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Methods in
Molecular Biology 2382

Marie-Cécile Caillaud Editor

Plant
Cell Division
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY

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

For further volumes:

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For over 35 years, biological scientists have come to rely on the research protocols and
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 Plant Cell Division

Methods and Protocols

Second Edition

Edited by

Marie-Cécile Caillaud
SiCE, Laboratoire Reproduction et Développement des Plantes, ENS Lyon, Lyon, France
Editor
Marie-Cécile Caillaud
SiCE, Laboratoire Reproduction et
Développement des Plantes
ENS Lyon
Lyon, France

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1743-4 ISBN 978-1-0716-1744-1 (eBook)
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Preface

Plant cell division is a highly regulated process by which a parent cell divides into two
daughter cells. Recent advances in the field have enabled us to study this fascinating process
in a quantitative manner, at high resolution both in space and time using cell biology,
biochemistry, and molecular biology.
In this second edition of Plant Cell Division: Methods and Protocols for MiMB, a
collection of innovative approaches and keystone methodologies are presented. This book
aims to allow the researcher to visualize, quantify, and modify cell division during cell cycle
progression.
After a review of the methods used to visualize the actin cytoskeleton during plant cell
division, the book focuses first on methodology to address mitosis progression as a part of
the cell cycle. It includes updated protocols already presented in the previous edition, such as
the immunolabeling of nuclei/chromosomes, along with a new approach for genome-wide
analysis of DNA replication timing. The book also sheds light on an interesting model to
study cell division: algae. Two chapters are dedicated to this model, depicting methodolo-
gies to assess commitment point attainment and cyclin-dependent kinase activity in syn-
chronized algal cultures.
The second part of the book presents cutting-edge approaches to manipulate cell
division, including the long-missing fast and easy system for transient localization of
fluorescent-tagged protein in dividing tobacco cells, as well as recent advances in automated
time-lapse imaging coupled with manipulation of cell divisions by vertical stage confocal
microscopy.
Analysis of the constantly growing volume of images generated by improved microscop-
ical technology is now more than ever a crucial step of our daily work. The third part of this
edition gathers, for the first time, chapters to quantify cell file numbers, defaults in root
architecture, cell division angles, and kinematic growth. I am certain that these quantifica-
tion methods will spread as standards in the field and will become the reference for future
researchers.
Finally, the book ends with a focus on the final step of the cell division, cytokinesis. A
quantitative method for evaluating phragmoplast morphology, and the analysis of protein
turnover by Fluorescence Recovery After Photobleaching during cytokinesis, are disclosed.
One chapter is dedicated to the well-known use of BY-2 synchronized tobacco cells for the
time-lapse imaging of cytokinesis, while the last chapter depicts a method to visualize one of
the most fascinating plant-specific structures, plasmodesmata, during the cell plate
expansion.

Lyon, France Marie-Cécile Caillaud

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Methods to Visualize the Actin Cytoskeleton During Plant Cell Division . . . . . . 1
Marie-Cécile Caillaud

PART I MITOSIS AND CELL CYCLE PROGRESSION

2 Immunolabeling of Nuclei/Chromosomes in Arabidopsis thaliana. . . . . . . . . . . . 19

Ulkar Ahmadli, Michael Sandmann, Joerg Fuchs, and Inna Lermontova
3 A Protocol for Genome-Wide Analysis of DNA Replication Timing
in Intact Root Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Leigh Mickelson-Young, Emily E. Wear, Jawon Song, Gregory J. Zynda,
Linda Hanley-Bowdoin, and William F. Thompson
4 Assaying Cyclin-Dependent Kinase Activity in Synchronized Algal Cultures . . . . 73
Kateřina Bišová
5 Analysis of Commitment Point Attainment in Algae Dividing
by Multiple Fission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Veronika Kselı́ková, Vilém Zachleder, and Kateřina Bišová

PART II MANIPULATION OF CELL DIVISION

6 Automated Time-Lapse Imaging and Manipulation of Cell Divisions
in Arabidopsis Roots by Vertical-Stage Confocal Microscopy . . . . . . . . . . . . . . . . . 105
Lukas Hoermayer, Jiřı́ Friml, and Matouš Glanc
7 Transient Overexpression of E2Fb to Induce Cell Divisions
in Nicotiana benthamiana Pavement Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Tamara Jimenez-G
ongora, Huang Tan, and Rosa Lozano-Durán

PART III QUANTIFICATION OF CELL DIVISION PATTERNING

8 Kinematic Growth Analysis of Rice (Oryza sativa) Leaf . . . . . . . . . . . . . . . . . . . . . . 131

Jingjing Fang and Xueyong Li
9 Studying Cell Division Plane Positioning in Early-Stage Embryos. . . . . . . . . . . . . 141
Katia Belcram, Jean-Christophe Palauqui, and Martine Pastuglia
10 Means to Quantify Vascular Cell File Numbers in Different Tissues . . . . . . . . . . . 155
Helena E. Arents, Gugan Eswaran, Matouš Glanc,
Ari Pekka M€ a hönen, and Bert De Rybel
11 Confocal Analysis of Arabidopsis Root Cell Divisions in 3D: A Focus
on the Endodermis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Roya Campos and Jaimie M. Van Norman
12 Quantification of Cell Division Angles in the Arabidopsis Root . . . . . . . . . . . . . . . 209
Katia Belcram, David Legland, and Martine Pastuglia
vii
viii Contents

PART IV IMAGING AND QUANTIFYING PLANT CYTOKINESIS

13 A Quantitative Method for Evaluating Phragmoplast Morphology . . . . . . . . . . . . 225

Takema Sasaki and Yoshihisa Oda
14 Analyses of Protein Turnover at the Cell Plate by Fluorescence Recovery
After Photobleaching During Cytokinesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Alexandra Deli, Leda-Eleni Tympa, and Panagiotis N. Moschou
15 Cell Cycle Synchronization and Time-Lapse Imaging of Cytokinetic
Tobacco BY-2 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Keisho Maeda and Takumi Higaki
16 Ultrastructural Observation of Cytokinesis and Plasmodesmata
Formation in Brown Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Chikako Nagasato, Rina Yonamine, and Taizo Motomura

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Contributors

ULKAR AHMADLI • Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),
Gatersleben, Germany
HELENA E. ARENTS • Department of Plant Biotechnology and Bioinformatics, Ghent
University, Ghent, Belgium; VIB Center for Plant Systems Biology, Ghent, Belgium
KATIA BELCRAM • Institut Jean-Pierre Bourgin, INRAE, AgroParisTech, Université Paris-
Saclay, Versailles, France
KATEŘINA BIŠOVÁ • Centre Algatech, Laboratory of Cell Cycles of Algae, Institute of
Microbiology of the Czech Academy of Sciences, Třeboň, Czech Republic
MARIE-CÉCILE CAILLAUD • Laboratoire Reproduction et Développement des Plantes,
Université de Lyon, UMR5667, ENS de Lyon, UCB Lyon 1, CNRS, INRA, Lyon, France
ROYA CAMPOS • Department of Botany and Plant Sciences, Center for Plant Cell Biology,
Institute of Integrative Genome Biology, University of California, Riverside, CA, USA
BERT DE RYBEL • Department of Plant Biotechnology and Bioinformatics, Ghent University,
Ghent, Belgium; VIB Center for Plant Systems Biology, Ghent, Belgium
ALEXANDRA DELI • Department of Biology, University of Crete, Heraklion, Greece; Institute
of Molecular Biology and Biotechnology, Foundation for Research and Technology—Hellas,
Heraklion, Greece
GUGAN ESWARAN • Department of Biological and Environmental Sciences, Institute of
Biotechnology, University of Helsinki, Helsinki, Finland
JINGJING FANG • National Key Facility for Crop Gene Resources and Genetic Improvement,
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
JIŘÍ FRIML • Institute of Science and Technology Austria (IST Austria), Klosterneuburg,
Austria
JOERG FUCHS • Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),
Gatersleben, Germany
MATOUŠ GLANC • Institute of Science and Technology Austria (IST Austria),
Klosterneuburg, Austria; Department of Experimental Plant Biology, Faculty of Science,
Charles University, Prague, Czechia; Department of Plant Biotechnology and
Bioinformatics, Ghent University, Ghent, Belgium; VIB Center for Plant Systems Biology,
Ghent, Belgium
LINDA HANLEY-BOWDOIN • Department of Plant and Microbial Biology, North Carolina
State University, Raleigh, NC, USA
TAKUMI HIGAKI • International Research Organization for Advanced Science and
Technology, Kumamoto University, Chuo-ku, Kumamoto, Japan
LUKAS HOERMAYER • Institute of Science and Technology Austria (IST Austria),
Klosterneuburg, Austria
TAMARA JIMENEZ-GÓNGORA • Shanghai Center for Plant Stress Biology, CAS Center of
Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China;
Center for Research in Agricultural Genomics (CRAG), Barcelona, Spain
VERONIKA KSELÍKOVÁ • Centre Algatech, Laboratory of Cell Cycles of Algae, Institute of
Microbiology of the Czech Academy of Sciences, Třeboň, Czech Republic; Faculty of Science,
University of South Bohemia, České Budějovice, Czech Republic

ix
x Contributors

DAVID LEGLAND • UR1268 Biopolymères, Interactions et Assemblages, INRAE, Nantes,

France; BIBS Facility, INRAE, Nantes, France
INNA LERMONTOVA • Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),
Gatersleben, Germany
XUEYONG LI • National Key Facility for Crop Gene Resources and Genetic Improvement,
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
ROSA LOZANO-DURÁN • Shanghai Center for Plant Stress Biology, CAS Center of Excellence
in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
KEISHO MAEDA • Graduate School of Science and Technology, Kumamoto University, Chuo-
ku, Kumamoto, Japan
ARI PEKKA MA€ HÖNEN • Department of Biological and Environmental Sciences, Institute of
Biotechnology, University of Helsinki, Helsinki, Finland
LEIGH MICKELSON-YOUNG • Department of Plant and Microbial Biology, North Carolina
State University, Raleigh, NC, USA
PANAGIOTIS N. MOSCHOU • Department of Biology, University of Crete, Heraklion, Greece;
Institute of Molecular Biology and Biotechnology, Foundation for Research and
Technology—Hellas, Heraklion, Greece; Department of Plant Biology, Uppsala BioCenter,
Swedish University of Agricultural Sciences and Linnean Center for Plant Biology,
Uppsala, Sweden
TAIZO MOTOMURA • Muroran Marine Station, Field Science Center for Northern Biosphere,
Hokkaido University, Muroran, Japan
CHIKAKO NAGASATO • Muroran Marine Station, Field Science Center for Northern Biosphere,
Hokkaido University, Muroran, Japan
YOSHIHISA ODA • Department of Gene Function and Phenomics, National Institute of
Genetics, Mishima, Shizuoka, Japan; Department of Genetics, School of Life Science, The
Graduate University for Advanced Studies, SOKENDAI, Mishima, Shizuoka, Japan
JEAN-CHRISTOPHE PALAUQUI • Institut Jean-Pierre Bourgin, INRAE, AgroParisTech,
Université Paris-Saclay, Versailles, France
MARTINE PASTUGLIA • Institut Jean-Pierre Bourgin, INRAE, AgroParisTech, Université
Paris-Saclay, Versailles, France
MICHAEL SANDMANN • Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),
Gatersleben, Germany
TAKEMA SASAKI • Department of Gene Function and Phenomics, National Institute of
Genetics, Mishima, Shizuoka, Japan; Department of Genetics, School of Life Science, The
Graduate University for Advanced Studies, SOKENDAI, Mishima, Shizuoka, Japan
JAWON SONG • Texas Advanced Computing Center, University of Texas, Austin, TX, USA
HUANG TAN • Shanghai Center for Plant Stress Biology, CAS Center of Excellence in
Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
WILLIAM F. THOMPSON • Department of Plant and Microbial Biology, North Carolina State
University, Raleigh, NC, USA
LEDA-ELENI TYMPA • Department of Biology, University of Crete, Heraklion, Greece;
Institute of Molecular Biology and Biotechnology, Foundation for Research and
Technology—Hellas, Heraklion, Greece
JAIMIE M. VAN NORMAN • Department of Botany and Plant Sciences, Center for Plant Cell
Biology, Institute of Integrative Genome Biology, University of California, Riverside, CA,
USA
EMILY E. WEAR • Department of Plant and Microbial Biology, North Carolina State
University, Raleigh, NC, USA
 Contributors xi

RINA YONAMINE • Graduate School of Environmental Science, Hokkaido University, Sapporo,

Japan
VILÉM ZACHLEDER • Centre Algatech, Laboratory of Cell Cycles of Algae, Institute of
Microbiology of the Czech Academy of Sciences, Třeboň, Czech Republic
GREGORY J. ZYNDA • NVIDIA, Santa Clara, CA, USA
 Chapter 1

Methods to Visualize the Actin Cytoskeleton During Plant

Cell Division
Marie-Cécile Caillaud

Abstract
Cell division in plants consists of separating the mother cell in two daughter cells by the centrifugal growth
of a new wall. This process involves the reorganization of the structural elements of the cell, namely the
microtubules and actin cytoskeleton which allow the coordination, the orientation, and the progression of
mitosis. In addition to its implication in those plant-specific structures, the actin cytoskeleton, in close
association with the plasma membrane, exhibits specific patterning at the cortex of the dividing cells, and
might act as a signaling component. This review proposes an overview of the techniques available to
visualize the actin cytoskeleton in fixed tissues or living cells during division, including electron, fluorescent,
and super-resolution microscopy techniques.

Key words Actin, Cell biology, Plant, Fluorescent-tagged reporters, Electron tomography, Immu-
nogold labeling, Phallotoxins, Super-resolution microscopy

1 Introduction

The topology of the tissue in the green lineage relies deeply on a

precise spatio-temporal control and coordination of division and
growth. As plant cells cannot relocate by migration, the orientation
of the cell division is brought about by a complex interplay between
the cell cycle machinery and other cellular structures, including the
microtubule and actin cytoskeletons as well as the plasma mem-
brane. When the cell cycle is progressing toward mitosis, most
cellular microtubules together with the actin filaments (F-actin)
relocate at the cell’s cortex in close association with the plasma
membrane forming the preprophase band (PPB) at the cortical
division zone (CDZ), marking the future site of division. In acen-
trosomal plant cells, the mitotic spindle allows the separation of the
genetic material during metaphase/anaphase transition while the
plant-specific structure called phragmoplast drives the formation of
a new cell wall separating the daughter cells in telophase at the

Marie-Cécile Caillaud (ed.), Plant Cell Division: Methods and Protocols, Methods in Molecular Biology,
vol. 2382, https://doi.org/10.1007/978-1-0716-1744-1_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

1
2 Marie-Cécile Caillaud

earlier defined cortical division site (CDS) [1]. Microtubule and

F-actin cytoskeletons are therefore closely associated and regulated
to form and orient these three—PPB, spindle, and phragmoplast—
distinct structures. During this process, the actin cytoskeleton, in
close association with the plasma membrane, adopts specific pat-
terning at the cortex of the dividing cells, leading to the formation
of F-actin enriched (actin twin peaks) and depleted zone (ADZ,
Fig. 1). This review proposes an overview of the techniques avail-
able to visualize the actin cytoskeleton in fixed tissues or living cells
during division, including electron, fluorescent, and super-resolu-
tion microscopy techniques.

