Table Of ContentMethods in
Molecular Biology 1744
Yongfeng Guo Editor
Plant
Senescence
Methods and Protocols
M M B
ethods in olecular iology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
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Plant Senescence
Methods and Protocols
Edited by
Yongfeng Guo
Tobacco Research Institute, Chinese Academy of Agricultural Sciences,
Qingdao, Shandong, China
Editor
Yongfeng Guo
Tobacco Research Institute
Chinese Academy of Agricultural Sciences
Qingdao, Shandong, China
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-7670-6 ISBN 978-1-4939-7672-0 (eBook)
https://doi.org/10.1007/978-1-4939-7672-0
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Preface
As the final phase of plant development, senescence is of great significance for the life cycle
of plants as well as for agriculture. Plant senescence is a type of senescence distinct from
senescence processes in animals and other systems (Chapter 1), mainly because of the nutri-
ent remobilization process which, as a result of evolutionary fitness, recycles nutrients in
senescing tissues to sink organs, a critical step for yield formation in agriculture. Nutrients
in senescing organs are released through massive degradation of macromolecules such as
proteins, polysaccharides, lipids, and nucleotides. The macromolecule degradation and
nutrient remobilization processes are driven by active expression of thousands of senes-
cence-associated genes (SAGs) (Chapter 2), which makes plant senescence distinct from
many other plant developmental processes.
Besides age, senescence of plants can be induced by a great number of factors including
reproductive growth, phytohormones, and environmental cues. There are also many factors
which function to inhibit plant senescence (Chapter 2). Thus, a complex regulatory net-
work involving transduction of various signals, gene expression, and metabolism is respon-
sible for the initiation of plant senescence. Protocols related to plant senescence induced by
ethylene (Chapter 8), abscisic acid (Chapters 9 and 11), jasmonic acid, salicylic acid, strigo-
lactones, peptide hormones (Chapter 11), shade/darkness (Chapter 12), salt (Chapter 13),
pathogen (Chapter 15), oxidative stress (Chapter 16), nutritional status (Chapter 14), and
postharvest conditions (Chapter 17) are summarized in this volume.
Once senescence is initiated, plants undergo a similar senescence execution process, the
so-called senescence syndrome. Protocols commonly used in characterizing molecular,
cellular, and biochemical aspects of the senescence syndrome are included in this volume.
These are methods in studying chlorophyll catabolism (Chapter 18), programmed cell death
(Chapter 21), cell wall polysaccharides (Chapter 25), vacuolar proteases (Chapter 22),
chloroplast protein degradation (Chapter 24), and autophagy (Chapter 23). Methods for
studying different types of senescence, including leaf senescence (Chapter 3), flower
senescence (Chapter 4), fruit ripening (Chapter 7), nodule senescence (Chapter 5), and
floral organ abscission (Chapters 6 and 26), are described. Also included are techniques for
studying plant senescence in general, such as gene identification and functional analysis
(Chapter 19), MicroRNAs (Chapter 20), map-based cloning (Chapter 17), virus-induced
gene silencing (Chapter 4), electron microscopy (Chapter 26), transcriptome (Chapter 27),
and metabolome (Chapter 28).
The techniques covered in this volume are either developed specially for plant senes-
cence studies or optimized for studying senescing plants. Every method includes not only
a detailed description of the procedures but also thoughtful notes for correct interpreta-
tion, special cautions, or alternative options. The aim is to provide a useful handbook of
standard protocols for plant molecular biologists working on senescence.
