Characterization of ethylene-induced abscission in miniature rose (Rosa hybrida L.) [Elektronische Ressource] / von Noorollah Ahmadi

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Characterization of Ethylene-Induced Abscission in Miniature Rose (Rosa hybrida L.) Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades Doktor der Gartenbauwissenschaften (Dr. rer. hort.) genehmigte Dissertation von Noorollah Ahmadi, M. Sc. Horticulture geboren am 11. September 1969 in Eghlid, IRAN 2008 Referent: Prof. Dr. Margrethe Serek Korreferent: Prof. Dr. Traud Winkelmann Tag der Promotion: 04. 12. 2008 Dedicated to my family Acknowledgements To the Almighty God, “ALLAH” Who have granted me everything and Who have blessed and supported me by His power in all my life, as well as giving me the opportunity to continue the study and complete this dissertation. During my stay in Germany for doing my PhD, I got an opportunity to study and learn many things. This dissertation could have never been completed without the help of so many generous people. I express my thanks and gratitude to all people who helped me, but their names are not mentioned here. I would like to express my upmost gratitude to my supervisor, Prof. Dr. Margrethe Serek. The excellent supervision and supports that she gave truly help the progression of this research project. Her poignant comments throughout the preparation of this thesis will never be forgotten. My deepest thanks go to my co-supervisor, Dr.

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Characterization of Ethylene-Induced Abscission in
Miniature Rose (Rosa hybrida L.)



Von der
Naturwissenschaftlichen Fakultät der
Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades



Doktor der Gartenbauwissenschaften
(Dr. rer. hort.)



genehmigte Dissertation


von
Noorollah Ahmadi, M. Sc. Horticulture
geboren am 11. September 1969 in Eghlid, IRAN


2008














Referent: Prof. Dr. Margrethe Serek
Korreferent: Prof. Dr. Traud Winkelmann
Tag der Promotion: 04. 12. 2008














Dedicated to my family
Acknowledgements

To the Almighty God, “ALLAH” Who have granted me everything and Who have blessed
and supported me by His power in all my life, as well as giving me the opportunity to
continue the study and complete this dissertation. During my stay in Germany for doing
my PhD, I got an opportunity to study and learn many things. This dissertation could have
never been completed without the help of so many generous people. I express my thanks
and gratitude to all people who helped me, but their names are not mentioned here.
I would like to express my upmost gratitude to my supervisor, Prof. Dr. Margrethe Serek.
The excellent supervision and supports that she gave truly help the progression of this
research project. Her poignant comments throughout the preparation of this thesis will
never be forgotten.
My deepest thanks go to my co-supervisor, Dr. Heiko Mibus-Schoppe, for his masterful
guidance and patience getting me introduced to the field of molecular genetics. I can say
without any doubt that his supports and suggestions inspired me to pursue a high level of
rigor during the course of this research.
I am grateful to Prof. Dr. Traud Winkelmann for consenting to be a co-referee as well as
for her support and comments. Thanks also go to Prof. Dr. Hans-Michael Poehling and
Prof. Dr. Bernhard Beßler for their kindness of being members of examination committee.
I am indebted to Iranian Ministry of Science, Research and Technology for awarding the
grant for this Ph.D. project. The technical assistance of Annette Steding, Wiltrud von
Oertzen, Gisela Bölsing, staff members of Floriculture Section, is sincerely appreciated.
Additionally, I am thankful to Herbert Geyer and his team in the greenhouse for supplying
and taking care of the plant material. Many thanks go to all my colleagues and office mates
who have been around these five years for their friendship and providing great atmosphere
to work in laboratory and office.
I am deeply grateful to my parents, Ebrahim Ahmadi and Golafshan Nasiri, for everything
they have done to support me in achieving my goal. I should also like to express my great
gratitude to my brothers and sisters who had to deal without me, with several family
occasions.
Last but not least, my sincere gratitude is extended to my wife, Narges Nasiri, for her
support and patience, especially for caring Navid and Nastaran who were born during my
study in Germany.
i
Table of Contents

