10.0 mg pyri-
doxine HCl, 10.0 mg nicotinic acid, and 40.0 mg glycine.
Store at 4  C.
5. Naphthalene acetic acid (NAA), 1 mg/mL (in 0.1 M NaOH).
6. 6-benzylaminopurine (6-BA), 1 mg/mL (in 0.1 M HCl).
7. Spectinomycin, 100 mg/mL.
8. Streptomycin, 100 mg/mL.
9. MS medium for growth of plants in sterile culture (1 L): 20
Macro salts (50 mL), 200 Micro salts (10 mL), 200 iron
solution (10 mL), 200 vitamins/organic compounds
(10 mL), pH 5.6–5.8 (adjust with 0.2 M KOH).
10. Regeneration medium of plants (RMOP) medium for shoot
regeneration: MS medium, 0.1 mg/L NAA, 1.0 mg/L 6-BA,
30 g sucrose, pH 5.7, 8.0 g/L agar.
11. Rooting medium: MS medium, 0.1 mg/L NAA, 30 g sucrose,
pH 5.7, 8.0 g/L agar.
Shoot regeneration medium or rooting medium is steri-
lized by autoclaving and cooled to approximately 60  C.

2.3 Vector for 1. RNA isolation reagent (e.g., TRIzol).

Tobacco Plastid 2. Restriction enzymes.
Transformation
3. PCR Mix (containing Pfu DNA polymerase, dNTPs, and reac-
tion buffer).
4. Milli-Q water.
5. Gene-specific primer pairs (see Note 1).
6. Gel Extraction Kit.
7. Plasmid Extraction Kit.
8. Competent cells of Escherichia coli.
9. Luria-Bertani (LB) solid or liquid medium: 1% tryptone, 0.5%
yeast extract, 1% NaCl, with or without 1% agar.
10. Mortar, pestle, liquid nitrogen.
6 Shengchun Li et al.

11. PCR Thermal Cycler.

12. NanoDrop spectrophotometer.
13. Shaking incubator.
14. Tobacco plastid transformation vector: pYY12 [20] (see
Note 2).

2.4 DNA-Coated Gold 1. Gold particles (0.6 μm).

Particles and Biolistic 2. Plasmid Midiprep Kit.
Delivery System
3. 2.5 M CaCl2 solution.
4. 0.1 M spermidine (free base).
5. Ice-cold 100% ethanol.
6. Sterile ultrapure water.
7. PDS-1000/He Biolistic Particle Delivery System.
8. Hepta adaptor, rupture disks (1100 psi), stopping screen,
macro-carriers, and macro-carrier holders for biolistic gun.
9. Vacuum pump for biolistic gun.
10. Pressurized helium in tank, 99.999% pure.
11. Laminar flow hood for bombardment.

2.5 Identification of 1. DNA extraction reagent (e.g., cetyltrimethylammonium bro-

Transplastomic Lines mide (CTAB)).
2. Oligonucleotides for hybridization probe: psaB-Fwd, TTAGC-
CAAAGGTGTAC GTTCATGAG, psaB-Rev:
TTGCCCGGCTGGTTAAATGC.
3. Restriction enzyme (Bgl II, see Note 3).
4. Hybridization and detection kit with digoxigenin-labelled
probes.
5. Positively charged nylon membranes.
6. UV crosslinker.

2.6 GR Enzyme 1. Protein extraction buffer (0.1 M NaH2PO4, 1 mM EDTA,

Activity Measurement pH 7.5).
2. Polyvinylpyrrolidone.
3. 10 mM NADPH.
4. 50 mM oxidized glutathione (GSSG).
5. Bicinchoninic Acid (BCA) Kit.
6. Spectrophotometer.
 Antioxidant and Plastid Transformation 7

3 Methods

3.1 Preparation of 1. Surface sterilize tobacco seeds in a 1.5 mL microcentrifuge

Tobacco Plant tube with 1 mL of dilute sodium hypochlorite for 8 min.
Materials Remove the sodium hypochlorite with a sterile pipette and
rinse the seeds with 1 mL of sterile Milli-Q water for 1 min.
Repeat rinsing with sterile Milli-Q water three times to remove
all traces of sodium hypochlorite.
2. Sprinkle about tobacco 100 seeds on the surface of MS
medium containing 3% sucrose in a Petri dish. When seedlings
reach 1 cm (about 2 weeks) in size, transfer seedlings to
Magenta boxes (one shoot per box) containing MS medium
plus 3% sucrose, and let them grow 3–4 weeks (see Note 4).
The plants are grown in a growth room in a 16 h photoperiod,
25  C/22  C, and an irradiance of 50–75 μmol m2 s1.

3.2 Construction of 1. Amplify the coding region (see Note 5) of any gene of interest
Tobacco Plastid (for instance, AtGR2, AT3G54660) using gene-specific
Transformation Vector primers.
2. Digest the pYY12 using the restriction enzymes Nco I and
Xba I.
3. Transform the digested pYY12 without GFP gene and targeted
gene (e.g., AtGR2) PCR products simultaneously into E. coli
competent cells to generate plastid transformation construct
pLSC5 via homologous recombination [13, 20, 21] (see
Fig. 1a).

3.3 Particle 1. Perform the following steps on ice (at 4  C). Sterile ultrapure
Preparation and DNA water and 100% ethanol must be ice-cold.
Coating 2. Use 1.5 mg of gold particles (0.6 μm diameter) per seven shots
(Hepta Adaptor).
3. Transfer the gold particles to 1.5 mL Eppendorf tube, add
600 μL 100% ice-cold ethanol in tube, and vortex for 1 min
at maximum power.
4. Spin down the tube in microcentrifuge at 5000 g.
5. Remove the ethanol completely, and resuspend the gold parti-
cles in 600 μL of sterile ultrapure water by vigorous vortex.
6. Spin down the tube in microcentrifuge at 5000 g, and discard
the supernatant.
7. Resuspend the gold particles in 175 μL sterile ultrapure water
by vortex.
8. Add to the gold preparation in the following order: 20 μg
plasmid DNA for transformation (concentration: ~2 μg/μL),
175 μL 2.5 M CaCl2, 35 μL 0.1 M spermidine.
8 Shengchun Li et al.

rps14
trnfM
ptDNA psaB

probe

trnG
psbZ
Bgl II

Bgl II
3.5 kb

Nco I

Xba I
Nt-Prrn:T7g10

CrPpsbA
B

CrTrbcl
rps14

TrrnB
loxP

loxP
pLSC5 psaB
GR2 aadA

probe

trnG
psbZ
Bgl II

Bgl II
7.1 kb

C bp

8000

5000

3000
Nt-WT

Nt-pLSC5#3

Nt-pLSC5#8
Marker

Fig. 1 Generation of transplastomic tobacco plants. (a) Physical map of plastid genome region (ptDNA) used for
integration of transgene and the map of tobacco plastid transformation vector pLSC5. AtGR2 is targeted to the
intergenic region between trnfM and trnG. The selectable marker gene aadA is controlled by psbA promoter
(CrPpsbA) and fused to rbcL 30 UTR (CrTrbcL) from Chlamydomonas reinhardtii, and the AtGR2 is driven by the
tobacco plastid rRNA operon promoter combined with the 50 UTR from gene10 of bacteriophage T7 (NtPrrn:
T7g10) and the 30 UTR from the E. coli ribosomal RNA operon rrnB (TrrnB). (b) RFLP analysis of transplastomic
tobacco lines (fragment sizes: 3.5 kb in the wild type, Nt-wt; 7.1 kb in Nt-pLSC5). (The figure is from Ref. [13]
with modification)
 Antioxidant and Plastid Transformation 9

9. Incubate on ice for 10 min, and vortex for few seconds every
minute.
10. Spin down the tube in microcentrifuge at 3500 g, and remove
supernatant completely.
11. Add 600 μL of 100% ethanol, and carefully resuspend the
particles by pipetting up and down.
12. Spin down the tube in microcentrifuge at 5000 g, and remove
supernatant completely.
13. Repeat steps 10 and 11 one more time.
14. Resuspend the particles carefully in 50 μL of 100% ethanol by
pipetting up and down.
15. Use 6.5 μL per bombardment shot, and carefully resuspend
particles by pipetting immediately before use.

3.4 Bombardment 1. Place abaxial side of sterile leaf samples (see Note 6) up on a
Procedure thin layer of RMOP medium without antibiotics in a Petri dish.
2. Sterilize rupture disks (1100 psi) for 1 min, macrocarriers for
5 min, and stopping screens for 10 min by soaking in 100%
ethanol, and then air-dry them thoroughly in an open Petri
dish in the laminar flow hood.
3. Turn on helium tank and set helium pressure at regulator for
1300–1400 (200–300 psi above rupture disk value).
4. Place the Petri dish holder 9 cm (first shelf from the bottom)
below the microcarrier assembly, and close the door.
5. Turn on the vacuum button VAC position, and allow the
vacuum pressure to reach 27–28 in. of Hg, switch the vacuum
button on HOLD position, and press the FIRE button until
the rupture disk is burst.
6. After bombardment, press the VENT switch to release the
vacuum. When the vacuum gauge shows zero, open the door
and take out the bombarded samples from the chamber.
7. Incubate the bombarded leaves in dark in culture room for
2 days (see Note 7).

3.5 Identification of 1. After 2 days, cut bombarded leaves into 5 mm  5 mm pieces,

Transplastomic Lines and place 12 pieces per plate abaxial side up on RMOP medium
supplemented with 500 mg/L spectinomycin (see Note 8).
The spectinomycin-resistant calli or shoots appear 4–12 weeks
after bombardment (see Note 9).
2. Screen plastid transformants by transferring small
(5 mm  5 mm) leaf sections of the regenerated shoots onto
RMOP medium containing (i) 500 mg/L spectinomycin, and
(ii) 500 mg/L each of spectinomycin plus streptomycin (see
Note 10).
10 Shengchun Li et al.

3. Verify authentic transplastomic plants via Southern blot (see

Note 11) if selected plastid transformant candidates can resist
upon above antibiotic treatments.
4. Repeat plant regeneration on the RMOP medium containing
500 mg/L spectinomycin, and confirm uniform transforma-
tion of plastid DNA by Southern blot analyses. Plants regener-
ated twice on selective spectinomycin medium can normally
achieve homoplastomic status.
5. Transfer the homoplasmic transplastomic tobacco shoots
regenerated on rooting medium in boxes.
6. Once roots have developed, transfer the plants to soil, and
grow them to maturity and harvest seeds in the greenhouse.

3.6 GR Enzyme 1. Select nodes 3 and 4 of 6-week-old soil grown plants for
Activity Assay measurement of enzyme activity.
2. Grind 100–150 mg leaf tissue in liquid nitrogen, and then add
approximately 50 mg insoluble polyvinylpyrrolidone followed
by 1.5 mL protein extraction buffer. Continue to grind during
thawing until a homogenous suspension is obtained.
3. Centrifuge 10 min at 4  C and 15,000 g, and transfer 1 mL
supernatant to a fresh tube.
4. Add 10 μL of 10 mM NADPH and 100 μL of extract 880 μL of
assay buffer (the same as extraction buffer) in cuvette at 25  C.
5. Start reaction by addition of 10 μL of 50 mM GSSG, and
monitor decrease in A340 (ε340NADPH ¼ 6200 M1cm1)
for 2–3 min at 10 s intervals.
6. Determine protein concentration using the commercial BCA
kit following the manufacturer’s instructions.
7. Calculate the amount of enzyme in the sample using the fol-
lowing formula (see Note 12):
GR activity ðnmol:mg protein  1: min  1Þ
sample rate Δ340 = min  blank rate Δ340 = min
¼
6220 M  1cm  1  mg protein in sample

4 Notes

1. Primers include gene-specific sequence and 20–30 bp exten-

sions homologous to vector ends. The primers harbor (under-
lined) a short homologous sequence to the vector pYY12
[20]. For instance, oligonucleotides used to amplify coding
region of the cDNA of Arabidopsis GR2 as follows: AtGR2-
Fwd-1,
 Antioxidant and Plastid Transformation 11

TTTAAGAAGGAGATATACCCATGAGTACCGATAATG-
GAGCTGAATC; AtGR2-Rev-1, GCCTTTCGTTTTATTTG
ATTCTAGATTCTACACCCCAGCAGCTGTTTTAG.
2. The plastid transformation vector pYY12 contains a green
fluorescent protein (GFP) expression cassette and a selectable
spectinomycin resistance gene (aadA) cassette [20].
3. Apart from Bgl II, alternatively chosen restriction enzyme
(s) should cut the DNA creating fragment(s) that consent to
clearly distinguish between wild type, heteroplastic, and homo-
plastic lines.
4. Leaf samples for bombardment can be taken when shoot tip
reaches half the height of the Magenta box.
5. Transit peptide sequence is not required for chloroplast-
targeted protein.
6. Only the youngest 2–3 leaves are used for bombardment.
7. Incubation allows time for marker gene expression before
selection is started.
8. The growth conditions for the whole selection procedure are
16 h light 20–25 μmol m2 s1 at 25  C and 8 h dark at 20  C.
9. The spectinomycin-resistant events are not always true plastid
transformants because spontaneous point mutations in the
plastid 16S rRNA also confer a similar spectinomycin resistance
phenotype.
10. Authentic transplastomic clones carrying an aadA gene are
resistant to both spectinomycin and streptomycin, whereas
spontaneous spectinomycin-resistant mutants are resistant
only to spectinomycin. While resistance is manifested as forma-
tion of green calli with regenerating shoots, sensitivity is indi-
cated by formation of scanty white callus in the leaves.
Resistance to streptomycin and spectinomycin indicates the
presence of selectable aadA gene. However, double selection
delays shoot formation. Hence, we score the presence of aadA
gene by resistance to streptomycin plus spectinomycin, but
screen homoplastic transplastomic plants on spectinomycin.
11. Labeling of the probe and hybridization are performed with
the commercial kit containing digoxigenin-labelling. Total cel-
lular DNA (5 μg) from leaves of wild type and spectinomycin-
resistant plants is digested using restriction enzyme (e.g., Bgl
II). Separate the digested DNA by electrophoresis in 1% aga-
rose gel, blot it onto a positively charged nylon membranes
through semi-dry capillary transfer method, and cross-link it to
the membrane by UV light. A 587 bp fragment of the psaB
gene amplified with primer pair psaB-Fwd/psaB-Rev is used as
a hybridization probe to verify plastid transformants (see
Fig. 1b).
12 Shengchun Li et al.