Qingdao, Shandong, China Yongfeng Guo
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Part I IntroductIon of Plant SeneScence
1 Concepts and Types of Senescence in Plants .............................. 3
Susheng Gan
2 Initiation, Progression, and Genetic Manipulation of Leaf Senescence........... 9
Akhtar Ali, Xiaoming Gao, and Yongfeng Guo
Part II P henotyPIc analySIS and Molecular MarkerS
of Plant organ SeneScence
3 Phenotypic Analysis and Molecular Markers of Leaf Senescence................ 35
Liming Zhao, Yan Xia, Xiao-Yuan Wu, Jos H.M. Schippers,
and Hai-Chun Jing
4 Investigation of Petal Senescence by TRV-Mediated Virus-Induced
Gene Silencing in Rose.............................................. 49
Chenxia Cheng, Junping Gao, and Nan Ma
5 Phenotypic Analysis and Molecular Markers of Plant Nodule Senescence......... 65
Xiuying Xia
6 Quantitative Analysis of Floral Organ Abscission in Arabidopsis
Via a Petal Breakstrength Assay........................................ 81
Chun-Lin Shi and Melinka A. Butenko
7 Characterization of Climacteric and Non-Climacteric Fruit Ripening ........... 89
Xiaohong Kou and Mengshi Wu
Part III horMonal control of Plant SeneScence
8 Ethylene Treatment in Studying Leaf Senescence in Arabidopsis ............... 105
Zhonghai Li and Hongwei Guo
9 The Assay of Abscisic Acid-Induced Stomatal Movement in Leaf Senescence...... 113
Yanyan Zhang and Kewei Zhang
10 The EPR Method for Detecting Nitric Oxide in Plant Senescence.............. 119
Aizhen Sun
11 Hormone Treatments in Studying Leaf Senescence......................... 125
Zenglin Zhang and Yongfeng Guo
Part IV StreSS-Induced SeneScence In PlantS
12 Methods to Study Darkness-Induced Leaf Senescence....................... 135
Yi Song and Lin Li
vii
viii Contents
13 Salt Treatments and Induction of Senescence ............................. 141
Yasuhito Sakuraba, Dami Kim, and Nam-Chon Paek
14 Methods for Elucidation of Plant Senescence in Response
to C/N-Nutrient Balance............................................ 151
Shoki Aoyama, Junji Yamaguchi, and Takeo Sato
15 Study of Cotton Leaf Senescence Induced by Alternaria alternata Infection ..... 161
Wei Liu, Wenwei Zhang, Na Zheng, Weibo Zhai, and Fangjun Qi
16 Study of Hydrogen Peroxide as a Senescence-Inducing Signal................. 173
Stefan Bieker, Maren Potschin, and Ulrike Zentgraf
17 Identification of Postharvest Senescence Regulators Through Map-Based
Cloning Using Detached Arabidopsis Inflorescences as a Model Tissue .......... 195
Donald A. Hunter, Rubina Jibran, Paul Dijkwel, David Chagné,
Kerry Sullivan, Aakansha Kanojia, and Ross Crowhurst
Part V Molecular and cellular ProceSSeS In Plant SeneScence
18 Chlorophyll and Chlorophyll Catabolite Analysis by HPLC................... 223
Aditi Das, Luzia Guyer, and Stefan Hörtensteiner
19 Identification and Functional Analysis of Senescence-Associated Genes in Wheat... 237
Geng Wang, Ke Li, and Chunjiang Zhou
20 Isolation, Purification, and Detection of Micro RNAs in Plant Senescence........ 247
Yujun Ren and Ying Miao
21 In Situ Detection of Programmed Cell Death in Senescing
Nicotiana tabacum Leaves Using TUNEL Assay........................... 267
Branka Uzelac, Dušica Janošević, and Snežana Budimir
22 Activities of Vacuolar Cysteine Proteases in Plant Senescence.................. 283
Dana E. Martínez, Lorenza Costa, and Juan José Guiamét
23 Study of Autophagy in Plant Senescence................................. 299
Xuefei Cui, Jing Zheng, Jinxin Zheng, and Qingqiu Gong
24 Isolation of Chloroplasts for In Organelle Protein Degradation Assay ........... 307
Scott W. Vande Wetering and Judy A. Brusslan
25 Plant Cell Walls: Isolation and Monosaccharide Composition Analysis........... 313
Yingzhen Kong, Malcolm O’Neill, and Gongke Zhou
26 Visualizing Morphological Changes of Abscission Zone Cells
in Arabidopsis by Scanning Electron Microscope........................... 321
Chun-Lin Shi and Melinka A. Butenko
Part VI SySteMS BIology In Plant SeneScence
27 RNA-Seq Analysis of the Transcriptome of Leaf Senescence in Tobacco ......... 331
Wei Li and Yongfeng Guo
28 Comprehensive Metabolomics Studies of Plant Developmental Senescence....... 339