Acknowledgements.........................................................................................................i
List of figures................................................................................................................ iv
Abbreviations................................................................................................................ v
Abstract........................................................................................................................ vii
1. Introduction..................................................................................1
2. Literature Review.........................................................................5
2.1 Senescence processes..............................................................................................5
2.2 Abscission...............................................................................................................6
2.2.1 Abscission phenomena.......................................................................................6
2.2.2 Abscission behavior of different plant organs...................................................7
2.2.2.1 Leaf and leaflet abscission.........................................................................7
2.2.2.2 Flower and flower parts abscission............................................................9
2.2.2.3 Fruit abscission...........................................................................................9
2.2.2.4 Other organs abscission............................................................................10
2.3 Ethylene synthesis and signal transduction pathway.......................................11
2.3.1 Ethylene synthesis............................................................................................11
2.3.2 Ethylene perception and signal transduction pathway.....................................12
2.3.3 Induction and regulation of ethylene biosynthesis...........................................15
2.3.3.1 Plant growth regulators............................................................................16
2.3.3.2 Environmental stresses.............................................................................16
2.3.3.3 Climacteric ethylene.................................................................................17
2.3.3.4 Pollination-induced senescence................................................................17
2.4 Ethylene, senescence and abscission.....................................................................20
2.4.1 Ethylene and senescence..................................................................................20
2.4.2 Ethylene and abscission....................................................................................21
ii
2.5 Non ethylene related senescence...........................................................................23
3. Publications....................................................................................26
Paper ..............................................................................................................................26
Ahmadi, N., Mibus, H. and Serek, M. (2008). Isolation of an ethylene induced
putative nucleotide laccase in miniature roses (Rosa hybrida L.). J. Plant Growth
Regul. 27:320–330.
Manuscript 1...................................................................................................................37
Characterization of ethylene-induced organ abscission in F1 breeding lines of
miniature roses (Rosa hybrida L.). Postharvest Biology and Technology (in press)
Manuscript 2...................................................................................................................52
Functional analysis of rose laccase (RhLAC) gene in tobacco plant by virus-induced
gene silencing technique. (in preparation for submission)
Conference paper...........................................................................................................61
Identification of ethylene induced genes in abscission zone of Rosa hybrida L. by use
thof differential display. 9 International Symposium on Postharvest Quality of
Ornamental Plants 10-14.06.2008, Odense, Denmark. Acta Horticulture (in press)
Poster...............................................................................................................................66
Isolation of ethylene induced putative nucleotide laccase in miniature roses (Rosa
hybrida L.). Gordon Research Conference on Plant Senescence 15-20.06.2008, Mount
Holyoke College South Hadley, MA, USA.
4. Discussion and Outlook................................................................68
4.1 Evaluation of leaves and buds abscission induced by exogenous ethylene.........68
4.2 Leaves yellowing and chlorophyll degradation.....................................................69
4.3 Expression of RhETR1, RhETR3, RhCTR1 and RhCTR2...................................70
4.4 Isolation of ethylene induced cDNAs......................................................................72
4.5 Induction, isolation and characterization of laccase gene....................................73
4.6 Laccase expression in various organs and genotypes...........................................74
4.7 Functional analysis of RhLAC by virus-induced gene silencing...........................75
4.8 Outlook......................................................................................................................79
5. References.......................................................................................82
iii
List of Figures
Fig. 1. Diagrammatic view of tissues in a typical leaf abscission zone............................7
Fig. 2. Biosynthesis pathway and regulation of ethylene................................................11
Fig. 3. The structural features of ethylene receptor gene family.....................................13
Fig. 4. Model for the transduction of the ethylene signal................................................14
Fig. 5. Mechanisms to control of ethylene effects..........................................................15


iv

Abbreviations

ABA abscisic acid
ACC 1-amino cyclopropane-1-carboxylic acid
ACO 1-aminocyclopropane-1-carboxylate oxidase
ACS 1-aminocyclopropane-1-carboxylate synthase
AVG aminoethoxyvinylglycine
bp base pair
°C degree Celsius
cDNA complementary DNA
CTR constitutive triple response
cv. cultivar
DDRT-PCR differential-display RT-PCR
EIL EIN3-like
EIN ethylene insensitive
ERF ethylene response factor
ERS ethylene response sensor
ETR ethylene resistant
etr ethylene resistant mutant
h hour
IAA indoleacetic acid
LAC laccase
1-MCP 1-methylcyclopropene
MAP mitogen-activated protein
min minute
mRNA messenger RNA
v