A B
250 600

Glutathione (nmol/g FW)

* * 500
(nmol/mg protein/min)
200
400 * *
GR Activity

150
300
100
200
50 100

0 0
Nt-pLSC5#3

Nt-pLSC5#8

Nt-pLSC5#3

Nt-pLSC5#8
Nt-WT

Nt-WT
Fig. 2 Glutathione reductase activity and glutathione analysis. (a) GR activity and (b) glutathione content in
wild type and transplastomic lines Nt-pLSC5. White bars, reduced forms. Red bars, oxidized forms. Data are
means  SE of four independent extracts. Asterisks indicate significant differences from WT at P<0.05. (The
figure is from Ref. [13] with modification)

12. As well as glutathione, the GR activities in both transplastomic

lines are significantly more increased than wild-type plants (see
Fig. 2), demonstrating that introducing gene(s) of key antioxi-
dant enzyme in chloroplasts is an efficient approach to increase
tolerance to oxidative stress. Glutathione content is measured
by enzyme-dependent recycling assay, as previously described
in Noctor et al. [22]).

Acknowledgments

This work was supported by grants from the National Natural

Science Foundation of China (32071477, 31700227, and
31300225), Innovation Base for Introducing Talents of Discipline
of Hubei Province (2019BJH021), and start-up funding of Anhui
Agricultural University.

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 Chapter 2

Analysis of Ascorbate Metabolism in Arabidopsis Under

High-Light Stress
Takanori Maruta and Takahiro Ishikawa

Abstract
Ascorbate is the most abundant soluble antioxidant in plants, and its concentration is enhanced under high-
light and other abiotic stresses. One of the main functions of ascorbate is the detoxification of reactive
oxygen species, as ascorbate-deficient plants are highly sensitive to high-light-induced photooxidative
stress. Its antioxidative role in plants is further complemented by the presence of ascorbate peroxidases,
as well as enzymes that recycle ascorbate from its oxidized forms. In parallel with ascorbate biosynthesis, the
expression and activity of these enzymes are enhanced by photooxidative stress. Thus, ascorbate metabolism
plays a key role in photooxidative stress acclimation. Herein, the present authors’ preferred protocols for
the application of high-light stress and the measurement of ascorbate and the activity of related enzymes are
described.

Key words Ascorbate, Dehydroascorbate, Photooxidative stress, High light, Chloroplast

1 Introduction

Light intensity is one of the most variable environmental cues.

Although plants require light to drive photosynthesis, their sole
energy source, high irradiance often exceeds the capacity of photo-
synthesis and results in the enhanced production of reactive oxygen
species (ROS), such as H2O2, in chloroplasts [1]. The activation of
photorespiratory flux by high irradiance also facilitates H2O2 pro-
duction in peroxisomes through glycolate oxidation [2, 3]. Further-
more, high irradiance is often concomitant with overheating
and/or drought, and these stress combinations further stimulate
ROS production [4]. It is now widely accepted that ROS are
signaling molecules that trigger the stress acclimation response as
well as other biological processes [5–7]. However, ROS are poten-
tially toxic, and their overaccumulation can cause oxidative inacti-
vation of cellular functions and can ultimately kill the cell
[8]. Plants have acquired multiple antioxidant systems to minimize
light-induced oxidative stress (photooxidative stress) [1, 9,

Amna Mhamdi (ed.), Reactive Oxygen Species in Plants: Methods and Protocols, Methods in Molecular Biology, vol. 2526,
https://doi.org/10.1007/978-1-0716-2469-2_2, © Springer Science+Business Media, LLC, part of Springer Nature 2022

15
16 Takanori Maruta and Takahiro Ishikawa

10]. Regulation of cellular ROS levels through antioxidant systems

is therefore essential to plant stress tolerance mechanisms, and this
regulation also allows plants to use ROS as signaling molecules in a
controlled manner.
Ascorbate is the most abundant water-soluble antioxidant in
plants, and it can remove ROS such as superoxide radical, hydroxyl
radical, and singlet oxygen in a non-enzymatic manner
[11]. Although ascorbate cannot efficiently react with H2O2, plants
have acquired ascorbate peroxidases (APXs), which convert H2O2
into water using ascorbate as an electron donor, allowing plants to
use ascorbate for H2O2 scavenging [11]. In addition to APXs,
enzymes that recycle ascorbate from its oxidized forms, monode-
hydroascorbate reductases (MDARs) and dehydroascorbate reduc-
tases (DHARs), are also present in plants [12]. MDARs use NAD
(P)H as an electron donor, while DHARs use glutathione (GSH) as
an electron donor. The latter reaction is coupled with glutathione
reductases (GRs), which recycle GSH from the oxidized form
(GSSG). Ascorbate peroxidases and these recycling enzymes con-
stitute the ascorbate-glutathione cycle (see Fig. 1), which is targeted
to the cytosol, chloroplasts, mitochondria, and/or peroxisomes
[8, 13].
Ascorbate metabolism is regulated by light intensity
[14, 15]. High irradiance enhances ascorbate concentrations
through the activation of ascorbate biosynthesis, the D-mannose/
L-galactose pathway [16], and the ascorbate-glutathione cycle
enzymes. Vitamin C-defective Arabidopsis mutants (vtc), in which
ascorbate biosynthesis capacity is limited [17], are highly sensitive
to high-light stress [15, 18]. A recent discovery revealed that
ascorbate recycling through GSH-dependent enzymatic and
non-enzymatic reactions is required for ascorbate accumulation
and high-light stress tolerance in Arabidopsis [15]. Thus, ascorbate
accumulation through ascorbate biosynthesis and recycling is criti-
cal in protecting cells from photooxidative stress.
Although several methods are available for measuring ascor-
bate, two representative methods are (1) high-performance liquid
chromatography (HPLC)-based (e.g., [19]) and (2) ascorbate oxi-
dase (AO)-based enzymatic assays (e.g., [20]). As compared to
other colorimetric assays, such as the bipyridyl method based on
iron (III) reduction [21], these two methods are more specific to
ascorbate. We have used both methods in previous studies [19, 22]
and, based on our own experiences, consider that the HPLC-based
assay is more accurate and reliable. One reason for this is likely to be
that ascorbate is very stable in metaphosphate buffers owing to its
metal chelating ability, which is used as the extraction buffer and as
the mobile phase for the HPLC assays in the protocol outlined
herein. In the case of AO-based assays, HCl or HClO4 is used for
ascorbate extraction to inactivate enzymes that use ascorbate and,
then, the extract needs to be diluted with a certain buffer and
 Analysis of Ascorbate Metabolism in Arabidopsis Under High-Light Stress 17

H2O2 2 ASC NAD+

APX MDAR
H2O 2 MDHA NADH

ASC DHA 2 GSH NADP+

(disproportionation)
DHAR GR
ASC GSSG NADPH

Fig. 1 The ascorbate-glutathione cycle in plants. APX ascorbate peroxidase, ASC

ascorbate (reduced form), DHA dehydroascorbate, DHAR dehydroascorbate
reductase, GR glutathione reductase, GSH glutathione (reduced form), GSSG
oxidized glutathione, MDHA monodehydroascorbate radical, MDAR
monodehydroascorbate reductase

neutralized for the subsequent AO reaction (e.g., [22]). These

processes not only complicate the method, but also may potentially
affect ascorbate stability and turnover. It should be noted that
AO-based assays are actually specific to ascorbate and more accessi-
ble in the absence of the HPLC system. Herein, the present
authors’ preferred laboratory protocols for the application of
high-light stress, measurement of ascorbate, and measurement of
APX, DHAR, and MDAR enzyme activities are described. Methods
for enzyme assays are largely according to a previous report [20].

2 Materials

1. White LED lighting unit.

2. 2% metaphosphate in water (v/v) as a buffer solution.
3. 1% metaphosphate in water (v/v) as the mobile phase, filtered
on two layers of filter paper with 4 μm pores (ADVANTEC).
4. 0.22 μm filter (Merck Millipore).
5. 350 mM Tris (2-carboxyethyl) phosphine hydrochloride
(TCEP) in water.
6. Ascorbate standards (0.125–1 mM) in 2% metaphosphate.
7. Ultra-fast liquid chromatography (UFLC) system (Prominence
UFLC, Shimadzu) equipped with an SPD-20A UV-Vis detec-
tor (Shimadzu).
8. C-18 column (LUNA C18(2), column 150
4.6 nm,
Shimadzu).
9. Potassium phosphate buffer. KH2PO4 (500 mM) and
K2HPO4 (500 mM) in water are to be combined and pH
adjusted to 7.6 (for APX activity) or 7.0 (for DHAR activity).
18 Takanori Maruta and Takahiro Ishikawa

10. 50 mM Ethylenediaminetetraacetic acid (EDTA) in water,

adjusted to pH 8.0 by addition of NaOH.
11. 50 mM MES/KOH buffer (pH 7.6) in water.
12. Ascorbate oxidase.
13. Spectrophotometer.

3 Methods

3.1 High-Light 1. Place and rotate plants on a turntable under an LED panel to
Exposure and Sample provide uniformity of irradiation (see Note 1).
Collection 2. After high-light (approximately 1500 μmol photons m2 s1)
exposure (see Note 2), excise leaves, and immediately immerse
them in liquid nitrogen in 15 or 50 mL tubes.
3. Prepare liquid nitrogen in a mortar, and immerse leaves in
mortars containing liquid nitrogen.
4. Grind plant tissues using a pestle, and carefully transfer the
powdered tissue (approximately 100 mg) into a 1.5 mL tube
(see Note 3). Samples should be stored at 80  C until use.

3.2 Ascorbate 1. Carefully add 1 mL of 2% metaphosphate buffer to frozen

Measurement tubes containing the powdered tissue. Keep tubes on ice cov-
ered with aluminum foil to provide shade because ascorbate is
unstable in the light. During thawing, gently shake the tubes to
mix the tissue with the buffer.
2. Centrifuge tubes for 10 min at 15,300
g (4  C).
3. Filter the supernatant using a 0.22 μm membrane filter.
4. Dilute the filtered samples in 2% metaphosphate buffer. In our
cases, samples are diluted 1/5 or 1/10 depending on plant
materials (e.g., high-light-exposed leaves contain a high con-
centration of ascorbate; see Note 4). Diluted samples can then
be used to measure the reduced form of ascorbate.
5. To measure and calculate total ascorbate (sum of reduced and
oxidized forms), transfer 360 μL of the diluted solution to a
fresh 1.5 mL tube. Next, add 40 μL of 350 mM TCEP (see
Note 5) to reduce oxidized ascorbate (DHA). Incubate on ice
or at 4  C for at least 2 h (see Note 6).
6. Prepare ascorbate standards (ranging from 0.125 to 1 mM) in
2% metaphosphate.
7. Inject 5 uL of the samples onto the UFLC. The mobile phase
consists of 1% metaphosphate and the flow rate is 0.5 mL
min1.
8. Measure the peak areas from standards (automatically by an
integrator of the HPLC instrument), and plot them against the
 Analysis of Ascorbate Metabolism in Arabidopsis Under High-Light Stress 19

400

350

300
Absorbance (mAU)

250

200

150

100

50

0 1 2 3 4 5 6 7 8 9 10
Time (min)

Fig. 2 Representative chromatogram of ascorbate standard

respective concentrations for ascorbate to obtain the calibra-

tion curve. Calculate ascorbate content using the peak area
from extracts and the calibration curve. In our system, the
retention time of ascorbate is about 8.5 min (see Fig. 2).
9. Calculate DHA content as the difference between total
(TCEP-treated) and reduced (untreated) ascorbate.
10. Calculate ascorbate redox state (reduced ascorbate/total ascor-
bate
100 [%]), which can be an oxidative damage marker (see
Note 7).

