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Methods in
Molecular Biology 2686

José Luis Riechmann

Cristina Ferrándiz Editors

Flower
Development
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:

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

Methods and Protocols

Second Edition

Edited by

José Luis Riechmann

Centre for Research in Agricultural Genomics CSIC-IRTA-UAB-UB, Barcelona, Spain; Institució Catalana de
Recerca i Estudis Avancats (ICREA), Barcelona, Spain

Cristina Ferrándiz
Instituto de Biología Molecular y Celular de Plantas CSIC-UPV, Valencia, Spain
Editors
José Luis Riechmann Cristina Ferrándiz
Centre for Research in Agricultural Instituto de Biologı́a Molecular y Celular de Plantas
Genomics CSIC-IRTA-UAB-UB CSIC-UPV
Barcelona, Spain Valencia, Spain
Institució Catalana de Recerca i Estudis Avancats (ICREA)
Barcelona, Spain

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-3298-7 ISBN 978-1-0716-3299-4 (eBook)
https://doi.org/10.1007/978-1-0716-3299-4

© Springer Science+Business Media, LLC, part of Springer Nature 2023

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Preface

Over the past 35 years, detailed insights into the genetic and molecular mechanisms that
control flower development in different angiosperm species have been obtained, making
great progress in the identification of key regulators of flower morphogenesis, in elucidating
their organization in pathways and networks, and in characterizing the developmental
process at multiple levels, from organismal to single cells. These advances have relied on
the continuous development of methodologies and techniques, from molecular genetics to
advanced microscopy or single-cell biology, and their implementation into plant research.
To facilitate further progress in the field of flower development, this book provides a
collection of protocols for many of the experimental approaches that are currently used to
study the formation of flowers, from genetic methods and phenotypic analyses, to genome-
wide experiments, modeling, and system-wide approaches. An effort has been made to
facilitate the incorporation of non-model species that can be useful to study specific devel-
opmental processes or the origin of evolutionary innovations. In addition, several introduc-
tory chapters provide an overview of the current status on the field of flower development,
also highlighting open questions and future directions. Methods chapters are organized in
three major sections: genetic and phenotypic analyses; experimental systems; and molecular
biology, genomics, and systems biology. Each chapter contains a brief introduction, step-by-
step methods, a list of necessary materials, and a Notes section with tips on troubleshooting,
as well as extensive reference lists. Comprehensive and up to date, we hope that this book on
flower development will become an essential guide for plant developmental biologists, from
the more novice to the experienced researcher, and for those considering venturing into the
field.

Barcelona, Spain José Luis Riechmann

Valencia, Spain Cristina Ferrándiz

v
Contents

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

PART I REVIEW AND OVERVIEW CHAPTERS

1 Flower Development in Arabidopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Hicham Chahtane, Xuelei Lai, Gabrielle Tichtinsky, Philippe Rieu,
Moı̈ra Arnoux-Courseaux, Coralie Cancé, Claudius Marondedze,
and François Parcy
2 Flower Development in the Solanaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Marie Monniaux and Michiel Vandenbussche
3 The ABC of Flower Development in Monocots: The Model of Rice
Spikelet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Ludovico Dreni
4 Model Species to Investigate the Origin of Flowers . . . . . . . . . . . . . . . . . . . . . . . . . 83
Charles P. Scutt
5 Hormones and Flower Development in Arabidopsis. . . . . . . . . . . . . . . . . . . . . . . . . 111
Victor M. Zúñiga-Mayo, Yolanda Durán-Medina,
Nayelli Marsch-Martı́nez, and Stefan de Folter

PART II GENETIC AND PHENOTYPIC ANALYSES

6 Genetic Screens for Floral Mutants in Arabidopsis thaliana: Enhancers

and Suppressors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Zhigang Huang, Thanh Theresa Dinh, Elizabeth Luscher,
Shaofang Li, Xigang Liu, So Youn Won, and Xuemei Chen
7 Genetic and Phenotypic Analysis of Shoot Apical and Floral Meristem
Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Mona M. Monfared, Thai Q. Dao, and Jennifer C. Fletcher
8 Cell Biological Analyses of Anther Morphogenesis and Pollen Viability
in Arabidopsis and Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Fang Chang, Shuangshuang Wang, Zesen Lai, Zaibao Zhang,
Yue Jin, and Hong Ma
9 Isolation of Meiocytes and Cytological Analyses of Male Meiotic
Chromosomes in Soybean, Lettuce, and Maize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Cong Wang, Xiang Li, Jiyue Huang, Hong Ma,
Chung-Ju Rachel Wang, and Yingxiang Wang
10 Genetic and Phenotypic Analyses of Carpel Development
in Arabidopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Vicente Balanzà, Patricia Ballester, Monica Colombo, Chloé Fourquin,
Irene Martı́nez-Fernández, Clara I. Ortiz-Ramı́rez,
and Cristina Ferrándiz

vii
viii Contents

11 Genetic and Phenotypic Analysis of Ovule Development

in Arabidopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Dayton C. Bird, Chao Ma, Sara Pinto, Weng Herng Leong,
and Matthew R. Tucker

PART III EXPERIMENTAL SYSTEMS

12 Floral Induction Systems for the Study of Arabidopsis Flower

Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Diarmuid Ó’Maoiléidigh, Bennett Thomson, and Frank Wellmer
13 Protoplasting and Fluorescence-Activated Cell Sorting of the Shoot
Apical Meristem Cell Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
G. Venugopala Reddy
14 Protoplast Isolation for Plant Single-Cell RNA-seq . . . . . . . . . . . . . . . . . . . . . . . . . 301
Shulin Ren and Ying Wang
15 Plant Nuclei Isolation for Single-Nucleus RNA Sequencing . . . . . . . . . . . . . . . . . . 307
Xu Xin, Fei Du, and Yuling Jiao
16 Isolation of Nuclei Tagged in Specific Cell Types (INTACT)
in Arabidopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Ruben M. Benstein, Markus Schmid, and Yuan You

PART IV MOLECULAR BIOLOGY, GENOMICS, AND SYSTEMS BIOLOGY

17 RNA In Situ Hybridization on Plant Tissue Sections: Expression

Analysis at Cellular Resolution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Vladislav Gramma and Vanessa Wahl
18 The GUS Reporter System in Flower Development Studies . . . . . . . . . . . . . . . . . . 351
Janaki S. Mudunkothge, C. Nathan Hancock, and Beth A. Krizek
19 Expression and Functional Studies of Leaf, Floral, and Fruit
Developmental Genes in Non-model Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
Natalia Pab
on-Mora, Harold Suárez-Baron, Yesenia Madrigal,
Juan F. Alzate, and Favio González
20 Gene Expression Analysis by Quantitative Real-Time PCR for Floral
Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
Raquel Álvarez-Urdiola, Mariana Bustamante, Joana Ribes,
and José Luis Riechmann
21 Misexpression Approaches for the Manipulation of Flower Development . . . . . . 429
Yifeng Xu, Eng-Seng Gan, and Toshiro Ito
22 Genomic Approaches for the Study of Flower Development
in Floriculture Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
Tomas Hasing and Aureliano Bombarely
23 Multi-Omics Methods Applied to Flower Development . . . . . . . . . . . . . . . . . . . . . 495
Raquel Álvarez-Urdiola, José Tomás Matus, and José Luis Riechmann
24 Peptidomics Methods Applied to the Study of Flower Development . . . . . . . . . . 509
Raquel Álvarez-Urdiola, Eva Borràs, Federico Valverde, José Tomás Matus,
Eduard Sabid
o , and José Luis Riechmann
 Contents ix

25 Quantifying Gene Expression Domains in Plant Shoot Apical

Meristems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537
Pau Formosa-Jordan and Benoit Landrein
26 A NanoLuc-Based Transactivation Assay in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . 553
Rosa Esmeralda Becerra-Garcı́a, José Erik Cruz-Valderrama,
Vincent E. Cerbantez-Bueno, Nayelli Marsch-Martı́nez,
and Stefan de Folter
27 A Hands-On Guide to Generate Spatial Gene Expression Profiles
by Integrating scRNA-seq and 3D-Reconstructed Microscope-Based
Plant Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567
Manuel Neumann and Jose M. Muino

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
Contributors

RAQUEL ÁLVAREZ-URDIOLA • Centre for Research in Agricultural Genomics (CRAG) CSIC-