NCED 9-cis-epoxycarotenoid dioxygenase
PCD programmed cell death
PCR polymerase chain reaction
PDS phytoene desaturase
PVX potato virus X
QRT-PCR quantitative reverse transcriptase real-time PCR
RACE rapid amplification of cDNA ends
RNA ribose nucleic acid
RT-PCR reverse transcriptase PCR
s second
SAG senescence-associated gene
SAM S-adenosyl-methionine
STS silver thiosulfate
TDZ thidiazuron
TRV tobacco rattle virus
VIGS virus-induced gene silencing


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Zusammenfassung

Charakterisierung der ethyleninduzierten Abscission bei
Topfrosen (Rosa hybrida L.)
Ethylen zeigt bei Topfrosen viele Effekte, die zu einer Verringerung der
Nacherntequalität führen: zum Beispiel das Abwerfen von Blättern, die
Blütenseneszenz und den Verlust von Knospen und Blüten. Um ethyleninduzierte Gene
zu isolieren wurde eine Differential Display PCR bei ethylenbehandelten Topfrosen der
Sorte ’Lavender’ durchführt. Insgesamt konnten 88 hochregulierte und 72
herunterregulierte Gene detektiert werden. 17 der hochregulierten cDNA Fragmente
konnten nach Klonierung und anschließender Sequenzierung verifiziert werden. Sieben
cDNA Fragmente zeigten keine Homologie zu bekannten Sequenzen aus Datenbanken
und fünf Fragmente zeigten eine Homologie zu Genen aus Bakterien, Pilzen oder
Menschen. Von den insgesamt fünf ethyleninduzierten cDNAs wurden drei cDNAs aus
Blattstielen und zwei cDNAs aus Blütenstielen isoliert. Zur Analyse der relativen
Expression dieser Gene wurde die qRT-PCR eingesetzt. Die relative Expression von
allen cDNAs war in den Blütenstielen höher als in den Blattstielen. Die vorliegende
Arbeit beschreibt ein ethyleninduziertes, laccasehomologes Gen, das als RhLAC
bezeichnet wurde. Die komplette cDNA Sequenz des Gens RhLAC beträgt 2005 bp und
codiert für ein mögliches Protein mit 573 Aminosäuren, die 3 konservierte Sequenzen
einer Multi-Kupfer-Oxidase Familien aufweisen. Die Analyse der abgeleiteten
Aminosäuresequenz dieses Gens zeigt eine Homologie von 58% zu einer möglichen
Laccase aus Zea mays (Akzessionsnummer CAJ30499) und eine Homologie von 56%
zu der Laccase 15 (Akzessionsnummer NP_100621) aus Arabidopsis thaliana. Durch
Southern Hybridisierungen konnten mehrere Kopien des Gens RhLAC in diploiden und
tetraploiden Rosen nachgewiesen werden. Die Expression des Gens RhLAC wurde in
unterschiedlichen Geweben untersucht. Die höchste relative Expression des Gens
RhLAC konnte in der Abscissionszone der Blätter nachgewiesen werden, und zwar in
dem basalen Bereich des Blattstiels (Pulvinus) (LANZ) und in der Abscissionszone der
Knospe, einem kleinen Segment des Blütenstiels (BANZ). Die geringste relative
Expression wurde in den Blättern, in den Blattstielen bzw. den Blütenstielen gefunden.
Um die Funktion der Laccase zu charakterisieren wurde die Translation des RhLAC-
homologen Gens bei Nicotiana benthamiana mittels Virus-Induced Gene Silencing
(VIGS), durch die Inokulation mit Agrobacterium tumefaciens, die mit einem Tabbacco
Rattle Virus (TRV) transformiert wurden, unterdrückt. Fünf Tage nach der Infiltration
wurden die Pflanzen mit Ethylen behandelt. Sowohl die Unterdrückung der
Transkription des laccasehomologen Gens als auch die Ethylenbehandlung reduzierten
die Pflanzenhöhe, die gesamte Blattfläche und die Anzahl der Blätter pro Pflanze. Die
Ethylenbehandlung beeinflusste nicht die Anzahl der Blätter bei den Pflanzen mit
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