2 Ultrastructural Studies

Actin cytoskeleton and its interaction with microtubule cytoskele-

ton were visualized using chemical fixed plant cells [2–4]. However,
chemical fixation and dehydration techniques were later revealed to
be inadequate for the preservation of the cytoskeleton [5, 6]. The
efficacy of the technique of freeze substitution for the preservation
of cytoskeletal details is now widely recognized [5–7]. Using such
approach, the actin cytoskeleton at the cortex of the cell was
described to be associated together with the plasma membrane,
but also with the endoplasmic reticulum beneath the cortical
microtubules [5]. More recently, the ultrastructure of the actin
cytoskeleton in the PPB in onion cotyledon epidermal cells was
achieved using high-pressure freezing/freeze substitution techni-
ques. This approach allowed the collection of quantitative informa-
tion on the number and length of F-actin inside the PPB. Using this
approach, F-actin cables 170-nm-long were observed to bind and
to cross-link microtubules [8]. Electron microscopic observations
of glycerinated Haemanthus cells indicate the presence of actin-F in
the mitotic spindle, in particular in the kinetochore fibers which
link the spindle to the chromosomes [9]. But these data are still
debated because the preparative techniques used might have
induced changes in actin-F distribution [10].

3 Immunogold Labeling of F-Actin

Using actin monoclonal antibodies, which gave the finest resolu-

tion with immunogold staining, it was shown that F-actin was often
seen in the near vicinity of another bundle or microtubule, in an
end to side or Y-shaped connection, closely parallel or juxtaposed to
microtubules. Actin-F bundle length often reached 10–20 μm
[11]. The distribution of F-actin cables was later visualized using
an immunogold-silver-enhanced method and compared with the
arrangement of immunogold-stained microtubules in the same
 Methods to Visualize the Actin Cytoskeleton During Plant Cell Division 3

preprophase metaphase anaphase polar F-actin accumulation

anaphase spindle
preprophase band
mitotic spindle actin twin peaks
actin depleted zone

cortical
division
zone

early cytokinesis late cytokinesis interphase

cell plate
insertion
zone
cell plate plasmodesmata

phragmoplast

Fig. 1 Schematic representation of the different steps of the plant cell division, with a focus on the actin
cytoskeleton (red)

cells [12]. Using this approach in the dividing endosperm cells of

the monocot Haemanthus, F-actin was observed on the spindle
surface and those present within the spindle are arranged parallel to
the long axis of the spindle. F-actin was found to not reorient
outside the spindle, while in the polar region of the spindle,
F-actin appears to be compressed by the elongating spindle
[12]. In anaphase, shorter F-actin orients in various directions
accumulate at the equator, where some of them are incorporated
into the phragmoplast [12].

4 Actin Staining with Phallotoxins

The biologically active components of poisonous Amanita mush-

rooms such as phallotoxins are known to be specific for the F-actin
[13–15]. Phallotoxins are low in molecular weight, and thus can
penetrate plant cells without prior cell wall digestion, and can also
be used with or without fixation before the staining step. The first
attempt to use phallotoxin to stain actin in the phragmoplast and
cell cortex was unsuccessful [16, 17]. Later, staining the unfixed
carrot suspension cells with rhodaminyl lysine phallotoxin
(rhodamine-phalloidin) in detergent extraction buffer set up the
protocol to visualize the actin cytoskeleton during cell division
[18, 19]. Using this approach, the organization of F-actin in the
PPB, mitotic spindle, and phragmoplast was described in various
4 Marie-Cécile Caillaud

plant species including maize, Allium, Tradescantia, and BY-2

tobacco cells [16, 20–22]. Using the BY-2 cell culture stably
expressing a fluorescent reporter for microtubules, the behavior
of the F-actin cytoskeleton was assessed by staining with
rhodamine-phalloidin [23]. Using this approach, the actin depleted
zone (ADZ) which is set up before the prophase, was reported to
appear first from one side of the dividing cell. Such behavior may be
a consequence of “polar” cytokinesis—the cell plate is attaching to
one side of the cell before the other end—as is often observed in
BY-2 cells [23] and as was reported in Arabidopsis cells [24]. The
ADZ finally extends to the other side of the dividing cell forming a
ring deprived of F-actin at the division plane [23].
Despite the handiness of using phallotoxins and the option of
removing possible damage to F-actin from the fixation procedure,
it has been questioned whether unfixed cells stained with phallo-
toxins could provide a misleading picture of F-actin configurations
in vivo [25–27]. Moreover, labeling procedures without fixation
cannot be used for double-labeling and therefore reduce the possi-
bility to study the dynamics of the F-actin together with the micro-
tubule cytoskeleton, for instance.

5 Immunolabeling in Fixed Tissues

Immunofluorescence labeling using a monoclonal antibody devel-

oped against actin is often used to visualize F-actin in fixed tissues.
Using this technique, the formation of an actin ring at the cortex of
the cell in preprophase, the “actin PPB” was visualized [28]. Dou-
ble-labeling of both microtubules and F-actin was therefore possi-
ble with this method, permitting to correlate the F-actin
organization in regard to the known organization of the microtu-
bule rearrangement that occurs throughout division [29]. As the
PPB narrows, the F-actin is also confined to an increasingly
restricted zone usually wider than the PPB in Allium [30]. In
synchronized tobacco BY2 cells, the triple staining of microtubules,
F-actin, and DNA was performed and allowed a better resolution in
time and space on the relationship between the mitosis progression
and formation of the PPB [31]. In wheat root tip cells, the immu-
nolocalization of actin revealed the absence of F-actin at the cortical
division zone, thereby revealing the Actin Depletion Zone (ADZ)
[32]. However, depending on the antibody, such a zone was more
or less wide [32, 33]. Early work using immunolocalization of actin
cytoskeleton in rye leaf tissue revealed the presence of a meshwork
of actin that concentrated near the end walls that face the spindle
poles [34]. Consistently, immunofluorescence labeling of F-actin
revealed a more intense reactivity to the actin antibody in the cell
pole facing the spindle than the longitudinal side walls [35–37]
(Fig. 1). Furthermore, the ADZ is short-lived from metaphase
 Methods to Visualize the Actin Cytoskeleton During Plant Cell Division 5

through the anaphase/telophase transition [16, 25]. The presence

of actin filaments before phragmoplast formation in endosperm
cells suggests that microfilaments could act as a substitute for cell
plate “memory” [11]. Co-visualization of F-actin and microtubules
by dual immunolabeling in BY-2 showed that the length of phrag-
moplast F-actin is shorter than that of microtubules [36, 38]. Dur-
ing telophase, the two zones of aligned cortical F-actin over the
ends of the cell gradually disappear and are replaced by new inter-
phase networks [37].
F-actin staining and immunolabeling approaches proved useful
when the study takes place in an organism that cannot be trans-
formed, or in multiple mutants for which crosses could be difficult.
In these cases, the staining techniques described above are still used
today. In parallel, advances in live imaging have led to the use of
fluorescent-tagged actin-binding domains (ABD) or proteins inter-
acting with the actin cytoskeleton [36]. These methods allow us to
visualize the dynamics of the F-actin cytoskeleton during plant
mitosis in vivo using for example confocal microscopy, tracking
system, and super-resolution microscopy (Table 1).

6 Fluorescent-Tagged Actin Reporters

6.1 The Talin is a cytoskeletal associated protein that localizes specifically in

Fluorescent-Tagged adherents-type junctions with the extracellular matrix [56] and is
C-Terminal Part absent from the plant genome. The amino acids 2345–2541 in the
of the Mouse Talin C-terminus of the mouse Talin (mTalin2345–2541) are responsible
(GFP-mTn2345–2541) for its actin-binding capacity [57]. Unlike the full-length protein,
mTalin2345–2541 is unable to nucleate actin polymerization and was,
therefore, used as a reporter to visualize F-actin in plants
[57, 58]. In tobacco BY-2 suspension cells, GFP-mTalin2345–2541
colocalizes with rhodamine-phalloidin [58]. Stable and ubiquitous
expression of GFP-mTalin2345–2541 in Arabidopsis transgenic lines
was used to visualize F-actin in all tissues with no apparent pheno-
typic effects [58]. While using GFP-mTalin2345–2541, the F-actin
behaviors in different cell types and organisms were described in
interphasic cells and in pollen tubes of different species [58–60]. To
my knowledge, F-actin dynamics during cell division was not ana-
lyzed using this reporter. Later study revealed that while GFP-mTa-
lin2345–2541 does not affect actin polymerization, it inhibits its
depolymerization by affecting the actin-depolymerizing factor
[61]. These findings led to the dropping of this reporter to study
F-actin in plants.

6.2 The Fluorescent- Fimbrin is a cytoskeletal protein associated with F-actin in various
Tagged Fimbrin metazoan structures, such as in microvilli, microspikes, membrane
ruffles, and cell-substrate attachment [62]. The first plant fimbrin-
like gene, AtFIM1, was identified in Arabidopsis [63] and since
 6
Table 1
Examples of fluorescent marker available to visualize F-actin in vivo

Name Localization Remarks Organism Vector/resistance References

35Spro:GFP-Talin All arrays Prevent F-actin Col-0 pCAMBIA-1390 (hygro [39, 40]
depolymerization & kana)
35Spro:DsRed2-Talin All arrays Prevent F-actin Col-0 pCAMBIA-1390 (hygro [41]
depolymerization & kana)
35Spro:DsRed2-Talin All arrays BY-2 pGWB2 (basta) [42]
Marie-Cécile Caillaud

35Spro:CFP-fABD2-CFP All arrays Morphological defects Col-0 pCAMBIA-1390 (hygro [41]

& kana)
35Spro:GFP-fABD2-GFP All arrays Morphological defects Col-0 pCAMBIA-1390 (hygro [39, 40]
& kana)
35Spro:GFP–fABD2 All arrays Col-0 pCAMBIA-1300 (hygro [43]
& kana)
UBQ10pro:GFP-fABD2- All arrays Col-0 pCAMBIA-1390 (hygro [41]
GFP & kana)
35Spro:GFP-Fim1 All arrays Col-0 pCB302 (hygro) [44]
35Spro:GFP-fABD2 All arrays Col-0 pCB302 (hygro) [44]
35Spro:YFP-fABD2-YFP All arrays Morphological defects Col-0 pCAMBIA-1390 (hygro [41]
& kana)
35Spro:mOrange-fABD2- All arrays Morphological defects Col-0 pCAMBIA-1390 (hygro [41]
mOrange & kana)
35Spro:tdTom-fABD2 All arrays BY-2 pMDC100 (kana) [45, 46]
35Spro:mCh-fABD2- All arrays Morphological defects Col-0 pCAMBIA-1390 (hygro [41]
mCherry & kana)
35Spro:RFP-fABD2 All arrays BY-2 pH7WGR2 (hygro) [47]
35Spro:YFP- MTs and F-actin BY-2 pMDC101 (kana) [45, 46]
TUB–tdTomato–ABD2
UBQ10pro:mCh-ABD2- All arrays Col-0 pCAMBIA-1390 (hygro [41]
mCh & kana)
Ub10pro:Lifeact-2mTU2 All arrays Col-0 pK7m34GW (kana) [48]
35Spro:Lifeact-YFPv All arrays Morphological defects Col-0; pGWB2 (basta) [41, 49]
Marchantia
Ub10pro:Lifeact-YFPv All arrays Col-0 pB7m34GW (basta) [50]
RPS5apro:Lifeact-tdTom All arrays Col-0 [48]
UB10pro:Lifeact-mCh All arrays Moss pTHUbi-gate (hygro) [51]
35Spro:Lifeact–psRFP All arrays BY-2 pH7WG2 (hygro) [52]
35Spro:AtKinG-GFP PPB and phragmoplast Minus-end directed kinesin BY-2 pGFP-N-bin [42]
35Spro:GFP-NtKCH PPB and phragmoplast Associate with both MTS and Tobacco pK7WGF2 (kana) [47]
AFs
35Spro:Myo8A-3XmEGFP Spindle, center of phragmoplast Actin-based molecular motors BY-2 pDC32 [53]
& CDS
PAtFH8:AtFH8–GFP Newly formed cell wall Too weak to be detected At pCAMBIA1300 template [43]
(kana)
XVEpro-AtFH8-GFP Newly formed cell wall At pER8 (hygro) [43]
XVEpro:AtFH14-GFP PPB, spindle and phragmoplast Bundle MTs and AFs and cross- BY-2 pER8 (hygro) [36]
link them
UB10pro:For2A-3XmEGFP Center of the growing Moss pTHUbi-gate (hygro) [53]
phragmoplast
35Spro:AtFH5-eGFP Center of the growing At pK7WGF2 (kana) [54]
phragmoplast
35Spro:AtFH4-GFP Associate with both MTS and BY-2 pMDC43 (kana) [55]
AFs
Methods to Visualize the Actin Cytoskeleton During Plant Cell Division

UB10pro:Myo8A-3XmEGF Spindle, center of phragmoplast Actin-based molecular motors Moss pTKUbi-gate (kana) [53]
& CDS
7
8 Marie-Cécile Caillaud

then has been extensively used as a reporter to visualize F-actin

cytoskeleton inside plant cells [39, 40, 64–66]. The Fimbrin
1 actin-binding domain 2 fused to GFP (GFP-fABD2) corresponds
to the N-terminal fusion of GFP to the C-terminal half of AtFim1
(aa 325–687), which includes the second ABD and the C-terminal
end of Arabidopsis Fimbrin 1 [63]. Compared with GFP fusions to
full-length AtFim1, GFP-FABD2 is more visibly labeling the native
F-actin organization in Arabidopsis [65] and rapidly became a
standard to visualize F-actin in plants [44].
In the tobacco BY-2 cell line stably transformed with a
GFP-fABD2, the appearance of a broad F-actin band in the late
G2 phase that later separates to form a structure corresponding to
the so-called ADZ in mitosis was confirmed [67]. Intriguingly, the
GFP-fABD2 labeled structure at the cell cortex in mitosis appeared
to form two bands rather than the ADZ. Measurements of fluores-
cent intensities indicated a GFP-fABD2 distribution that resembled
two peaks, and we therefore named the structure actin “twin peaks”
(Fig. 1) [67]. The cell plate was shown to form exactly in the
middle of the actin “twin peaks” suggesting that these structures
act as a landmark for cytokinesis orientation [67]. Live-cell imaging
in BY2 of the GFP-fABD2 distribution was next monitored
together with the endocytic tracer, FM4–64, that labels the cell
plate [68]. During cytokinesis, GFP-fABD2 accumulates at the
phragmoplast and subsequently approaches the expanding cell
plate edges [68]. In vivo visualization of F-actin in Arabidopsis
plants stably expressing 35Spro:GFP-fABD2 showed that mitotic
cells have an F-actin-depleted zone around the equator, whereas
F-actin accumulates at the plasma membrane which is facing the
spindle poles [44]. During cytokinesis, this overall actin polarity is
still maintained [44]. Using this reporter, the phragmoplast F-actin
could be observed at different stages of cytokinesis [44].
In Arabidopsis, the expression of the GFP-fABD2-GFP
reporter under the 35S promoter [39] has inhibitory effects on
cell and organ growth [41]. The use of the ubiquitin10 (UBQ10)
promoter to drive expression of the GFP-fABD2-GFP reporter
minimized some of the artifacts that researchers might encounter
when using fABD2-based and other F-actin reporters for studying
F-actin dynamics in living plants [41].

