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Unformatted text preview: Efficacy of new inhibitors of ethylene perception in improvement of
the display quality of miniature potted roses (Rosa hybrida L.) Der Naturwissenschaftlichen Fakultät
der Universität Hannover
des akademischen Grades eines Doktors der Gartenbauwissenschaften
-Dr. rer. hort.- genehmigte
Mantana BUANONG, M. Sc. Postharvest Technology
geboren am 8. November 1973 in Khonkaen, Thailand Oktober 2005 Referent: Prof. Dr. Margrethe Serek
Korreferent: Prof. Dr. Thomas Debener
Date of oral examination: 12th January 2006 Dedicated to
my beloved brother Acknowledgements
This thesis presents work supported by a grant from The Asian Development Bank (ADB),
Thailand and I am very grateful that I stood a chance to conduct research in the postharvest field
combined with the modern methods of molecular biology.
I would like to express my special thanks to my supervisor Prof. Dr. Margrethe Serek, a
distinguished professor in the field of postharvest physiology of ornamental crops, for her
untiring guidance and her constructive attitude throughout the course of study and during the
preparation of this thesis.
I would also like to thank Prof. Ed Sisler – University of North Carolina, USA, for kindly
providing the chemicals (1-OCP and 1-DCP) used in this research and his suggestion about
preparing these substances.
I am very grateful to Dr. Heiko Mibus, who introduced me to this part of molecular biology. His
insight into molecular biology and many fruitful discussions led to the idea of the molecular part
of the study; his help and advice during my study was invaluable. Special thanks to other staff
members of the Floriculture Section for their kind assistance and creating a positive atmosphere
during the three-year period. Moreover, I thank my friends for their support that made this study
and the entire stay in Germany enjoyable.
Finally, I would like to thank my parents, my brother and sister, together with my boyfriend, who
contributed to the success of my research. Their encouragement enabled me to successfully
complete this Doctoral study. ii Table of contents
List of Figures…………………………………………………………………………………..….vi
List of Appendices………………………………………………………………..........................viii
1. General introduction………………………………………………………………..……….…..1
1.1. Ethylene and flower senescence……...………………………………………….....................1
1.2. Exogenous ethylene……………………...……………………………………........................2
1.3. Endogenous ethylene……………………………...………………………………………......3
1.4. Pollination-induced senescence…………………………...………………………………......3
1.5. Non-ethylene mediated senescence…………...…………………………………………....…4
1.6. Ethylene biosynthesis……...……………………………………………………………….…5
1.6.2. ACC synthase and ACC oxidase............................................................................................6
1.7. Strategies to breed ethylene-insensitive flower….………………….....……………………...7
1.8. Ethylene sensitivity…………………………………………………………………………....8
1.9. Statement of problem…..…………...………………………………………………………9
2. Efficacy of new inhibitors of ethylene perception in improvement of display quality of
miniature potted roses (Rosa hybrida L.)………………………………………………………...11
2.1.1. Compounds interacting with the ethylene receptor in plants…………………………........12
18.104.22.168. Ethylene and ethylene analogues……………...…………………………………………12
22.214.171.124. Competitive ethylene antagonists……………………...……………...…………………13
2.1.2. Mode of action of compounds blocking the receptor…………………………………...…13
2.1.3. Ethylene antagonists inactivating the receptor for an extend period of time…………...….14
2.2. Materials and Methods………………...……………………………………………………..19