IRTA-UAB-UB, Edifici CRAG, Campus UAB, Cerdanyola del Vallès, Barcelona, Spain
JUAN F. ALZATE • Centro Nacional de Secuenciaci
on Gen
omica–CNSG, Sede de
Investigaci
on Universitaria–SIU, Medellı́n, Antioquia, Colombia
MOÏRA ARNOUX-COURSEAUX • CNRS, Université Grenoble Alpes, CEA, INRAE, IRIG,
BIG-LPCV, Grenoble, France
VICENTE BALANZÀ • Instituto de Biologı́a Molecular y Celular de Plantas CSIC-UPV,
Campus de la Universidad Politécnica de Valencia, Valencia, Spain
PATRICIA BALLESTER • Instituto de Biologı́a Molecular y Celular de Plantas CSIC-UPV,
Campus de la Universidad Politécnica de Valencia, Valencia, Spain
ROSA ESMERALDA BECERRA-GARCÍA • Unidad de Gen
omica Avanzada (UGA-LANGEBIO),
Centro de Investigaci
on y de Estudios Avanzados del Instituto Politécnico Nacional
(CINVESTAV-IPN), Irapuato, Guanajuato, Mexico; Departamento de Biotecnologı́a y
Bioquı́mica, Unidad Irapuato, CINVESTAV-IPN, Irapuato, Guanajuato, Mexico
RUBEN M. BENSTEIN • Umeå Plant Science Centre, Department of Plant Physiology, Umeå
University, Umeå, Sweden
DAYTON C. BIRD • School of Agriculture, Food and Wine, University of Adelaide, Waite
Campus, Urrbrae, SA, Australia
AURELIANO BOMBARELY • Instituto de Biologı́a Molecular y Celular de Plantas (IBMCP)
(UPV-CSIC), Valencia, Spain
EVA BORRÀS • Centre for Genomic Regulation (CRG), Barcelona Institute of Science and
Technology, Barcelona, Spain; Universitat Pompeu Fabra, Barcelona, Spain
MARIANA BUSTAMANTE • Centre for Research in Agricultural Genomics (CRAG) CSIC-
IRTA-UAB-UB, Edifici CRAG, Campus UAB, Cerdanyola del Vallès, Barcelona, Spain;
Roche Pharma Research and Early Development, Roche Innovation Center Basel,
F. Hoffmann-La Roche Ltd., Basel, Switzerland
CORALIE CANCÉ • CNRS, Université Grenoble Alpes, CEA, INRAE, IRIG, BIG-LPCV,
Grenoble, France
VINCENT E. CERBANTEZ-BUENO • Unidad de Gen
omica Avanzada (UGA-LANGEBIO),
Centro de Investigaci
on y de Estudios Avanzados del Instituto Politécnico Nacional
(CINVESTAV-IPN), Irapuato, Guanajuato, Mexico
HICHAM CHAHTANE • CNRS, Université Grenoble Alpes, CEA, INRAE, IRIG, BIG-LPCV,
Grenoble, France; Institut de Recherche Pierre Fabre, Green Mission Pierre Fabre,
Conservatoire Botanique Pierre Fabre, Soual, France
FANG CHANG • State Key Laboratory of Genetic Engineering, Ministry of Education Key
Laboratory of Biodiversity Sciences and Ecological Engineering and Institute of Biodiversity
Sciences, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai,
China
XUEMEI CHEN • Department of Botany and Plant Sciences, University of California,
Riverside, CA, USA
MONICA COLOMBO • Instituto de Biologı́a Molecular y Celular de Plantas CSIC-UPV,
Campus de la Universidad Politécnica de Valencia, Valencia, Spain; CREA Research
Centre for Genomics and Bioinformatics, Fiorenzuola d’Arda, Italy

xi
xii Contributors

JOSÉ ERIK CRUZ-VALDERRAMA • Unidad de Gen
omica Avanzada (UGA-LANGEBIO),

Centro de Investigaci
on y de Estudios Avanzados del Instituto Politécnico Nacional
(CINVESTAV-IPN), Irapuato, Guanajuato, Mexico
THAI Q. DAO • Plant Gene Expression Center, USDA-ARS/UC Berkeley, Albany, CA, USA;
Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA;
Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, USA
STEFAN DE FOLTER • Unidad de Gen
omica Avanzada (UGA-LANGEBIO), Centro de
Investigaci
on y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-
IPN), Irapuato, Guanajuato, Mexico
THANH THERESA DINH • Department of Botany and Plant Sciences, University of California,
Riverside, CA, USA
LUDOVICO DRENI • Instituto de Biologı́a Molecular y Celular de Plantas (IBMCP), Consejo
Superior de Investigaciones Cientı́ficas-Universidad Politécnica de Valencia, Valencia,
Spain
FEI DU • State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental
Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing,
China
YOLANDA DURÁN-MEDINA • Departamento de Biotecnologı́a y Bioquı́mica, Centro de
Investigaci
on y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-
IPN), Irapuato, Guanajuato, Mexico
CRISTINA FERRÁNDIZ • Instituto de Biologı́a Molecular y Celular de Plantas CSIC-UPV,
Campus de la Universidad Politécnica de Valencia, Valencia, Spain
JENNIFER C. FLETCHER • Plant Gene Expression Center, USDA-ARS/UC Berkeley, Albany,
CA, USA; Department of Plant and Microbial Biology, University of California, Berkeley,
CA, USA
PAU FORMOSA-JORDAN • Sainsbury Laboratory, University of Cambridge, Cambridge, UK;
Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding
Research, Cologne, Germany; Cluster of Excellence on Plant Science (CEPLAS), Max
Planck Institute for Plant Breeding Research, Cologne, Germany
CHLOÉ FOURQUIN • Instituto de Biologı́a Molecular y Celular de Plantas CSIC-UPV,
Campus de la Universidad Politécnica de Valencia, Valencia, Spain
ENG-SENG GAN • Republic Polytechnic, School of Applied Science (SAS), Singapore, Singapore
FAVIO GONZÁLEZ • Instituto de Ciencias Naturales, Universidad Nacional de Colombia,
Sede Bogotá, Bogotá, Colombia
VLADISLAV GRAMMA • Max Planck Institute of Plant Physiology, Potsdam, Germany
C. NATHAN HANCOCK • Department of Biology and Geology, University of South Carolina
Aiken, Aiken, SC, USA
TOMAS HASING • ELO Life Systems, Durham, NC, USA
JIYUE HUANG • College of Life Sciences, South China Agricultural University, Guangzhou,
China; Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China
ZHIGANG HUANG • Hunan Provincial Key Laboratory of Phytohormones and Growth
Development, Hunan Agricultural University, Changsha, China
TOSHIRO ITO • Nara Institute of Science and Technology, Biological Sciences, Plant Stem Cell
Regulation and Floral Patterning Laboratory, Ikoma, Nara, Japan
YULING JIAO • State Key Laboratory of Plant Genomics, Institute of Genetics and
Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of
Sciences, Beijing, China; State Key Laboratory of Protein and Plant Gene Research, Peking-
 Contributors xiii

Tsinghua Center for Life Sciences, Center for Quantitative Biology, School of Life Sciences,
Peking University, Beijing, China
YUE JIN • State Key Laboratory of Genetic Engineering, Ministry of Education Key
Laboratory of Biodiversity Sciences and Ecological Engineering and Institute of Biodiversity
Sciences, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai,
China
BETH A. KRIZEK • Department of Biological Sciences, University of South Carolina,
Columbia, SC, USA
XUELEI LAI • CNRS, Université Grenoble Alpes, CEA, INRAE, IRIG, BIG-LPCV,
Grenoble, France; Huazhong Agricultural University, National Key Laboratory of Crop
Genetic Improvement, Hubei Hongshan Laboratory, Wuhan, China
ZESEN LAI • State Key Laboratory of Genetic Engineering, Ministry of Education Key
Laboratory of Biodiversity Sciences and Ecological Engineering and Institute of Biodiversity
Sciences, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai,
China
BENOIT LANDREIN • Sainsbury Laboratory, University of Cambridge, Cambridge, UK;
Laboratoire Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon,
UCB Lyon 1, CNRS, INRAE, INRIA, Lyon, France
WENG HERNG LEONG • School of Agriculture, Food and Wine, University of Adelaide, Waite
Campus, Urrbrae, SA, Australia
SHAOFANG LI • Department of Botany and Plant Sciences, University of California,
Riverside, CA, USA
XIANG LI • College of Horticulture, Henan Agricultural University, Zhengzhou, China
XIGANG LIU • Department of Botany and Plant Sciences, University of California, Riverside,
CA, USA
ELIZABETH LUSCHER • Department of Botany and Plant Sciences, University of California,
Riverside, CA, USA
CHAO MA • School of Agriculture, Food and Wine, University of Adelaide, Waite Campus,
Urrbrae, SA, Australia
HONG MA • Department of Biology, The Huck Institutes of the Life Sciences, The
Pennsylvania State University, University Park, PA, USA
YESENIA MADRIGAL • Instituto de Biologı́a, Universidad de Antioquia, Medellı́n, Colombia
CLAUDIUS MARONDEDZE • CNRS, Université Grenoble Alpes, CEA, INRAE, IRIG,
BIG-LPCV, Grenoble, France; Department of Biochemistry, Faculty of Medicine, Midlands
State University, Senga, Gweru, Zimbabwe
NAYELLI MARSCH-MARTÍNEZ • Departamento de Biotecnologı́a y Bioquı́mica, Centro de
Investigaci
on y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-
IPN), Irapuato, Guanajuato, Mexico
IRENE MARTÍNEZ-FERNÁNDEZ • Instituto de Biologı́a Molecular y Celular de Plantas CSIC-
UPV, Campus de la Universidad Politécnica de Valencia, Valencia, Spain
JOSÉ TOMÁS MATUS • Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-
UAB-UB, Edifici CRAG, Campus UAB, Cerdanyola del Vallès, Barcelona, Spain;
Institute for Integrative Systems Biology (I2SysBio), Universitat de València-CSIC,
Paterna, Valencia, Spain
MONA M. MONFARED • Plant Gene Expression Center, USDA-ARS/UC Berkeley, Albany,
CA, USA; Department of Plant and Microbial Biology, University of California, Berkeley,
CA, USA; Department of Molecular and Cellular Biology, University of California, Davis,
CA, USA
xiv Contributors

MARIE MONNIAUX • Laboratoire Reproduction et Développement des Plantes, Université de

Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, Lyon, France
JANAKI S. MUDUNKOTHGE • Department of Biological Sciences, University of South Carolina,
Columbia, SC, USA
JOSE M. MUINO • Institute for Biology, Humboldt-Universit€ a t zu Berlin, Berlin, Germany
MANUEL NEUMANN • Institute for Biology, Humboldt-Universit€ a t zu Berlin, Berlin,
Germany
DIARMUID Ó’MAOILÉIDIGH • Department of Biology, Maynooth University, Maynooth,
County Kildare, Ireland
CLARA I. ORTIZ-RAMÍREZ • Instituto de Biologı́a Molecular y Celular de Plantas CSIC-UPV,
Campus de la Universidad Politécnica de Valencia, Valencia, Spain
NATALIA PABÓN-MORA • Instituto de Biologı́a, Universidad de Antioquia, Medellı́n,
Colombia
FRANÇOIS PARCY • CNRS, Université Grenoble Alpes, CEA, INRAE, IRIG, BIG-LPCV,
Grenoble, France
SARA PINTO • LAQV REQUIMTE, Departamento de Biologia, Faculdade de Ciências da
Universidade do Porto, Porto, Portugal
G. VENUGOPALA REDDY • Department of Botany and Plant Sciences, Center for Plant Cell
Biology (CEPCEB), Institute of Integrative Genome Biology (IIGB), University of
California, Riverside, CA, USA
SHULIN REN • College of Life Sciences, University of Chinese Academy of Sciences, Beijing,
China
JOANA RIBES • Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-
UB, Edifici CRAG, Campus UAB, Cerdanyola del Vallès, Barcelona, Spain
JOSÉ LUIS RIECHMANN • Centre for Research in Agricultural Genomics (CRAG) CSIC-
IRTA-UAB-UB, Edifici CRAG, Campus UAB, Cerdanyola del Vallès, Barcelona, Spain;
Instituci
o Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
PHILIPPE RIEU • CNRS, Université Grenoble Alpes, CEA, INRAE, IRIG, BIG-LPCV,
Grenoble, France; Structural Plant Biology Laboratory, Department of Botany and Plant
Biology, University of Geneva, Geneva, Switzerland
EDUARD SABIDÓ • Centre for Genomic Regulation (CRG), Barcelona Institute of Science
and Technology, Barcelona, Spain; Universitat Pompeu Fabra, Barcelona, Spain
MARKUS SCHMID • Umeå Plant Science Centre, Department of Plant Physiology, Umeå
University, Umeå, Sweden; Department of Plant Biology, Linnean Centre for Plant
Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden
CHARLES P. SCUTT • Laboratoire Reproduction et Développement des Plantes, Université de
Lyon, ENS de Lyon, UCB Lyon-1, CNRS, INRA, Lyon, France
HAROLD SUÁREZ-BARON • Instituto de Biologı́a, Universidad de Antioquia, Medellı́n,
Colombia; Departamento de Ciencias Naturales y Matemáticas, Pontificia Universidad
Javeriana, Cali, Colombia
BENNETT THOMSON • Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland
GABRIELLE TICHTINSKY • CNRS, Université Grenoble Alpes, CEA, INRAE, IRIG,
BIG-LPCV, Grenoble, France
MATTHEW R. TUCKER • School of Agriculture, Food and Wine, University of Adelaide, Waite
Campus, Urrbrae, SA, Australia
FEDERICO VALVERDE • Institute for Plant Biochemistry and Photosynthesis CSIC – University
of Seville, Seville, Spain
 Contributors xv

MICHIEL VANDENBUSSCHE • Laboratoire Reproduction et Développement des Plantes,

Université de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, Lyon, France
VANESSA WAHL • Max Planck Institute of Plant Physiology, Potsdam, Germany; The James
Hutton Institute, Dundee, UK
CHUNG-JU RACHEL WANG • Institute of Plant and Microbial Biology, Academia Sinica,
Taipei, Taiwan
CONG WANG • College of Life Sciences, South China Agricultural University, Guangzhou,
China; Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China
SHUANGSHUANG WANG • State Key Laboratory of Genetic Engineering, Ministry of Education
Key Laboratory of Biodiversity Sciences and Ecological Engineering and Institute of
Biodiversity Sciences, Institute of Plant Biology, School of Life Sciences, Fudan University,
Shanghai, China; School of Life Sciences, East China Normal University, Shanghai, China
YING WANG • College of Life Sciences, University of Chinese Academy of Sciences, Beijing,
China
YINGXIANG WANG • College of Life Sciences, South China Agricultural University,
Guangzhou, China; Guangdong Laboratory for Lingnan Modern Agriculture,
Guangzhou, China
FRANK WELLMER • Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland
SO YOUN WON • Department of Botany and Plant Sciences, University of California,
Riverside, CA, USA
XU XIN • State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental
Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing,
China; College of Advanced Agricultural Sciences, University of Chinese Academy of
Sciences, Beijing, China
YIFENG XU • College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu,
China
YUAN YOU • Center for Plant Molecular Biology (ZMBP), Department of General Genetics,
Eberhard Karls University of Tübingen, Tübingen, Germany; Department of Molecular
Life Sciences, Technical University of Munich, Freising, Germany
ZAIBAO ZHANG • State Key Laboratory of Genetic Engineering, Ministry of Education Key
Laboratory of Biodiversity Sciences and Ecological Engineering and Institute of Biodiversity
Sciences, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai,
China
VICTOR M. ZÚÑIGA-MAYO • CONACyT – Postgrado en Fitosanidad-Fitopatologı́a, Colegio
de Postgraduados, Campus Montecillo, Montecillo, Estado de México, Mexico
 Part I

Review and Overview Chapters

 Chapter 1

Flower Development in Arabidopsis

Hicham Chahtane, Xuelei Lai, Gabrielle Tichtinsky, Philippe Rieu,
Moı̈ra Arnoux-Courseaux, Coralie Cancé, Claudius Marondedze,
and François Parcy

Abstract
Like in other angiosperms, the development of flowers in Arabidopsis starts right after the floral transition,
when the shoot apical meristem (SAM) stops producing leaves and makes flowers instead. On the flanks of
the SAM emerge the flower meristems (FM) that will soon differentiate into the four main floral organs,
sepals, petals, stamens, and pistil, stereotypically arranged in concentric whorls. Each phase of flower
development—floral transition, floral bud initiation, and floral organ development—is under the control
of specific gene networks. In this chapter, we describe these different phases and the gene regulatory
networks involved, from the floral transition to the floral termination.

Key words Flower development, Arabidopsis, Meristem, MADS-box

1 Part 1: The Floral Transition

The moment at which the floral transition occurs in plants is key for
reproductive success and is fine-tuned by external and internal cues.
Eventually, the signaling pathways of these cues converge on a
handful of genes called floral integrators that make the link between
the regulation of flowering time and the triggering of flower devel-
opment [1]. Among these integrators, the gene FLOWERING
LOCUS T (FT) plays a fundamental role [2] (see Fig. 1). It encodes
a mobile protein produced in the leaves and that travels through the
phloem to activate other floral integrators at the SAM [3]. These
include LEAFY (LFY), an orphan and plant-specific transcription
factor (TF) of the Helix-Loop-Helix class and SUPPRESSOR OF
CONSTANS OVEREXPRESSION 1 (SOC1), a TF belonging to
the MADS-box TF family [4, 5]. As a result, the onset of expression
of the florigen FT greatly influences the passage from the vegetative

José Luis Riechmann and Cristina Ferrándiz (eds.), Flower Development: Methods and Protocols,
Methods in Molecular Biology, vol. 2686, https://doi.org/10.1007/978-1-0716-3299-4_1,
© Springer Science+Business Media, LLC, part of Springer Nature 2023

3
4 Hicham Chahtane et al.

Fig. 1 Simplified representation of the gene network involved in floral transition. Close up on the vegetative
SAM containing leaves (in green) before the floral transition. Internal (grey panel) and external (yellow panel)
cues regulate the expression of the florigen FT. Sugar content represented as T6P, and nitrates, are both
positive regulators of flowering and activate indirectly FT expression. The age-dependent pathway is under the
control of SPL and miRNA156. When the plant ages, SPL accumulates and negatively regulates AP2-like
transcription through mir172 to promote FT transcription. Temperature responses are mediated at least by
SVP, FLC, and PIF4. Increased ambient temperature induces chromatin accessibility allowing PIF4 to activate
FT. Upon cold response, SVP (in complex with FLMβ) represses FT transcription. Vernalization response is
mediated by the repressive activity of FLC on FT. In daylight conditions such as in the afternoon, GI promotes
CO by modulating CDF activity. PHYA promotes CO stability during the day. During the night, the complex
COP1/SPA/ELF3 represses CO activity. Once FT is promoted, the florigen complex FT/FD acts in the SAM to
activate several floral integrators such as SOC1, AP1 and indirectly LFY. Accumulation of the floral integrators
induces the SAM to switch to the reproductive phase. Please note that the crosstalk between pathways exist
and that this simplified representation does not include all the regulation processes discussed in the text
 Flower Development in Arabidopsis 5

to the reproductive stage. This peculiar gene acts as a hub to

integrate several external and internal signals and its regulation is
highly controlled by a myriad of mechanisms [6] (see Fig. 1). We
will mention a non-exhaustive list of the major actors controlling
FT expression.