6.3 Lifeact Reporter An important development is the identification of a 17-amino-acid

peptide (Lifeact) derived from the N-terminus of yeast Abp140
protein that binds specifically to F-actin with a low affinity and
appears to be a faithful reporter of actin dynamics without disrupt-
ing them [69]. While Lifeact decorates F-actin with minimum
perturbation of actin dynamics in plants, its expression needs to
be optimized to reach a lower expression level than fADB2 in plants
[49]. Indeed, it was reported that Arabidopsis seedlings overex-
pressing at high level Lifeact-YFPv reporter under the 35S
Methods to Visualize the Actin Cytoskeleton During Plant Cell Division 9

promoter had less dense and more bundled F-actin in some cell
types compared with fABD2-based reporter [41].
To test whether Lifeact–psRFP affects the dynamics of actin-F
in BY-2 stable line, the cells were challenged with Latrunculin B, a
drug which eliminates actin-F depending on their innate turnover.
The authors assumed that if the expression of Lifeact–psRFP
increases the stability of actin-F, the cells expressing Lifeact–
psRFP should be less sensitive to Latrunculin B. However, the
Lifeact–psRFP expressing cell line was more sensitive, which argues
against reduced dynamics of actin-F [52].
Using Arabidopsis plants, stably expressing Lifeact as a
reporter, we tracked the behaviors of the F-actin cytoskeleton in
time with an unpreceded precision [48]. To follow in vivo division
in the root meristem overtime, manual adjustment is needed, oth-
erwise the area under study will grow out of the field of view (the
root tip grows out of the field of view in about 20 min). By
developing an unsupervised approach to automatically track cells
using spinning disk confocal microscopy [70], we are now able to
automatically track the region under study in real time during
acquisition so that root growth can be compensated. This keeps
the region under study within frame for the duration of the experi-
ment. By imaging in vivo, the fluorescent-tagged Lifeact under the
control of UB10 and RPS5a promoters—both thought to be less
strong than 35S promoter—we confirmed earlier reports describ-
ing an apical-basal accumulation of the actin filaments in the late
stage of cell division and quantified this behavior (Fig. 1) [30, 35,
44, 48, 71]. We also confirmed this finding using another fluores-
cent reported GFP-FABD [48]. By visualizing fluorescent-tagged
Lifeact together with cell division markers in the dividing root tip
cells, we observed that the F-actin polarization occurs at the meta-
phase/anaphase transition and persists in time throughout cytoki-
nesis [48]. In this study, we generated a set of fluorescent-tagged
Lifeact markers in cyan, yellow, and red [48], that should be helpful
in performing colocalization analysis in Arabidopsis.
To validate the use of this reporter to visualize the F-actin in
plant, BY-2 cells stably expressing Lifeact–psRFP were
paraformaldehyde-fixed, and stained using Alexa-Fluor®488
labeled phalloidin. This experiment showed a clear colocalization
of Alexa-Fluor®488-phalloidin and Lifeact–psRFP for the perinuc-
lear actin. However, in the cell cortex, only the green fluorescence
of Alexa-Fluor®488-phalloidin could be detected, whereas the red
Lifeact–psRFP signal was absent, indicating that there is a clear
structural difference between these actin subpopulations [52].
While studies relying on fluorescent-based reporters multi-
plied, it is important to keep in mind that these biosensors might
not label the entire population of F-actin filaments in cells due to
competition with endogenous actin regulatory proteins. As such,
10 Marie-Cécile Caillaud

accurate detection of F-actin by immunolocalization might be

considered as a complementary approach in certain cell types [71].

7 MT–Actin Interactions and How to Image Both at the Same Time

Interaction between microtubules and F-actin has been reported in

various plant systems, and proteins connecting these two cytoskel-
etal networks were identified, even if the molecular mechanism is
still elusive [11, 72, 73]. Among them, kinesins with a calponin
homology domain (KCHs) have been identified recently as a plant-
specific subgroup of the kinesin-14 family and are suspected to act
as microtubule–actin filament cross-linkers. The Cotton kinesin
GhKCH2, which also contains an actin-binding calponin homol-
ogy (CH) domain in the N-terminus, decorates the phragmoplast
midzone in dividing root tip cells; it was shown using immunolo-
calization assays [74].
Similarly, NtKCH, a KCH homolog of tobacco BY-2 cells,
accumulates in the PPB [47]. Using the transient Agrobacterium-
mediated transformation of BY-2 cells (TAMBY2), the Arabidopsis
kinesin 14-type molecular motor (AtKinG) was found to strongly
label the PPB [42]. Fluorescence double-labeling in BY-2 cells
showed that AtKinG localizes to both microtubules and F-actin
[42]. In BY-2 cells, the fluorescent-tagged kinesin-like AtKCA1
protein from Arabidopsis that interacts with the Cyclin-dependent
kinases AtCDKA labels the plasma membrane in a non-homoge-
nous manner [75, 76]. In a stable BY-2 line, GFP-AtKCA1 is
excluded from the cortical division zone (CDZ) forming the
“KCA1 depleted zone” (KDZ) [76].
This finding contrasts with what was observed for the
fluorescent-tagged actin nucleator FORMIN 6 (AtFH6-GFP)
which labeled the plasma membrane without showing regions of
reduced fluorescence [76]. To visualize F-actin together with the
AtKCA1 fusion protein, fABD2 was fused N-terminally to RFP
(fABD2-RFP) and stably transformed in BY-2 cells expressing
GFP-AtKCA1 [76]. Dual live-cell imaging showed that the ADZ
and the KDZ colocalize [76]. However, the localization of
AtKCA1 in its native system was not yet reported and therefore
the existence of the KDZ in a dividing plant tissue still needs to be
confirmed.
The myosins which constitute a large superfamily of proteins
that share a common domain of interaction with F-actin, were also
reported to colocalize with F-actin and microtubules. The most
studied of them, the moss Myo8, was shown to interact with both
F-actin and microtubules. In Physcomitrella, Myo8 localizes to the
division site and the phragmoplast during cell division and may
promote phragmoplast guidance via F-actin to the division
site [53].
 Methods to Visualize the Actin Cytoskeleton During Plant Cell Division 11

While microtubule-associated proteins are rarely reported to

also bind to F-actin, a 190 kDa Microtubules Associated Protein
(NtMAP-190) isolated from mini-protoplasts of tobacco cultured
cells was shown to bind to both F-actin and microtubules and to
localize at the mitotic spindle [77].
The Formins are involved in the polymerization of actin and
associate with the fast-growing end of the F-actin. In moss, the
fluorescent-tagged class II Formin 2A (For2A) strongly labels the
center of the expanding phragmoplast, as it was also reported for
the type I Arabidopsis Formins AtFH5 and AtFH8 [43, 53,
54]. These observations suggest that the fast-growing ends of
F-actin are anchored at its midzone and the pointed ends are in
the cytoplasm [53]. By contrast, the type II Arabidopsis Formin
AtFH14 that, like For2A, acquires an N-terminal PTEN-like
domain [78] colocalizes with α-tubulin at the entire PPB, spindle,
and phragmoplast when expressed in BY-2 and Arabidopsis suspen-
sion cells [36]. These findings highlight specificity among the For-
mins in their interaction with the cytoskeleton and anionic lipids.
Indeed, the type I Arabidopsis Formin AtFH4 is able to bind
microtubules in vitro and in vivo [55], suggesting that specific
Formins could have an impact on both the F-actin and microtubule
cytoskeleton during cell division.
While these fluorescent-tagged actin-binding proteins are avail-
able (Table 1), the fact that they often localize to both actin and
microtubules can make the interpretation of labeling experiments
difficult. However, peculiar staining patterns such as the one
observed for Myo8 during cytokinesis will be key to study in detail
in time and space the events occurring for example at the cortex of
the cell in late telophase.

8 Visualization of F-Actin In Vivo by Super-Resolution Microscopy

As electron microscopy did in the past, super-resolution micros-

copy provides the ability to see details of cellular and even macro-
molecular structures that were not possible to see before. Photo-
Activated Localization Microscopy (PALM) has attracted consider-
able attention because they allow structures below the Abbe limit to
be resolved and to image single molecules [79]. In recent years, a
growing interest to use this method in plant research is obvious
even though only a few laboratories are equipped with a super-
resolution microscope which allows the detection of the emission
from individual fluorophores. Importantly, these emitters must be
photoactivatable or photoconvertible by irradiation with visible
light. By activating them sparsely and stochastically, each CCD
camera frame contains only a few spots of the size of the point-
spread function, which allows each molecule to be precisely loca-
lized and followed.
12 Marie-Cécile Caillaud

Using BY-2 cells stably transformed with photoactivatable fluo-

rescent proteins fused to the actin-binding probe Lifeact, the
dynamic reorganization of the actin cytoskeleton during the cell
cycle was recently assessed by PALM with optical sectioning
[52]. While the Lifeact–monomeric variant fused to IrisFP was
found to indiscriminately label central and cortical actin filaments,
the tetrameric Lifeact–photoswitchable red fluorescent protein
(Lifeact–psRFP) labeled only a specific perinuclear subpopulation
of actin called the “nuclear basket” [52]. It is not clear why such
difference exists; however, this experiment highlights how difficult
it is to have a full picture of the different subpopulation of F-actin in
the cell. Researcher should be therefore cautious in the interpreta-
tion of the results, in particular when talking about “depletion” of
the fluorescent signal. During mitosis, Lifeact–psRFP was exclu-
sively localizing at the nuclear basket, arranged in a fine mesh-like
structure until the end of metaphase and excluded from the spindle.
At the beginning of the anaphase, Lifeact–psRFP signal in the
nuclear basket was reduced. In telophase, while the new nuclear
envelope was formed, a clear gradient of the Lifeact–psRFP was
observed toward the cell poles [52].

9 Conclusion

In the past few years, the increasing interest in the visualization of

the actin cytoskeleton is illustrated by the number of different
techniques and fluorescent reporter lines generated. Yet, so far,
the behaviors of the actin cytoskeleton during mitosis have been
mostly studied in a single cell context such as the BY-2 cell line. The
dynamics of the actin cytoskeleton in growing tissues remain par-
tial, even if recent studies started to address it. To date, there are
still difficulties to visualize dividing cells in the meristematic part of
the plant at super-resolution because of the topology of the tissues
or the presence of the root cap. Many other technical challenges are
still blocking progress in the field, to address for example whether
or not the ADZ is also observed in a dividing cell in its physiological
context. In a tissue context, the fluorescence observed in the sur-
rounding interphase cells might mask potential domains at the
cortex of the dividing cell such as the ADZ. The development of
an inducible promoter or artificial system to visualize the dividing
cell at better resolution might be the next task. Furthermore, while
many zonation in the cortex of the dividing cells (CDZ, ADZ, actin
twin peaks, KDZ for example) were described in different systems,
the temporal and spatial relationship between them is still cryp-
tic. The integration of the localization of the F-actin cytoskeleton in
a referential together with the key player of the cell division seems
to be the only way to assemble the pieces of the puzzle during this
crucial process. In animals, actin mutants have strong phenotypes.
 Methods to Visualize the Actin Cytoskeleton During Plant Cell Division 13

It’s more difficult to understand the role of F-actin in plants, simply

because the phenotype of single knock-outs are weak (or because of
the high redundancy in F-actin regulators). It would be therefore
helpful to obtain more quantitative data about the localization of
the actin during cell division and to develop systems to modify the
actin cytoskeleton in order to build up a model in the light of what
was done for the microtubules cytoskeleton in plants.

Acknowledgments

We are grateful to the SiCE group (RDP, Lyon, France), in partic-

ular Yvon Jaillais and Isabelle Fobis-Loisy; to Oliver Hamant and
Jan Traas (RDP, Lyon, France) for comments and discussions. This
work was supported by ANRJC/JC INTERPLAY (ANR-16-
CE13-0021) and ANR PRC PlantScape (ANR-20-CE13-
0026) and a SEED FUND ENS LYON-2020.