3.3 APX Activity 1. Carefully add 1 mL of 50 mM potassium phosphate buffer pH

7.6 containing 20% sorbitol (w/v), 1 mM EDTA, and 1 mM
ascorbate, to a frozen tube containing the powdered tissue.
Keep the tube on ice and, during thawing, gently shake the
tube to mix the tissue with the buffer.
2. Centrifuge the tubes for 20 min at 15,300
g (4  C).
3. Transfer the supernatant to a new 1.5 mL tube (extract for
soluble APX activity).
4. Wash precipitate with 1 mL of the same potassium phosphate
buffer twice.
5. Solubilize the washed precipitate with 500 μL of 50 mM potas-
sium phosphate buffer containing 1% 3-[(3-Cholamidopropyl)
dimethylammonio]propanesulfonate (CHAPS) (w/v)
(in addition to sorbitol, EDTA, and ascorbate), and incubate
for 1 h at 4  C with rotation (see Note 8).
20 Takanori Maruta and Takahiro Ishikawa

6. Centrifuge for 20 min at 15,300
g (4  C).

7. Transfer the supernatant to a new 1.5 mL tube (extract for
insoluble APX activity).
8. Add 10 μL of 40 mM ascorbate, 20 μL of 50 mM EDTA,
910 μL potassium phosphate buffer (50 mM, pH 7.6), and
50 μL extract to a quartz cuvette (path length ¼ 1 cm). Incu-
bate the cuvette for 1 min at 25  C.
9. Start the reaction by adding 10 μL H2O2 (50 mM), and
monitor the decrease in A290 for 3 min at 25  C (e290 ascor-
bate ¼ 2800 M1 cm1).
10. Perform a control experiment without the extract to correct for
chemical scavenging of H2O2.
11. Subtract control rates from rates obtained with the extract.
12. Measure protein concentration (per mL of extracts) of the
extracts (see Note 9).
13. Calculate APX activity using the formula described in Note 10.

3.4 DHAR Activity 1. Carefully add 1 mL of potassium phosphate buffer (50 mM,
pH 7.0) containing 1 mM EDTA to a frozen tube containing
the powdered tissue. Keep the tube on ice, and, during thaw-
ing, gently shake the tube to mix the tissue with the buffer.
2. Centrifuge for 15 min at 15,300
g (4  C).
3. Transfer the supernatant to a new 1.5 mL tube.
4. Add 50 μL DHA (2 mM), 50 μL GSH (50 mM), 500 μL
potassium phosphate buffer (100 mM), and 350 μL water to
a quartz cuvette (path length ¼ 1 cm). Incubate the cuvette for
1 min at 25  C.
5. Start the reaction by adding 50 μL extract, and monitor the
decrease in A265 for 1 min at 25  C (e265 ascor-
bate ¼ 14,000 M1 cm1).
6. Perform a control experiment without the extract to correct for
GSH-dependent non-enzymatic reduction of DHA.
7. Subtract control rates from rates obtained with the extract.
8. Measure protein concentration (per mL of extracts) of the
extracts (see Note 9).
9. Calculate DHAR activity using the formula described in
Note 10.

3.5 MDAR Activity 1. Carefully add 1 mL MES/KOH buffer (50 mM, pH 6.0)
containing 1 mM ascorbate, 2 mM CaCl2, and 40 mM KCl,
to a frozen tube containing the powdered tissue. Keep the tube
on ice, and, during thawing, gently shake the tube to mix the
tissue with the buffer.
 Analysis of Ascorbate Metabolism in Arabidopsis Under High-Light Stress 21

2. Centrifuge for 15 min at 15,300
g (4  C).

3. Transfer the supernatant to a new 1.5 mL tube.
4. Add 10 μL NADH (25 mM), 10 μL ascorbate (250 mM),
920 μL HEPES buffer (50 mM), and 50 μL extract to a quartz
cuvette (path length ¼ 1 cm). Incubate the cuvette for 1 min at
25  C.
5. Start the reaction by adding 10 μL of ascorbate oxidase (20 U/
mL) to generate MDHA, and monitor the decrease in A340 for
3 min at 25  C (e340 NADH ¼ 6200 M1 cm1).
6. Measure protein concentration (per mL of extracts) of the
extracts (see Note 9).
7. Calculate MDAR activity using the formula described in Note
10.

4 Notes

1. Even if plants are exposed to light stress under a large LED

panel, the intensity of the light that plants receive varies
depending on their position under the panel. To homogenize
light intensity from the light source, plants are placed on a
turntable and rotated during light exposure.
2. High-light stress refers to the detrimental effects of excess light
energy on plant function and growth through damaging the
photosynthetic apparatus. The light intensity that damages
photosynthesis and inhibits growth varies depending on plant
species. In general, Arabidopsis plants are grown under
50–200 μmol photons m-2 s-1, and they do not show any
inhibition of growth and development. However, plants
exhibit obvious light stress phenotypes, such as anthocyanin
accumulation and/or photo-bleaching, at high-light intensities
(1000–2000 μmol photons m2 s1). Growth of Arabidopsis
plants can be inhibited at light intensities (lower than high
light, e.g., 400–800 μmol photons m2 s1), which is often
called moderate light stress to distinguish from high light. The
quality of light should be also taken into consideration for light
stress assays. The white light from the specific LED panel that
we use does not contain the full color spectrum and mainly
consists of blue and red light. Generally, this drives photosyn-
thesis effectively and can limit potential secondary effects, such
as UV and heat stresses. However, green light has previously
been shown to be efficient for photosynthesis, especially in the
deeper parts of the leaf tissue [23]. It has also been recently
reported that far red light, which cannot drive photosynthesis,
protects photosystem I against fluctuating light stress [24].
22 Takanori Maruta and Takahiro Ishikawa

3. This sample collection method might be technical and not an

easy way for measurements of ascorbate as well as other redox
metabolites, because ascorbate oxidation can be caused during
thawing in the absence of buffer. Thus, it is very important that
the sample should be frozen at all times in liquid nitrogen and
should not be thawed in the absence of buffer. Despite the
difficulty, this method can provide many samples from one pool
of plant materials for distinct assays.
4. When Arabidopsis plants are grown under optimal growth
conditions (50–100 μmol photons m2 s1), foliar ascorbate
level is ranged from 3 to 5 μmol g1 FW. The ascorbate level is
increased by high-light exposure. In our hand, the maximum
foliar ascorbate level is about 12 μmol g1 FW after 48 h of
continuous high-light (1500 μmol photons m2 s1) exposure.
5. Prepare the solutions containing the following redox com-
pounds freshly each day: ascorbate, DHA, GSH, NADH,
and TCEP.
6. Although dithiothreitol (DTT) is generally used to reduce
DHA, the protocol outlined herein employs TCEP as a reduc-
tant. To reduce DHA using DTT, the sample solution must be
neutralized and incubated for an amount of time (e.g., 30 min)
at room temperature, under which conditions ascorbate and
DHA are not stable. In contrast to DTT, TCEP has a broad
effective pH range (pH 1.5–8.5), and, in the conditions
recounted herein, 35 mM TCEP can completely reduce DHA
on ice within 2 h. In our own hand, the treatment can be
extended for up to 12 h or more. Incubation of samples with
TCEP can be performed in the sample drawer of the HPLC
machine at 4  C during the measurements of reduced ascorbate
and standards.
7. Since ascorbate oxidation is enhanced by oxidative stress, the
ascorbate redox state has been used as an oxidative stress
marker. However, we have never detected the significant
increase in DHA content in leaves exposed to high light even
when ascorbate recycling capacity is severely compromised
[15], suggesting that DHA is highly unstable and does not
accumulate at high levels in Arabidopsis leaves even under
oxidative stress conditions.
8. For APX activity measurements, the soluble fraction contains
cytosolic and stromal isoforms, while the insoluble fraction
consists of peroxisomal membrane- and thylakoid membrane-
bound isoforms [25]. In our own hand, almost negligible
DHAR and MDAR activities can be detected in the insoluble
fraction, although Arabidopsis MDAR4 is a peroxisomal
membrane-bound isoform [26].
 Analysis of Ascorbate Metabolism in Arabidopsis Under High-Light Stress 23

9. We usually measure protein content using a Bradford Protein

Assay Kit according to the manufacturer protocol.
10. When assayed in 1 mL final volume (using 50 μL of extract) in a
spectrophotometer (path length ¼ 1 cm), the following sim-
plified formulas can be used for enzyme activity calculations.
APX activity (μmol min1 mg1 protein) ¼ (Δ290 min1)

(1000/2800)
(1000/50 [extract volume])
(1/protein
concentration [mg/mL]. DHAR activity (μmol min1 mg1
protein) ¼ (Δ265 min1)
(1000/14,000)
(1000/50
[extract volume])
(1/protein concentration [mg/mL].
MDAR activity (μmol min1 mg1 protein) ¼ (Δ340
min1)
(1000/6200)
(1000/50 [extract vol-
ume])
(1/protein concentration [mg/mL]. For DHAR
activity calculations, since GSSG has a low absorbance at
265 nm (180 mM1 cm1 at pH 7.0), the reaction rate should
be corrected by multiplying by a factor of 0.98 (assuming the
formation of 1 mol of GSSG for 1 mol of ascorbate formed)
[27]. In our own hand, activities of soluble and insoluble
APXs, DHAR, and MDAR in Arabidopsis leaves grown under
normal growth conditions are 1.0–1.5, 0.2–0.3, 0.2–0.25, and
0.15–0.25 μmol min1 mg1 protein, respectively.

Acknowledgments

This work was supported by JSPS KAKENHI Grant Numbers

18K19179 (T.M), 17H03807 (T.I. and T.M.), and 19K22284
(T.I. and T.M.)

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 Chapter 3

Genetic Manipulation of Reactive Oxygen Species (ROS)

Homeostasis Utilizing CRISPR/Cas9-Based Gene Editing
in Rice
Sheng Xu, Tao Chen, Mimi Tian, Marie-Sylviane Rahantaniaina,
Linlin Zhang, Ren Wang, Wei Xuan, and Yi Han

Abstract
Reactive oxygen species (ROS) are now recognized as key signals in plant stress responses. Adverse
environmental conditions can either promote ROS production or downregulate antioxidative enzymes,
leading to the alteration of redox homeostasis and activation of ROS-linked stress signaling. To uncover
their signaling mechanisms and to characterize related components, genetic modification of ROS homeo-
stasis is a central approach. CRISPR/Cas9-based genome editing system has become a powerful tool for
gene mutation in a variety of organisms, including plants. Within this chapter, we describe a method that
can be applied to manipulate ROS homeostasis in rice (Oryza sativa L.) utilizing CRISPR/Cas9 technol-
ogy. Step-by-step protocols including the design and construction of Cas9/sgRNA, agrobacterium-
mediated transformation, and mutation characterization are described. Application of this system in editing
a rice catalase gene CatC, a key antioxidative enzyme in controlling ROS homeostasis, is also presented.

Key words ROS homeostasis, H2O2, Antioxidant, Catalase, CRISPR/Cas9 system, Gene editing

1 Introduction

Abiotic and biotic stresses undoubtedly can lead to enhanced pro-

duction and accumulation of reactive oxygen species (ROS) in
photosynthetic organisms. ROS are defined as oxygen (O2) deriva-
tives exhibiting more chemically reactive than O2 itself [1]. They
are more widely recognized as important signal molecules instead
of its traditional concept of toxicity even in plant development as
well as environmental responses [2–4]. In plants, superoxide,
hydroxyl radical, singlet oxygen, and hydrogen peroxide (H2O2)
are main forms of ROS which are generated from several essential
metabolic pathways such as during photosynthesis, respiration, and
photorespiration, as well as NADPH oxidases and certain perox-
idases [2]. H2O2 is one of the most stable of ROS; it can be

Amna Mhamdi (ed.), Reactive Oxygen Species in Plants: Methods and Protocols, Methods in Molecular Biology, vol. 2526,
https://doi.org/10.1007/978-1-0716-2469-2_3, © Springer Science+Business Media, LLC, part of Springer Nature 2022

25
26 Sheng Xu et al.