2.2.1. Chemicals……………………………………...…………………………………………...19 iii Table of contents
2.2.2. Plant material………………………………………………………………………………19
2.2.3. Effects of concentration of 1-OCP and 1-DCP on display quality………………………...20
2.2.4. Effects of treatment time on 1-OCP and 1-DCP on display quality……………………….20
2.2.5. Effects of temperature on efficacy of 1-OCP and 1-DCP on display quality……...………21
2.2.6. Comparison of effectiveness of 1-MCP, 1-OCP and 1-DCP between ‘Lavender’ and
2.2.7. Experimental design and statistics…...…………………………………………………….21
2.3.1. Effect of concentrations of 1-OCP and 1-DCP on display quality…………...……………22
2.3.2. Effect of exposure time of 1-OCP and 1-DCP on display quality…………………...…….24
2.3.3. Effect of temperature of 1-OCP and 1-DCP on display quality……………………......….26
2.3.4. Efficacy of 1-MCP, 1-OCP and 1-DCP in cultivars ‘Lavender’ and ‘Vanilla’...………….28
3. Expression analysis of genes for ethylene biosynthesis enzyme, ethylene perception and signal
transduction pathway after pretreatment with ethylene inhibitors of miniature potted roses (Rosa
3.1.1. Expression regulation of genes for ethylene biosynthesis enzyme…...………………...….35
3.1.2. Ethylene receptor gene expression………………….....……………..……………………36
3.1.3. Ethylene insensitive mutant of ethylene receptor……………………………...…………..40
3.1.4. Expression regulation of ethylene signal transduction pathway…………...………………42
3.2. Materials and Methods…...…………………………………………………………………..49
3.2.1. Plant material…...………………………………………………………………………….49
3.2.2. Database analyses and primer design…...…………………………………………………49
3.2.3. DNA isolation and PCR……………..…………………………………….……………49
3.2.4. Cloning, DNA sequencing and sequence Analysis……………...………………………...50
3.2.5. RNA Isolation and RT-PCR…………………...…………………………………………..51
3.2.6. Northern blot hybridization……………...………………………………………………...53
3.2.7. DIG labeling of the probes for northern blot hybridization………………………………..54
iv Table of contents
3.2.8. Hybridization and washing conditions for northern blots…………………………………55
3.3.1 Cloning and sequence analysis of RhERF1……………………………………………...…56
3.3.2 Expression patterns of genes for the ethylene biosynthesis enzyme, ethylene perception and
signal transduction pathway after pretreatment with ethylene receptor inhibitors……………….57
Appendices…………………………………………………………………………………......…91 v List of Figures
Fig. 1. Ethylene biosynthesis pathway…………………………………………………………….5
Fig. 2. Propose model of action of ethylene and 1-methylcyclopropene (1-MCP) on the ethylene
Fig. 3. Chemical structure of compounds interacting with the ethylene receptor………………..18
Fig. 4. Mean of percent leaf drop (A, B) and total bud and flower drop (C, D) of miniature potted
roses cultivar ‘Lavender’ pretreated with 1-OCP and 1-DCP at concentrations of 0, 200, 500,
1000, 2000 nl l-1, 1-MCP (200 nl l-1) for 6 h at 20 oC and untreated controls….......…..………...23
Fig. 5. Mean of percent leaf drop (A, B) and total bud and flower drop (C, D) of miniature potted
roses cultivar ‘Lavender’ pretreated with 1-OCP and 1-DCP at exposure time of 1-OCP and
1-DCP (1000 nl l-1) 0, 2, 4, 6, 12 h, 1-MCP (200 nl l-1) for 6 h at 20 oC and untreated controls.
Fig. 6. Mean of percent leaf drop (A, B) and total bud and flower drop (C, D) of miniature potted
roses cultivar ‘Lavender’ pretreated with 1-OCP and 1-DCP at temperatures of 5 oC, 10 oC, 15 oC,
20 oC, 1-MCP (200 nl l-1) for 6 h at 20 oC and untreated controls……………...………………..27
Fig. 7. Mean of percent leaf drop and days of total bud and flower drop of miniature potted roses
cultivar ‘Vanilla’ (A, C) and ‘Lavender’ (B, D) pre-treated with 1-OCP, 1-DCP (1000 nl l-1),
respectively, and 1-MCP (200 nl l-1) for 4 h at 20 oC and the control……………………………29