1.1 External Temperature is a major determinant of flowering and is effective

Parameters through two distinct mechanisms (see review by Susila, et al. [7] for
Influencing details). One is a prolonged exposure of seedlings to cold called
Flowering—Tempera- vernalization, whereas the other results in the internalization of the
ture and Photoperiod ambient temperature [8–10].
The central gene in vernalization is FLOWERING LOCUS C
1.1.1 Temperature (FLC), which encodes a MADS-box TF [11] (see Fig. 1). Genetic,
transcriptomic and genome-wide binding profiles of FLC revealed
its main activities in the direct repression of two major floral inte-
grators, FT and SOC1 [12]. The action of FLC on FT and SOC1 is
partially under the control of gibberellic acid (GA) sensitive pro-
teins DELLA, where DELLA and FLC form a repressor complex to
downregulate the expression of floral integrators, revealing the
crosstalk between vernalization and hormonal pathways [13]. The
expression of FLC is strongly and negatively correlated with flower-
ing time and FT expression [11]. The main positive regulator of
FLC is FRIGIDA (FRI), which maintains high FLC mRNA level
and conditions the plant to remain in the vegetative state. On the
other hand, FLC expression is suppressed by the chromatin remo-
delling protein VERNALIZATION INSENSITIVE3 (VIN3),
whose expression is rapidly induced after a cold period
[14]. More recently, another mode of regulation of FLC has been
discovered, involving post-transcriptional mechanisms (see review
for details [15, 16]). It has been shown that cold exposure induces
the formation of two types of non-coding RNA variants of FLC,
called COOLAIR (for COLD INDUCED LONG ANTISENSE
INTRAGENIC RNA) and COLDAIR (for COLD ASSISTED
INTRONIC NONCODING RNA) [17, 18]. These two
non-coding RNAs repress FLC by either preventing its translation
(in the case of COOLAIR) or by promoting epigenetic repression
at the locus [18–21].
In addition to the effect of temperature through vernalization,
the ambient temperature also strongly affects flowering time. This
control likely involves a wealth of pathways and mechanisms and
research is still ongoing to understand what are the major ones
[10]. For example, a recent study described how ambient tempera-
ture controls the degree of unsaturation of phosphatidyl glycerol,
that in turn affects the retention of the FT protein in leaf cells
[22]. Higher temperature reduces unsaturation leading to
increased release of FT in phloem companion cells and accelerated
flowering [22, 23].
6 Hicham Chahtane et al.

The SHORT VEGETATIVE PHASE (SVP) gene, which also

belongs to the MADS-box family, acts as a repressor of flowering,
particularly on FT transcription [24, 25]. How ambient tempera-
ture regulates SVP has been recently unravelled but is still under
debate (see discussion in [26]). Like other MIKC-type MADS box
proteins, SVP acts in complex with homologous proteins to repress
FT transcription [27]. One of them is FLOWERING LOCUS M
(FLM) that is transcribed as several splice variants in a temperature-
dependent manner [27, 28]. One splice variant, called FLM-β, is
preferentially produced when the temperature is cooler. At low
temperature, SVP preferentially interacts with FLM-β, which allows
SVP to be translocated to the nucleus and to inhibit suppresses FT
expression [27–29] (see Fig. 1). In addition to the transcriptional
regulation of FT by SVP, the stability of SVP protein is also
temperature-dependent: it is degraded via the proteasome when
the ambient temperature increases, reducing further the SVP
repressive activity on FT level under elevated temperature [30].
PHYTOCHROME INTERACTING FACTOR4 (PIF4) pro-
tein has also been proposed to regulate FT expression in a
temperature-dependent manner [31]. PIF4 binds directly to a
regulatory region of the FT promoter that is only accessible at
high temperature (see Fig. 1). More recent shreds of evidence
suggest that PIF4 activity is highly dependent on external tempera-
ture, mainly due to its interaction with the thermosensor protein
PHYTOCHROME B (PHYB) (see review for details [32]). In
addition, others temperature-dependent mechanism regulating
PIF4 expressions have recently been identified in response to
warm temperature. One involved the TF CYCLOIDEA AND
PCF TRANSCRIPTION FACTOR 5 (TCP5), which is stabilized
under heat stress, and both positively regulates PIF4 expression and
modulates its activity to integrate thermomorphogenesis responses
[33]. Other mechanisms mediated by the chromatin remodelling
POWERDRESS protein participates in the regulation of histone
accessibility at the PIF4 locus after elevated temperature and thus is
a critical component of the temperature responses [34].
Recently, it has been shown that EARLY FLOWERING3
(ELF3), a member of the evening complex (EC; Consists of
ELF3, ELF4 and LUX ARRYTHMO (LUX)) involved in circadian
clock regulation, also controls the response to ambient tempera-
ture. elf3 mutant display a strong early flowering phenotype, due to
ELF3 repressive action on FT expression [35, 36]. The activity of
the prion-like domain protein ELF3, and its binding to specific
genomic regions is dependent on temperature, suggesting that
the EC could act as a sensor of temperature to control flowering
[35, 37–40]. Interestingly, several light signaling components such
as PHYB and ELF3 bind common genomic regions, suggesting
that these two proteins act additively to integrate external cues
(temperature and light) to modulate flowering [38].
 Flower Development in Arabidopsis 7

1.1.2 Photoperiod The onset of flowering is also largely regulated by the duration of
light exposure, a mechanism called photoperiod perception, as
revealed by the earlier Arabidopsis blooming in long day
(LD) relative to short day (SD) conditions (see review [41]). The
major actor of the photoperiodic pathway is the CONSTANS
(CO) gene, which encodes a B-box TF directly responsible for the
transcription of FT. In growth chamber conditions, the CO gene is
repressed in the early morning, then its transcription increases at
the end of the day and during the night [42, 43]. This rhythmic
regulation is controlled by two complexes: CYCLING DOF FAC-
TOR (CDF) and GIGANTEA (GI)/FLAVIN-BINDING KELCH
REPEAT-F-BOX 1 (FKF1). During the morning, the CDFs
directly represses the transcription of CO while the rest of the day
and night the GI/FKF1 complex strongly represses the CDFs
transcription, allowing indirect activation of CO [44] (see Fig. 1).
Other factors such as light quality influence the activity of
GI/FKF1 complex and add another layer of complexity to the
regulation of CO (see review for details [41, 44]).
Regulation of CO also occurs at the post-transcriptional level.
During the day, perception of light involves PHYTOCHROMES
(PHY) such as PHYA, which stabilizes CO and thus affects FT
regulation [42] (see Fig. 1). During the night, CO protein is
degraded by the proteasome through the action of an E3 ubiquitin
ligase complex involving CONSTITUTIVE PHOTOMORPHO-
GENESIS1 (COP1) and SUPPRESSOR OF PHYA-105 protein
(SPA1) [43]. ELF3 also physically interacts with COP1/SPA com-
plex and is key to negatively affect CO stability [36, 45] (see Fig. 1).
Importantly, several studies highlight different genetic
responses to flowering when plants were grown in artificially con-
trolled conditions compared to natural conditions. For instance, a
morning peak of FT expression is observed only under natural
environments but not in lab-controlled conditions [36]. This is
probably due to changes in CO stability, where the protein is slowly
degraded in natural environments due to different light and circa-
dian clock crosstalk [36]. Another example is illustrated by the light
spectrum used in classic lab growth conditions, which most of the
time lack ultraviolet-B (UV-B) wavelength. Plants sense UV-B
thanks to UV RESISTANT LOCUS 8 (UVR8) photoreceptor
and respond by upregulating several genes, including the REPRES-
SOR OF UV-B PHOTOMORPHOGENESIS 2 (RUP2) (see
review for details [46]). Under SD conditions and in response to
light containing UV-B, RUP2 is induced and acts as a repressor of
flowering by preventing FT expression via physical interaction with
CO and inactivation of CO activity [47]. This study reveals an
UVR8-dependent flowering pathway (under specific photoperiod
conditions) and again challenges the classical use of standardized
conditions found in many labs to study flowering time regulation.
8 Hicham Chahtane et al.

1.2 Internal Cues— Besides the above-mentioned external factors that play an impor-
Metabolism and Age- tant role in flowering, plants also use endogenous signals such as
Dependent Pathways metabolic cues to trigger the energy-consuming production of
flowers, fruits, and seeds. As a result, internal signals such as energy
reserves or the perception of age allow a genetic control of genes
involved in flowering.

1.2.1 Sugar and Nitrate Trehalose 6-phosphate (T6P) is a disaccharide that acts as a sensor
Metabolism of the plant’s energy reserves [48]. T6P is synthesized by TREHA-
LOSE-6-PHOSPHATE SYNTHASE1 (TPS1), an enzyme pre-
sents in leaves and SAM. Mutation in the TPS1 gene leads to the
death of the embryo, demonstrating the key function of this gene in
the plant life cycle [49]. An ingenious complementation experi-
ment to produce plants with reduced T6P level by driving TPS1
expression under a seed-specific promoter to promote embryogen-
esis and seedling development shows that those plants are late
flowering [49]. This experiment strongly suggests that T6P is an
endogenous signal that positively regulates flowering. Further
study confirmed the fundamental role of T6P in the positive regu-
lation of FT and also genes involved in the age-dependent pathway
(which will be discussed later in the review) [50] (see Fig. 1).
However, it is still unclear how T6P triggers downstream events
leading to gene regulation of FT and other targets, including its
perception, integration, and activation of transcriptional targets.
Nitrate availability is another key component that positively
regulates floral transition, for example, a delayed of flowering is
often observed when a suboptimal concentration of nitrate is used
[51]. Master regulators of nitrates signaling, the TFs NIN-LIKE
PROTEIN 6 and 7 (NLP6 and 7) probably directly regulate
SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3
(SPL3) expression in a nitrate-dependent manner. This leads to
the upregulation of SOC1 expression, a key floral integrator in the
nitrate response, thus promoting bolting [51] (see Fig. 1). Interest-
ingly, crosstalk between nitrate signaling, T6P metabolism and
photoperiod pathway exists, highlighting the importance of the
metabolic state to flowering regulation [51].