perceived as a signal through interactions with components such as

phytohormones, ion signaling, transcription factors, and coactiva-
tors that are redox sensitive. H2O2 has a strong oxidizing potential
and can oxidize proteinaceous cysteinyl thiols to sulfenic acid that
thereby may affect the protein conformation and functionality [5].
Apart from inducing ROS production, alteration in ROS avail-
ability can also result from decreased metabolism. As other living
organisms, plants have evolved a set of complicated antioxidative
systems to finely control the dynamic change of cellular ROS levels.
These mainly involve ascorbate-glutathione pathway, thiol-depen-
dent peroxidases, as well as catalase (CAT). Hence, they are key
regulators involved in ROS homeostasis and signaling cascades
[6]. Among them, CAT was shown to play a crucial role in redox
homeostasis in plants [7]. CAT can limit the accumulation of H2O2
that is produced directly from peroxisomal glycolate oxidase reac-
tion during photorespiration. Increased H2O2 availability in perox-
isome has been reported to function in spontaneous cell death,
activation of defense phytohormone-dependent pathways includ-
ing salicylic acid (SA), jasmonic acid and ethylene, pathogen resis-
tance, and transcriptome reprogramming in Arabidopsis [8–
15]. Therefore, catalase-deficient mutants provide an excellent
genetic tool leading to endogenous H2O2 accumulation that con-
tributes to elucidating the molecular mechanisms of H2O2 signal-
ing transduction. For instance, a direct investigation through
genetically blocking glutathione accumulation in Arabidopsis
catalase-deficient mutant cat2 showed that glutathione plays a key
role in transmitting oxidative signals [16, 17]. Subsequent work
further found that S-nitrosoglutathione reductase (GSNOR) acts
downstream of H2O2 to promote SA-dependent pathways. More-
over, glutathione-dependent denitrosation of GSNOR was shown
to maintain this biological function, establishing the missing link
between glutathione and GSNOR in the complex framework of
H2O2 signaling [18]. Additionally, introducing secondary muta-
tions lacking cytosolic ascorbate peroxidase, cytosolic/peroxisomal
glutathione reductase, and all three dehydroascorbate reductases
into the Arabidopsis cat2 mutant can abolish or decrease H2O2-
mediated oxidative stress responses [19–21], emphasizing the
unexpectable but crucial roles of antioxidant enzymes in regulating
ROS homeostasis.
The clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated protein 9 (Cas9) system has
emerged as an efficient and powerful technology for targeted
genome editing in many higher plants such as Arabidopsis
[22, 23], tobacco [24, 25], rice [26–28], sorghum [29], maize
[30], wheat [31], grape [32], tomato [33, 34], soybean [35], citrus
[36, 37], and other species [38]. CRISPR/Cas9 technology
involves two major components including the Cas9 protein and a
short guide RNA (sgRNA) [39]. The 20 nucleotides at the 50 end of
 ROS Homeostasis and Gene Editing 27

a sgRNA can precisely identify the target sequence site according to

the base-pairing rules. The target site must be located immediately
upstream of a protospacer adjacent motif (PAM), which is often the
consensus NGG (N, any nucleotide; G, guanine). This accurate
binding can activate the endonuclease activity of Cas9 to produce
a double-strand break in the target sequence that stimulate DNA
repair mechanisms in vivo, leading to gene mutation (e.g., inser-
tion, deletion, and replacement). Compared with previously devel-
oped genome-editing technologies such as zinc-finger nucleases
(ZFNs) and transcription activator–like effector nucleases
(TALENs), CRISPR/Cas9 system has much higher editing effi-
ciency, and multiple sgRNAs with different target sequences can be
designed to direct the Cas9 protein to multiple genomic sites for
simultaneous multiplex editing [27]. Thus, CRISPR/Cas9 system
has become an essential genome editing technology and provides a
convenient and vital tool for genetic engineering in plant
biology [39].
Although crucial roles are established for ROS during plant-
environment interactions, outstanding questions remain
concerning exactly how the signaling functions of these reactive
species are mediated in plants especially for crops. Furthermore,
while progress in understanding ROS-mediated redox homeostasis
and signaling has been greatly accelerated by work on the model
plant, Arabidopsis thaliana, yet the specific roles of each compo-
nent in antioxidant pathways and their interactions in the regula-
tion of ROS homeostasis and associated stress responses in crop
plants such as Oryza sativa remain scarce. Here, we describe the
basic procedures that can be applied to manipulate ROS homeosta-
sis in rice via utilizing the agrobacterium-mediated transformation
of plasmid DNA harboring a CRISPR/Cas9 system.

2 Materials

2.1 Generation of 1. Mature dry rice (Oryza sativa L. subsp. japonica cv. Wuyunjing
Gene-Edited Rice with 27) seeds.
Callus Method (See 2. 70% ethanol (vol/vol).
Note 1)
3. 2.5% sodium hypochlorite (vol/vol).
4. Sterilized distilled water.
5. Tween-20.
6. Sterilized forceps.
7. Sterilized filter paper.
8. Parafilm.
28 Sheng Xu et al.

Table 1
N6 medium

Name Reagent Concentration Stock concentration

1 (g/L) 10 (g/L)
N6 major salts KNO3 2.83 28.3
(NH4)2SO4 0.463 4.63
CaCl2 l 2H2O 0.166 1.66
MgSO4 l 7H2O 0.185 1.85
KH2PO4 0.40 4.0
1 (mg/L) 100 (mg/L)
N6 minor salts MnSO4 l 4H2O, 4.4 440
ZnSO4 l 7H2O, 1.5 150
H3BO3 1.6 160
KI 0.8 80
1 (mg/L) 100 (mg/L)
N6 vitamins Thiamine hydrochloride 1 100
Pyridoxine hydrochloride 0.5 50
Nicotinic acid 0.5 50
Glycine 2 200
1 (mg/L) 100 (g/L)
Fe-EDTAa FeSO4 l 7H2O 27.8 2.78
EDTA-Na2 37.3 3.73
1  (g/L)
Other Sucrose 30
Casamino acid 0.3
Agar or Gelrite 4.0
a
For 100  Fe-EDTA: dissolve 2.78 g FeSO4 l 7H2O in 900 mL of hot distilled water and add 3.73 g EDTA-Na2. Cool
to room temperature and make up the volume to 1000 mL. Store at 4  C

9. Callus induction medium (pH 5.8): N6 medium (see Table 1)

containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D),
0.1 g/L myo-inositol, and 2.9 g/L proline.
10. Callus infection medium (pH 5.2): AAM medium (see Table 2)
containing 100 μmol/L acetosyringone (see Note 2).
11. Co-cultivation medium (pH 5.2): N6 medium containing
2.0 mg/L 2,4-D, 10 g/L glucose, and 100 μmol/L
acetosyringone.
12. Selection medium: N6 medium containing 2.0 mg/L 2,4-D,
0.1 g/L myo-inositol, 2.9 g/L proline, 0.25 g/L carbenicillin,
and 50 mg/L hygromycin.
13. Regeneration medium: N6 medium containing 25 g/L sorbi-
tol, 2.0 mg/L 2,4-D, 2.0 mg/L kinetin, and 0.05 mg/L
α-naphthalene acetic acid (NAA).
 ROS Homeostasis and Gene Editing 29

Table 2
AAM mediuma

Name Reagent Concentration Stock concentration

1 (g/L) 10 (g/L)
AA major salts KCl 2.83 28.3
CaCl2 l 2H2O 0.463 4.63
MgSO4 l 7H2O 0.166 1.66
KH2PO4 0.185 1.85
1 (mg/L) 100 (mg/L)
B5 minor salts MnSO4 l 4H2O 13.2 1320
ZnSO4 l 7H2O 2 200
CuSO4 l 5H2O 0.025 2.5
Na2MoO4 l 2H2O 0.25 25
CoCl2 l 6H2O 0.025 2.5
H3BO3 3 300
KI 0.75 75
1 (mg/L) 100 (g/L)
B5 vitamins Thiamine hydrochloride 10 1
Pyridoxine hydrochloride 1 0.1
Nicotinic acid 1 0.1
Myo-inositol 100 10
1 (mg/L) 100 (g/L)
Fe-EDTA FeSO4 l 7H2O 27.8 2.78
EDTA-Na2 37.3 3.73
1 (mg/L) 10 (g/L)
AA amino acidsa L-Glutamine 876 8.76
L-Aspartatic acid 266 2.66
L-Arginine 174 1.74
Glycine 7.5 0.075
1 (g/L)
Other Sucrose 68.5
Casamino acid 0.5
Glucose 36
Sterilize with a 0.22 μm cellulose acetate syringe filter
a

14. Rooting medium: Murashige and Skoog (MS) medium (see

Table 3) containing 0.05 mg/L NAA.
15. Growth chamber: continuous light.

2.2 Cas9/Target- 1. pYLsgRNA intermediates (PYLsgRNA-OsU6a/Lac,

sgRNA Vector PYLsgRNA-OsU6b, PYLsgRNA-OsU6c, and PYLsgRNA-
Construction OsU3).
2. pYLCRISPR/Cas9Pubi-H binary vector.
3. Restriction enzymes.
4. T4 DNA ligase.
30 Sheng Xu et al.

Table 3
MS medium

Name Reagent Concentration Stock concentration

1 (g/L) 10 (g/L)
MS major salts KNO3 1.9 19
NH4NO3 1.65 16.5
CaCl2 l 2H2O 0.44 4.4
MgSO4 l 7H2O 0.37 3.7
KH2PO4 0.17 1.7
1 (mg/L) 100 (mg/L)
MS minor salts MnSO4 l 4H2O 22.3 2230
ZnSO4 l 7H2O 1.06 1060
CuSO4 l 5H2O 0.025 2.5
Na2MoO4 l 2H2O 0.25 25
CoCl2 l 6H2O 0.025 2.5
H3BO3 6.2 620
KI 0.83 83
1 (mg/L) 100 (g/L)
MS vitamins Thiamine hydrochloride 10 1
Pyridoxine hydrochloride 0.5 0.05
Nicotinic acid 0.5 0.05
Myo-inositol 100 10
Glycine 2 0.2
1 (mg/L) 100 (g/L)
Fe-EDTA FeSO4 l 7H2O 27.8 2.78
EDTA-Na2 37.3 3.73
1 (g/L)
Other Sucrose 15
Casamino acid 1

5. Gibson Assembly Cloning Kit.

6. 2  Taq Master Mix.
7. Chimeric primers with target sequence and universal primer set
for amplifying the sgRNA constructs (see Tables 4 and 5).
8. Milli-Q water.
9. PCR Thermal Cycler.
10. DNA Gel Extraction Kit.
11. Appropriate percentage of agarose in 1  TAE buffer (40 mM
Tris-acetate, 1 mM EDTA-Na2, pH 8.3).
12. NanoDrop spectrophotometer.
13. Competent cells of Escherichia coli.
14. Luria-Bertani (LB) solid or liquid medium: 1% tryptone, 0.5%
yeast extract, 1% NaCl, with or without 1% agar.
 ROS Homeostasis and Gene Editing 31

Table 4
Primers used for preparation of sgRNA expression cassette(s) for Golden Gate Cloning

Position Primer set Sequence (5’-3’)

1st PCR U-F CTCCGTTTTACCTGTGGAATCG
GR-R CGGAGGAAAATTCCATCCAC
OsU6a- CGACATCACCCACCTCACCTcggcagccaagccagca
CatC
gR-CatC AGGTGAGGTGGGTGATGTCGgttttagagctagaaat
2nd PCR
Site B-L Pps-L TTCAGAggtctcTctcgACTAGTATGGAATCGGCAGCAAAGG
Site 2 Pgs-2 AGCGTGggtctcGtcagggTCCATCCACTCCAAGCTC
Site 2 Pps-2 TTCAGAggtctcTctgacacTGGAATCGGCAGCAAAGG
Site 3 Pgs-3 AGCGTGggtctcGtcttcacTCCATCCACTCCAAGCTC
Site 3 Pps-3 TTCAGAggtctcTaagacttTGGAATCGGCAGCAAAGG
Site 4 Pgs-4 AGCGTGggtctcGagtccttTCCATCCACTCCAAGCTC
Site 4 Pps-4 TTCAGAggtctcTgactacaTGGAATCGGCAGCAAAGG
Site B-R Pgs-R AGCGTGggtctcGCTCGACGCGTATCCATCCACTCCAAGCTC
Flanking primers PB-L GCGCGCgGTctcGCTCGACTAGTATGG
PB-R GCGCGCggtctcTACCGACGCGTATCC
Vector sequencing SP-L GCGGTGTCATCTATGTTACTAG
primers SP-R TGCAATAACTTCGTATAGGCT
Transgenic plant Cas9-F CTGACGCTAACCTCGACAAG
testing primers Cas9-R CCGATCTAGTAACATAGATGACACC

15. Sterile LB solid medium containing 50 mg/L kanamycin,

100 mg/L X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galacto-
pyranoside), and 125 mg/L IPTG (Isopropyl β-D-1-
thiogalactopyranoside).
16. Shaking incubator.
17. Plasmid Extraction Kit.

2.3 Agrobacterium 1. Agrobacterium tumefaciens competent cells GV3101 (see Note

Transformation and 3).
Infection of Callus 2. Liquid nitrogen.
3. Water bath.
4. Sterile LB liquid and solid medium containing 50 mg/L kana-
mycin and 25 mg/L rifampicin.
32 Sheng Xu et al.