Fig.8. Basis scheme of the two-component system…………………………………….………...37
Fig. 9. The Arabidopsis ethylene receptor family……………………………………..………......38
Fig.10. A current view of the ethylene signal transduction pathway formulated on the basis of clone
Fig. 11. A model for the role of kinase activity in ethylene signaling……………………………44
Fig. 12. Alignment of the DNA-binding domains of RhERF1 in comparison with other ERF
Fig. 13. Percent leaf drop of miniature potted roses cultivar ‘Vanilla’ (A) and ‘Lavender’ (B)
after day 9 pretreated with 200 nl l -1 f or 6 h and 1000 nl l -1 1 -OCP and 1-DCP for 4 h at
20oC………………………………………………………………………………………………57 vi List of Figures
Fig.14. Expression of genes for ethylene biosynthesis enzyme, ethylene perception and other
ethylene related genes in miniature potted roses cultivar ‘Vanilla’ in untreated plant and
pretreatment with 1-MCP, 1-OCP and 1-DCP after 9 days of continuous exposure of exogenous
ethylene and without ethylene treatment…………………………………………………………58
Fig.15. Expression of genes for ethylene biosynthesis enzyme, ethylene perception and other
ethylene related genes in miniature potted roses cultivar ‘Vanilla’ in untreated plant and
pretreatment with 1-MCP, 1-OCP and 1-DCP after 9 days of continuous exposure of exogenous
ethylene and without ethylene treatment…………………………………………………………60
Fig.15. Expression of genes for ethylene biosynthesis enzyme, ethylene perception and other
ethylene related genes in miniature potted roses cultivar ‘Lavender’ in untreated plant and
pretreatment with 1-MCP, 1-OCP and 1-DCP after 9 days of continuous exposure of exogenous
ethylene and without ethylene treatment…………………………………………………………62 vii List of Appendices
A 1 Degenerate primer pairs for RhERF1 using partial DNA fragment of Arabidopsis
A 2 Sequence analysis of Rosa hybrida 92 bp partial DNA fragment…………………………...91
A 3 Sequence analysis for specific primers of Rosa hybrida………………………………….....92
A 4 Computer programs used for analysing gene sequences………………………..…………...98
A 5 The principle of ligation into a Topo TA Cloning Kit……………………………………...101 viii Abbreviations
ACC 1-Aminocyclopropane-1-carboxylic acid AdoMed Adenosylmethionine ACO ACC oxidase ACS ACC synthase AOA Aminooxyacetic acid AVG Aminoethoxyvinylglycine BA Benzyladenine CO2 Carbondioxide CP Cyclopropene 3,3-DMCP 3,3-dimethylcyclopropene DACP Diazocyclopentadine 1-DCP 1-Decylcyclopropene DNA Deoxyribose nucleic acid DNAase Deoxyribonuclease dNTP Deoxyribonucleotide tri-phosphate E. coli Escherichia coli 1-ECP 1-Ethylcyclopropene EDTA Ethylene diamine tetra acetate GC Gas chromatograph 1-HCP 1-Hexylcyclopropene LB medium Luria-Bertani medium 1-MCP 1-Methylcyclopropene 3-MCP 3-Methylcyclopropene NCBI National center for biotechnology information Nr Never ripe (LeETR3) 1-OCP 1-Octylcyclopropene RH Relative humidity RNA Ribose nucleic acid RNase Ribonuclease RT-PCR Reverse transcriptase polymerase chain reaction
ix SAM S-adenosyl methionine STS Silver thiosulfate TAE buffer Tris-Acetate-EDTA buffer Taq Thermus aquaticus Tris tris-(hydroxymethyl) aminomethane Genes/protein:
The symbol for genes is shown in italics and capitals (e.g. ETR1). The name of a mutation in a
gene is italicized but not capitalized, e.g. etr1-1. The symbol for the protein encoded by the gene
uses the same letters and numbers and is capitalized, but not italicized (e.g. ETR1)
ACO ACC oxidase ACS ACC synthase CTR Constitutive triple response EIL EIN-like EIN Ethylene insensitive ERF Ethylene response factor ERS Ethylene response sensor ETR Ethylene resistant x Kurzfassung
Wirkung von neuen Inhibitoren der Ethlyenperzeption zur Verbesserung der
Haltbarkeit von Topfrosen (Rosa hybrida L.)