1.2.2 Age-Dependent During the vegetative growth, the morphology of successive leaves
Pathway evolves with age, with changes in size, shape, petiole length, and
trichome distribution [52]. This is due to a juvenile-to-adult phase
transition conditioning flowering (see [53] for details). This transi-
tion is mainly under the control of an interplay between micro-
RNAs and TFs. Vegetative growth is controlled by miR156 and its
targets, the members of the SPL TF family [54]. SPL protein levels
increase with leaf age, whereas the miR156 is abundant during the
juvenile phase. In young leaves, high levels of miR156 repress SPL
translation [55]. Thus, the miR156/SPL ratio represents a sensor
of the age of the plant. Upon miR156 decrease, SPL proteins are
 Flower Development in Arabidopsis 9

produced and induce the synthesis of miR172, which targets several

members of the APETALA2 (AP2) TF family [56] (see Fig. 1).
Among them, TARGET OF EAT 1 binds to CO and inhibits its
activity, leading to FT repression [57]. Some SPLs, such as SPL9 or
SPL3, also activate flowering by acting directly on the transcription
of integrators such as LFY and SOC1 in the SAM [54, 55, 58].
Genetic analyses revealed that the age-dependent pathway
controlling flowering is highly dependent on environmental condi-
tions or plant species. For instance, multiple spl mutants flowered
similarly later in both LD or SD conditions in Arabidopsis Col-0
ecotype [54], whereas in others plants such as Arabis alpina or
Cardamine flexuosa the interplay between the photoperiod or ver-
nalization pathways revealed the importance of the
SPL/miRNA156/miR172 pathway on flowering [59, 60].

1.3 Integration of the The different signaling pathways described above converge towards
Different Input to the regulation of FT either in leaves or in the SAM. Signals in favor
Promote Flowering of blooming result in an accumulation of FT protein in leaves,
and Flower Formation where it is loaded via the phloem companion cells and transported
to the SAM. In association with the bZIP TF FLOWERING
LOCUS D (FD), the complex FT/FD activates the transcription
of other floral integrators such as SOC1 and APETALA1 (AP1) [2]
(see Fig. 1). Using genome-wide analyses, it has been shown that
FD binds not only G-boxes (as expected for a bZIP TF) but also
non-canonical DNA motifs [61]. Once activated by FT/FD, AP1
and SOC1 (in association with AGL24) directly activate LFY in the
SAM to promote flowering [62–64] (see Fig. 1).
The FT/FD complex must act transiently to promote the
expression of the floral integrators AP1 and LFY since a prolonged
FT/FD activity prevents the development of normal flowers
[65]. Once the floral integrators accumulate in SAM, the plant
starts to produce floral primordia, the earliest recognizable stage
of a flower.

2 Part 2: The First Hours of Arabidopsis Flowers

Whether a primordium will become a leaf or a flower, its emergence

involves a set of basic regulators including the auxin phytohor-
mone. In both cases, auxin distribution and signaling are critical
but the auxin-driven floral primordia emergence requires the action
of a few floral regulators, which will confer the floral fate (reviewed
in [66]).

2.1 Primordium Clear evidence from the role of auxin in flower development came
Positioning and Auxin from the study of the pin-formed 1 (pin1) and pinoid (pid) mutants
Signaling that lack floral primordia: they are a phenocopy of auxin polar
transport inhibitor application and can be rescued by exogenous
10 Hicham Chahtane et al.

auxin application [67–70]. Auxin indeed controls the regular and

predictable arrangement of leaves and flowers around the stem,
called phyllotaxis. Several genetic and mathematical evidences
showed that the regulation of auxin localization leads to a self-
organized pattern involving local auxin accumulation surrounded
by auxin depletion, thereby creating the heterogeneity triggering
organ initiation [70–73]. This process is made more robust by the
interplay between auxin and cytokinin signaling [74]. Deciphering
the roles of auxin distribution in flower initiation implies to detail
auxin biosynthesis, transport, and signaling.

2.2 Auxin Local auxin synthesis, in addition to polar auxin transport, also
Biosynthesis and contributes to flower emergence (see [75] for review). The major
Transport actors in the tryptophan-dependent auxin biosynthesis are proteins
encoded by TRYPTOPHAN AMINOTRANSFERASE OF ARA-
BIDOPSIS/YUCCA (YUC) genes. Their role is most obvious in
the yuc1 yuc2 yuc4 yuc6 quadruple mutant, which makes almost no
floral bud or only rudimentary incomplete flowers [76].
Auxin dynamic transport, named polar auxin transport, is
driven at the plasma membrane by the specific influx and efflux
carriers, respectively encoded by the AUXIN RESISTANT1
(AUX1) and PIN FORMED1 (PIN1) gene families [77] (see
Fig. 2). Local auxin accumulation and depletion are mediated by
asymmetric localization of PIN proteins, which drive auxin in the
epidermis and generate an auxin sink below the primordia after they
have formed [70, 78]. PIN1 polarity undergoes a dynamic change
during organ initiation due to the action of PID, a serine-threonine
protein kinase, and of a PP2A-phosphatase responsible for the
phosphorylation status and the asymmetric distribution of PIN1
in the young floral meristem [79, 80]. More recently, D6 PRO-
TEIN KINASE (D6PK) has been shown to also phosphorylate
PIN1 proteins and be required for efficient PIN1 distribution in
the inflorescence [81]. However, the two kinases have
non-redundant roles as their phosphosites on PIN1 are different
and d6pk and pid display distinct phenotypes. PIN1 dynamics in the
cell also involve the MACCHI-BOU4 (MAB4) gene family, which
encode NONPHOTOTROPIC HYPOCOTYL 3-like proteins and
regulate PIN1 endocytosis. Multiple mutants for MAB4 genes
(such as the triple mab4 mel1 mel2 mutant) have pin-like
inflorescences [82].

2.3 Downstream of Cells transcriptionally respond to auxin through the nuclear auxin
Auxin Signaling pathway, which involves AUXIN RESPONSE FACTORs (ARFs),
Aux/IAA repressors, and the TIR1/AFB auxin co-receptors
[70, 83]. ARF5 (also referred to as MONOPTEROS, MP) is the
major ARF acting in the floral meristem initiation, as some mp/arf5
mutant alleles lack floral buds on the stem [84, 85] (see Fig. 2). MP
induces floral meristem emergence in various ways, such as
 Flower Development in Arabidopsis 11

Fig. 2 Key regulators involved in floral meristem initiation. Close up on the nascent FM (stage 2) on the flank of
the SAM. Locally produced auxin (for instance IAA) by YUC biosynthesis enzymes is canalized in the epidermis
(L1) of the flank of the SAM by the PIN transporters (under the control of PID kinase) to form an auxin
maximum. This auxin peak triggers the activation of ARF5/MP TF through degradation of the associated
Aux/IAA repressors. ARF5/MP is the main factor between auxin and downstream events. Associated with SYD
and BRM chromatin remodelers, it activates the AINT/AIL6 and LFY/RAX1 cascades that control redundantly
meristem initiation. ARF5/MP also induces FIL, involved in meristem initiation and in specifying the abaxial
territory of the FM. The WUS/CLV meristem homeostasis loop is indicated as central to allow the formation of
the floral meristem, but the way it is controlled by upstream actors is still unclear. In collaboration with ARF3
and HDA19, FIL also represses the expression of the SAM marker STM. On the other hand, STM is induced by
mechanical stress at the border between the SAM and the FM. This spatial regulation of STM, in collaboration
with CUC genes and other partners mentioned in the text, allows the establishment of a clear border between
the SAM and the FM
12 Hicham Chahtane et al.

reinforcing local auxin maxima by orienting PIN polarity in a

non-cell autonomous manner, possibly via the activation of
MAB4 [82, 86].
Auxin activated ARF5 also regulates the expression of two early
acting regulators of floral meristem identity, AINTEGUMENTA
(ANT), an AP2/ERF TF, and LFY [87–90]. ARF5 recruits the
chromatin remodelers BRAHMA (BRM) or SPLAYED (SYD),
which are subunits of the SWI/SNF (SWITCHING DEFEC-
TIVE/SUCROSE NON FERMENTING) complex that enables
chromatin opening, thus leading to gene activation of ANT and
LFY [91] (see Fig. 2). Both ANT and LFY participate in floral
primordium emergence, a role that remains cryptic in ant or lfy
single mutants but is revealed in mutant combinations such as pin
ant ail, lfy pid or lfy pin [92–94]. Moreover, ANT and its homolog
AINTEGUMENTA like 6 (AIL6) bind common genomic regions
to activate (likely directly) LFY expression in parallel to ARF5 as
well as a wide range of genes involved in floral development
[95, 96]. The few ARF5 direct targets identified in FM include
MAB4 and ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER
PROTEIN6 (AHP6), a negative regulator of cytokinin signaling
that reinforces the auxin pattern adding robustness to the phyllo-
taxis [5, 74]. Further efforts such as elucidating the ARF5 binding
landscape in floral tissue will shed light on the gene network
involved in this step [97].

2.4 Boundaries and Genes involved in abaxial/adaxial polarity such as REVOLUTA

Polarization Set the (REV) and FILAMENTOUS FLOWER (FIL) are also necessary
Limit of the Floral during flower meristem development as attested by floral defects in
Primordium the corresponding mutants or the fil rev double [98–101]. REV
may function by modulating local response to auxin during floral
primordium formation [102]. Other important factors involved in
early meristem development include several TFs such as LATERAL
SUPPRESSOR (LAS), CUP-SHAPED COTYLEDON 1 to
3 (CUC1–3), or REGULATOR OF AXILLARY MERISTEMS
1 (RAX1) [103–107]. Particularly, RAX1 is expressed in the ear-
liest stage of floral primordium and directly activated by LFY [108].
Conversely, the expression of the SHOOT MERISTEMLESS
(STM), a SAM marker gene, is repressed at the site of auxin maxima,
where the floral bud forms. This repression is important to promote
the initiation of floral primordium and is controlled by at least two
convergent pathways [109] (see Fig. 2). First, STM expression is
downregulated directly by the repressive class B ARF3 TF that
binds to STM second intron. In parallel, ARF5 directly activates
FIL, which in turn participates in STM repression [91, 109]. Based
on protein-protein interactions and genetic data, it has been pro-
posed that FIL/ARF3 complex jointly recruits the histone deace-
tylase HDA19 to promote chromatin compaction on STM locus
 Flower Development in Arabidopsis 13

and ensure its silencing [109]. This dual function of ARF proteins
warrants stable repression of STM in the early floral primordium
steps, to allow proper development of floral primordia initiation.