Table 5
Primers used for preparation of sgRNA expression cassette(s) for Gibson Assembly

Position Primer set Sequence (50 -30 )

1st U-F CTCCGTTTTACCTGTGGAATCG
PCR GR-R CGGAGGAAAATTCCATCCAC
OsU6a- CGACATCACCCACCTCACCTcggcagccaagccagca
CatC
gR-CatC AGGTGAGGTGGGTGATGTCGgttttagagctagaaat
2nd
PCR
Site B-L Pps-GAL ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAA
TCGGCAGCAAAGG
Site 2 Pgs-GA2 CAGGGAGCGGATAACAATTTCACACAGGCACATCCACTCCAAGCTC
TTG
Site 2 Pps-GA2 GTGCCTGTGTGAAATTGTTATCCGCTCCCTGGAATCGGCAGCAAAGG
Site 3 Pgs-GA3 CCACGCATACGATTTAGGTGACACTATAGCGCATCCACTCCAAGCTC
TTG
Site 3 Pps-GA3 CGCTATAGTGTCACCTAAATCGTATGCGTGGTGGAA
TCGGCAGCAAAGG
Site 4 Pgs-GA4 GTCGCTAGTTATTGCTCAGCGGCCAAGCTCATCCACTCCAAGCTC
TTG
Site 4 Pps-GA4 GAGCTTGGCCGCTGAGCAATAACTAGCGACTGGAA
TCGGCAGCAAAGG
Site B-R Pgs-GAR TAGCTCGAGAGGCGCGCCAATGATACCGACGCGTATCCATCCAC
TCCAAGCTCTTG

5. Agrobacterium cultivation medium: AB medium (see Table 6)

containing 50 mg/L kanamycin and 25 mg/L rifampicin.
6. Spectrophotometer.

2.4 Validation of 1. Plant Genomic DNA Purification Kit.

Targeted Mutations in 2. 2  Taq Master Mix.
Transgenic Plants
3. Testing primers.
4. PCR Thermal Cycler.
5. Direct Sanger sequencing (see Note 4).

3 Methods

3.1 Induction of Calli 1. Surface sterilize dehusked seeds with 70% ethanol for 30 s and
2.5% sodium hypochlorite containing 1 drop per 50 mL of
Tween-20 for 20 min with stirring.
 ROS Homeostasis and Gene Editing 33

Table 6
AB medium

Name Reagent Concentration Stock concentration

1 (g/L) 20 (g/L)
a
AB salts KCl 0.15 3
NH4Cl 1 20
CaCl2 l 2H2O 0.01325 0.265
MgSO4 l 7H2O 0.3 6
FeSO4 l 7H2O 0.0025 0.05
1 (g/L) 20 (g/L)
AB bufferb NaH2PO4 1 20
K2HPO4 3 60
1 (g/L)
Other Glucose 5
Agar 15
Autoclave at 121  C for 20 min. Store at 4  C
a

Adjust pH to 7.0 and autoclave at 121  C for 20 min. Store at 4  C

b

2. Rinse the seeds with sterile distilled water for five times.
3. Place the sterilized seeds on callus induction medium in a Petri
dish (twenty-five seeds per single plate), and seal the plate with
parafilm.
4. Incubate the seeds at 28  C with continuous illumination with
100 μmol m2 s1 irradiation for 3–4 weeks to form calli.
5. Collect the actively growing calli (about 1–3 mm in diameter),
which are yellowish white and relatively dry, and subculture on
fresh callus induction medium at 28  C for 3 days.

3.2 Vector 1. Select the appropriate target site(s) and design of chimeric
Construction primers with target sequence strands. Use online toolkit
CRISPR-GE (Genome Editing) (http://skl.scau.edu.cn/)
[40] to expedite all experimental designs (see Note 5). Firstly,
design all possible target sites with the defined PAM in a given
sequence of interest (or in rice genome with gene locus name)
by using the targeDesign program. Subsequently, predict the
potential off-target sites with the off-Target algorithm. After
selecting appropriate target site(s), generate primers for the
target-sgRNA expression cassette(s) construction with the pri-
merDesign program (see Note 6).
2. Generate the target-sgRNA expression cassette(s). Firstly,
determine the arrangement of multiple U3/U6 promoters
among the sgRNA expression cassette(s) (see Note 7), and
the corresponding primer sets (see Tables 4 and 5) (see Note
8). Then, prepare the first and second (overlapping) PCR
34 Sheng Xu et al.

pYLsgRNA-OsU6a/LacZ AmpR
pYLsgRNA-OsU6b pUC18 backbone
pYLsgRNA-OsU6c Bsa I
pYLsgRNA-OsU3
U-F Pps
5’-sgRNA-3’ OsU6/U3 promoter
BsaI(2) Pgs gR-R BsaI(1)
LacZ

Target sequence (+)

U-F gRT#+
pUC18 backbone
OsU6/U3 promoter sgRNA

First PCR U#T#- First PCR gR-R

Target sequence (-)

Second (overlapping) PCR Annealing

BsaI Pps
LB Extension
hP35S
2h HPT T35S KanR pBR322 ori/bom
pVS1 replicon Pgs
PCR BsaI
BsaI
PUbi

pYLCRISPR/Cas9Pubi-H

BsaI
SP-L RB
NLS NLS
Cas9p Tnos Placz:ccdB
SP-R
OsU6a sgRNA + OsU6b sgRNA + OsU6c sgRNA + OsU3 sgRNA …
BsaI (B-L) BsaI (B-R)
Target 1 (T1) Target 2 (T2) Target 3 (T3) Target 4 (T4)

Golden Gate Cloning

or Gibson Assembly

2hP35S HPT T35S KanR pBR322 ori/bom

pVS1 replicon
LB
PUbi

pYLCRISPR/Cas9Pubi-H/Target-sgRNA

NLS NLS RB
Cas9p Tnos
BsaI BsaI
(B-L) (B-R)

BsaI Golden Gate Cloning MluI

PB-L MluI T1 T2 T3 T4 (B-R)
ctcg ctga aaga gact cggt
gagc OsU6a sgRNA gact OsU6b sgRNA ttct OsU6c sgRNA ctga OsU3 sgRNA gcca
(B-L) LacZ
PB-R
BsaI
PB-L MluI Gibson Assembly MluI
GA-R
|||||||||| OsU6a sgRNA |||||||||| OsU6b sgRNA |||||||||| OsU6c sgRNA |||||||||| OsU3 sgRNA ||||||||||
GA-L LacZ
PB-R

Fig. 1 Construction of the Cas9/Target-sgRNA vector. The overlapping PCR method was used for generation of
sgRNA expression cassette(s) containing target sequence. The chimeric primers with target sequence strands
are given in Tables 5 and 6. The first PCR is carried out in two separate reactions with U-F/U#T#- and gRT#+/
gR-R primer pair, respectively. U# indicates a given promoter, and T#+ and T#- indicate forward and reverse
strands of a target sequence, respectively

reaction for each sgRNA cassette (see Fig. 1). Here, we make a
schematic diagram of one sgRNA designed to target the rice
catalase gene CatC (see Fig. 2a).
3. Make the Cas9/target-sgRNA constructs. Separate the PCR
product by agarose gel electrophoresis, and purify the DNA
fragment. Quantify the concentration of PCR product and
pYLCRISPR/Cas9Pubi-H binary vector with NanoDrop spec-
trophotometry. Set up a BsaI-digestion/ligation reaction or a
Gibson Assembly reaction (see Note 9).
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 So we strolled over to the young men's quarters, and while I wrapped
myself in a gloomy atmosphere that I considered was typical of a
temperamental killer, Nikka swapped anecdotes of crime with the others
who drifted in and out. I looked for Kara, but she was nowhere in view.
After Nikka had once established my character, the Gypsies gave me a wide
berth, and I had nothing to do but smoke and appear murderous. And I must
say I got sick of the part. I was the first man up when Mother Kathene
swung the stew-pot out of the chimney and old Zitzi and Lilli began to
distribute tin plates and cups in an irregular circle on the floor. It was poor
food, but plenty, and anyway, it broke the monotony of being an abandoned
criminal.

With the passing of the twilight the young men moved to the courtyard.
In the middle of the open space was a black smirch on the paving, and here
they built a fire of driftwood collected from the beach under the wall. It was
a tribute to the immemorial habits of their race. Even here in the crowded
city they must close the day with a discussion of its events around a tribal
blaze, exactly as they would have done upon the road, exactly as thousands
of other Gypsy tribes were doing at that very moment on the slopes of the
Caucasus, in the recesses of the Kilo Dagh, in the pine forests of the
Carpathians, on the alien flanks of the Appalachians far across the sea.

A buzz of talk arose. The primitive Gypsy fiddles and guitars began to
twang softly. Nikka was the center of a gossiping group. Men and women
from the opposite side of the court joined the circle. Young girls, with the
lithe grace of the Gypsy, as unselfconscious as animals, sifted through the
ranks of the bachelors. Beran Tokalji, himself, a cigarette drooping
sardonically from the corner of his mouth, stalked out and sat down with
Nikka.

In the changing shadows beyond the range of the firelight children

dodged and played unhindered by their elders. High overhead the stars
shone like fireflies under a purple vault. And from the spreading mass of
Stamboul echoed a gentle hum, the hum of a giant hive, a myriad voices
talking, singing, praying, laughing, shouting, cursing, screaming. None of
the discordant night noises of the West. No whistle-blasts, no shrieking of
flat wheels on tortured rails, no honking of motor-horns, no clamor of
machinery. Only the drone of the hive.

A man raised his voice in a song, and the exultantly melancholy pæan to
beauty blended with the other sounds like a skillfully woven thread in a
tapestry. It died away so gradually as to seem as if it had never been. The
fiddles sighed to silence in a burst of expiring passion.

Nobody spoke for several moments. Music was bred in the bone of these
wild folk. It held them as could nothing else.

"What of Giorgi Bordu?" said Tokalji presently. "Does he sing or play or

dance?"

Nikka reached out his hand almost eagerly.

"I will play, if you wish. I vowed not to touch the fiddle again, but—"

His fingers closed lovingly on the crude instrument, and he cuddled it

under his chin. His bow swept the strings in a torrent of arpeggios. He stood
up and strode into the firelight as if upon a stage. And then he began to play,
plaintively, at first, in a minor key. There were the noises of the night, a
crackling fire, animals stirring, the cry of a child, awakening. The music
brightened, quickened, became joyous. You felt the rays of the sun, and
comfort of work. Men and women danced and sang. A harsh note
intervened. There was a quarrel. Anger yelled from the strings. Turmoil
ensued. Faster and faster went the tune. And then peace, and the measure
became slower, almost stately.

The caravan had passed on. A forest encompassed it. Boughs clashed
overhead, birds twittered and sang. Cool shadows fell athwart the path. But
the way grew steep. The music told of the rocks and the slippery mud where
a stream had overflowed, of the steady climb, of the endurance required.
The caravan reached the height. A chill wind blew, but fair before them
stretched a pleasant land, and the descent was easy to the warm, brown road
that wound across the plain. Sunset and camp again, firelight, the moon
overhead, talk of love, the sensuous movement of a dance. Then,
languorous and slow, the coming of sleep.
 I did not know it, but I was listening to the composition of Zaranko's
Gypsy Sonata Op. 27, which some day, I suppose, will be as famous as the
Revolutionary Etude or the Hungarian Rhapsody or Beethoven's dream of
the moonlight. But no audience will ever hear it with greater appreciation
than those ragged Gypsies who sat around the fire in the dirty courtyard of
the house in Sokaki Masyeri. As Nikka resumed his place in the outer
circle, only the whispering of the flames broke the stillness. The very
children were frozen on their knees, drunk with the ecstasy of melody.

"Heh!" called Beran Tokalji, first to shake off the spell. "I do not wonder
you vowed not to touch the fiddle, if you like the open road. With that bow
of yours, Giorgi Bordu, you could wring hundreds of gold pieces from the
Franks. You play like the Redcoats in the khans in Buda and Bucharest.
Heh-heh! I have heard Niketu and Stoyan Mirko and Karaji, and they were
not to be compared with you. It is seldom the bravest men have the touch of
the fiddler."

Others spoke up readily in praise or asked questions as to Nikka's

opinion on moot points of harmony and the desirable methods of
interpreting various Gipsy songs. They would have had him play again, but
he refused. I think he was emotionally exhausted.

"We have no fiddler to match with you," remarked Tokalji, "and the
gaida[1] and the flute are not fit for real music. But our maidens can dance.
Heh, girls, come out, shy ones! Let the strangers view your grace."

[1] Bagpipes.

They giggled amongst themselves, and swayed into a group that was as
spontaneously instinct with rhythm as an old Greek temple frieze. But
suddenly they split apart.

"Kara will dance," they cried. "Let Kara dance for the strangers."
 And Kara floated into the circle of firelight like a spirit of the forest. She
still wore only the scanty madder-red skirt and torn bodice. The cloud of her
hair tumbled below her waist. Her tiny naked feet barely touched the
ground. Slowly she whirled, and the Gipsy fiddles caught her time. A man
with cymbals clashed an accompaniment. A flute whistled soprano. She
increased the tempo; she varied her steps. She was a flower shrinking
beneath the grass. She was a dove pursued by a falcon. She was a maiden
deserted by her lover. She was a fairy hovering above the world.