Ethylen ist ein wichtiges Nachernteproblem bei Topfrosen. So kommt es bei der Vermarktung durch das
Verwelken der Blüten oder durch den Abwurf der Knospen, der Blüten und der Blätter zu
Qualitätseinbußen. In dieser Arbeit wird die Wirkung und ein möglicher praktischer Einsatz von 1-MCP
(1-Methylcyclopropen) Analogen, wie 1-OCP mit einer 8er-Carbon-Kette in der ersten Position und
1-DCP mit einer 10er-Carbon-Kette in der ersten Position für Topfrosen getestet. Die Sorte ’Lavender’
Kordana mit einer hohen Ethylensitivität von Rosen Kordes, Sparrishoop, wurde, um die besten
Bedingungen zu finden, im verkaufsfertigen Stadium mit unterschiedlichen Konzentrationen von 1-OCP,
1-DCP oder 1-MCP behandelt und anschließend, für das gesamte Experiment, kontinuierlich exogenem
Ethylen ausgesetzt. Die Sorte ’Lavender’ wurde dann mit der ethylen-tolerante Sorte ’Vanilla’ verglichen.
Der Versuch wurde vollkommen randomisiert mit 3 Wiederholungen und je 3 Töpfen pro Wiederholung
durchgeführt. Ausgewertet wurde der Verlust der Blüten, Knospen und Blätter. Alle getesteten
Konzentrationen der Chemikalien zeigten eine Wirkung im Vergleich zu den unbehandelten
Kontrollpflanzen. 1-OCP und 1-DCP zeigte die beste Wirkung bei einer Konzentration von 1000 nl l-1.
Damit war eine fünfmal höhere Konzentration, als die zum Vergleich genutzte optimale Konzentration
von 1-MCP (200 nl l-1), notwendig. Die Wirksamkeit von 1-OCP und DCP wurde in Abhängigkeit zur
Behandlungszeit und der Temperatur untersucht. Eine Behandlungszeit von 4 Stunden reichte sowohl für
1-OCP als auch 1-DCP aus, um eine optimale Haltbarkeit der Topfrosen zu erreichen. Eine längere
Behandlungszeit führte zu keiner Verbesserung. Offenbar hatte auch die Behandlung der Topfrosen bei
unterschiedlichen Temperaturen keinen Einfluss auf die Wirkung von 1-OCP und 1-DCP. So zeigten sich
keine Unterschiede in einem Temperaturbereich zwischen 10o C und 20o C. Für die molekulargenetischen
Untersuchungen wurden von der Sorte ‘Vanilla’ und ‘Lavender’ Blüten- und Blattproben nach der
Behandlung mit 1-OCP, 1-DCP und 1-MCP nach 9 Tagen entnommen. Anschließend wurde die
Expression von Genen der Ethylensynthese (5 unterschiedliche ACS Gene), der Ethylenperzeption
(5 unterschiedliche Rezeptoren) und der Signaltransduktion (4 unterschiedliche Signaltransduktrionsgene)
untersucht. Ein Signaltransduktionsgen (RhERF) wurde bei den Topfrosen erstmals isoliert. Das mögliche
Polypeptid von RhERF1 besitzt eine ERF Domain mit einer hohen Homologie zu bereits bekannten ERF
Domains. Northernhybridisierungen wurden mit den Ergebnissen der RT-PCR verglichen. Jedoch zeigte
sich, dass aufgrund der hohen Homologie zwischen den unterschiedlichen Ethylenrezeptoren eine
Unterscheidung des Hybridisierungssignals schwierig war. Die RT-PCR Analysen zeigten, dass die
Expression von allen untersuchten Genen, mit Ausnahme der Gene RhACS1-2 und RhETR4 in den
Blütenblätter der Kontrollpflanzen ’Vanilla’ und ‘Lavender’ durch Ethylen induziert wird. Dahingegen
wurde nach einer Behandlung mit den Ethylenrezeptorinhibitoren (1-OCP und 1-MCP) eine
Unterdrückung der Expression bei allen untersuchten Genen, mit und ohne Ethylen nachgewiesen. Jedoch
konnte eine starke Expression der Gene RhETR3, RhEIN3 und RhEIL nach einer Behandlung mit 1-DCP
nachgewiesen werden. In der Abwesenheit von Ethylen zeigte sich eine starke Expression in den Blättern
der ‘Vanilla’ Kontrollpflanzen bei allen Genen mit Ausnahme des Gens RhACS1. Nach der Behandlung
mit den Ethylenrezeptorinhibitoren konnten keine Transkripte des untersuchten Gens in den Blättern von
‘Vanilla’ nachgewiesen werden. In den ‘Vanilla’ Blättern konnte eine Erhöhung der RhEIN3 Transkripte
nach der Behandlung mit den Ethylenrtezeptorinhiobitoren bei exogenem Ethylen nachgewiesen.