References

1. Livanos P, Muller S (2019) Division plane
establishment and cytokinesis. Annu Rev
Plant Biol 70:239–267
2. Seagull R, Heath I (1979) The effects of tannic
acid on the in vivo preservation of microfila-
ments. Eur J Cell Biol 20(2):184–188
3. Hardham A, Gunning B (1980) Some effects
of colchicine on microtubules and cell division
in roots of Azolla pinnata. Protoplasma 102
(1–2):31–51
4. Heath I, Seagull R (1982) Oriented cellulose
fibrils and the cytoskeleton: a critical compari-
son of models. In: Lloyd CW (ed) The cyto-
skeleton in plant growth and development.
Academic, pp 163–182
5. Tiwari SC, Polito VS (1988) Organization of
the cytoskeleton in pollen tubes of Pyrus com-
munis: a study employing conventional and
freeze-substitution electron microscopy,
immunofluorescence, and rhodamine-
phalloidin. Protoplasma 147(2–3):100–112
6. Lancelle S, Cresti M, Hepler P (1987) Ultra-
structure of the cytoskeleton in freeze-
substituted pollen tubes of Nicotiana alata.
Protoplasma 140(2–3):141–150
7. Tiwari SC et al (1984) Cytoskeleton and inte-
gration of cellular function in cells of higher
plants. J Cell Biol 99(1 Pt 2):63s
8. Takeuchi M et al (2016) Single microfilaments
mediate the early steps of microtubule bun-
dling during preprophase band formation in
onion cotyledon epidermal cells. Mol Biol
Cell 27(11):1809–1820
9. Forer A, Jackson WT, Engberg A (1979) Actin
in spindles of Haemanthus katherinae endo-
sperm. II. Distribution of actin in chromo-
somal spindle fibres, determined by analysis of
serial sections. J Cell Sci 37:349–371
10. Schroeder TE (1976) Actin in dividing cells:
evidence for its roles in cleavage but not mito-
sis. Cell Motil:265–278
11. Schmit AC, Lambert AM (1988) Plant actin
filament and microtubule interactions during
anaphase-telophase transition: effects of antag-
onist drugs. Biol Cell 64(3):309–319
12. Mole-Bajer J, Bajer AS, Inoue S (1988) Three-
dimensional localization and redistribution of
F-actin in higher plant mitosis and cell plate
formation. Cell Motil Cytoskeleton 10
(1–2):217–228
13. Dancker P et al (1975) Interaction of actin with
phalloidin: polymerization and stabilization of
F-actin. Biochim Biophys Acta 400
(2):407–414
14. De Vries J, Wieland T (1978) Influence of
phallotoxins and metal ions on the rate of pro-
teolysis of actin. Biochemistry 17
(10):1965–1968
15. Wulf E et al (1979) Fluorescent phallotoxin, a
tool for the visualization of cellular actin. Proc
Natl Acad Sci 76(9):4498–4502
16. Clayton L, Lloyd CW (1985) Actin organiza-
tion during the cell cycle in meristematic plant
cells. Actin is present in the cytokinetic phrag-
moplast. Exp Cell Res 156(1):231–238
14 Marie-Cécile Caillaud
17. Gunning B, Wick S (1985) Preprophase bands,
phragmoplasts, and spatial control of cytokine-
sis. J Cell Sci Suppl 2:157–179
18. Lloyd CW, Traas J (1988) The role of F-actin
in determining the division plane of carrot sus-
pension cells. Drug studies. Development 102
(1):211–221
19. Traas JA et al (1987) An actin network is pres-
ent in the cytoplasm throughout the cell cycle
of carrot cells and associates with the dividing
nucleus. J Cell Biol 105(1):387–395
20. Panteris E, Adamakis I-DS, Tzioutziou NA
(2009) Abundance of actin filaments in the
preprophase band and mitotic spindle of
brick1 Zea mays mutant. Protoplasma 236
(1–4):103–106
21. Yasuda H et al (2005) Localization of actin
filaments on mitotic apparatus in tobacco
BY-2 cells. Planta 222(1):118–129
22. Valster AH et al (1997) Probing the plant actin
cytoskeleton during cytokinesis and interphase
by profilin microinjection. Plant Cell 9
(10):1815–1824
23. Hoshino H et al (2003) Roles of actin-depleted
zone and preprophase band in determining the
division site of higher-plant cells, a tobacco
BY-2 cell line expressing GFP-tubulin. Proto-
plasma 222(3–4):157–165
24. Cutler SR, Ehrhardt DW (2002) Polarized
cytokinesis in vacuolate cells of Arabidopsis.
Proc Natl Acad Sci U S A 99(5):2812–2817
25. Kakimoto T, Shibaoka H (1987) Actin fila-
ments and microtubules in the preprophase
band and phragmoplast of tobacco cells. Pro-
toplasma 140(2–3):151–156
26. Kakimoto T, Shibaoka H (1987) A new
method for preservation of actin filaments in
higher plant cells. Plant Cell Physiol 28
(8):1581–1585
27. Sonobe S, Shibaoka H (1989) Cortical fine
actin filaments in higher plant cells visualized
by rhodamine-phalloidin after pretreatment
with m-maleimidobenzoyl N-hydroxysuccini-
mide ester. Protoplasma 148(2–3):80–86
28. Palevitz BA (1987) Actin in the preprophase
band of Allium cepa. J Cell Biol 104
(6):1515–1519
29. McCurdy DW, Gunning BE (1990) Reorgani-
zation of cortical actin microfilaments and
microtubules at preprophase and mitosis in
wheat root-tip cells: a double label immunoflu-
orescence study. Cell Motil Cytoskeleton 15
(2):76–87
30. Bo L, Palevitz BA (1992) Organization of cor-
tical microfilaments in dividing root cells. Cell
Motil 23(4):252–264
31. Katsuta J, Hashiguchi Y, Shibaoka H (1990)
The role of the cytoskeleton in positioning of
the nucleus in premitotic tobacco BY-2 cells. J
Cell Sci 95:413–422
32. McCurdy DW, Sammut M, Gunning BES
(1988) Immunofluorescent visualization of
arrays of transverse cortical actin microfila-
ments in wheat root-tip cells. Protoplasma
147(2):204–206
33. Panteris E (2008) Cortical actin filaments at
the division site of mitotic plant cells: a recon-
sideration of the ‘actin-depleted zone’. New
Phytol 179(2):334–341
34. Cho SO, Wick SM (1991) Actin in the devel-
oping stomatal complex of winter rye: a com-
parison of actin antibodies and Rh-phalloidin
labeling of control and CB-treated tissues. Cell
Motil Cytoskeleton 19(1):25–36
35. Baluska F et al (1997) Rearrangements of
F-actin arrays in growing cells of intact maize
root apex tissues: a major developmental switch
occurs in the postmitotic transition region. Eur
J Cell Biol 72(2):113–121
36. Li Y et al (2010) The type II Arabidopsis for-
min14 interacts with microtubules and micro-
filaments to regulate cell division. Plant Cell 22
(8):2710–2726
37. Liu B, Palevitz BA (1992) Organization of
cortical microfilaments in dividing root cells.
Cell Motil Cytoskeleton 23(4):252–264
38. Yoneda A et al (2004) Disruption of actin
microfilaments causes cortical microtubule dis-
organization and extra-phragmoplast forma-
tion at M/G1 interface in synchronized
tobacco cells. Plant Cell Physiol 45
(6):761–769
39. Wang YS, Yoo CM, Blancaflor EB (2008)
Improved imaging of actin filaments in trans-
genic Arabidopsis plants expressing a green
fluorescent protein fusion to the C-and N-ter-
mini of the fimbrin actin-binding domain
2. New Phytol 177(2):525–536
40. Wang YS et al (2004) Green fluorescent pro-
tein fusions to Arabidopsis fimbrin 1 for spatio-
temporal imaging of F-actin dynamics in roots.
Cell Motil Cytoskeleton 59(2):79–93
41. Dyachok J et al (2014) Fluorescent protein-
based reporters of the actin cytoskeleton in
living plant cells: fluorophore variant, actin
binding domain, and promoter considerations.
Cytoskeleton 71(5):311–327
42. Buschmann H et al (2011) Cytoskeletal
dynamics in interphase, mitosis and cytokinesis
analysed through Agrobacterium-mediated
transient transformation of tobacco BY-2
cells. New Phytol 190(1):258–267
 Methods to Visualize the Actin Cytoskeleton During Plant Cell Division
43. Xue XH et al (2011) AtFH8 is involved in root
development under effect of low-dose latrun-
culin B in dividing cells. Mol Plant 4
(2):264–278
44. Voigt B et al (2005) GFP-FABD2 fusion con-
struct allows in vivo visualization of the
dynamic actin cytoskeleton in all cells of Arabi-
dopsis seedlings. Eur J Cell Biol 84
(6):595–608
45. Kojo KH, Yasuhara H, Hasezawa S (2014)
Time-sequential observation of spindle and
phragmoplast orientation in BY-2 cells with
altered cortical actin microfilament patterning.
Plant Signal Behav 9
46. Kojo KH et al (2013) Dual observation of
microtubules and actin microfilaments during
cell cycle progression in BY-YTRF1 cells. Cyto-
logia 78(3):203–204
47. Klotz J, Nick P (2012) A novel actin-
microtubule cross-linking kinesin, NtKCH,
functions in cell expansion and division. New
Phytol 193(3):576–589
48. Lebecq A, Fangain A, Boussaroque A, Caillaud
M-C (2021) Dynamic apical-basal enrichment
of the F-Actin during cytokinesis in arabidopsis
cells embedded in their tissues. bioRxiv:
2021.2007.2007.451432
49. Era A et al (2009) Application of lifeact reveals
F-actin dynamics in Arabidopsis thaliana and
the liverwort, Marchantia polymorpha. Plant
Cell Physiol 50(6):1041–1048
50. Doumane M et al (2020) iDePP: a genetically
encoded system for the inducible depletion of
PI(4,5)P2 in Arabidopsis thaliana.
bioRxiv:2020.05.13.091470
51. van Gisbergen PA et al (2012) Class II formin
targeting to the cell cortex by binding PI (3, 5)
P2 is essential for polarized growth. J Cell Biol
198(2):235–250
52. Durst S et al (2014) Organization of perinuc-
lear actin in live tobacco cells observed by
PALM with optical sectioning. J Plant Physiol
171(2):97–108
53. Wu SZ, Bezanilla M (2014) Myosin VIII
associates with microtubule ends and together
with actin plays a role in guiding plant cell
division. elife 3
54. Ingouff M et al (2005) Plant formin AtFH5 is
an evolutionarily conserved actin nucleator
involved in cytokinesis. Nat Cell Biol 7
(4):374–380
55. Deeks MJ et al (2010) The plant formin
AtFH4 interacts with both actin and microtu-
bules, and contains a newly identified
microtubule-binding domain. J Cell Sci 123
(Pt 8):1209–1215
56. Burridge K, Connell L (1983) Talin: a cyto-
skeletal component concentrated in adhesion
15
plaques and other sites of actin-membrane
interaction. Cell Motil 3(5):405–417
57. Kost B, Spielhofer P, Chua NH (1998) A
GFP-mouse talin fusion protein labels plant
actin filaments in vivo and visualizes the actin
cytoskeleton in growing pollen tubes. Plant J
16(3):393–401
58. Kost B et al (2000) Non-invasive F-actin visu-
alization in living plant cells using a
GFP-mouse talin fusion protein. In: Actin: a
dynamic framework for multiple plant cell
functions. Springer, pp 637–659
59. Holweg C, Süßlin C, Nick P (2004) Capturing
in vivo dynamics of the actin cytoskeleton sti-
mulated by auxin or light. Plant Cell Physiol 45
(7):855–863
60. Hoffmann A, Nebenführ A (2004) Dynamic
rearrangements of transvacuolar strands in
BY-2 cells imply a role of myosin in remodeling
the plant actin cytoskeleton. Protoplasma 224
(3–4):201–210
61. Ketelaar T, Anthony RG, Hussey PJ (2004)
Green fluorescent protein-mTalin causes
defects in actin organization and cell expansion
in Arabidopsis and inhibits actin depolymeriz-
ing factor’s actin depolymerizing activity
in vitro. Plant Physiol 136(4):3990–3998
62. Bretscher A (1981) Fimbrin is a cytoskeletal
protein that crosslinks F-actin in vitro. Proc
Natl Acad Sci 78(11):6849–6853
63. McCurdy DW, Kim M (1998) Molecular clon-
ing of a novel fimbrin-like cDNA from Arabi-
dopsis thaliana. Plant Mol Biol 36(1):23–31
64. Kovar DR et al (2001) Fluorescently-labeled
fimbrin decorates a dynamic actin filament net-
work in live plant cells. Planta 213(3):390–395
65. Sheahan MB et al (2004) A green fluorescent
protein fusion to actin-binding domain 2 of
Arabidopsis fimbrin highlights new features of
a dynamic actin cytoskeleton in live plant cells.
Plant Physiol 136(4):3968–3978
66. Yoneda A et al (2007) Recent progress in living
cell imaging of plant cytoskeleton and vacuole
using fluorescent-protein transgenic lines and
three-dimensional imaging. Protoplasma 230
(3–4):129–139
67. Sano T et al (2005) Appearance of actin micro-
filament ‘twin peaks’ in mitosis and their func-
tion in cell plate formation, as visualized in
tobacco BY-2 cells expressing GFP–fimbrin.
Plant J 44(4):595–605
68. Higaki T et al (2008) Quantitative analysis of
changes in actin microfilament contribution to
cell plate development in plant cytokinesis.
BMC Plant Biol 8:80
69. Riedl J et al (2008) Lifeact: a versatile marker
to visualize F-actin. Nat Methods 5
(7):605–607
16 Marie-Cécile Caillaud
70. Doumane M et al (2017) Automated tracking
of root for confocal time-lapse imaging of cel-
lular processes. Bio Protoc 7(8):e2245
71. Sadot E, Blancaflor EB (2019) The actomyosin
system in plant cell division: lessons learned
from microscopy and pharmacology. In: Sahi
VP, Baluška F (eds) The cytoskeleton: diverse
roles in a plant’s life. Springer, pp 85–100
72. Takeuchi M, Staehelin A, Mineyuki Y (2017)
Actin-microtubule interaction in plants. In:
Jimenez-Lopez JC (ed) Cytoskeleton-
structure, dynamics, function and disease.
InTechOpen, pp 33–54
73. Sampathkumar A et al (2011) Live cell imaging
reveals structural associations between the actin
and microtubule cytoskeleton in Arabidopsis.
Plant Cell 23(6):2302–2313
74. Xu T et al (2009) A cotton kinesin GhKCH2
interacts with both microtubules and microfila-
ments. Biochem J 421(2):171–180
75. Vanstraelen M et al (2004) A plant-specific
subclass of C-terminal kinesins contains a con-
served a-type cyclin-dependent kinase site
implicated in folding and dimerization. Plant
Physiol 135(3):1417–1429
76. Vanstraelen M et al (2006) Cell cycle-
dependent targeting of a kinesin at the plasma
membrane demarcates the division site in plant
cells. Curr Biol 16(3):308–314
77. Igarashi H et al (2000) Isolation of a novel
190 kDa protein from tobacco BY-2 cells: pos-
sible involvement in the interaction between
actin filaments and microtubules. Plant Cell
Physiol 41(8):920–931
78. van Gisbergen PA, Bezanilla M (2013) Plant
formins: membrane anchors for actin polymer-
ization. Trends Cell Biol 23(5):227–233
79. Hedde PN, Nienhaus GU (2014) Super-
resolution localization microscopy with photo-
activatable fluorescent marker proteins. Proto-
plasma 251(2):349–362
 Part I

Mitosis and Cell Cycle Progression

 Chapter 2

Immunolabeling of Nuclei/Chromosomes in Arabidopsis

thaliana
Ulkar Ahmadli, Michael Sandmann, Joerg Fuchs, and Inna Lermontova

Abstract
The cell cycle is a complex sequence of events by which cells grow and divide mitotically or meiotically.
Mitosis results in the generation of two identical daughter cells, while meiosis generates gametes as a
prerequisite for sexual reproduction. To study the localization and dynamics of proteins involved in the
regulation and proceeding of the cell cycle, life cell imaging of proteins fused to fluorescent tags can be
performed. However, in some cases this approach cannot be applied, e.g., due to low fluorescence intensity,
fast bleaching, or degradation of recombinant proteins by the proteasome pathway. Instead, immunolabel-
ing with protein-specific antibodies offers a useful approach for the analysis of intact cells. Alternatively,
immunolabeling can also be applied to isolated and/or flow-sorted nuclei of particular cell cycle stages
(G1, S, and G2) or of different endopolyploidy levels. The following chapter will detail indirect immuno-
labeling protocols to analyze the subcellular localization and distribution of cell cycle-specific proteins in
Arabidopsis thaliana.

Key words A. thaliana, Immunolabeling, Chromosomes, Nuclei, Mitosis, Meiosis

1 Introduction

To understand the functional importance of proteins involved in

the control of cell cycle dependent processes, studies of their sub-
cellular localization are required. In some cases, these analyses are
done by life cell imaging of proteins fused to fluorescent tags (e.g.,
GFP or YFP). However, in many cases the expression level of cell
cycle dependent proteins is very low and, consequently, the expres-
sion of the fusion constructs under control of an endogenous
promoter not strong enough to generate detectable fluorescence
signals. On the other hand, the expression under control of a
constitutive promoter might result in unspecific localization pat-
terns. In these cases, the immunolabeling approach might be a
useful alternative and/or in the latter case could be used to confirm

Marie-Cécile Caillaud (ed.), Plant Cell Division: Methods and Protocols, Methods in Molecular Biology,
vol. 2382, https://doi.org/10.1007/978-1-0716-1744-1_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

19
20 Ulkar Ahmadli et al.

results obtained by expression of GFP fusion constructs under

control of a constitutive promoter.
Immunolabeling is a biochemical process allowing the spatial
and temporal localization of antigens (usually proteins) within
nuclei, cells, tissues, or organs. Immunolabeling can be applied
either directly or indirectly. In the direct variant, the protein-
specific primary antibody is directly linked to a tag (most frequently
fluorescent compounds). Although this method guarantees a mini-
mum of cross-reaction, it is less frequently used compared to the
indirect method. The indirect method employs a two-step protocol
in which the unlabeled protein-specific primary antibody is
detected by a tagged secondary antibody in a second step
(Fig. 1). This allows the universal use of commercially available
secondary antibodies of any available color. Usually the indirect
method results in stronger signals per antigen.
The application of immunolabeling allows the analyses of cells
at different mitotic (Fig. 2a) and meiotic stages in high numbers as
a prerequisite for statistical analysis. Furthermore, quantification of
immunosignal intensities can be used to study the dynamics of
proteins during mitotic [1, 2] and meiotic [3] cell cycles. Double
immunostaining experiments (Fig. 2b) can be applied to visualize
and localize two proteins simultaneously [4], provided that they are
raised in different animals.
Here we describe detailed protocols on the indirect immuno-
labeling of cell cycle-specific proteins on nuclei of A. thaliana
prepared either by squashing of root tips or flower buds or by
flow sorting or spinning of nuclei suspensions from leaf tissue.

Secondary Antibody

Primary Antibody

Antigen

Nuclei/Chromosomes

Slide

Fig. 1 Schematic view of immunolabeling procedure

 Immunolabeling of Nuclei/Chromosomes in Arabidopsis thaliana 21

a
cenH3

early G2 late G2 Prometaphase Anaphase/Telophase

CENP-C EYFP-cenH3 DAPI Merge

cenH3 Merge cenH3 Merge

haploid diploid

Fig. 2 (a) Immunolabeling of centromeric histone H3 (cenH3) at centromeres of Arabidopsis thaliana with anti-
cenH3 antibody through the mitotic cell cycle. (b) Double immunolabeling of centromeric protein CENP-C (red)
and EYFP-cenH3 (green) at chromocenters of a meristematic nucleus of EYFP-cenH3 transformant with anti-
CENP-C and anti-GFP antibodies, respectively. (c) Immunolabeling of cenH3 at centromeres of nuclei isolated
from young leaves of 4-week-old Arabidopsis haploid and diploid plants prepared with the cytospin method.
DNA is counterstained with DAPI (blue). Bar ¼ 2μm

2 Materials

2.1 Preparation 1. Three-days-old seedlings or inflorescences of 4–6 weeks old

of Slides (Squashing plants of A. thaliana.
Method) 2. MTSB (MicroTubule-Stabilizing Buffer): 50 mM PIPES,
5 mM MgSO4, 5 mM EGTA, pH 6.9.
3. 1
PBS (Phosphate Buffered Saline): 137 mM NaCl, 2.7 mM
KCl, 10 mM Na2PO4·2H2O, 1.8 mM KH2PO4, pH 7.4.
4. Fixation buffer: 4% (w/v) paraformaldehyde in MTSB (or 1

PBS) buffer.
22 Ulkar Ahmadli et al.