We who watched her were breathless with the joy of the spectacle, and
when she sank to the ground in a little pile of rags and hair as the music
ended, I thought she must be worn out. But she bounded up at once,
breathing regularly, radiating vitality.

"Now I will dance the Knife Dance!" she exclaimed. "Who will dance
with me?" And before any could answer her, she seized a blazing stick from
the fire, and ran around the circle waving it overhead until she came to
where Nikka sat. "Ho, Giorgi Bordu, you who do not fear the knife, will
you dance the Knife Dance with me?"

Every eye in the circle was fixed on Nikka, for, although I did not know
it then, to have refused her invitation would have been a deadly insult,
equivalent to a declaration of enmity toward her family and tribe. Similarly,
acceptance of it amounted to an admission that he considered her favorably
as a wife, without definitely committing him to matrimony.

Nikka did not hesitate. He stepped to her side. She slipped one arm
around his waist, and with the other swung her torch in air until it showered
sparks over the circle.

"Hi!" she cried.

"Hi!" echoed Nikka.

And they pranced around the fire while the music commenced an air so
fiercely wild that it made the blood tingle to listen to it. Then she flung
down her torch, and tore free from Nikka's arm. He followed her. She
eluded him. Bound and round they tore, keeping step the while. Now she
accepted him, now she rejected him. At last he turned from her, arms
folded, contemptuously unmoved. She wooed him with rhythmic ardor. He
denied her. She drew her knife; he drew his. Eyes glaring, lips pinched, they
circled one another, feinting, striking, leaping, posturing.

"Click!" The blades struck together.

"Hi! Hi!" they cried.

"Click! Clack! Click!" went the knife-blades.

"Ho! Ho!" they shouted.

The game was to see how near you could come without cutting. To avoid
hurt the dancers required quick eyes and agile bodies. The blades flashed
like meteors in the shifting light, wheeling and slashing and stabbing. In the
beginning Kara forced the pace. Nikka retired before her, rather than risk
doing her harm. But slowly he assumed the mastery. His knife was always
at her throat, and active as she was, he refused to be shaken off. She fended
desperately, panting now, bright-eyed and flushed. But he pressed her. Their
blades clashed, he gave his a twist and hers dropped from her hand.

He seized her, forcing her back across his knee, knife up-raised to strike,
while the fiddles clutched at one's nerves and the cymbals clanged with
wicked glee. The scene—Nikka's tall figure, with the poised knife, and the
lithe, slender form he held, expressing in every curve and line its
tempestuous, untamed soul—brought to my memory the song I had heard
him sing one morning in the music-room at Chesby:

And best of all, I shall hear

The wild, mad Tzigane songs,
Cruel and gay and lustful,
Like fiddles and clanging gongs.

And in the glare of the campfires

I shall see the Tziganes dance—
Women with lithe, round bodies,
Men straight as a heiduck's lance.
 And perhaps a wild brown maiden
Will seek me—

Crash! boomed a knock on the street-door. And rap-rap-rap! it was

repeated. Crash! again.

The music stopped. Nikka released his partner, and Kara stooped quickly
and snatched up her knife, tossing the hair out of her eyes, heedless as usual
of the rags that slipped off her shoulders.

Men looked at each other uncertainly. Hands crept to waist-sashes.

"Heh!" said Tokalji. "Who can it be in such a hurry at this hour?"'

Crash! The door resounded under the battering of a pistol-butt.

CHAPTER XVIII

THE BIG SHOW BEGINS

The women and children—all save Kara—withdrew into the shadows.

The men gathered together. Tokalji crossed to the entrance.

"Less noise there!" he shouted threateningly. "This is a peaceful house."

But his manner changed the moment he opened the wicket. What he said
we could not hear, but we saw him quickly turn the lock and throw back a
leaf of the door, salaaming low as he stepped aside. Six men burst in, four
of them in European clothes, and Nikka and I exchanged a glance of
apprehension as we recognized the broad shoulders of their leader and
heard his snarling voice.
 Toutou LaFitte had arrived. With him were Hilyer, Serge Vassilievich
and Hilmi Bey. The two who brought up the rear, somewhat sulky and
fearful, were the spies we had seen in front of the Pera Palace that morning.

"Can I trust nobody to fulfill my orders?" whined Toutou, striding

toward the fire. "I tell you to spare no efforts—and I come to find you
singing and dancing around a fire! Is that working? Is that carrying out our
treaty? But all are the same! My best people fail me."

His green eyes shone evilly; his hands writhed with suppressed ferocity.
Tokalji, having refastened the door, followed him across the courtyard. The
Gypsy looked uncomfortable, but showed no fear.

"What could we have done that we have not done?" he retorted. "Was it
our fault that you lost track of the two missing ones? As for the English lord
and his servant, my two men that I see with you have shadowed them day
and night."

"And lost them to-day, as they admit," snarled Toutou. "Lost them for a
whole day! Who knows what has been accomplished in that time?"

"You are right there," agreed Tokalji coolly, "and I have just picked two
new men to take their places. Zlacho and Petko are good enough for
ordinary thievery, but this job seems to be above them."

"That is well," said Toutou, partly mollified. "There must be a change in

our methods or we shall fail in this coup. I decided to hasten on to
Constantinople with my colleagues because I was sure the two who have
escaped us must come here sooner or later, and whenever they come we
shall find them. But I cannot do everything. It is for you to follow their
trails."

"Never fear! We shall," replied Tokalji. "My new men start out at once.
One of them is a Frank like yourself; the other is a Tzigane."

"Ha; let me see that Frank," exclaimed Toutou. "I know many of the
Franks who live with the Tziganes."
 "Step out, Giorgi Bordu and Jakka," called Tokalji.

Nikka sunk his fingers in my arm in a warning grip, and we stepped

forth from the group of Tziganes clustered in front of the fire. There was at
least a chance that we should not be identified—but its value was
demonstrated the instant the firelight splashed over Nikka's aquiline face
and tense, febrile body.

"Surely, I have seen that lean fellow before," piped Hilmi Bey, pointing
at Nikka.

"I saw them standing near the Frank lord and his servant in Pera this
morning," said one of the spies.

"What of that?" shouted Tokalji angrily. "It is true they followed the
Franks—which was more than you could do, Petko—and robbed them."

"No, the Franks followed them," protested Zlacho, the other spy.

"You lie, you dog!" bellowed Tokalji. "You think to discredit them
because they will do the work you bungled."

Vassilievich pushed in front of the newcomers.

"Is it my imagination," he inquired softly, "or does the stocky one bear a
resemblance to the Americansky, Nash?"

"By jove, I think you're right!" exclaimed Hilyer, speaking for the first
time.

"Be ready," hissed Nikka from the corner of his mouth, without shifting
his eyes from our enemies.

His right hand was thrust into his waist-sash.

"I do not like this business," rasped Toutou, pulling a knife from inside
his vest. "Somebody shall be tortured until he tells the truth."

I felt a pressure between Nikka and myself, and Kara's voice whispered:
 "Run, you fools! To the House of the Married!"

Nikka's pistol flashed blue in the firelight.

"Shoot, Jack!" he cried.

A ruddy flame jetted from his muzzle, and the spy Petko dropped dead.
Toutou LaFitte pushed Zlacho in the line of fire before himself, and dived
into the encircling shadows as Zlacho crumpled up with a broken leg.
Tokalji, Hilyer, Vassilievich and Hilmi scattered. I swung on my heel and
shot twice over the group of Gypsies by the fire. I could not bring myself to
shoot at them, for there were women and children close by. Then a bullet
whistled past my ear, and Toutou's voice whined:

"No shooting! Use your knives! Take them alive!"

I had a fleeting glimpse of Kara, running at me with her knife raised.

"There are only two!" roared Tokalji. "Pull them down!"

"Run!" I heard Nikka shout.

We pelted for the house on our left, the House of the Married, as Kara
had called it. Despite Toutou's warning, a second bullet spattered on the
stones between Nikka and me; but we were poor marks in the half-light,
with people running in every direction, many of them uncertain who were
friends or foes. I turned as I ran, and fired into the ground in front of Kara,
who was the closest of our pursuers; but she refused to be frightened and
actually plunged through the doorway on our heels.

"I'll tend to her," panted Nikka. "You fasten the door, Jack."

There was a wooden bar, which I dropped into place, and the next
minute the framework groaned under a weight of bodies.

"No shooting," yelled Tokalji. "You fools, you'll have the Frank police in
here!"
 "One hundred Napoleons a head for them," barked Toutou. "Dead or
alive."

The uproar redoubled, and then Tokalji evidently invaded the throng
hammering at the door.

"Leave that door alone," he snapped. "You're wasting time. Go through

the windows."

"Come on, Nikka," I urged. "We can't guard every point. We must run
for it."

"But what about this?" demanded Nikka whimsically. He jerked his

pistol muzzle at Kara sitting demurely on the floor, playing with her knife.
"If we show our backs, she'll knife us or open the door—and besides, where
shall we go?"

"Tie her up," I answered impatiently.

Kara, who, of course, could not understand a word of what we were

saying, laughed with glee.

"Do you think I am your enemy?" she demanded in the Tzigane dialect.
"I tell you I am your friend. See!"

And she tossed her knife across the room.

"I came with you to help you, Giorgi Bordu."

"My name is Nikka Zaranko," he answered shortly.

"What matters your name?" She leaped up and flung her arms around his
neck. "It is you I love—not your name."

Nikka eyed me sheepishly across her shoulder.

"See you, little one," he remonstrated, "this is no time for talking of love.
We may be dead in five minutes."
 "Oh, no," she said, releasing him, nevertheless, "you shall be off and
away. I, Kara—" and it was ridiculous how she strutted in the manner of
Tokalji, himself—"will set you free—because I love you."

"But I am the enemy of your tribe—your enemy," replied Nikka. "You

do not realize what you do."

"I care not who you are," she insisted. "I love you. I care that for the
tribe!"

She snapped her fingers.

"But come," she added as a crash sounded outside. "They have broken in
a window. Follow me."

She led us into an adjoining room, where in the thickness of the wall a
narrow stairway corkscrewed upward, debouching on the upper floor. Here
was a long hall, with rooms opening off it, their windows usually on the
inner courtyard, the Garden of the Cedars of the First Hugh's Instructions.
She turned to the right, and entered one of the rooms. A ladder leaned
against the wall below a trap-door in the roof. In a corner stood a bedstead,
which she stripped of its clothes, revealing the cords that served for springs.

"Cut those with your knife," she said. "When we take to the roofs we
will need them to help us down again."

Nikka did as she directed, while I shut the door, and piled the few
articles of furniture against it. Tokalji's men were in full cry downstairs.

"There is more than enough rope here," said Nikka, coiling it on his arm.
"Some of it I am going to use for you."

"What?"

Passion dawned in her big eyes.

"You cannot go with us, little one. We have no place to take you. And
you do not know me. To-morrow you would cry your eyes out."
 "I tell you I love you," she answered proudly. "I, Kara Tokalji."

"The daughter of my deadly enemy," reiterated Nikka.

"Oh, he is not my father," she said lightly. "No, I think I will go with
you, Nikka."

"And I think you won't," retorted Nikka, gritting his teeth. "Here, Jack,
catch hold."

He cut the rope in two, gave me half, and with the remaining section,
approached her. She backed away from him.

"I'm not going to hurt you," pleaded Nikka. "But I must bind you so they
will not suspect that you aided us. Don't you see? And we could not run so
fast with you."

"I can run as fast as the Frank," she declared. "But—"

"Our enemies will be here in a moment," warned Nikka.

She extended her hands, wrists joined together.

"Bind me," she said wearily. "I love you, Nikka Zaranko. If I can help
you in no other way, then, I will help you by staying here."

He bound her gently, hand and foot, without a word, and laid her on the
floor by the bed. I ascended the ladder, and pushed back the trapdoor.

"You will come again?" she asked, looking up at him with mournful
eyes.

"If I do, it will be as an enemy," he returned.

"Your enemies are my enemies," she cried, struggling to a sitting

position. "With a woman it is her man who counts. She cares nothing for the
tribe—unless it be her man's. Now, you are my man, Nikka Zaranko."
 Nikka stooped over her, and I scrambled up on the roof. I believe he
kissed her. I heard his feet on the ladder-rungs, and his voice calling back:

"You are a brave girl. We will talk about this some other time, if the stars
are kind."

"Oh, we shall meet again," she replied, her cords creaking as she
dropped flat on the floor. "I am as sure of it as if Mother Kathene had told
me when the sight was on her."

To me he merely said:

"Hurry, Jack! We've lost too much time. Which way?"

But I reached down first, and hauled up the ladder. The door was shaking
under a shower of blows. Kara looked interested as my arm appeared, and
her lips shaped themselves for a kiss. Then she saw it was I, and scowled.

"Next house," I panted, and we set off across the roof.

To our left was the inner courtyard, a well of darkness in which tinkled
the Fountain of the Lion. To our right lay Sokaki Masyeri. Ahead was a
drop of ten feet on to the adjoining roof, the difference in height
representing the declining slope of the ground. We made it without any
difficulty. The people in this house had been aroused by the shooting, and
we could hear their voices and movements. But we shuffled on cautiously,
until we came to their courtyard, which ran clear from the street-front to the
old sea-wall.