Hingegen wurde bei den identischen Bedingungen eine Unterdrückung der RhEIN3 Expression in den
Blättern der Sorte ’Lavender’ nachgewiesen werden. Möglicherweise sind die RhEIN3 Transkripte ein
limitierender Faktor bei der Ethylensignaltransduktion. Diese könnten dann während der Regulation der
Blütenseneszenz auf transkriptionaler Ebene reguliert werden. In der Abwesenheit von Ethylen wird die
Expression der Gene RhACS1, RhETR3 und RhERF1 unterdrückt. Eine ähnliche Wirkung zeigt auch
1-OCP, es inhibiert die Expression der Gene RhACS1, RhCTR2 and RhERF1. 1-DCP inhibiert ebenfalls
die Expression der Gene RhEIN3 und RhEIL. Jedoch zeigt sich bei der Anwesenheit von Ethylen, nach
einer Behandlung mit 1-MCP eine Expressionshemmung der Gene RhACS1, RhETR4, RhCTR1-2, RhEIL xi und RhERF1. Eine ähnliche Wirkung zeigt 1-OCP. Diese Behandlung führt zu der Expressionsinhibierung
der Gene RhACS1, RhETR2, RhETR4, RhCTR1-2, RhEIN3 und RhERF1. Die Belandung mit 1-DCP
unterdrückt die Expression der Gene RhACS1 und RhETR4. Die Ergebnisse zeigen, dass die Expression
der Gene der Ethylensynthese, der Ethylenperzeption und der Signaltransduktion nach einer Behandlung
mit Ethylenrezeptorinihibitoren und bei Anwesenheit und nicht Anwesenheit von Ethylen, sowohl über
eine positive als auch negative Feedbackreaktion bei den Sorten ‘Vanilla’ and Lavender’, reguliert wird.
Dies könnte sowohl durch die Pflanzenart als auch durch das untersuchte Gewebe beeinflusst werden.
Zusammenfassend zeigen die Ergebnisse (physiologisch und molekulargenetisch), dass eine Behandlung
mit Ethylenrezeptorinihibitoren die Haltbarkeit bei Topfrosen, durch die Verhinderung des Verlustes der
Knospen, der Blüten und der Blätter, verlängert. Wobei jedoch 1-MCP eine bessere Wirkung, durch eine
mögliche Unterdrückung der Gene der Ethylensynthese, der Ethylenperzeption und der Signaltransduktion
zeigt, als 1-OCP und 1-DCP.
Schlüsselwörter: ACC Synthase, Ethylenperzeption und Signaltransduktion, Ethylen, Ethyleninhibitoren,
Haltbarkeit, Genexpression, Rosa hybrida, Seneszenz xii Abstract
Efficacy of new inhibitors of ethylene perception in improvement of display
quality of miniature potted roses (Rosa hybrida L.)
Ethylene is an important postharvest problem in miniature potted roses that leads to loss of quality during
marketing by accelerating flower senescence, and bud, flower and leaf drop. The effect of pretreatments
with analogues of 1-MCP, 1-OCP substituted with an 8-carbon chain in the 1-position and 1-DCP with a 10carbon chain, and 1-MCP were studied to possibly come up with a potential commercial approach for
improving display quality of miniature potted roses. ‘Lavender’ Kordana breeding line from Rosen
Kordes, Sparrishoop, which is sensitive to ethylene, were grown and pretreated after harvest with different
levels of 1-OCP, 1-DCP, 1-MCP or air and continuously exposed to exogenous ethylene throughout the
experimental period to find the best conditions for ‘Lavender’ as compared with ‘Vanilla’, which is
insensitive to ethylene. They were then arranged in a completely randomized design comprising 3
replications per treatment of 3 pots per replication and evaluated for bud and flower drop as well as leaf
drop. All tested levels of all chemicals were effective when compared to untreated (control) plants. 1-OCP
and 1-DCP were the most effective at concentrations 1000 nl l -1 , which was five times higher than
the concentration of 1-methylcyclopropene (1-MCP) (200 nl l-1) used as a standard. Exposure time of 4 h
for both 1-OCP and 1-DCP was sufficient to improve display life of miniature roses and longer exposures
did not have any additional beneficial effect. Apparently, exposing miniature potted roses to various
temperatures did not have an influence on the performance of both 1-OCP and 1-DCP, which were
effective at temperatures of 10oC to 20oC. Samples of ‘Vanilla’ and ‘Lavender’ petals and leaves from the
best condition of pretreatment of 1-OCP, 1-DCP and 1-MCP or air were taken for molecular studies in