5. Enzymes mixture: 2.5% (w/v) pectinase (1 u/mg), 2.5% (w/v)

cellulase (10 u/mg), 2.5% (w/v) pectolyase (0.3 u/mg, Sigma
Aldrich, St. Louis, USA) in MTSB (or 1
PBS) buffer.
6. Embryo dishes.
7. Stereomicroscope.
8. Dissection needles and a fine forceps.
9. Polysine-coated slides.
10. Coverslips 24
24.
11. Coplin jar.
12. Platform shaker.

2.2 Preparation Common material for both techniques:

of Slides Using A Flow
1. Leaves of 4–6 weeks old A. thaliana plants (3–4 young leaves).
Sorter or a Cytospin
2. Tris buffer: 10 mM Tris–HCl pH 7.5, 10 mM Na2-EDTA,
100 mM NaCl, 0.1% Triton X-100, pH 7.5. Store at 4  C.
3. Fixation buffer: 4% ice-cold formaldehyde in Tris buffer.
4. Nuclei isolation buffer: 15 mM Tris–HCl, 2 mM Na2EDTA,
0.5 mM Spermin, 80 mM KCl, 20 mM NaCl, 15 mM Mer-
captoethanol, 0.1% Triton X100, pH 7.5.
5. Speed vacuum concentrator or exsiccator.
6. Petri dishes.
7. Razor blade.
8. Standard microscope slides.
9. Coplin jar.
10. Platform shaker.
11. Light microscope.
Material specific for the cytospin technique:
12. (a) 1
PBS (Phosphate Buffered Saline) 137 mM NaCl,
2.7 mM KCl, 10 mM Na2PO4·2H2O, 1.8 mM KH2PO4,
pH 7.4.
13. (a) 35 μm filter mesh.
14. (a) Cytospin.
Material specific for flow sorting:
12. (b) Sucrose buffer: 100 mM Tris–HCl, 50 mM KCl, 2 mM
MgCl2, 0.05% Tween 20, and 5% sucrose, pH 7.9.
13. (b) 0.5–2 μg/ml 40 ,6-diamidino-2-phenylindole (DAPI).
14. (b) Flow sorter.
 Immunolabeling of Nuclei/Chromosomes in Arabidopsis thaliana 23

2.3 Immunolabeling 1. Blocking solution: 8% BSA, 0.1% Triton X100 in MTSB buffer.
2. Antibody dilution buffer: 1% BSA in MTSB buffer
(or 1
PBS).
3. Primary and secondary antibodies.
4. 0.5–2 μg/ml 40 , 6-diamidino-2-phenylindole (DAPI).
5. Coplin jar.
6. Humidity chamber: a hermetic container (e. g., plastic box for
microscope glass slides) with wet paper on the bottom.
7. Fluorescence microscope equipped with a high resolution
CDD camera.

2.4 Sources Antibodies against various antigens, including plant-specific anti-

of Primary gens, are produced worldwide by many different companies. The
and Secondary following link compiles a brought collection of primary antibodies
Antibodies that can be purchased from different companies: http://www.
for Immunolabeling antibodies-online.com/search.php. The antibodies are available as
Experiments polyclonal and/or monoclonal once. While a polyclonal antibody
represents a mixture of antibodies from different B cells that are
2.4.1 Commercially able to recognize multiple epitopes on the same antigen, a mono-
Available Antibodies clonal antibody is an antibody from a single B cell and binds only to
one unique epitope. Polyclonal antibodies are faster to generate
and less expensive but also less specific with a higher potential for
cross-reactivity. Monoclonal antibodies in contrast are highly spe-
cific with a reduced probability for cross-reactivity but significantly
more expensive and more time consuming to generate. While
monoclonal antibodies are usually generated in mice, a broad
range of animals (e.g., rabbit, rat, goat, sheep, and guinea pig) is
available for the production of polyclonal antibodies.
Before selecting commercial antibodies for immunolabeling
experiments, it has to be checked whether these antibodies are
suitable for this application, usually this information is given in
the data sheet of the product. It is easily possible that antibodies
which give specific signals on Western blot are not suitable for the
immunolabeling experiments or vice versa.

2.4.2 Custom-Made Although the list of commercially available antibodies against plant
Antibodies antigens is constantly increasing, it is still mainly restricted to anti-
bodies against commonly used marker proteins. Antibodies against
specific proteins of interest have to be generated either from
isolated and purified proteins or from synthetic peptides. Using
full-length recombinant proteins as antigens for raising antibodies
enables the production of a conformational epitope which might be
useful for the recognition of folded proteins. However, the purifi-
cation of recombinant proteins is time consuming and expensive.
The use of synthetic peptides requires less effort and is cheaper but
bears the risk of a lower antigenicity.
24 Ulkar Ahmadli et al.

2.4.3 Antibodies Against Antibodies against synthetic peptides can be raised using programs
Synthetic Peptides offered by different companies. These programs include the syn-
thesis of peptides, their conjugation to carriers and the immuniza-
tion of animals of your choice (http://www.eurogentec.com,
http://www.genscript.com, http://www.abcam.com). Alterna-
tively, peptides synthesized by companies can be used for immuni-
zation using facilities of your home institution if available.

2.4.4 Antibodies Against To generate antibodies against recombinant proteins, either the
Recombinant Proteins whole protein or only certain protein domains can be used as
antigens. It is preferable to generate and use for the immunization
soluble recombinant protein. To achieve this task, several references
can be used as guidelines [5, 6]. Depending on the protein of
interest, an optimization of the protocol might be required. This
includes optimizations on the genetic/vector level (gene/codon
usage engineering, choice of vector and solubility promoting tags
like MBP [Maltose binding protein]) as well as on the host/induc-
tion level (choice of host [Escherichia coli, yeast, etc.] including
choice of strains, choice of expression conditions [temperature,
concentration of inducer, etc.]).
Furthermore, if some parts of the protein are soluble while
others are not, then it is possible to express only the soluble part.
Smaller protein antigens often deliver more specific antiserum than
larger proteins.

3 Methods

3.1 Preparation 1. For immunostaining of mitotic chromosomes and meriste-

of Slides (Squashing matic nuclei germinate seeds (100–200) of A. thaliana by
Method) placing them in a row on wet filter paper in Petri dishes.
Incubate Petri dishes vertically for 3 days at 21  C (16 h
light/8 h dark) in a growth chamber. For the analysis of meiosis
(and mitosis) in flower buds, use 4–6 weeks old A. thaliana
plants grown in soil.
2. Collect seedlings or inflorescences into an embryo dish with
MTSB (or 1
PBS) buffer.
3. Remove MTSB (or 1
PBS) and add 2 ml of freshly prepared
fixation solution (4% (w/v) paraformaldehyde in MTSB or
1
PBS), apply vacuum (using a speed vacuum concentrator)
for 1–2 min in case of seedlings or 20 min (see Note 1) for
flower buds and incubate the samples afterwards for 20 min on
ice with constant gentle shaking on a platform shaker.
4. Remove the fixation solution, briefly rinse and afterwards wash
material 3
5 min in MTSB (or 1
PBS) on ice with constant
shaking.
 Immunolabeling of Nuclei/Chromosomes in Arabidopsis thaliana 25

5. Remove MTSB (or 1
PBS) and add ~150 μl of enzyme mix-

ture and incubate seedlings for 20 min at 37  C and flower
buds for 25 min (see Note 2).
6. Stop the reaction by adding 1–2 ml of MTSB (or 1
PBS)
(plant material has to be covered with buffer). Remove the
solution using a pipet and add 1–2 ml of fresh MTSB buffer
(or 1
PBS).
7. Cut root tips (5–6 per slide) (see Note 3) or isolate anthers and
ovaries (from 5 to 6 flower buds of appropriate stage
(0.5–1 mm)) under a stereomicroscope on polysine-coated
slides (see Note 4) in a drop of MTSB buffer (or 1
PBS),
cover the material with a coverslip, and remove excess of liquid
by putting the slides between two sheets of filter paper. After-
wards, squash the samples (with thumb and/or preparation
needle) (check in addition under light microscope) and put
the slides to liquid nitrogen. Remove the cover slips using a
razorblade, put the slides into a coplin jar with MTSB (or 1

PBS) and use them for immunolabeling as described below.

3.2 Preparation Common steps for both techniques:

of Slides Using a Flow
1. For isolation of leaf nuclei, grow plants in soil in a cultivation
Sorter or a Cytospin
room for 4–6 weeks.
2. Fix 3–4 young leaves either for 20 min in 10 ml freshly
prepared 4% formaldehyde/Tris buffer on ice under vacuum
in an exsiccator or for 5 min in a speed vacuum concentrator
and subsequently for 15–25 min on ice without vacuum.
3. Wash leaves 2
10 min in Tris buffer on ice.
4. Use a paper tissue to remove the excess of buffer from the leaf
material.
5. To isolate the nuclei, chop leaves using a sharp razorblade in
400–500 μl (for the cytospin) or 1 ml (for flow sorting)
ice-cold nuclei isolation buffer in a pre-cooled Petri dish (see
Note 5).
6. Filter the nuclei suspension using, e.g., 5 ml polystyrene
round-bottom tube with cell-strainer cap 35 μm
(BD Biosciences) to remove cell debris.
Steps specific for the cytospin technique [7]:
7. (a) Dilute extracted nuclei with nuclei isolation buffer 1:10 (see
Note 6).
8. (a) Use 100 μl of diluted nuclei suspension to spin down nuclei
on slide using cytospin (700 rpm for 5 min).
9. (a) Transfer slides in a Coplin jar with 1
PBS and store them
on ice until use (see Note 7).
26 Ulkar Ahmadli et al.

Steps specific for the flow sorting technique:

7. (b) Stain nuclei with 1 μg/ml 40 ,6-diamidino-2-phenylindole
(DAPI) and sort them according to their different fluorescence
intensities corresponding to the different ploidy levels into
separate collection tubes using a flow sorting facility [8]. For
flow sorting each flow cytometer with a sorting device and
appropriate light source and filter equipment for DAPI excita-
tion and emission can be used.
8. (b) Pipette 10–15 μl of sucrose buffer onto microscopic slides
and add equivalent amounts of sorted nuclei, gently mix it and
air dry the slides overnight.
9. (b) Use slides for immunolabeling or transfer them for longer
storage to 20  C.

3.3 Immunolabeling 1. Add ~100 μl of blocking solution per slide (Subheading 3.1,
step 7; Subheading 3.2, step 9a), cover it with parafilm and
incubate it for 1 h at room temperature in a humidity chamber.
Before applying the blocking solution to slides with flow-
sorted nuclei (see Note 8) (Subheading 3.2, step 9b), wash
them for 5 min in MTSB (or 1
PBS) in a coplin jar, fix them
for 10 min in 4% (w/v) paraformaldehyde/MTSB (or 1
PBS)
on ice and wash them as described above (Subheading 3.1, step
4). Perform washing and fixation steps on a platform shaker
with constant gentle shaking.
2. Dilute primary antibodies in dilution buffer (see Notes 9 and
10), remove the blocking solution, add ~40 μl of the diluted
primary antibodies per slide, cover slides with parafilm and
incubate them overnight at 10  C or 1 h at room temperature
in a humidity chamber (see Note 11).
3. Wash the slides 3
5 min in MTSB (or 1
PBS) in a coplin jar
with constant shaking.
4. Add ~40 μl of diluted secondary antibodies (see Notes 12 and
13) to each slide, cover the slides with parafilm and incubate
them 1 h at room temperature (to obtain stronger signals, the
slides were incubated at 37  C) in a humidity chamber.
5. Wash the slides 3
5 min in MTSB (or 1
PBS) in a coplin jar
with constant shaking.
6. Add a drop (8 μl) of antifade (Vectashield) containing 1 μg/ml
DAPI as a DNA counterstain to each slide and cover it with a
coverslip (24
24).
7. Perform microscopic analysis.
 Immunolabeling of Nuclei/Chromosomes in Arabidopsis thaliana 27

4 Notes

1. Apply vacuum in 5 min intervals to allow a better penetration

of the fixation solution into the plant material.
2. Incubation time has to be optimized each time for the newly
prepared enzymes mixture. For seedlings it can vary from 5 to
25 min and for flower buds from 25 to 30 min.
3. After incubation in the enzyme mixture root tips very often
separate from seedling and can be collected by pipet with a
small volume pipet tip (2–10 μl) under binocular.
4. It is recommended to use polysine-coated slides which allow
better adhesion of plant material.
5. Chop material softly to prevent the destruction of nuclei
structure.
6. To adjust the dilution, check the concentration of nuclei sus-
pension under a microscope.
7. Use for immunolabeling freshly prepared slides (store in 1

PBS max 3–4 days).
8. Slides with flow-sorted nuclei stored at 20  C should be
equilibrated to room temperature for ~30 min and then dried
for 30–60 min at 60  C.
9. The optimal dilution of antibodies has to be determined empir-
ically. When a new antibody has to be tested, it is advisable to
try different dilutions, starting with 1:100, 1:500, and 1:1000
and afterwards using smaller dilution steps if required.
10. For double immunolabeling experiment, dilute the two
selected primary antibodies to the optimal concentration in
dilution buffer (see Note 9). It is important that these anti-
bodies have been raised in different animals. Alternatively,
double immunostaining experiment can be performed using a
sequential approach: incubation with first primary and the
corresponding secondary antibody (see Subheading 3.3, steps
2–5), fixation for 10 min in 4% paraformaldehyde following by
washing in MTSB (or 1
PBS) and then incubation with
second primary and the corresponding secondary antibody
(see Subheading 3.3, steps 2–6).
11. Incubation time and temperature have a severe impact on the
quality of staining. These conditions need to be optimized for
each antibody and sample to achieve a staining of high specific-
ity with low cross-reactivity. Usually the specificity of the
immunolabeling is higher by incubating the primary antibody
overnight at low temperatures (i.e., 10  C).
28 Ulkar Ahmadli et al.

12. For the dilution of secondary antibodies, follow manufac-

turer’s instruction. For optimal results, an adjustment of the
antibody dilution might be required.
13. For the double immunostaining experiments, combine sec-
ondary antibodies conjugated to different fluorescent dyes:
for instance, green (Alexa 488; FITC) and red (Rhodamine,
Alexa 594).

Acknowledgments

U.A. is supported by a grant from the Deutsche Forschungsge-

meinschaft (LE2299/3-1).