"No choice," grunted Nikka. "Here's a chimney. Knot your rope. It can't
be more than twenty-five feet to the ground.'

"Why not slide directly into the street?" I argued.

"They might catch us coming down. Do as I say, and we can make sure
whether the coast is clear before we leave the courtyard."

He went down first, and I followed him, scorching my hands, for the
rope was thin and had no knots to check one's descent. I was in mid-air
when I heard an exclamation beneath me, and a thud.

"What the devil—" I started to whisper.

"Hsst!" came from Nikka. "Don't say anything."

He was standing over an inert figure lying on the ground beside a half-
opened door.

"Did you—"

"No, only belted him over the head with my pistol."

A woman's voice sounded inside the house, aggressively inquisitive.

"My God!" breathed Nikka. "She'll be out in a minute, and I can't hit her.
We've got to try the street."

We stole through the courtyard to the street-door. Behind us Toutou's

house was seething with activity. Somebody, apparently, had just gained the
roof. The woman inside the house we had invaded became impatient, and a
light showed. My fingers fumbled for the latch; it seemed to me I should
never find it. The light wavered into the doorway, and a scream rose shrilly.

"Let me try," said Nikka. "Here it is!"

He pulled the door toward us very slowly, and we peered into the street.
Not a figure showed in the direction of Tokalji's house. Ahead of us only a
kerosene lantern burned in front of a coffee-shop on the corner where
Sokaki Masyeri curved to the north. And the woman in the doorway of the
house behind us was shrieking for dear life.

We sped out into the street, letting the door slam behind us. The noise
distracted the attention of the woman from her unconscious husband, and
she left him to run after us. We also made the mistake of taking the middle
of the way instead of sticking to the shadows under the walls. And we had
not gone fifty feet when we were seen by Gypsies on the roof of Tokalji's
house, and they, with the woman to help them, cried the rest of the pack hot
on our trail.
 At the corner by the coffee-shop I looked back and counted six in a
tapering string, with more emerging from the courtyard or climbing over the
roofs. Luckily for us, however, there was a four-way crossing a hundred
yards beyond the coffee-shop, and Nikka turned left, away from Pera,
toward which they would expect us to head. We would have been safe then
if we had not blundered into a Turkish gendarme. He was naturally
suspicious of our haste, and blocked the narrow way; but I gave him a
terrific punch in his fat stomach before he could pull his gun.

We got by, of course, but his roars put the Tziganes right, and they
followed the scent instead of losing it as we reckoned they would. The only
thing for Nikka to do in the circumstances was to twist and turn without
heed to direction and lose both pursuers and ourselves in the breakneck
purlieus of Stamboul. He succeeded in shaking off the Gypsies finally, but
we were hopelessly astray, and it was past midnight when we found the
Khan of the Georgians and staggered through the gate to thread a precarious
path between sleeping men, camels, bullocks, asses and horses.

Wasso Mikali awakened with the first knock on his door, and admitted
us. Smoking cigarette after cigarette as rapidly as he could roll them, he
listened to the story of our adventures with avidity,—although I discovered
later that Nikka had suppressed Kara's part—and immediately dispatched
his young men to spy around Tokalji's house, and learn the dispositions the
enemy were taking. Then he insisted that we should sleep while he kept
watch, and the last memory I have of that awful night is of the old Gypsy's
figure stretched out on the floor, his back against the bolted door and a
cigarette in his mouth.

When we awakened the sun was streaming in through the open door
along with all the noises of the Khan and many of its smells. Our guardian
had coffee ready for us in a pot on the brazier, and his young men had sent
in a report. The women and children had left Tokalji's house under escort of
several of the men shortly after dawn. A vigilant guard was being
maintained on the entrance, and nobody had come or gone—aside from the
party of women and children—since observation had been established.
Before sunrise our spies had heard the sounds of digging inside the
premises.
 Wasso Mikali looked doubtful as he imparted this last information.

"Perhaps they, too, have discovered the location of the treasure," he

suggested.

"No," said Nikka, smiling. "They are burying their dead."

"Ha, that is a good thought to hold in the mind," exclaimed the old
Gypsy, immensely pleased. "What better pleasure could a man ask than to
contemplate his enemies burying their brother that he slew!"

But instead of indulging in this Tzigane pastime we decided to take our

European clothing and adjourn to a neighboring Turkish bath where we
could remove the evidence of our Gypsy life. Wasso Mikali went with us to
carry back to the khan our discarded Gipsy costumes. I urged him to join us
in the pool after we had soaked off the top layer of iniquities in a private
room; but he shook his head with a grimace of disgust.

"Tell Jakka, O son of my sister," he said, "that I marvel at the way you
risk your naked skins. How can a man hope to withstand the cold and heat
if he has nothing but clothing to cover him? Too much water is bad for the
strongest. It weakens the muscles."

CHAPTER XIX

FIRST CRUISE OF THE CURLEW

"So far, Jack, you and Mr. Zaranko seem to have had most of the fun,"
pronounced my cousin Betty, as we sat at luncheon in the Kings' private
sittingroom in the Pera Palace.

Watkins for the moment acted as butler, and we were safe from
inquisitive ears and could talk with freedom.
 "What interests me," said Hugh thoughtfully, "is how many of those
Johnnies you scragged last night."

"Only the one, I think," replied Nikka.

"You hit another chap," I reminded him.

"Yes, but two off their strength doesn't mean any great reduction in their
fighting force."

"Still, counting in those two and the men they sent off with their women,
as Nikka's pals reported, they'll be a good bit weaker than they were,"
argued Hugh.

"Just the same," insisted Betty, "we ought not to run any unnecessary
risks."

"Who's we?" I inquired.

"See here, Jack," she flashed, "because you're my cousin is no reason

why you can bully me. You might as well understand that I am in this, and I
am going to have my part in whatever we do."

"Hear, hear," Hugh applauded servilely.

Nikka laughed.

"How about it, Vernon?" I demanded of my uncle.

He spread his hands in a gesture of depreciation.

"My dear Jack," he said, "you evidently have small acquaintance with
the younger feminine generation. Betty is of legal age—I trust, my dear,
you have no objection to the revelation of an intimate detail your sex are
supposed to cherish in secret?—"

"Not a particle, dad," Betty responded cheerfully.

 "—and within reasonable limits, her judgment is to be depended upon.
Moreover, a not unimportant consideration is that she knows how to run a
motor, and in our excursions in the Curlew her aid has been of some
value.",

"Don't be stuffy, Jack," urged Hugh. "Give the girl a chance. There are
lots of things she can do, short of mixing it with your friend Toutou. I
gather that Nikka's lady friend in the hostile camp was not averse—"

"That's a different matter," I interrupted, perceiving the embarrassment

on Nikka's face.

We had slurred over Kara's personal interest in his fortunes, but even so,
the incident, to quote Betty's analysis, was "romantic to the nth degree."

"I don't see that it is," asserted Betty stubbornly, "and I intend to play my
part. You are short-handed—"

"You forget that Nikka has seven men hidden away in Stamboul," I
reminded her.

"On the contrary, I take them into account," she retorted. "But you have
all been saying that it is advisable not to use them, except in a final
emergency."

"That is true," agreed Nikka. "The more we bring into this row, the
noisier it will become. Also, as we decided before, we ought to have an ace
or two in the hole. Take my advice, and hang on to Wasso Mikali and his
young men to the last."

"I'm not disputing you," said Betty, still belligerent. "What you say is
only what I've been saying. But would you mind telling me why you are so
set against using your Gypsies?"

"If we use them there will be killing on a big scale," said Nikka
succinctly. "That sort of thing is bound to become known."
 "I met Riley-Gratton, the O.C. of the M.P.s this morning, and he gave me
a wad of town gossip," cut in Hugh, "but he didn't say anything about our
lads' scrap at Tokalji's house."

"Oh, we can get away with it once or maybe twice," returned Nikka, "but
if we keep it up we'll run into trouble."

"No question of it," I said.

"Then what are we arguing about?"' demanded Betty.

I laughed.

"Darn it all," I confessed. "You won't let up, will you? Well, have it your
own way. What do you want to do?"

"Run you down the Bosphorus after dark for a look at Tokalji's house
from the water side," she answered promptly.

Hugh intervened.

"There's no question in the minds of you two chaps but that any attack
ought to come from the water front, is there?" he asked.

"It couldn't very well come from the street," replied Nikka. "There's a
high, windowless wall and a strong door, and even in that lawless quarter
publicity would attend an armed invasion of private property."

"Of course," said Betty, her head in the air, "it couldn't be any other way.
Now tell us some more about the hiding-place of the treasure."

Nikka shrugged his shoulders and looked at me.

"What more can we say?" I answered. "There's the courtyard and the red
stone."

"It's not hollow, you said?" spoke up King.

"No."
 "That would indicate a task of some difficulty in prying loose the
covering of the treasure chamber," he remarked. "We have—or rather, I
should say, Betty has—taken precautions to install on board the Curlew an
equipment of crowbars, pick-axes, shovels, chisels and other tools—"

"—and a knotted rope with a grapnel on the end to help in going up the
sea-wall," reminded Betty.

"True, my dear. Your forethought has been admirable. What I was about
to say, however, was that a certain amount of time—I fear, perhaps, an
inordinate amount of time—will be required to pry loose the covering of the
vault. How are we to secure ourselves such an opportunity?"

"By choosing a time when the occupants of the house are off-watch and
their numbers diminished," declared Hugh.

"True," agreed Nikka, "yet I confess I don't see how—"

And to make a long story short we hashed it over all afternoon until tea-
time, without arriving at any clearer view of the outlook before us. By that
time we were sick of the discussion, and voted to suspend. Vernon King and
Betty went to a reception at the British High Commissioner's, and the rest
of us planned to take a walk on the chance of running into Wasso Mikali,
who had promised to come over to Pera in the afternoon if his spies picked
up any additional information.

The first person we saw in the hotel lobby was Montey Hilyer. He hailed
us in front of the booking-office.

"I say, Chesby," he drawled in tones that reached all the bystanders, "I
don't know what sort of a lark you fellows were up to last night, but really,
you know, you can't take liberties with natives in the East—and especially,
with their women. Really, old chap, you ought to be careful. In your place, I
think I'd clear out of Constantinople. No knowing what kind of trouble you
may get into."

Hugh was furious. He looked Hilyer up and down with cold scorn.
 "Are you taking a flyer in blackmail, by any chance?" he asked
deliberately.

"Not yet," answered Hilyer cheerfully. "No knowing, though. Matter of

fact, at present, I'm protecting some poor natives who fear they are going to
be victimized by a gang of foreigners."

"Well, whatever you are doing, I should prefer that you keep away from
me in the future," said Hugh. "I can't afford to have the Jockey Club
stewards hear that I've been talking to you."

As it happened, the one episode in Hilyer's piebald past that irked his
pride and aroused sore memories was his suspension from the privileges of
the turf. He was cynically indifferent to every other charge brought against
him. But the man was a sincere horseman, his racing ventures had been the
breath of life to him, his disgrace and compulsion to enter his
thoroughbreds under other men's colors had been a bitter blow. And he
showed this feeling now. His face went dead-white; his nostrils pinched in.

"All right, Chesby," he said curtly, "I won't forget that."

And he disappeared into the bar.

"Curse the rotter," muttered Hugh. "I'm glad something will flick him on
the raw."

"You were hard on him," said Nikka seriously. "After all, why should
you mind anything that he can say?"

"He was hoping that Miss King was within hearing distance," retorted
Hugh. "He said what he did deliberately to smear smut on all of us. A dog
like that doesn't deserve consideration."

"Some people believe a dog does deserve consideration, Lord Chesby,"

said a feminine voice behind us.

We turned to face Hélène de Cespedes. The Countess Sandra Vassilievna

was with her. Maude Hilyer, her face as ghastly as her husband's, was
hurrying away from them.

"You may be enemies, but why should you make a woman cry?" added
the Russian girl. "She will be unhappy for the rest of the day."

"I'm very sorry," answered Hugh stiffly, "but do you sincerely believe
that her husband is entitled to insult me in public?"

"It was a rotten thing he said," admitted Hélène frankly. "And of course,
he is a rotter. But as I told you boys once, they are a queer pair, and Maudey
—well, she really thinks that if they ever get to a state of affluence, they can
both turn around and live straight. It's damned silly, but—do you believe in
fairies? Those who don't, generally envy those who do."

"We don't believe in fairies," I answered good-temperedly, "and we also

don't believe in letting a man who is a thief get away with a gratuitous
insult."

"Oh, you're right," said Sandra Vassilievna impartially, "from your own
point of view. But I'm going up to tell Maudey that she'll only ruin her
complexion if she weeps for what an offensively honest man says to her."

Hélène laughed as the Russian walked off.

"Women are almost as funny as men, aren't they?" she said. "Oh, say,
before I forget it, Mr. Nash, you want to look out for that girl's brother. You
slammed him one or two in that fight at Chesby, and he's had it in for you
ever since. And after last night, all the men are wild. If that Gypsy Tokalji
catches you—phew! Oh, boy! And Toutou!"