investigating the expression of genes responsible for ethylene biosynthesis enzyme (5 different ACS
genes), ethylene perception (5 different receptors) and signal transduction pathway (4 different
transcription regulating genes) after 9 days of continuous exposure to exogenous ethylene. One
transcription regulating gene (RhERF1) was isolated from miniature rose. The putative polypeptide of
RhERF1 had an ERF domain, which shares high homology with other reported ERF domains. Northern
blot hybridization analysis was used to compare with results of RT-PCR. However, it was difficult to
distinguish the hybridization signal of ethylene receptors between RhETR1 and RhETR2, which were high
homolog. RT-PCR analysis revealed that ethylene induced the expression of all genes investigated in
control ‘Vanilla’ and ‘Lavender’ petals except RhACS1-2 and RhETR4, respectively, while pretreatments
of ethylene receptor inhibitors (1-OCP and 1-MCP) suppressed the expression of all genes in the presence
or absence of ethylene. However, strong expression of RhETR3, RhEIN3 and RhEIL transcripts was
detectable in ‘Lavender’ petals pretreated with 1-DCP. In the absence of ethylene, there was strong
expression of all genes in control ‘Vanilla’ leaves but no expression of RhACS1, while the accumulation of
all genes was eliminated after pretreatments of ‘Vanilla’ leaves with ethylene receptor inhibitors.
However, the level of RhEIN3 mRNA was upregulated by pretreatment of ethylene receptor inhibitors in
‘Vanilla’ leaves in the presence of ethylene while the accumulation of RhEIN3 mRNA was suppressed in
‘Lavender’ leaves. RhEIN3 transcript is probably rate-limiting for ethylene perception and signal
transduction pathway, which is regulated in leaves during flower senescence at the transcriptional level. In
the absence of ethylene, 1-MCP suppressed the expression of RhACS1, RhETR3 and RhERF1 transcripts.
Similarly, 1-OCP inhibited the expression of RhACS1, RhCTR2 and RhERF1 transcripts. Likewise, 1-DCP
inhibited the expression of RhEIN3, RhEIL genes. However, in the presence of ethylene, pretreatment
of ‘Lavender’ leaves with 1-MCP suppressed the expression of RhACS1, RhETR4, RhCTR1-2, RhEIL and
RhERF1 transcripts. Similarly, 1-OCP treatment inhibited the expression of RhACS1, RhETR2, RhETR4,
RhCTR1-2, RhEIN3 and RhERF1 transcripts. Pretreating ‘Lavender’ leaves with 1-DCP suppressed the
expression of RhACS1 and RhETR4 transcripts. These results indicated that the expression of genes for
ethylene biosynthesis enzyme, ethylene perception and signal transduction pathway was regulated by both
positive and negative feedback regulation mechanism in ‘Vanilla’ and Lavender’ pretreated with ethylene
receptor inhibitors in the presence or absence of ethylene. This might depend on plant species and tissues
under investigation. Physiologically and molecularly, these results suggest that pretreatments with xiii inhibitor of ethylene receptor improved the display life of miniature roses by delaying bud, flower and leaf
drop, whereas 1-MCP is more effective than 1-OCP and 1-DCP, possibly by suppressing the expression of
some genes for ethylene biosynthesis enzyme, ethylene perception and signal transduction pathway.
Key words: ACC synthase, display quality, ethylene, ethylene perception and signal transduction, gene
expression, inhibitors of ethylene receptor, Rosa hybrida, senescence. xiv General Introduction 1 General Introduction
1.1 Ethylene and flower senescence
Senescence can widely be defined as the combination of events that lead to the death of cells,
tissues or organs (Reid and Wu, 1992). This process occurs at many stages during the
development of an organism and at many levels (Noodén et al., 1997). It is mediated by a series
of highly coordinated physiological and biochemical changes, such as increased activity of
hydrolytic enzymes, degradation of macromolecules, loss of cellular compartmentation and
increase in respiratory activity. These changes are related to changes in gene expression and
synthesis of protein (Borochov and Woodson, 1989; Van Altvorst and Bovy, 1995).