2.5 Establishment of Floral meristem initiation also involves re-establishing a stem cell
a Transient Stem Cell niche in the flower with a combined expression of CLAVATA3
Identity (CLV3), a stem cell marker gene, and WUSCHEL (WUS), a master
regulator of the organizing center [110, 111]. As in the SAM, these
two regulators form a minimal genetic circuitry that ensures a
transient stem cell niche formation. Models have been proposed
on how a combination of signals (hormonal or derived from the L1
layer) should re-establish an organizing center when the flower
meristem reaches a certain size [112]. However, the necessary
connection to the early FM regulators (MP, LFY, ANT) remains
elusive (see Fig. 2).

2.6 Modifications in Whereas gene regulatory networks between TFs have been unra-
Cell Wall Composition velled, few connections have been made with terminal actors bear-
Trigger Floral ing the enzymatic activities that shape cells. This gap started to be
Outgrowth filled when ANT and AIL6 were shown to promote cell wall poly-
saccharide modification leading to change in cell growth
[113]. The importance of cell wall composition is illustrated by
mutations in several GLYCOSYLTRANSFERASES OF THE CEL-
LULOSE SYNTHASE LIKE D (CSLD) genes that strongly affect
the number of floral primordia [114]. This likely affects auxin
distribution, as revealed by the reduced pattern of the auxin trans-
porter PIN1 in some cell wall cellulose biosynthesis mutant, such as
cesa3 [115]. Also, flower primordia express a specific set of glyco-
syltransferases, suggesting that the FM outgrowth is facilitated by
local modification of the physical properties of the cell wall
[114]. The regulation of plant cell growth is indeed closely linked
to the cell wall structure [116]. Combining use of the naked pin1
stem with chemical and physical treatments, Sassi et al. illustrated
how auxin affects cell wall stiffness and microtubule orientation.
Before floral outgrowth, cell cortical microtubules are anisotropic
thereby promoting vertical growth and inhibiting bulging. An
auxin maximum reduces anisotropy and cell wall stiffness, promot-
ing the emergence of the FM [117]. This change in cell wall
properties and tissue shape generates local mechanical perturba-
tions that rapidly induces STM in FM boundaries [118] (see Fig. 2).

3 Part 3: Determination of the Floral Primordia Identity

3.1 Activation of the While mutants affected in floral primordiaoutgrowth show naked
Floral Meristem stems, other mutants develop lateral structures but they lack a
Identity proper floral identity. This is typically the case of lfy, where flowers
are usually replaced by inflorescences subtended by leaves or by
14 Hicham Chahtane et al.

abnormal flowers often bearing bracts. LFY encodes an orphan

plant-specific TF which controls FM identity together with the
MADS-Box gene AP1 or CAULIFLOWER (CAL), a close homo-
log of AP1 [5, 119, 120]. They are both expressed early in the FM,
LFY from the floral stage 0 on and immediately inducing AP1. LFY
and AP1 orchestrate the robust acquisition of floral meristem iden-
tity thanks to a feed-forward and positive feedback loop between
them and with the class I HD-Zip TF LATE MERISTEM IDEN-
TITY2 (LMI2) [63, 121–123].
In parallel to the direct AP1 activation, LFY also modulates the
GA hormone pathway: it induces expression of ELA1, which
encodes a gibberellin catabolic enzyme thus leading to reduced
GA levels in the FM [124]. This GA reduction frees the AP1
activator SPL9 from the repression by the DELLA proteins. There-
fore, multiple activations of AP1 by LFY ensure an irreversible
acquisition of floral identity (see Fig. 3).
Several new LFY and AP1 upstream regulators have been
recently identified: the DORNRÖSCHEN (DRN), known as a
direct target of MP/ARF5 in the embryo [85], and DRNL and
PUCHI TFs (all belonging to the AP2/EREBP family) act redun-
dantly with the BTB/POZ proteins BOP1/2 to regulate LFY
expression. The quintuple drn drnl puchi bop1 bop2 exhibits such
a strong decrease in LFY expression that it phenocopies the lfy
mutant [125]. BOP1/2 proteins not only affect LFY expression,
but these components of an E3 ligase complex also seem to pro-
mote LFY activity through a post-transcriptional mechanism
[126, 127].

3.2 Repression of the The irreversible switch towards flower identity requires the repres-
Inflorescence Trait sion of the inflorescence identity promoted by TERMINAL
FLOWER 1 (TFL1) and other genes such as SOC1, SVP, and
AGL24. TFL1 belongs to the same family as FT but acts oppositely:
while FT promotes flowering, TFL1 represses flowering due to a
small difference in 4 residues critical for the protein function
[128, 129]. TFL1 plays a double role: it represses LFY and AP1
expression and promotes the vegetative state of the meristem (see
[130] for details) (see Fig. 3). As FT, TFL1 (devoid of DNA binding
capability) acts in complex with other TFs, such as the bZIP protein
FD to regulate gene expression [61, 131–133]. To allow a stable
switch to floral identity, AP1 directly binds CArG boxes in a remote
3′ regulatory region of TFL1 and represses its expression. AP1 also
represses the vegetative MADS-box TFs, including SOC1, AGL24,
and SVP [63, 134] (see Fig. 3). Until recently, LFY was also thought
to directly repress TFL1 [135]. However, recent evidence suggests
that LFY directly activates TFL1, and only represses it indirectly by
activating AP1 [5, 136, 137]. The functional relevance of this
incoherent feedback loop between LFY, AP1, and TFL1 is still
unclear but it has been proposed that it could act as a sensor of
 Flower Development in Arabidopsis 15

Fig. 3 Gene network specifying floral meristem identity. Close up on the nascent FM (stage 2–3) on the flank of
the SAM. Inflorescence and floral genetic identity are antagonistic. Whereas the inflorescence meristem is
specified by TFL1, as well as SVP and SOC1, the floral meristem is under the control of LFY and AP1 TFs. LFY is
early expressed in the floral anlagen below ARF5/MP (see Fig. 2), but is also controlled by other factors such
as PUC, DRN/DRNL, and BOP1/2. LFY induces the expression of AP1 directly, or indirectly through LMI2 and
the GA hormonal pathway. AP1 also induces LFY in a positive feedback regulatory loop stabilizing the genetic
floral identity. Once induced, AP1 inhibits the flowering time genes and the inflorescence identity genes.
Reciprocal inhibition between TFL1 and AP1 is considered as central to maintain exclusive inflorescence and
floral identities

floral meristem marker and make sure that floral patterning starts
only when the levels of LFY and AP1 are sufficient [136, 138]. A
model of the early floral meristem regulatory network has been
proposed that captures the different gene expressions observed in
wild-type and several mutant backgrounds [139].
16 Hicham Chahtane et al.

3.3 A Common Early Despite their completely different DNA binding specificity, LFY
Regulatory Network and AP1 share several common target genes [140, 141]. Almost
Between LFY and AP1 30% (769 genes) of all genomic regions bound by LFY are also
bound by AP1. Among them, 25% are both regulated by LFY and
AP1. Many of them are linked to flower development, such as genes
involved in auxin (MAB4, ARF6, ARF11, and IAA18), the GA
catabolism enzymes (ELA1, GA2ox20) and other regulators of
floral development (SEP3, RAX1, LMI1, FD, and TFL1)
[63, 140, 142, 143]. Another feature shared by LFY and AP1 is
their ability to bind to closed chromatin region, a property charac-
teristic of pioneer factors [144–148]. The fact that LFY and AP1
share many bound regions and regulate a common set of target
genes suggests that they may act in the same complex. This could
be direct physical interaction or indirect association mediated by
common co-factors, such as chromatin remodelers BRM and SYD,
which are recruited by LFY and to interact with AP1 as shown by
mass spectrometry experiments [149, 150]. However, the only
MADS-box TF interacting with LFY is SEP3, and available proteo-
mics data do not suggest a direct interaction between LFY and
other MADS-box TFs [150, 151].
Another possibility could explain why LFY and AP1 shared
common targets. Genes bound by multiple TFs show more
dynamic expression during flower development [152]. Thus,
genes with multiple different TF binding sites in their regulatory
sequence tend to be more rapidly and more efficiently regulated to
promote the switch to flower development.