"They weren't able to catch us last night," returned Nikka. "They aren't
likely to have as good a chance again."

"You put up a great fight," she agreed. "Oh, I'm handing it to you, all of
you! You're the best little bunch I ever ran across. Say, I wouldn't believe an
English lord could be as much of a hustler as you, Lord Chesby. Your uncle,
he—"
 She shrugged.

"What about my uncle?" asked Hugh eagerly. "D'you mind telling how
your push got on to him?"

"N-no, I suppose there's no harm now," she answered slowly. "Poor old
fellow! I was darned sorry he was croaked. We none of us— Well, what's
the use talking? That Toutou is a devil, Mr. Nash knows it. I only hope he
and the rest of you don't get to know him any better. But about your uncle,
Lord Chesby. He was a cinch. He ran around here like a kid in a game of
'Cops-and-thieves.' Everybody knew he was up to something. The
authorities thought he was just a nut. But when he took to snooping around
Tokalji's house, our folks got wise to it he might be on to something good.
Tokalji's tribe have always had this tradition of a treasure— But you know
about that. Tokalji had been working with us since before the War, and he
realized this was more than he could tackle by himself, so he called on
Toutou. The rest is what's going to happen."

"And that?" asked Hugh, grinning.

"My dear young lord, you'll lose your shirt—if not your life," she
retorted airily.

"Tough luck," said Hugh, "but your people have got to do better, in that
case."

"You're dead right," she agreed. "Say, Mr. Zaranko, on the level now, did
that girl of Tokalji's sell out to you last night?"

Nikka stared at her blankly, his face a perfect mask.

"We had a good deal of trouble with her," he returned. "Had to tie her up.
She was right on our heels, with her knife."

Hélène shook her head.

"Ye-es, that's true, but—I saw her this morning. Humph! Maybe I'm a
fool. I told Toutou to mind his own business, and not mix into the tribe's
affairs. Tokalji said she was all right, and that ought to be enough."

"God help Toutou if he went after her," I said facetiously.

Hélène gave me a quick glance.

"Maybe you're right," she said. "I've often wondered what Toutou would
do against a woman who used a knife. He—he gets 'em in a different way.
Well, I'm babbling, which is a sign of old age. Be good, boys, and give up
before you get into serious trouble. As ever, your well-wisher, Hélène."

And she tripped off.

"What a delightful criminal," I remarked. "Somehow I don't mind so

much the idea of being plucked by her."

"You're losing your perspective," growled Hugh, who was in a righteous

frame of mind, partly because he was in love and partly because of his clash
with Hilyer. "A crook is a crook. They're all against us. I don't know but that
the women are the most dangerous where you are concerned, Jack. Why are
you so damned susceptible?"

At which I laughed. Nikka, walking beside us, had no ears for our
conversation. His thoughts were on that slim, brown Tzigane maid about
whom Hélène de Cespedes had inquired. But he woke up a block farther on,
when a big, turbanned figure shambled past us, with a guttural exclamation
from the corner of his mouth. At the next corner there was a traffic block,
and we grouped casually around Wasso Mikali.

"Tokalji's women and children are in camp beyond Boghazkeui on the

edge of the Forest of Belgrade," he murmured, staring at a fat Turkish Pasha
who was rolling by in a Daimler. "There are five men with them. Five other
men have left Sokaki Masyeri since morning. If Franks were there they
have gone."

"It is good, my uncle," returned Nikka, affecting to speak to Hugh.

"Continue the watch. If there is more to report bid one of your young men
lounge before the khan where we are staying to-morrow in the forenoon."
 "It shall be done," said the old man, and he elbowed his way through our
ranks as though in haste to cross over.

I looked behind us for the inevitable spies. There were several

Levantines in European dress and tarboosh on the corner—and Hilmi Bey,
who pretended that he was not noticing us. His attitude was that of scorning
to spy and hating to have it supposed that he could demean himself to so
plebeian a phase of crime. I called a greeting to him in derision.

"Are you walking our way?" I asked.

"I have a house in the Rue Midhat Pasha," he answered effusively. "I am
going to visit my wives. It is a long time since I have seen them. Don't let
me detain you, gentlemen. I turn right at the opposite corner."

"A vain dog," commented Nikka, sourly watching Hilmi's plump back.
"He was afraid to be caught in such an ordinary undertaking.'

"Well," said Hugh, whose temper had improved, "it goes to show that
criminals are human beings. Every one of these birds seems to have some
sense of shame if you can only pick out the right point of contact."

We led our escorts—for we took it for granted that we were under

observation—a dilatory stroll, and arrived back at the Pera Palace in time
for dinner, which, as usual, we had served in the King's sitting room. It was
a leisurely meal, for we had time to kill. There was an early moon, and we
wanted it to set before the Curlew left the Golden Horn.

After Watkins had brought the coffee, Betty excused herself. She
returned in a quarter of an hour dressed in a warm sport suit instead of the
light evening frock she had worn, and carrying two boxes of cartridges.

"Have you all got your pistols loaded?" she inquired. "Watkins? Daddy?"

"I think so, my dear," answered her father absent-mindedly. "I wish,
Jack, that you had observed more carefully the carvings on that colonnade.
It may be truly ancient or— What? What is it, Betty?"
 She deftly frisked him, and examined his automatic.

"Yes, it's all right," she said, returning it to him. "And for Heaven's sake
remember, Dad, that the safety lock is on. Here's an extra clip. Watkins?"

Watkins set down the tray of coffee-cups, and cautiously hauled his
weapon from his hip-pocket.

"Quite right, I think, ma'am, Miss King," he replied.

"Here's an extra clip for you, too. Boys?"

"You don't catch old campaigners like us with empty weapons," I jeered.
"It isn't we who'll be getting into trouble."

"I wish I could be sure of that," she retorted. "Most likely I'll be trying to
pull you out of a scrape twenty-four hours from now. But let's get started.
We have a car at the side entrance to run us down to the Man-o'-war
Landing, where the Curlew is moored."

If the spies were still watching the hotel, as I have no doubt they were,
we gave them the slip. We went downstairs together, and shot into the
closed car which was in waiting, Watkins sitting beside the chauffeur. Ten
minutes later we drew up on the Curlew's dock, secure from observation
because of the British marine sentries who stood guard at the dock-gates.

The Curlew was a handy craft, decked over forward, with a roomy
cockpit and a good, heavy-duty Mercedes engine. She was nothing to look
at, but reliable and efficient. Betty, who was an experienced yachtswoman,
automatically assumed command, and Hugh and Watkins as automatically
accepted the rôle of crew. Vernon King, Nikka and I tried to be as
inconspicuous as possible.

"Lay for'ard, Hugh, and slack off that bow-line," ordered Betty
energetically. "How is the engine, Watkins? Very well, turn it over."

There was a splutter, and then the steady "put-put-put."

 "Cast off that bow-line, Hugh! Lay aft, Watkins. Is the stern-line slack?
Pay out! Let go! Get out from under my feet, Jack. No, Daddy, you can't
have a cigar—nothing but running-lights. I'd douse those if I weren't afraid
of the Navy people. Mr. Zaranko, d'you mind dropping into the cabin and
taking a look at the tools we laid in?"

We chugged slowly through the glut of shipping in the Golden Horn,

edging away from the Galata shore toward the picturesque bulk of
Stamboul. Seraglio Point loomed ahead of us, high, rugged, tree-covered,
dotted with infrequent lights. We rounded it, the lighthouse twinkling on
our starboard beam, and turned southwest into the Bosphorus, with the wide
sweep of the Marmora just ahead. To port the outline of Scutari and the
suburbs on the Asiatic shore showed dimly. To starboard Stamboul towered,
white and ghostly and serenely beautiful, more than ever the magic city of
the Arabian Nights. The steamer from Rodosto and other Marmoran ports
steamed past us with a swash and gurgle. A belated fishing-boat flapped by.
Then we had the waters to ourselves.

"Have you the night-glasses, Hugh?" questioned Betty. "See if you can
make out the St. Sophia minarets." And to us: "That's our first landfall in
making Tokalji's house. Watkins, I think it ought to be safe now to douse
the running-lights."

Hugh leaned forward across the cabin-roof, resting on his elbows, eyes
glued to the glasses.

"Right O," he called back. "I'm on them—and I can see that big old
tower of the sea-walls that lies this side of the jetty."

Betty cut off the engine.

"Fetch the sweeps, Watkins," she whispered. "We'll pull in. Quiet,
everybody."
 CHAPTER XX

OUT OF LUCK

Hugh and Watkins unlashed two heavy oars from the cabin roof and
thrust them outboard through oarlocks rivetted to the cockpit railing. Side
by side, in unison, they pulled with a long, deliberate stroke, while Betty
steered. It was no easy task to move that launch across the swift-flowing
tide of the Bosphorus, and it seemed an endless time before the blurred
mass of the shoreline, becoming visible to our unaided sight, furnished an
index to the progress we were making.

"Nikka and I can relieve them," I offered as the rowers began to pant.

"You haven't done it before," answered Betty shortly. "You might

splash."

Indeed, the oars made scarcely a ripple as they were lifted, feathered and
dipped, tedious as was the effort imposed both by their weight and the size
of the launch.

"Much farther?" Hugh gritted between clenched teeth.

"The jetty is right ahead," Betty reassured him. "You had better get
forward, Dad, and be ready to fend off the rocks."

Vernon King climbed up on the cabin roof and crawled into the bow.
Nikka and I strained our eyes endeavoring to identify the details of the
shore. To the right, and already a little astern of us, was a huge round tower,
one of the bulwarks of the ancient walls. Other than this there was only a
dim range of masonry, the city walls, for the most part, crowned by houses.
Not a light showed opposite to us.

Presently, letting our eyes drop lower, we descried immediately in front a

low breakwater, a jagged pile of rocks that ran out from the shore in the
form of a blunted hook. Betty, steering carefully, brought the Curlew inside
the hook and bow-on to the shore, so that the launch was protected from the
current that flowed through the Strait. King scrambled ashore and made fast
a line around one of the rocks, then felt his way back along the slippery
footing of the breakwater and stepped into the cockpit. Hugh and Watkins
unshipped the sweeps and laid them on the cabin roof.

All of us were staring at the blank darkness of the shoreline, tense and
watchful; but my uncle's interest was still largely of an antiquarian nature.

"Do you appreciate how extraordinarily fortunate we are to have this

ruined jetty to moor to?" he whispered excitedly. "No galleys in the old
days were ever able to assail these seaward walls because of the currents.
Without protection, we, too, should be smashed to pieces if we tried to lie
under them. But this place evidently was one of the walls of a harbor for the
Imperial galleys. It was, of course, fortified. This hook terminated in a
strong tower. A second hook—"

"Daddy, Daddy," remonstrated Betty, "you aren't lecturing to-night. We

—we're reconnoitering the enemy's position."

Hugh had been studying the shore again through the night-glasses.

"Not a sign of life," he murmured. "Now, you chaps, show us the lay of
the land."

Nikka and I, with the help of the glasses, plotted for the others the
arrangement of Tokalji's establishment. There was the brick extension of the
bachelors' quarters, crowning a part of the sea-wall. There was the gap
between this structure and the House of the Married, which was shut in only
by the crenellated height of the wall. And finally, there was the House of the
Married, with the Garden of the Cedars concealed within its heart, lifting its
solid bulk above all adjoining buildings. There were no windows on the
seaward face of Tokalji's house.

"The old wall between the two wings—between the bachelors' quarters
and warehouse and the House of the Married—ought to be easy to climb," I
concluded.
 "The wall of the House of the Married is very irregular, too," added
Betty. "We have passed it close in a number of times by daylight, and we all
agreed an active man could climb it."

"That's a good idea," approved Nikka. "If you could enter by the House
of the Married you could seize the valuable part of the position first. Sound
military strategy."

"Yes," assented Hugh, "you could consolidate your position—how the

old lingo comes back, though!—and then occupy the rest of the place as
convenient. By Jove, if you didn't want to occupy it, you could—"

"Oh, you'd have to occupy it," I interrupted. "I say, do you know that
place looks deserted?"

"There's somebody there, never fear," rejoined Betty.

"According to Nikka's uncle, a good part of the garrison were withdrawn

to-day," returned Hugh.

"There is no use hurrying," cautioned my uncle. "We shall have plenty of

opportunities."

"There is good reason for striking when you are not expected," retorted
Hugh.

Nobody answered him. We were all staring hungrily at the shadowy

shape of the House of the Married, towering above the seawall. It
hypnotized us. We were enthralled by the unfathomable mysteries it
suggested, by the knowledge of the mighty prize it contained.

"There's no time like the present," I said softly.

"Yes, they won't be looking for us so soon again," agreed Nikka. "They
will be figuring that we had enough of a fright last night."

"Perhaps you are right," surrendered Vernon King. "Audacity, we are

frequently told, is the favored bride of fortune. I must admit that this place
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