References

1. Lermontova I, Schubert V, Fuchs J, Klatte S,
Macas J, Schubert I (2006) Loading of Arabi-
dopsis centromeric histone CENH3 occurs
mainly during G2 and requires the presence of
the histone fold domain. Plant Cell
18:2443–2451
2. Lermontova I, Fuchs J, Schubert V, Schubert I
(2007) Loading time of the centromeric histone
H3 variant differs between plants and animals.
Chromosoma 116:507–510
3. Schubert V, Lermontova I, Schubert I (2014)
Loading of the centromeric histone H3 variant
during meiosis—how does it differ from mitosis?
Chromosoma 123:491–497
4. Lermontova I, Koroleva O, Rutten T, Fuchs J,
Schubert V, Moraes I, Koszegi D, Schubert I
(2011) Knockdown of CENH3 in Arabidopsis
reduces mitotic divisions and causes sterility by
disturbed meiotic chromosome segregation.
Plant J 68:40–50
5. Green MR, Sambrook J (2012) Molecular clon-
ing, 4th edn. Cold Spring Harbor Laboratory
Press, New York
6. Sorensen HP, Mortensen KK (2005) Soluble
expression of recombinant proteins in the cyto-
plasm of Escherichia coli. Microb Cell Factories 4
(1):1
7. Ishii T, Schubert V, Khosravi S, Dreissig S,
Metje-Sprink J, Sprink T, Fuchs J, Meister A,
Houben A (2019) RNA-guided endonucle-
ase—in situ labelling (RGEN-ISL): a fast
CRISPR/Cas9-based method to label genomic
sequences in various species. New Phytol 222
(3):1652–1661
8. Jasencakova Z, Meister A, Walter J, Turner BM,
Schubert I (2000) Histone H4 acetylation of
euchromatin and heterochromatin is cell cycle
dependent and correlated with replication rather
than with transcription. Plant Cell
12:2087–2100
 Chapter 3

A Protocol for Genome-Wide Analysis of DNA Replication

Timing in Intact Root Tips
Leigh Mickelson-Young, Emily E. Wear, Jawon Song, Gregory J. Zynda,
Linda Hanley-Bowdoin, and William F. Thompson

Abstract
DNA replication during S phase in eukaryotes is a highly regulated process that ensures the accurate
transmission of genetic material to daughter cells during cell division. Replication follows a well-defined
temporal program, which has been studied extensively in humans, Drosophila, and yeast, where it is clear
that the replication process is both temporally and spatially ordered. The replication timing (RT) program is
increasingly considered to be a functional readout of genomic features and chromatin organization.
Although there is increasing evidence that plants display important differences in their DNA replication
process compared to animals, RT programs in plants have not been extensively studied. To address this
deficiency, we developed an improved protocol for the genome-wide RT analysis by sequencing newly
replicated DNA (“Repli-seq”) and applied it to the characterization of RT in maize root tips. Our protocol
uses 5-ethynyl-20 -deoxyuridine (EdU) to label replicating DNA in vivo in intact roots. Our protocol also
eliminates the need for synchronization and frequently associated chemical perturbations as well as the need
for cell cultures, which can accumulate genetic and epigenetic differences over time. EdU can be fluores-
cently labeled under mild conditions and does not degrade subnuclear structure, allowing for the differen-
tiation of labeled and unlabeled nuclei by flow sorting, effectively eliminating contamination issues that can
result from sorting on DNA content alone. We also developed an analysis pipeline for analyzing and
classifying regions of replication and present it in a point-and-click application called Repliscan that
eliminates the need for command line programming.

Key words 5-Ethynyl-20 -deoxyuridine, Cell cycle, DNA replication, Flow cytometry, Repli-seq,
Replication timing, Maize, Plant

1 Introduction

The temporal program of DNA replication along the chromosomes

in eukaryotes is a highly regulated and reproducible process [1] that

Supplementary Information The online version of this chapter (https://doi.org/10.1007/978-1-0716-1744-

1_3) contains supplementary material, which is available to authorized users.

Marie-Cécile Caillaud (ed.), Plant Cell Division: Methods and Protocols, Methods in Molecular Biology,
vol. 2382, https://doi.org/10.1007/978-1-0716-1744-1_3,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

29
30 Leigh Mickelson-Young et al.

ensures the accurate transmission of genetic material to each

daughter cell during cell division. DNA replication timing (RT)
studies have revealed that replication is a temporally ordered pro-
cess that is coordinated with transcription, chromatin dynamics,
and the three-dimensional chromatin landscape [2, 3]. For exam-
ple, early replicating DNA is transcriptionally active, rich in genes
and contains euchromatic histone modifications [4–10], while late
replicating DNA is associated with heterochromatin and repetitive
elements [5, 11]. Furthermore, chromosome conformation cap-
ture studies revealed that early and late replication strongly corre-
lates with open and closed chromatin compartments, respectively
[12–14]. Increasingly, replication time is thought to be an impor-
tant functional readout of genomic features and chromatin
organization [15].
The information we have on RT programs comes mostly from
fungal and metazoan systems [2, 16] and, until recently, very little
was known about the RT programs in plants. Although the replica-
tion machinery is broadly conserved across eukaryotes, there are
numerous differences between plants and other eukaryotes. For
example, plants lack geminin [17, 18] and Rif1 [19, 20], two
proteins intimately involved in the regulation of DNA replication.
Plants also display differences in the spatiotemporal distribution of
initiation regions [21] and of replicating DNA within the nucleus
[22, 23]. Moreover, S-phase duration, on average, is shorter in
plants than that of mammals [24], indicating that plant replication
programs may be temporally compressed compared to those of
animals. These differences emphasize the importance of studying
DNA replication in plants, as replication programs are likely to
differ between kingdoms and phyla, and information specific to
replication in plant systems is needed to integrate with data on
chromatin dynamics and gene expression.
Our group has recently characterized RT programs in maize
and Arabidopsis [21, 25–28]. In this chapter, we present a Repli-
seq protocol (Fig. 1) that we developed to characterize DNA
replication timing (RT) programs in maize root tips. Although
our system is conceptually similar to the Repli-seq protocol first
described for human cells [8], our system takes advantage of a new
labeling method that allows the physical separation of labeled and
unlabeled nuclei. We used this protocol to provide the first whole
genome analysis of RT in a higher plant [26], and to extend our
original analysis of Arabidopsis chromosome 4 [25] to the entire
genome [27]. Additionally, we present a computational pipeline
called Repliscan [29] that we developed to analyze sequencing data
generated by the Repli-seq protocol, and present it here as a “point-
and-click” application hosted on CyVerse.
Our protocol uses 5-ethynyl-20 -deoxyuridine (EdU) to label
replicating DNA in vivo for 20 min in the maize root tip. After
labeling, we dissect the 0–1 mm portion of the root which contains
A Protocol for Genome-Wide Analysis of DNA Replication Timing in Intact. . . 31

Fig. 1 Repli-seq workflow. A simplified Repli-seq workflow, highlighting the

major steps in the protocol. The dashed lines connect four of the text boxes to a
picture or cartoon illustrating that step. The first picture is a 3-day-old B73 maize
seedling with a white box drawn around the 0–1 mm portion of the root tip,
which is dissected and used in this protocol. The second picture is an example of
a mini-food processor (not a full-sized blender) used for chopping fixed, frozen
root tips for bulk nuclei isolation. The third picture shows an example of a
bivariate plot from flow sorting EdU-labeled and DAPI stained nuclei, illustrating
the S-phase “arc” (see Fig. 2). The last picture is an example of the output of the
Repli-seq data analysis pipeline using Repliscan for a ~2 Mb region on maize
B73 chromosome 4. The blue, green, and red tracks represent normalized
replication signal in early (E), middle (M), and late (L) portions of S phase,
respectively. The bottom track shows the final classification (segmentation) of
replication time(s) at each locus (see Fig. 4)

the meristem where many cells undergo a conventional mitotic cell

cycle [30]. Dissected root tips are fixed, and nuclei are bulk isolated
by a method modified from the original chopping method of
Galbraith and colleagues [25, 31, 32] . Incorporated EdU is con-
jugated in situ to Alexa fluor (AF) 488 using click chemistry
32 Leigh Mickelson-Young et al.

[33]. Nuclei are counterstained with 4,6-diamidino-2-phenylin-

dole (DAPI) and fractionated into different portions of S phase
using two color flow cytometry and sorting. EdU-labeled nuclei are
cleanly separated from unlabeled G1 and G2 nuclei, and divided
into early, middle, and late S-phase populations based on DNA
content. DNA is extracted from the early, middle, and late fractions,
and the EdU-labeled DNA is immunoprecipitated (IP) using an
anti AF-488 antibody. IP DNA is sequenced, the sequencing reads
are mapped to the maize B73 reference genome, and analysis using
Repliscan classifies the B73 genome by replication time [26].
A major improvement of our Repli-seq protocol, as compared
to the first reported protocol [8], is that we use the thymidine
analog, EdU, instead of 5-bromo-20 -deoxyuridine (BrdU) to label
replicating DNA in vivo. Visualization of EdU using click chemistry
[33] is achieved rapidly and under mild conditions that do not
degrade nuclei or subnuclear structure [34], in contrast to the
harsh conditions needed to visualize BrdU by immunocytochemis-
try. As noted above, EdU labeling also permits intact, replicating
nuclei (S-phase nuclei containing EdU) to be unambiguously dis-
tinguished from non-replicating nuclei (G1 and G2 nuclei that do
not incorporate EdU) by flow cytometry. Our protocol thus pro-
vides pure populations of labeled, replicating nuclei, which cannot
be achieved when sorting based solely on DNA content
[26, 32]. The additional purification reduces the contamination
of unlabeled DNA during immunoprecipitations. The improved
separation is especially important in the early and late S-phase
fractions, which otherwise would contain a large excess of unla-
beled nuclei (Fig. 2c, d).
Another advantage of our protocol is that labeling occurs in an
intact organ. Maize root tips contain many proliferating cells that
efficiently label with EdU and can be easily isolated for analysis
[32]. EdU has also been used successfully to label DNA in other
plant systems, including plant cell cultures [24, 30]. The protocol is
easily applied to unsynchronized cell populations, as occurs in root
tips, avoiding disruptive synchronization treatments such as appli-
cation of chemical inhibitors and sucrose starvation [35–37]. RT
analysis of intact root tips also avoids any genetic or epigenetic
instability that can occur in plant cell cultures [38–40]. Although,
at this time, we cannot separate different cell types of the root tip,
our analysis pipeline identified RT differences between mitotic vs
endocycling cells, suggesting possible RT differences among cell
types [28].
One further benefit of this protocol is that we highlight the
Repliscan pipeline that we developed to detect and classify regions
of replication into discrete combinations of replication signatures.
The utility, accuracy, and general applicability of Repliscan has been
validated through reanalysis of existing BrdU-based, Repli-seq data
from human and Drosophila [29]. The point-and-click format of
 A Protocol for Genome-Wide Analysis of DNA Replication Timing in Intact. . . 33

Fig. 2 Flow sorting gating strategy to remove debris and doublets, final sorting gates, and reanalysis of sorted,
replicating nuclei. (a–c) The gating strategy used to separate debris and doublets from intact nuclei is
illustrated in three bivariate pseudo-colored dot plots. Each dot is a single event and the color gradient (blue to
red) represents increasing nuclear density. (a) Parent gate 1 (PG1) differentiates debris from intact nuclei
based on light scattering properties (side scatter pulse height; SSC-H) and DNA content (DAPI fluorescence;
DAPI-H). A gate (black polygon) is drawn around intact nuclei of all ploidy levels, excluding debris, and broken
nuclei. (b) The nuclei in PG1 are further gated using Parent gate 2 (PG2) to remove doublets (aggregates of two
or more nuclei). Doublets are differentiated from single nuclei using DAPI pulse width (DAPI-W) and DAPI pulse
area (DAPI-A) to reflect particle geometry and size. (c) Nuclei in the singlet gate (PG2) are represented based
on Alexa fluor 488 fluorescence (AF-488-H) and DNA content (DAPI-H). EdU/AF-488-labeled S-phase nuclei
form an “arc” between the G1 and G2 populations (2C and 4C DNA contents, respectively). Sorting gates
(black rectangles) identify populations separated for replication timing analysis: G1 (non-replicating nuclei
with 2C DNA content), early S phase (E; replicating nuclei with 2C DNA content), middle S phase (M; replicating
nuclei with DNA content between 2C and 4C), and late S phase (L; replicating nuclei with 4C DNA content). We
do not normally sort G2 nuclei, as the G1 population is sufficient to normalize for copy number and
sequencability. Nuclei in the four gated fractions are sorted into individual tubes and their DNA is sequenced
and analyzed as described in this protocol. (d) Overlaid univariate histograms of relative DNA content
expressed as DAPI pulse height (DAPI-H) showing a reanalysis of nuclei populations from the E (blue peak),
M (green peak), and L (red peak) sorted populations in panel c. The gray histogram shows all nuclei from PG2
for reference. The overlaid E, M, and L histograms demonstrate the relative purity of the sorted populations, as
there is very little overlap between them. The overlap of the E and L peaks with the gray G1 and G2 peaks,
respectively, emphasizes the benefit of using EdU labeling to differentiate replicating nuclei from
non-replicating nuclei, achieving a sort purity nearly impossible if sorting on DNA content alone
34 Leigh Mickelson-Young et al.

the Repliscan application presented in this protocol facilitates the

production of accurate RT profiles without the need for command
line programming. These RT profiles can be compared to other
genomic features (i.e., genes, GC content, nuclease sensitivity, and
transcriptional activity) [26].
The steps needed for successful production and analysis of
quality Repli-seq data for maize are presented below. This protocol
can be easily adapted to other plant systems.

2 Materials

Wear the appropriate personal protective equipment when

handling reagents that pose any hazard and dispose according to
local regulations. All chemicals used are reagent grade. The supplies
and equipment listed in Subheadings 2.1 and 2.2 are items that are
needed for several steps throughout the protocol and are listed here
to reduce repetition. All other supplies and equipment are listed
under each protocol step.

2.1 Supplies 1. 2-mL round-bottom microcentrifuge tubes.

2. 5-mL round-bottom polypropylene tubes.
3. Microscope slides.
4. Qubit dsDNA High Sensitivity Assay Kit and tubes.

2.2 Equipment 1. Refrigerated microcentrifuge.

2. Qubit fluorometer.
3. Refrigerator or cold room.
4. Heated water baths or incubators.
5. Fluorescence microscope.

2.3 Plant Material 1. Zea mays cv B73 seeds. Approximately 450–550 seeds per
and Growing experiment based on an 80% germination rate.
Conditions 2. 150–250 mL germicidal commercial bleach (without additives)
made to a 0.5% sodium hypochlorite solution containing 0.05%
Tween 20.
3. Magnetic stir plate.
4. Fish tank bubbler.
5. 1–2 L glass beaker or a 1 quart round, glass pyrex dish.
6. Magenta boxes.
7. Paper towels.
8. Growth chamber.
 A Protocol for Genome-Wide Analysis of DNA Replication Timing in Intact. . . 35

2.4 In Vivo EdU 1. 40 mM 5-ethynyl-20 -deoxyuridine (EdU) in dimethyl sulfoxide

Pulse-Labeling (DMSO), stored at 80  C.
of Replicating DNA, 2. 25 μM EdU solution in water made from the 40 mM EdU
Dissecting and Fixing stock solution.
Root Tips 3. 1 Phosphate buffered saline (PBS).
4. 1% paraformaldehyde in 1 PBS.
5. 2 M Glycine, filter sterilized.
6. Liquid nitrogen.
7. Large rectangular dish (similar to dimensions 7 in. long by 4 in.
wide by 4 in. deep).
8. #10 scalpel.
9. Fine-tipped forceps (curved and tapered similar to Fisher #12-
000-131).
10. Petri dishes.
11. Small glass beaker (25–50 mL).
12. Kimwipes.
13. Orbital shaker.
14. Desiccator attached to a vacuum pump.