3.4 The Genesis of Emerging flower primordia are separated from the SAM by a group
the Boundary FM-SAM of specialized cells that form a boundary, characterized by reduced
growth [153]. This boundary interface separates and influences
two territories of different developmental fates, the SAM and the
FP. Several TFs belonging to the TALE superfamily contribute
redundantly to SAM maintenance, including the KNOX class pro-
tein STM, and the BELL class proteins PENNYWISE (PNY) and
the closely related POUND-FOOLISH (PNF) [154–156]. Both
KNOX and BELL class proteins specify meristems and define
boundaries likely by forming functional heterodimers [156]. Several
genes involved in boundary formation are regulated, directly or
indirectly by the TALE proteins. Some of them are encoded by
NAC family TFs CUP-SHAPED COTYLEDON (CUC1–3).
CUC1 and 2 act redundantly to establish boundaries of floral
primordia [157, 158]. CUC1 directly activates STM expression in
a positive feedback loop to generate the boundary between the
SAM and the FP, a function probably shared by the closed relatives
CUC2 and CUC3 proteins [104, 105, 159]. Similarly to CUCs,
BOP1 and BOP2 are expressed in the boundary and also define
boundaries of the floral meristem in addition to their role in floral
identity [160]. PNY and PNF downregulate BOP1 and BOP2 to
Another random document with
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Ecribellatae, 385
Ectatosticta davidi, 393
Ectinosoma, 62
Edriophthalmata, 112, 121
Eggs, of Phyllopoda, 32;
of Cladocera, 44;
of Copepoda, 59, 62, 66, 67, 71, 74;
of Branchiura, 77;
of Syncarida, 114;
of Peracarida, 123;
of Hoplocarida, 141;
of Eucarida, 144;
of Trilobites, 238;
of Limulus, 275;
of Pedipalpi, 309;
of Spiders, 358;
of Solifugae, 424;
of Pseudoscorpions, 434;
of Phalangidea, 442;
of Acarina, 456;
of Tardigrada, 478;
of Pentastomida, 493;
of Pycnogons, 520
Ehrenberg, on systematic position of Tardigrada, 483
Eleleis crinita, 396
Ellipsocephalus, 224, 235, 247;
E. hoffi, 248
Embolobranchiata, 258, 259, 297 f.
Emmerich, on facial suture of Trinucleus, 226
Encephaloides, 193;
E. armstrongi, 192, 193;
habitat, 205
Encrinuridae, 251
Encrinurus, 227, 235, 251
Endeis didactyla, 534;
E. gracilis, 539;
E. spinosus, 541
Endite, 9, 10
Endopodite, 9, 10;
of Trilobites, 237
Endosternite, 257, 305, 330
Endostoma, of Eurypterus, 287
Engaeus, 157;
E. fossor, distribution, 213
Enoplectenus, 418
Enterocola, 67;
E. fulgens, 67
Entomostraca, defined, 6;
diagnosis, 18;
of littoral zone, 197;
fresh-water, of southern hemisphere, 216
Entoniscidae, 130, 134
Enyo, 400
Enyoidae, 399
Eoscorpius, 298
Epeira, 409;
E. angulata, 315, 409;
E. basilica, 350, 351;
web of, 351;
E. bifurcata, 359;
E. caudata, 359;
E. cornuta, 409;
E. cucurbitina, 372, 409;
E. diademata, 335, 340, 343, 345, 359, 366, 380, 409;
anatomy, 332;
 cocoon, 358;
silk, 360;
spinnerets, 325;
E. labyrinthea, 350;
E. madagascarensis, 360;
E. mauritia, 349;
E. pyramidata, 409;
E. quadrata, 366, 409;
E. triaranea, 350;
E. umbratica, 409
Epeiridae, 376, 377, 406
Epeirinae, 408
Ephippium, 48
Epiblemum, 420
Epicarida, 129;
sex in, 105
Epicaridian, larva of Epicarida, 130
Epicoxite, of Eurypterus, 287
Epidanus, 449
Epigyne, 319, 333, 378
Epipharynx, 459
Epipodite, 9, 10
Episininae, 402
Episinus truncatus, 403
Epistome, of Eurypterida, 291;
of Pseudoscorpions, 431, 436;
of Phalangidea, 443
Erber, 355, 356
Eremobates, 429
Eremobatinae, 429
Eresidae, 398
Eresus cinnaberinus, 398
Eriauchenus, 411
Erichthoidina, larva of Stomatopod, 143
Ericthus, larva of Stomatopod, 143
Erigone, 405
Erigoninae, 404
Eriophyes, 465;
E. ribis, 455, 465;
E. tiliae, 465
Eriophyidae, 464
Eriphia, 191;
E. spinifrons, 191
Erlanger, von, on development and position of Tardigrada, 483
Ero, 411;
E. furcata, 366, 411;
cocoon, 358;
E. tuberculata, 412
Eryonidae, 158;
habitat, 204
Eryonidea, 157
Erythraeinae, 473
Estheria, 21, 22, 23, 36;
E. gubernator and E. macgillivrayi, habitat, 33;
E. tetraceros, 36
Eucarida, 114, 144 f.
Euchaeta norwegica, 58
Eucopepoda, 57 f.
Eucopia australis, 119
Eucopiidae, 113, 114, 118
Eudendrium, Pycnogons on, 520
Eudorella, 121
Eukoenenia, 423;
E. augusta, 423;
E. florenciae, 423;
E. grassii, 423
Eulimnadia, 36;
E. mauritani, 36;
E. texana, 36
Euloma, 230
Eumalacostraca, 112 f.
Eupagurinae, 180
Eupagurus, 180;
E. bernhardus, commensalism, 172;
distribution, 199;
E. excavatus, parasitic castration of, 101;
E. longicarpus, metamorphosis, 179;
E. prideauxii, commensalism, 172;
E. pubescens, distribution, 199
Euphausia pellucida, 145, 146
Euphausiacea, 144
Euphausiidae, 113, 114, 144;
larval history, 145;
eyes, 150
Eupodes, 471
Euproöps, 278
Eurycare, 232, 247
Eurycercus, 53;
alimentary canal, 42;
E. lamellatus, habitat, 207
Eurycide, 505, 533;
E. hispida, 506, 507, 533
Eurycididae, 533
Eurydium, 485
Euryopis, 404
Eurypelma, 389;
E. hentzii, 361, 370
Euryplax, 195
Eurypterida, 258, 278, 283 f.
Eurypteridae, 290 f.
Eurypterus, 283 f., 290, 291, 292;
E. fischeri, 284, 286, 289
Eurytemora, 59;
E. affinis, habitat, 206
Eusarcus, 283, 291
Euscorpiinae, 308
Euscorpius, 298, 308;
E. carpathicus, 299
Eusimonia, 429
Euterpe acutifrons, 61, 61;
distribution, 203
Euthycoelus, 389
Evadne, 54;
young, 47
Excretory system (including Renal organs), in Crustacea, 12;
in Arachnids, 257;
in Limulus, 270;
in Tardigrada, 481;
in Pentastomida, 491
Exner, on mosaic vision, 148
Exopodite, 9, 10;
of Trilobites, 237
Eyes, compound, of Crustacea, 146, 147;
physiology of, 148;
 of deep-sea Crustacea, 149;
connexion with phosphorescent organs, 151;
regeneration of, 6;
of Trilobites, 227 f., 228;
of Limulus, 271;
of Eurypterida, 285;
of Scorpions, 301;
of Pedipalpi, 309;
of Spiders, 315, 334; of Solifugae, 426;
of Pseudoscorpions, 431;
of Phalangidea, 442;
of Acarina, 458;
of Pycnogons, 517

Fabre, on habits of Spiders, 298 f.;

of Tarantula, 361 f.;
on Wasp v. Spider, 368 f.
Facet, of Trilobites, 235
Facial suture, 225 f., 232
Falanga, 424
False articulations, 444
False-scorpions, 430
Fecenia, 399
Filistata, 391;
F. capitata, 392;
F. testacea, 392
Filistatidae, 319, 336, 391
Finger-keel, 303
Fixed cheek, 225, 226, 227
Flabellifera, 124 f.
Flabellum, 270
Flacourt, 363
Flagellum, in Solifugae, 426, 428;
in Pseudoscorpions, 433
Forbes, 374
Ford, S. W., on development of Trilobites, 238
Forel, on Lake of Geneva, 206
Formicina, 405
Formicinae, 405
Formicinoides brasiliana, 318
Fragilia, 535
Free cheek, 225, 226, 227
Fresh-water, Crustacea, 205 f.;
Spiders, 357
Furcilia (Metazoaea), larva of Euphausia, 145
Fusulae, 325, 335

Galathea, 169, 170;