Mutsumi Watanabe, Takayuki Tohge, Salma Balazadeh, Alexander Erban,
Patrick Giavalisco, Joachim Kopka, Bernd Mueller-Roeber,
Alisdair R. Fernie, and Rainer Hoefgen
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Contributors
akhtar alI • Tobacco Research Institute, Chinese Academy of Agricultural Sciences,
Qingdao, Shandong, China; Nuclear Institute for Food and Agriculture, Peshawar,
Pakistan
ShokI aoyaMa • Faculty of Science and Graduate School of Life Science, Hokkaido
University, Sapporo, Japan
SalMa Balazadeh • Max Planck Institute of Molecular Plant Physiology,
Potsdam-Golm, Germany; Institute of Biochemistry and Biology, University of Potsdam,
Potsdam-Golm, Germany
Stefan BIeker • ZMBP, Department of General Genetics, University Tuebingen,
Tuebingen, Germany
Judy a. BruSSlan • Department of Biological Sciences, California State University,
Long Beach, CA, USA
Snežana BudIMIr • Institute for Biological Research “Siniša Stanković”,
University of Belgrade, Belgrade, Serbia
MelInka a. Butenko • Department of Molecular Biosciences, University of Oslo, Oslo,
Norway
daVId chagné • The New Zealand Institute for Plant & Food Research Limited,
Food Industry Science Centre, Palmerston North, New Zealand
chenxIa cheng • Department of Ornamental Horticulture, China Agricultural
University, Beijing, China; Beijing Key Laboratory of Development and Quality Control
of Ornamental Crops, China Agricultural University, Beijing, China
lorenza coSta • Instituto de Fisiología Vegetal (INFIVE)—Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de La Plata,
Buenos Aires, Argentina
roSS crowhurSt • The New Zealand Institute for Plant & Food Research Limited, Mt.
Albert Research Centre, Auckland, New Zealand
xuefeI cuI • Tianjin Key Laboratory of Protein Science, College of Life Sciences,
Nankai University, Tianjin, China; Department of Plant Biology and Ecology, College of
Life Sciences, Nankai University, Tianjin, China
adItI daS • Institute of Plant and Microbial Biology, University of Zurich, Zurich,
Switzerland
Paul dIJkwel • Institute of Fundamental Sciences, Massey University, Palmerston North,
New Zealand
alexander erBan • Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm,
Germany
alISdaIr r. fernIe • Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm,
Germany
SuSheng gan • Plant Biology Section, School of Integrative Plant Science, College of
Agriculture and Life Sciences, Cornell University, Ithaca, NY, USA
xIaoMIng gao • Tobacco Research Institute, Chinese Academy of Agricultural Sciences,
Qingdao, Shandong, China
ix
x Contributors
JunPIng gao • Department of Ornamental Horticulture, China Agricultural University,
Beijing, China; Beijing Key Laboratory of Development and Quality Control of
Ornamental Crops, China Agricultural University, Beijing, China
PatrIck gIaValISco • Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm,
Germany
QIngQIu gong • Tianjin Key Laboratory of Protein Science, College of Life Sciences,
Nankai University, Tianjin, China; Department of Plant Biology and Ecology, College of
Life Sciences, Nankai University, Tianjin, China
Juan JoSé guIaMét • Instituto de Fisiología Vegetal (INFIVE)—Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de La Plata,
Buenos Aires, Argentina
yongfeng guo • Tobacco Research Institute, Chinese Academy of Agricultural Sciences,
Qingdao, Shandong, China
hongweI guo • The State Key Laboratory of Protein and Plant Gene Research, College of
Life Sciences, Peking-Tsinghua Center of Life Sciences, Peking University, Beijing, China;
Department of Biology, South University of Science and Technology of China, Shenzhen,
Guangdong, China
luzIa guyer • Institute of Plant and Microbial Biology, University of Zurich, Zurich,
Switzerland
raIner hoefgen • Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm,
Germany
Stefan hörtenSteIner • Institute of Plant and Microbial Biology, University of Zurich,
Zurich, Switzerland
donald a. hunter • The New Zealand Institute for Plant & Food Research Limited,
Food Industry Science Centre, Palmerston North, New Zealand
dušIca JanošeVIć • Institute of Botany and Botanical Garden “Jevremovac”, Faculty of