2.5 Isolating Nuclei 1. Cell lysis buffer (CLB): 15 mM Tris–HCl (pH 7.5), 2 mM
Na2EDTA, 80 mM KCl, 20 mM NaCl, 0.1% Triton X-100.
Adjust pH to 7.5 and then add 15 mM 2-mercaptoethanol
(buffer modified from LB01 in [41]).
2. cOmplete™ or cOmplete™ Mini protease inhibitor tablets
(Roche).
3. Miracloth.
4. Small plastic funnel.
5. 50 mL conical tubes.
6. Commercial food processor (like Cuisinart® Mini-Prep® 2.6-
cup food processor model DLC-1SS).
7. Refrigerated swing bucket centrifuge with rotor to hold 50 mL
tubes.

2.6 Clicking EdU 1. Click-iT® EdU Alexa Fluor® 488 Imaging Kit (Life
to Alexa Fluor 488 Technologies).
2. Modified CLB: 15 mM Tris–HCl (pH 7.5), 80 mM KCl,
20 mM NaCl, 0.1% Triton X-100, pH 7.5.
3. 4,6-diamidino-2-phenylindole (DAPI) stock solution: 1 mg/
mL in sterile H2O.
4. RNase A stock.
Another random document with
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 He felt himself held up by the language difficulty. Up to the
present he had had extraordinary luck in this respect, but then up to
the present he had been interviewing educated persons whose
business brought them in contact with foreigners. He doubted if he
could make boatmen and loafers about the quays understand what
he wanted.
A trial convinced him that his fears were well founded, and he lost
a solid hour in finding the Berlitz School and engaging a young
linguist with a reputation for discretion. Then, accompanied by M.
Jules Renard, he returned to the quays and set systematically to
work. He began by inquiring where boats might be hired, and where
there were steps at which ships’ boats might come alongside. Taking
these in turn he asked had the boatmen taken a passenger out to
the L’Escaut between 2:00 and 3:00 p.m. on the previous Thursday?
Or had the loafer, stevedore, shunter, or constable, as the case
might be, noticed if a boat had come ashore from the same vessel
on the same date and at the same time?
Though the work was easy it bade fair to be tedious, and
therefore for more than one reason French felt a glow of satisfaction
when at his fourth inquiry his question received an affirmative
answer. A wizened old man, one of a small knot of longshoremen
whom M. Renard addressed, separated himself from his companions
and came forward. He said that he was a boatman, and that he had
been hailed by a man—an Englishman, he believed—at the time
stated, and had rowed him out to the ship.
“Ask him if that’s the man,” French directed, producing Dangle’s
photograph, though he felt there could be no doubt as to the reply.
He was therefore immensely dashed when the boatman shook
his head. This was not the man at all. The traveler was a short,
rather stout man with a small fair mustache.
French gasped. The description sounded familiar. Taking out
Blessington’s photograph he passed it over.
This time the boatman nodded. Yes, that was the man he had
rowed out. He had no doubt of him whatever.
This was unexpected but most welcome news, though as French
thought over it, he saw that it was not so surprising after all. If
Dangle was in it, why not Blessington, and for the matter of that, why
not Sime also? In this case he wondered where Susan could be, and
more acutely, what had been the fate of Joan Merrill. Possibly, he
thought, his inquiries about Dangle would solve these questions
also.
Half an hour later he struck oil for the second time. Another
boatman, a little further along the quays, had also rowed a
passenger out to the L’Escaut, and this one, it appeared, was
Dangle. But though French kept working steadily away, he could
hear nothing of Sime.
In the end it was a suggestion of Renard’s that put him once
more on the trail. The interpreter proved an intelligent youth, and
when he had grasped the point at issue, he stopped and pointed to
the river.
“You say, monsieur, that the sheep, she lie there, opposite the
Musée Steen, is it not so? Bon! We haf walked along all the quays
near to that. Your friends would not haf hired boat from farther on—it
is too far. You say, too, they come from England secretly, is it not?
Bon! They would come to the other side.”
French did not understand.
“The other side?” he repeated questioningly.
“But yes, monsieur, the other side.” The young fellow’s eyes
flashed in his eagerness. “Over there, La Gare de Waes.” He pointed
out across the great stream to its west bank.
“I didn’t know there was a station across there,” French admitted.
“Where does the line go to?”
“Direct to Ghent. Your friends change trains at Ghent. It is a quiet
railway. They come unseen.”
“Good man,” said French heartily. “We’ll go and find out. How do
you get to the blessed place?”
M. Renard smiled delightedly.
“Ah yes, monsieur. You weesh to cross? Is it not?” he cried. “This
way. We take ferry from the Quai Van Dyck. It is near.”
Half an hour later they had reached the Tête de Flandre—the
low-lying western bank of the Scheldt. It bore a small but not
unpicturesque cluster of old-fashioned houses, nestling about one of
the historic Antwerp forts. Renard, now apparently quite as
interested in the chase as French, led the way along the river bank
from boatman to boatman, with the result that before very many
minutes had passed French had obtained the information he wanted.
It appeared that about 1:00 p.m. on the day in question, a
strapping young boatman had noticed three strangers approaching
from the direction of the Waes Station, a hundred yards or more
distant. They consisted of a tall, clean-shaven man of something
under middle age and two women, both young. One was tall and
strongly made and dark as to hair and eyes, the other was slighter
and with red gold hair. The smaller one seemed to be ill, and was
stumbling along between the other two, each of whom supported her
by an arm. None of the trio could speak French or Flemish, but they
managed by signs to convey the information that they wanted to be
put on board the L’Escaut, which was lying out in midstream. The
man had rowed them out, and they had been received on board by
an elderly gentleman with a dark beard.
Further questions produced the information that the fair lady
appeared to be seriously ill, though whether it was her mind or body
that was affected, the boatman couldn’t be sure. She was able to
walk, but would not do so unless urged on by the others. She had
not spoken or taken any interest in the journey. She had not
appeared even to look round her, but had sat gazing listlessly at
nothing, with a vacant expression in her eyes. Her companions had
had real difficulty in getting her up the short ladder on to the
L’Escaut’s deck.
The news was rather unexpected to French. About Joan Merrill it
was both disconcerting and reassuring; the former because he could
not see that the gang had anything but a sinister reason for
inveigling the young girl aboard the ship—probably she will fall
overboard at night, he thought; the latter because she was at least
still alive, or had been two days ago. It was quite evident that she
was drugged, probably with morphine or something similar. It might,
however, mean that while wishing Joan no harm, they were taking
her with them on their expedition to insure her silence as to their
movements.
As French returned across the ferry, he kept on puzzling as to
Lowenthal’s position. Could Lowenthal be arrested? Was he in
league with the gang? If so, could he be held responsible for the
abduction of Joan Merrill? French didn’t think the evidence would
justify drastic measures. He had, as a matter of fact, no actual
evidence against Lowenthal. Of his complicity he was satisfied, but
he doubted if he could prove it.
He got rid of the young interpreter, and strolling slowly along the
quays, thought the matter out. No, he had not a enough case with
which to go to the Belgian police. But he could do the next best
thing. He could call on M. Lowenthal for the second time, and try to
bluff an admission out of him.
As he walked to the Rue des Tanneurs, he felt his prospects were
not rosy. But at least he had no difficulty in obtaining his interview. M.
Lowenthal seemed surprised to see him so soon again, but received
him politely, and asked what he could do for him.
“I want to ask you another question, M. Lowenthal, if you please,”
French answered in his pleasantest manner, “and first I must tell you
that the agency I hold is that of Detective Inspector at New Scotland
Yard in London. My question is this: When you and M. Merkel
entered into relations with Blessington, Sime, and the Dangles, did
you know that they were dangerous criminals wanted by the English
police?”
In spite of the most evident efforts for self-control, Lowenthal was
so much taken aback that he could not for some moments speak.
His swarthy face turned a greenish hue and little drops of sweat
showed on his forehead. To the other pleasant characteristics with
which French had mentally endowed him, he now added that of
coward, and his hopes of his bluff succeeding grew brighter. He sat
waiting in silence for the other to recover himself, then said suavely:
“After that, M. Lowenthal, you will see for yourself that you cannot
plead ignorance of the affair. Let me advise you for your own sake to
be open with me.”
The man pulled himself together. He wiped his brow as he replied
earnestly, but in somewhat shaky accents:
“That I haf met Blessington, Sime, and Dangle I do not deny,
though they were Merkel’s friends—not mine. But I do not know that
they are criminal. Dangle, he called here and asked Merkel to take
him on the next”—he hesitated for a word—“next work, next sail of
the sheep. Merkel said that Dangle iss a writer—he writes books. He
weeshed to see the sail to Casablanca to deescribe it in hiss book.
Merkel said he would haf to pay fare, the firm could not afford it
unless. Dangle agreed. Merkel was going himself, and Dangle
suggested Sime and Blessington go also to make party—to play
cards. Of a second Dangle I know nothing. They went secretly—I
admit it—because the law forbids to take passengers for sail without
a certificate. That is all of the affair.”
Not a single word of this statement did French believe, but he
saw that unless he could get some further information, or surprise
this Lowenthal into some more damaging admission, he could not
have him arrested. After all, the story hung together. Merkel might
conceivably be playing his own game, and have pitched the yarn of
the author out for copy to his partner. The contravention of the
shipping laws would undoubtedly account for the secrecy with which
the start was made. Certainly there was no evidence to bring before
a jury.
French proceeded to question the junior partner with
considerable thoroughness, but he could not shake his statement.
The only additional facts he learned were that the L’Escaut was
going to Casablanca on the order of the Moroccan Government to
load up a cargo of agricultural samples for the Italian market, and
that M. Merkel was accompanying it simply as a holiday trip.
With this French had to be content, and he went to the post
office, and got through on the long distance telephone to his chief at
the Yard. To him he repeated the essentials of the tale, asking him to
inquire from the Moroccan authorities as to the truth of their portion
of it, as well as to endeavor to trace the L’Escaut.
On leaving the post office, it occurred to him that communication
with the L’Escaut should be possible by wireless, and he returned to
the Rue des Tanneurs to ascertain this point. There he was told that
just after he had left M. Lowenthal had received a telephone call,
requiring his immediate presence in Holland, and he had with a great
rush caught the afternoon express for the Dutch capital.
“Skedaddled, by Jove!” said French to himself. “Guess that lets in
the Belgian police.”
He called at headquarters, and saw the officer in charge, and
before he left to catch the connection for London, it had been
arranged that the movements of the junior partner should be gone
into, and a watch kept for the return of that enterprising weaver of
fairy tales.
 Chapter XVIII.
A Visitor from India
When French reached Victoria, the first person he saw on the
platform was Maxwell Cheyne.
“They told me at the Yard that you might be on this train,” the
young man said excitedly as he elbowed his way forward. “Any
news? Anything about Miss Merrill?”
He looked old and worn, and it was evident that his anxiety was
telling on him. In his eagerness he could scarcely wait for the
Inspector to dismount from his carriage, and his loud tones were
attracting curious looks from the bystanders.
“Get a taxi,” French answered quietly. “We can talk there.”
A few seconds later they found a vehicle, and Cheyne, gripping
the other by the arm, went on earnestly:
“Tell me. I can see you have learned something. Is she—all
right?”
“I got news of her on Thursday last. She was all right then,
though still under the influence of a drug. The whole party has gone
to sea.”
“To sea?”
“Yes, to sea in a small tramp. I don’t know what they are up to,
but there is no reason to suppose Miss Merrill is otherwise than well.
Probably they took her with them to prevent her giving them away.
They would drug her to get her to go along, but would cease it as
soon as she was on board. I wired for inquiries to be made at the
different signal stations, and news may be waiting for us at the Yard.”
A few seconds sufficed to put Cheyne in possession of the salient
facts which French had learned, and the latter in his turn asked for
news.
 “By Jove, yes!” Cheyne cried, “there is news. You remember that
Arnold Price had disappeared? Well, yesterday I had a letter from
him!”
“You don’t say so?” French rejoined in surprise. “Where did he
write from?”
“Bombay. He was shortly leaving for home. He expects to be
here in about a month.”
“And what about his disappearance?”
“He was ill in hospital. He had gone up to Agra on some private
business and met with an accident—was knocked down in the street
and was insensible for ages. He couldn’t say who he was, and the
hospital people in Agra couldn’t find out, and he hadn’t told the
Bombay people where he was going to spend his leave.”
“Did he mention the letter?”
“Yes, he thanked me for taking charge of it and said that when he
reached home he would relieve me of further trouble about it. He
little knows!”
“That’s so,” French assented.
Their taxi had been held up by a block at the end of Westminster
Bridge, but now the mass cleared and in a few seconds they
reached the Yard.
French’s first care was to get rid of Cheyne. He repeated what he
had learned about Joan Merrill, then, assuring him that the key of the
matter lay in the cipher, he advised him to go home and try it once
more. Directly any more news came in he would let him know.
Cheyne having reluctantly taken his departure, French made
inquiries as to what had been done in reference to his telephone
from Antwerp. It appeared that the Yard had not been idle. In the first
place an application had been made to the Moroccan Government,
who had replied that no ship had been chartered by them for freight
at Casablanca, nor was anything known of agricultural samples for
the Italian market. Lowenthal’s story must therefore have been an
absolute fabrication. He had, however, told it so readily that French
suspected it had been made up beforehand, so as to be ready to
serve up to any inquisitive policeman or detective who might come
along.
 Next Lloyd’s had been approached, as to the direction the
L’Escaut had taken, and a reply had shortly before come in from
them. It stated that up to noon on that day, the vessel had not been
reported from any of their stations. But this, French realized, might
not mean so much. If she had gone south down the English Channel
it would have been well on to dark before she reached the Straits of
Dover. In any case, had she wished to slip through unseen, she had
only to keep out to the middle of the passage, when in ordinary
weather she would have been invisible from either coast. On the
other hand, had she gone north, she would almost naturally have
kept out of sight of land. It was true that in either case she would
have been likely to pass some other vessel which would have
spoken her, and the fact that no news of such a recognition had
come to hand seemed to indicate that she was taking some unusual
course out of the track of regular shipping.
French wired this information to the Antwerp police, and then, his
chief being disengaged, went in and gave him a detailed account of
his adventures in Belgium.
Chief Inspector Mitchell was impressed by the story. He sat back
in his chair and treated French to a prolonged stare as the latter
talked. At the end of the recital he remained sitting motionless for
some moments, whistling gently below his breath.
“Any theories?” he said at last.
French shook his head.
“Well, no, sir,” he answered slowly. “It’s not easy to see what
they’re after. And it’s not easy to see, either, why the whole gang
wanted to go. It looked at first as if they were just clearing out
because of Cheyne’s coming to the Yard, but it’s more than that. The
arrangements were made too long ago. They have been dealing with
that Antwerp firm for several weeks.”
“The hard copper was all a story?”
“Looks like it, sir. As a matter of fact every single statement those
men made that could be tested has been proved false. Even when
there didn’t seem any great object in a yarn they pitched it. Lies
seemed to come easier to them.”
“Well, I’ve known a good few cases of that, and so have you,
French. It’s a habit that grows. Now, what’s your next move?”
 French hesitated.
“For the moment the outlook’s not very cheery,” he said at last.
“All the same I can’t believe that boat can go away out of the Scheldt
and disappear. In my judgment she’s bound to be reported before
long, and I’m looking forward to getting word of her within the next
day or so. Then I have no doubt that the tracing is some kind of
cipher, and if we could read it we should probably get light on the
whole affair.”
“Why shouldn’t you read it? Try it again.”
“I intend to, sir. But I don’t hope for much result, because I don’t
believe we’ve got the genuine document. I don’t believe they would
have handed it, nor a copy of it either, to a man they intended to
murder, lest it should be found on his body. I’d state long odds they
gave him a fake.”
“I think you’re probably right,” the chief admitted. “Try at all
events. You never know your luck.”
He bent over his desk, and French, realizing that the interview
had come to an end, quietly left the room. Then, seeing there was
nothing requiring his attention urgently, and tired after his journey, he
went home.
But contrary to his expectations, the next day passed without any
news of the L’Escaut, and the next, and many days after that. Nor
could all his efforts with the tracing throw any light on that mysterious