G. intermedia, Pleurocrypta parasitic on, 133;
G. strigosa, 170;
gut of, 15
Galatheidae, 169
Galatheidea, 169
Galea, 433, 436
Galena, 412
Galeodes, 429, 527;
nervous system, 428;
chelicera, 429;
G. arabs, 425;
G. araneoides, 425
Galeodidae, 428
Gall-mites, 455, 464
Gamasidae, 470
Gamasinae, 470
Gamasus, 460, 461, 463, 470;
G. coleoptratorum, 470;
G. crassipes, 470;
G. terribilis, 461
Gammaridae, 138
Gammarus, 137, 138;
of Lake Baikal, 212;
of Australia, 216;
G. locusta, 138, 138;
G. pulex, 138
Gampsonyx, 115, 118
Garstang, on respiration of crabs, 186 n.
Garypinae, 436, 437
Garypus, 431, 436, 437, 438;
chelicera, 432;
G. littoralis, 430
Gaskell, 270, 277, 334
Gasteracantha, 410;
G. minax, 410
Gasteracanthinae, 317, 409
Gastrodelphys, 73
Gastrolith, of Lobster, 155
Gaubert, 525 n.
Gebia littoralis, 167
Gecarcinidae, 196
Gecarcinus, 194, 195, 196
Gegenbaur, 523
Gelanor, 411, 412
Gelasimus, 194, 196;
habitat, 198;
distribution, 210;
G. annulipes, 194
Genal angle, 225
Gené, 461
Genital operculum, of Eurypterida, 288, 289, 291
Genysa, 388
Gerardia, Laura parasitic on, 93
Geryon, 195
Giardella callianassae, 73
Gibocellidae, 448
Gibocellum sudeticum, 447
Giesbrecht, on Copepoda, 57;
on phosphorescence, 59
Gigantostraca, 258, 283 f.
Gill-book, 270
Glabella, 223
Glabella-furrows, 223
Glands, of Tardigrada, 481;
of Pentastomida, 490, 491;
of Pycnogons, 511;
coxal, of Arachnids, 257, 270, 337;
green, of Malacostraca, 110;
poison-, of Arachnids, 337, 360;
spinning, of Spiders, 335;
of Pseudoscorpions, 434
Glaucothoe, larva of Eupagurus, 179, 180
Gluvia, 429
Glycyphagus, 466;
G. palmifer, 466;
 G. plumiger, 466
Glyphocrangon, 164;
G. spinulosa, 158, 164
Glyphocrangonidae, 164
Glyptoscorpius, 283, 291, 294
Gmelina, 138
Gmogala scarabaeus, 394
Gnamptorhynchus, 533
Gnaphosa, 397
Gnathia maxillaris, 124;
life-history of 125
Gnathiidae, 124
Gnathobase, 10, 264
Gnathophausia, 119, 256 n.;
maxillipede of, 10
Gnathostomata, 56
Gnosippus, 429
Goldsmith, 362
Gonads, = reproductive organs, q.v.
Gonodactylus, 143;
G. chiragra, 143
Gonoplacidae, 195
Gonoplax, 195;
G. rhomboides, 195
Gonyleptidae, 442, 448, 449
Goodsir, Harry, 535, 540
Gordius, parasitic in Spiders, 368
Gossamer, 342
Graells, 364
Graeophonus, 309
Graff, von, on position of Tardigrada, 483
Grapsidae, 193, 195;
habitat, 198, 201
Graptoleberis, 53
Grassi, 422
Green gland, 110 (= antennary gland, q.v.)
Gregarious Spiders, 340
Grenacher, 517
Griffithides, 251
Gruvel, on Cirripedia, 80, 86
Guérin-Méneville, 439
Gurney, on Copepoda, 62;
on Brachyuran metamorphosis, 181 n.
Gyas, 450
Gylippus, 429
Gymnolepas, 89
Gymnomera, 38, 54
Gymnoplea, 57

Hadrotarsidae, 394
Hadrotarsus babirusa, 394
Haeckel, on plankton, 203
Haemaphysalis, 469
Haematodocha, 322
Haemocera, 64;
H. danae, life-history, 64, 65
Haemocoel, 5, 11
Hahnia, 325, 416
Hahniinae, 416
Halacaridae, 472
Halocypridae, 108
Halosoma, 539
Hannonia typica, 533
Hansen, on Choniostomatidae, 76;
on Cirripede Nauplii, 94;
on classification of Malacostraca, 113
Hansen and Sörensen, 422, 439, 443, 448
Hapalogaster, 181;
H. cavicauda, 178
Hapalogasterinae, 181
Harpactes hombergii, 395
Harpacticidae, 61, 62;
habitat, 206
Harpedidae, 245
Harpes, 225, 226, 230, 231, 234, 246;
H. ungula, 248;
H. vittatus, eyes, 228
Harporhynchus, 53
Harvest-bugs, 454, 473
Harvestmen, 440, = Phalangidea, q.v.
Harvest-spiders, 440, = Phalangidea, q.v.
Harvesters, 440, = Phalangidea, q.v.
Hasarius falcatus, 421
Haustellata, 501 n.
Haustoriidae, 137
Haustorius arenarius, 137
Hay, on name Lydella, 486 n.
Heart, of Phyllopoda, 29;
of Cladocera, 43;
of Nebalia, 112;
of Syncarida, 115;
of Peracarida, 118;
of Isopoda, 122;
of Danalia, 132;
of Amphipoda, 136;
of Squilla, 142;
of Eucarida, 144;
of Limulus, 268;
of Scorpions, 305;
of Pedipalpi, 311;
of Spiders, 331;
of Solifugae, 427;
of Pseudoscorpions, 434;
of Phalangidea, 445;
of Acarina, 460;
of Pycnogons, 516
Heart-water, 470
Hedley, on home of cocoa-nut, 174
Heligmonerus, 388
Heller, 455
Hemeteles fasciatus, 367;
H. formosus, 367
Hemiaspis, 278;
H. limuloides, 278
Hemioniscidae, 130
Hemiscorpion lepturus, 307
Hemiscorpioninae, 306, 307
Henking, 447, 460
Hentz, 367
Herbst, on regeneration of eye, 6 n.
Hermacha, 388
Hermaphroditism, 15;
caused by parasite, 101, 102;
partial and temporary, 102;
normal, 105;
in Cymothoidae, 126;
in Isopoda Epicarida, 129;
in Entoniscidae, 135;
in Caprella, 140
Hermippus, 317, 399;
H. loricatus, 400
Hermit-crab, 167, 171;
commensalism, 172;
reacquisition of symmetry, 173;
regeneration of limbs, 156
Hermit-lobster, 167
Herrick, on the Lobster, 154
Hersilia (Araneae), 401;
H. caudata, 400
Hersiliidae (Araneae), 326, 400
Hersiliidae (Copepoda), 73
Hersiliola, 401
Heterarthrandria, 58
Heterocarpus alphonsi (Pandalidae), phosphorescence, 151
Heterochaeta papilligera, 60
Heterocope, 59
Heterogammarus, 138
Heterometrus, 307
Heterophrynus, 313
Heteropoda venatoria, 414
Heterostigmata, 471
Heterotanais, 123
Hexameridae, 91
Hexathele, 390
Hexisopodidae, 429
Hexisopus, 429, 429
Hexura, 391
Hippa, 171;
H. emerita, distribution, 202
Hippidae, 171
Hippidea, 170;
habitat, 198
Hippolyte, 164;
distribution, 200;
H. varians, 164
Hippolytidae, 164;
distribution, 199
Hodge, George, 523, 540
Hodgson, 508
Hoek, on Cirripedia, 80;
on Pycnogons, 505, 512, 513
Holm, G., on Agnostus, 225;
on Eurypterus, 285 n.
Holmia, 236, 242, 247;
H. kjerulfi, 242, 246
Holochroal eye, 228
Holopediidae, 51
Holopedium, 38, 51
Homalonotus, 222, 249;
H. delphinocephalus, 223
Homarus, 154;
habitat, 200;
excretory
glands, 13;
H. americanus, 154;
H. vulgaris, 154
Homoeoscelis, 76
Homola, 184;
distribution, 205
Homolidae, 184
Homolodromia, 184;
H. paradoxa, resemblance to Nephropsidae, 184
Hood, of Phalangidea, 442, 452
Hoplocarida, 114, 141
Hoploderma, 468;
H. magnum, 467
Hoplophora, 468
Horse-foot crab, = Limulus, q.v.
Hoyle, on classification of Pentastomids, 495
Hughmilleria, 283, 290, 292
Humboldt, on Porocephalus, 488 n.
Hutton, 424
Huttonia, 398
Hyale, 139
Hyalella, 137, 139;
distribution, 211, 217
Hyalomma, 469
Hyas, 192, 193;
 distribution, 200
Hyctia nivoyi, 421
Hydrachnidae, 472
Hydractinia, Pycnogons on, 523
Hydrallmania, Pycnogons on, 524
Hymenocaris, 112
Hymenodora, 163
Hymenosoma, 193;
distribution, 200
Hymenosomatidae, 193
Hyperina, 140
Hypochilidae, 393
Hypochilus, 336, 393;
H. thorelli, 393
Hypoctonus, 312
Hypoparia, 243
Hypopus, 463
Hypostome, of Trilobites, 233, 237;
of Bronteus, 233;
of Acarina, 469
Hyptiotes, 349, 411;
H. cavatus, snare, 350;
H. paradoxus, 350, 411

Iasus, 165, 167;

distribution, 200
Ibacus, 167
Ibla, 88;
I. cumingii, 88;
I. quadrivalvis, 88, 89
Ichneumon flies, and Spiders, 367
Icius, 421;
I. mitratus, 382
Idiops, 388
Idothea, habitat, 211
Idotheidae, 127
Ihle, J. E. W., 526 n.
Ilia, 188;
I. nucleus, 188;
respiration, 187
Illaenus, 229, 231, 235, 249;
I. dalmanni, 248
Ilyocryptus, 40, 53
Inachus, 192, 193;
I. mauritanicus, Sacculina parasitic on, 97 f.;
parasitic castration in, 101;
temporary hermaphroditism of, 103;
Danalia and Sacculina parasitic on, 131
Integument, of Pycnogons, 518
Irregular Spider-snares, 351
Ischnocolus, 389
Ischnothele dumicola, 390
Ischnurinae, 306, 307
Ischnurus ochropus, 307
Ischnyothyreus, 394
Ischyropsalidae, 451
Ischyropsalis, 444, 451
Isokerandria, 69 f.
Isometrus europaeus, 306
Isopoda, 121 f., 242
Ixodes, 469;
I. ricinus, 469
Ixodidae, 469
Ixodoidea, 455, 462, 468

Janulus, 403
Jaworowski, on vestigial antennae in a Spider, 263
Johnston, George, 540
Jumping-Spiders, 419

Karshia, 429
Karshiinae, 429
Katipo, 363, 403
King-crab, =Limulus, q.v.
Kingsley, on Trilobites, 239, 243 n.;
on breeding habits of Limulus, 271
Kishinouye, on Limulus, 274, 275
Klebs, on the frequency of human Pentastomids, 494
Knight Errant, 540
Koch, C., 397 n.
Koch, L., 397 n.
Kochlorine, 92;
K. hamata, 93
Koenenia, 422, 527, 528;
K. mirabilis, 423
Koltzoff, 15
König, 524