Biology, University of Belgrade, Belgrade, Serbia
ruBIna JIBran • The New Zealand Institute for Plant & Food Research Limited,
Food Industry Science Centre, Palmerston North, New Zealand; Institute of
Fundamental Sciences, Massey University, Palmerston North, New Zealand
haI-chun JIng • Key Laboratory of Plant Resources, Institute of Botany, Chinese
Academy of Sciences, Beijing, China
aakanSha kanoJIa • Institute of Fundamental Sciences, Massey University,
Palmerston North, New Zealand
daMI kIM • Department of Plant Science, Plant Genomics and Breeding Institute,
Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul,
Republic of Korea
yIngzhen kong • Tobacco Research Institute, Chinese Academy of Agricultural Sciences,
Qingdao, Shandong, China
JoachIM koPka • Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm,
Germany
xIaohong kou • School of Chemical Engineering and Technology, Tianjin University,
Tianjin, China
lIn lI • Institute of Plant Biology, State Key Laboratory of Genetic Engineering, School of
Life Sciences, Fudan University, Shanghai, China
ke lI • College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei, China
weI lI • Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao,
Shandong, China
Contributors xi
zhonghaI lI • The State Key Laboratory of Protein and Plant Gene Research, College of
Life Sciences, Peking-Tsinghua Center of Life Sciences, Peking University, Beijing, China
weI lIu • State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of
Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
nan Ma • Department of Ornamental Horticulture, China Agricultural University,
Beijing, China; Beijing Key Laboratory of Development and Quality Control of
Ornamental Crops, China Agricultural University, Beijing, China
dana e. Martínez • Instituto de Fisiología Vegetal (INFIVE)—Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de La Plata,
Buenos Aires, Argentina
yIng MIao • Center for Molecular Cell and Systems Biology, College of Life Sciences,
Fujian Agriculture and Forestry University, Fuzhou, Fujian, China
Bernd Mueller-roeBer • Max Planck Institute of Molecular Plant Physiology,
Potsdam-Golm, Germany; Institute of Biochemistry and Biology, University of Potsdam,
Potsdam-Golm, Germany
MalcolM o’neIll • Complex Carbohydrate Research Center, University of Georgia,
Athens, GA, USA
naM-chon Paek • Department of Plant Science, Plant Genomics and Breeding Institute,
Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul,
Republic of Korea
Maren PotSchIn • ZMBP, Department of General Genetics, University Tuebingen,
Tuebingen, Germany
fangJun QI • State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute
of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
yuJun ren • Center for Molecular Cell and Systems Biology, College of Life Sciences,
Fujian Agriculture and Forestry University, Fuzhou, Fujian, China
yaSuhIto SakuraBa • Department of Plant Science, Plant Genomics and Breeding
Institute, Research Institute of Agriculture and Life Sciences, Seoul National University,
Seoul, Republic of Korea
takeo Sato • Faculty of Science and Graduate School of Life Science, Hokkaido University,
Sapporo, Japan
JoS h.M. SchIPPerS • Key Laboratory of Plant Resources, Institute of Botany, Chinese
Academy of Sciences, Beijing, China; Molecular Ecology, Institute for Biology I, RWTH
Aachen University, Aachen, Germany
chun-lIn ShI • Department of Molecular Biosciences, University of Oslo, Oslo, Norway
yI Song • Institute of Plant Biology, State Key Laboratory of Genetic Engineering, School of
Life Sciences, Fudan University, Shanghai, China
kerry SullIVan • The New Zealand Institute for Plant & Food Research Limited, Food
Industry Science Centre, Palmerston North, New Zealand
aIzhen Sun • The National Key Laboratory of Plant Molecular Genetics and National
Center for Plant Gene Research (Shanghai), CAS Center for Excellence in Molecular
Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai, China
takayukI tohge • Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm,
Germany
Branka uzelac • Institute for Biological Research “Siniša Stanković”, University of
Belgrade, Belgrade, Serbia