document. As time passed he began to grow more and more
despondent, and the fear that he was going to make a mess of the
case grew steadily stronger. In vain he laid his difficulties before his
wife. For once that final source of inspiration failed him. Mrs. French
did not take even one illuminating notion. When the third week had
gone by, something akin to despair seized upon the Inspector. The
only possibility of hope now seemed to lie in the return of Arnold
Price, and French began counting the days until his arrival.
One night about three weeks after his return from Belgium he
settled down with a cigar after dinner, his thoughts running in their
familiar groove: What were these people engaged on? Was there
any way in which he could find out? Had he overlooked any
evidence or any inquiries? Had he neglected any possible line of
research?
 The more he considered the affair in all its bearings, the more
conscious he became of the soundness of the advice he had given
to Cheyne, and which in his turn he had received from his chief.
Unquestionably in the tracing lay the solution he required, and once
again he racked his brains to see if he could not by any means
devise a way to read its message.
On this point he concentrated, going over and over again
everything he had learned about it. For perhaps an hour he
remained motionless in his chair, while the smoke from his cigar
curled up and slowly dissolved into the blue haze with which the
room was becoming obscured. And then suddenly he sat up and
with a dawning, tremulous eagerness considered an idea which had
just leaped into his mind.
He had suddenly remembered a statement made by Cheyne
when he was giving his first rather incoherent account of his
adventures. The young man said that it had been arranged between
himself and Joan Merrill that if either were lucky enough to get the
tracing into his or her possession, the first thing he or she would do
would be to photograph it. Now, in juxtaposition with that statement,
French recalled the facts, first, that Joan must have reached her flat
on the night of her abduction at least several minutes before
Blessington and Sime arrived with their car; and secondly, that
during those minutes she had the tracing with her—the genuine
tracing, as there was every reason to believe. Had Joan
photographed it?
French was overwhelmed with amazement and chagrin at his
failure to think of this point before, nor could he acquit Cheyne of a
like astounding stupidity. For himself he felt there was no excuse
whatever. He had even specially noticed the girl’s camera and the
flashlight apparatus which she used for her architectural details
when he was searching her rooms, but he had then, and since then
up till this moment, entirely and completely forgotten the
arrangement made between the partners.
Late as it was, French decided to go then and there to ascertain
the point. The key of Joan’s flat was at the Yard, and twenty minutes
later he had obtained it and was in a taxi bowling towards Horne
Terrace.
 He kept the vehicle while he ran up the ten flights to No. 12 and
secured the camera. Then hastening down, he was driven back to
the Yard.
By a piece of good luck he found a photographer who had been
delayed by other important work, and him he pressed into the
service forthwith. With some grumbling the man returned to his dark
room. French, too eager to await his report, accompanying him.
A few moments sufficed to settle the question. The camera
contained a roll of films of which the first seven had been exposed,
and a short immersion in the developer showed that numbers 5, 6,
and 7 bore the hoped for impress.
Gone was French’s despondency and the weariness caused by
his heavy day, and instead he was once more the embodiment of
enthusiasm and cheery optimism. He had it now! At last the secret
was within his grasp! Of his ability to read the message, now that he
was sure he had the genuine one, he had no doubt. He had always
liked working out ciphers, and since he had succeeded in extracting
the hidden meaning from the stock and share list which had been
sent to the elusive Mrs. X in the Gething murder case, his belief in
his own powers had become almost an obsession. He could hardly
restrain his eagerness to get to grips with this new problem until the
negatives should be dry and prints made.
The photographer was able to promise these for the following
day, and till then French had to possess his soul in patience. But on
his return from lunch he found on his desk three excellent prints of
the document.
They were only half-plate size, or about one-third that of the
tracing which had been given to Cheyne. He therefore instructed the
photographer to prepare enlargements which would bring the
document up to more nearly the size of the original. These were
ready before it was time for him to leave for home, and he sat down
with ill-controlled excitement to compare them with the document at
which he had already spent so much time.
And then he suddenly experienced one of the most bitter
disappointments of his life. To all intents and purposes the two were
the same! There were the same circles, the same numbers, letters,
and signs enclosed therein, the same phrase, “England expects
every man to do his duty,” spaced round in the same way! The
tracing had not been very accurately done, as some of the circles
seemed slightly out of place, but the discrepancies were trifling, and
seemed obviously due to careless copying. He gave vent to a single
bitter oath, then sat motionless, wrapped in the most profound
gloom.
He took tracing and photographs home with him, and spent the
greater part of the evening making a minute comparison between the
two. The enlargement unfortunately was not exactly the same size
as the tracing, and he therefore began his work by covering the
surfaces of both with proportionate squares.
Taking the tracing first he drew parallel lines one inch apart both
up and down it and across, thus covering its whole surface with inch
squares. Then he divided the prints into the same number of equal
parts both vertically and horizontally and ruled them up in squares
also. These squares were slightly smaller than the others—about
seven-eighths of an inch only—but relatively the lines fell on each in
the same positions. A comparison according to the squares thus
showed at a glance similarity or otherwise between the two
documents.
As he examined them in detail certain interesting facts began to
emerge. The general appearance, the words “England expects every
man to do his duty,” and the circles with their attendant letters and
numbers were identical on both sheets. But there were striking
variations. The position of certain of the circles was different. Those
containing numbers and crooked lines were all slightly out of place,
while those containing letters remained unmoved. Moreover, the little
crooked lines, while preserving a rough resemblance to the originals,
were altered in shape. The more he considered the matter the more
evident it became to French that these divergences were intentional.
The tracing which had been given to Cheyne was intended to
resemble the other superficially—and did so resemble it, but it had
clearly been faked to make it valueless.
 If French were right so far, and he had but little doubt of it, it
followed that the essential feature of the circles and crooked lines
was position. This, he felt, should be a useful hint, but as yet he
could not see where it led.
He pondered fruitlessly over the problem till the small hours, and
next morning he took the documents back to the Yard to continue his
studies. But he did not have an opportunity to do so. Other work was
waiting for him. To his delight he found that Arnold Price had reached
home, and that he and Cheyne were waiting to see him.
Price proved to be a lanky and rather despondent-looking
individual with a skin burned to the color of copper and a pair of
exceedingly shrewd blue eyes. He dropped into the chair French
indicated, and instantly pulled out and lit a well-blackened cutty pipe.
“Got in yesterday morning,” he announced laconically, “and wired
Torquay I was going down. By the merest luck I got a reply before I
started that Cheyne was in town. I looked him up and here I am.”
French smiled pleasantly. Though interested in the man, he could
not help noting with some amusement at once the restraint and the
completeness of his statement. How refreshing, he thought, and how
rare, to meet some one who will give you the pith of a story without
frills!
“I’m glad to meet you, Mr. Price,” he said cordially. “I suppose Mr.
Cheyne has told you the effect that your letter has had on us all?”
The other nodded.
“Not altogether surprising,” he declared. “There’s money in the
thing—or so I always believed, and this other crowd must believe it
too; though how they got on to the affair licks me.”
“We shall be very much interested to hear what you can tell us
about it,” French prompted. “Will you smoke, Mr. Cheyne?” He held
out his cigar case.
“I can’t tell you much,” Price returned, “and nothing that will clear
up this blessed mystery that seems to have started up. But this is my
story for what it’s worth. Before the war I was on one of the Hudson
and Spence boats and I had the luck to get into the R.N.R. when
hostilities broke out. I stayed on in my old ship till she was torpedoed
a couple of years later, then I was appointed third officer on the
Maurania. We were on a trip from South Africa to Brest with army
stores, when one day, just as we came into the English Channel, we
were attacked by a U-boat. We had an 18-pounder forward, and by a
stroke of luck we gave old Fritz one on the knob that did him in. The
boat went down and a dozen of the crew were left swimming. We put
out a boat and picked one or two of them up. The skipper was
clinging on to a lifebelt, but just as we came up he let go and began
to sink. I was in charge of the boat, and some fool notion came over
me—I think in the hurry I forgot he was a U-boat skipper—but
anyhow like a fool I got overboard and got hold of him. It was nothing
like a dramatic rescue—there was no danger to me—and we were
back on board inside fifteen minutes.”
French and Cheyne were listening intently to this familiar story.
So far it was almost word for word that told by Dangle. Apparently,
then, there was at least one point on which the latter had told the
truth.
“We weren’t out of trouble,” Price resumed, “and next day we
came up against another submarine. We exchanged a few shots and
then a British destroyer came up and drove him off. But I had the
luck to stop a splinter of shell, and when we got to Brest I was sent
to hospital. The U-boat skipper had got a crack on the head when his
boat went down, and he was sent in too. By a chance we got side by
side beds in the same ward, and used to talk a bit, though he was a
rotter, even for a Boche.”
Price paused to draw on his cutty pipe, expelling great clouds of
smoke of a peculiarly acrid and penetrating quality. Then, the others
not speaking, he went on:
“It turned out that the wound on Schulz’s head—his name was
Schulz—was serious, and he grew steadily worse. Then one night
when the ward was quiet, he woke me and said he knew his number
was up and that he had a secret to tell me. We listened, but all the
other fellows seemed asleep, and then he told me he could put me in
the way of a fortune—that he had hoped to get it himself after the
war, but now that it would be a job for someone else. He said he
would tell me the whole thing, and that I might make what I could out
of it, if only I would pledge myself to give one-eighth of what I got to
his wife. He gave me the address—somewhere in Breslau. He asked
me to swear this and I did, and then he took a packet from under his
pillow and handed it to me. ‘There,’ he said, ‘the whole thing’s there.
I put it in cipher for safety, but I’ll tell you how to read it.’ Well, he
began to do so, but just then a sister came in, and he shut up till she
would leave. But the excitement of talking about the thing must have
been too much for him. He got a weak turn and never spoke again.”
“But,” Cheyne interposed, “what about the hard copper? Dangle
told us about Schulz’s discovery.”
Price gazed at him vacantly for some moments and then
suddenly smote the table.
“I’ve got it!” he cried with an oath. “Dangle! I remember that chap
now! He was in the next bed on the other side of Schulz. That’s right!
I couldn’t call him to mind when you mentioned him before. Of
course! He heard the whole tale, and that’s what started him on this
do.”
“I know,” Cheyne returned. “He admitted that all right. But he told
us about the hard copper. You haven’t mentioned that.”
Price shook his head.
“Don’t know what you’re talking about,” he declared. “What do
you mean by hard copper?”
“Dangle mentioned it. He was listening to the conversation. He
told us all that about Schulz’s story of the fortune, and about his wife
and all that, just as you have, but he said Schulz went on to explain
what the fortune was: that he had hit on a way of treating copper that
made it as hard as steel. The cipher contained the formula.”
Again Price shook his head.
“All spoof,” he observed. “Not a word of truth in it. Schulz never
mentioned copper or said anything more than I’ve told you.”
French spoke for the first time.
“We found this Dangle a man of imagination, all through, and it is
easy to see why he invented that particular yarn. By that time he had
undoubtedly read the cipher, and he wanted something to mislead
Mr. Cheyne as to its contents. The story of the hard copper would
start a bias in Mr. Cheyne’s mind which would tend to keep him off
the real scent.” He paused, but his companions not speaking,
continued: “Now we have that bias cleared away, at least one
interesting fact emerges. The whole business starts with the sea—
the U-boat commander, Schulz, and it looks as if it was going to end
up with the sea, the tramp, the L’Escaut.”
 As French said these words an idea flashed into his mind, and he
went on deliberately, but with growing excitement:
“And when we connect the idea of a U-boat commander giving a
message which ends with a sea expedition, with the fact, which I
have just discovered, that the essence of his cipher is the position of
the markings on it, we seem to be getting somewhere.”
Price smote his thigh.
“By Jemima!” he cried. “I’ve got you. That blessed tracing is a
map!”
“A map, yes. That’s what I think,” French answered eagerly, and
then as suddenly he saw the possible significance of Nelson’s
exhortation, he went on dramatically: “A map of England!”
Cheyne swore softly.
“My word, if we aren’t a set of blithering idiots!” he exclaimed. “Of
course! ‘England’ is the title. That’s as clear as day! The other words
are added as a blind. Let’s have the thing out, Inspector, and see if
we can’t make something of it now.”
As French produced his enlarged photographs not one of the
three men doubted that they were at last well on the way towards
wresting the secret from the document which had so long baffled
them.
 Chapter XIX.
The Message of the Tracing
Inspector French spread the photograph on his desk, and
Cheyne and Price having drawn up chairs, all three gazed at it as if
expecting that in the light of their great idea its message would have
become obvious.
But in this they were disappointed. The suggestion did not seem
in any way to help either French or Cheyne, and Price, who of
course had not seen the document before, was satisfactorily
mystified. Granted that the thing was a map, granted even that it was
a map of England, its meaning remained just as provokingly hidden
as ever.
Presently Price gave vent to an exclamation. “Hang it all!” he
cried irritably, and then: “I suppose those numbers couldn’t be
soundings? Could they give depths at the circles?”
“That’s an idea,” Cheyne cried, but French shook his head.
“I think there’s more in it than that,” he observed. “If you examine
those numbers you’ll find that they’re consecutive, they run from one
to thirty-six. Soundings wouldn’t lend themselves to such an
arrangement. You may be right, Mr. Price, and we must keep your
idea in view, but I don’t see it working out for the moment.”
Silence reigned for a few moments, then Price sat back from the
table and spoke again.
“Look here, Inspector,” he said, knocking the ashes out of his
pipe and beginning to fill it with his strong, black mixture, “you said
something just now I didn’t quite follow. Let’s get your notion clear.
You talked of this thing beginning with the sea—at Schulz, and
ending with the sea—at L’Escaut, and Schulz’s message being a
map. Just what was in your mind?”
 “Only the obvious suggestion that if you leave a message which
provokes an expedition, you must also convey in your message the
destination of that expedition, and a map seems the simplest way of
doing it. But on second thoughts I question my first conclusion.
There must be an explanation of the secret as well as a direction of
how to profit by it, and it would seem to me doubtful that such an
explanation could be covered by a map.”
“Sounds all right, that,” Price admitted. “Have you any idea what
the secret might be? Sounds like treasure or salvage or something of
that kind.”
“I scarcely think salvage,” French answered. “The L’Escaut is not
a salvage boat, and a boat not specially fitted for the purpose would
be of little use. But I thought of treasure all right. This Schulz might
have robbed his ships—there would always be money aboard, and
even during the war many women traveled with jewelry. The man
might easily have made a cache of valuables somewhere round the
coast.”
“Easily,” Cheyne intervened, “or he might have learned of some
valuable deposit in some out of the way cove round the coast, like
those chaps in that clinking tale of Maurice Drake’s, WO₂.”
“As at Terneuzen?” said French. “I read that book—one of the
best I ever came across. It’s a possibility, of course.”
The talk here became somewhat rambling, Price not having read
WO₂ and wanting to know what it was about, but French soon
reverted to his photograph. He reminded his hearers that they were
all interested in its elucidation. Miss Merrill’s safety, his own
professional credit, Cheyne’s peace of mind, and Price’s fortune, all
were at stake.
“We have,” he went on, “evolved the idea that perhaps this
tracing may be a map of England. On further thought that suggestion
does not seem promising, but as we have no other let us work on it.
Assume it is a map of England, and let us see if it leads us
anywhere.” There were murmurs of assent from his hearers, and he
continued: “Now it seems to me the first thing to do is to try if we can
fit these circles and lines into the map of England. Is there anything
corresponding to them in English geography?”