Flower senescence is a common cause of quality loss and reduced vase life of flowering plants
and cut flowers (Serek and Reid, 2000a). Senescence in many flowers is accompanied by
pollination promoting the production of ethylene which ultimately causes petal wilting (Nichol,
1977), abscission and sleepiness (florets failed to re-open) of petals (Marousky and Harbaugh,
1979) and a climacteric increase in ethylene production (Nichols, 1968; Singh and Moore, 1994).
This is induced by several factors, e.g., water stress (Sankat and Mujaffar, 1994), carbohydrate
depletion (Ketsa, 1989), microorganisms (Witte and Van Doorn, 1991), and ethylene effects
(Wu et al., 1991a, b). The phytohormone ethylene plays a vital role in regulation of flower
senescence in many species. Additionally, ethylene-induced senescence in some sensitive species
may result from the production of endogenous ethylene or from exposure to exogenous
ethylene (Van Altvorst and Bovy, 1995; Halevy and Mayak, 1979). The action of ethylene is
based on two types of responses: the response to a change in the concentration of ethylene, and
the response to a change in the sensitivity of tissue to ethylene (Sato-Nara et al., 1999). Certain
plants are ethylene-sensitive and flowers of these plants deteriorate rapidly in the presence of
ethylene. Ethylene can be produced by almost all parts of plants, although the production relies
on the type of tissue and stage of development (Müller and Stummann, 2003). In carnation, a
large amount of ethylene is synthesized several days after full opening of the flower during
natural senescence (Manning, 1985; Peiser, 1986; Woodson et al., 1992), or several hours after
compatible pollination (Nichols, 1977; Nichols et al., 1983; Larsen et al., 1995) or treatment with
exogenous ethylene (Borochov and Woodson, 1989; Wang and Woodson, 1989). The increased
1 General Introduction
ethylene production accelerates in-roll of petals resulting in wilting of the flower. Flowers
develop the early stages of wilting symptoms when treated with ethylene and later disappear
when ethylene treatment is stopped. This suggests that ethylene is required beyond the
appearance of early visual symptoms to uphold the “senescence syndrome” (Mayak and
Kofranek, 1976). 1.2 Exogenous ethylene
Senescence of climacteric flower petals is associated with an increased rate of ethylene
production and respiration, concomitant with the onset of petal wilting. Exposure to exogenous
ethylene promotes senescence of climacteric flower petals with activation of ACC synthase
and/or ACC oxidase (Borochov and Woodson, 1989).
Woltering and Doorn (1988) studied petal senescence in mature flowers of 93 species from 22
families. Most of the flower families (Geraniaceae, Libiatae, Ranunculaceae, Rosaceae and
Scrophulariaceae) showed initial abscission in response to ethylene except for a few families
(Caryophyllaceae, Campanulaceae, Malvaceae and Orchidaceae), which showed wilting as their
primary senescence symptom.
Moreover, Woltering and Doorn (1988) observed that abscission in Rosaceae is the initial
symptom of flower senescence. Further evidence supporting this view has found that bud
abscission of ‘Victory Parade’ roses occurs when plants are sensitive to ethylene (Serek, 1993).
However, in miniature potted roses, petal wilting as a form of flower senescence is more obvious
than petal abscission (Müller et al. 1998). The aging process of the flowers is apparently
accelerated by exogenous ethylene. Increasing amounts of exogenous ethylene in postharvest
environments also induces a variety of symptoms (petal abscission, leaf and bud drop) in rose
cultivars, and there are marked differences in sensitivity and response to ethylene among cultivars
(Müller et al., 1998). 2 General Introduction
1.3 Endogenous ethylene
Ethylene clearly influences natural flower senescence as an integral part of the aging process in
Ipomoea and Hibicus and serves to accelerate aging but may not be the initial causative agent.
This accelerating effect of ethylene is apparently related to the autocatalytic nature of the
ethylene biosynthetic system where ethylene stimulates its own synthesis in both flowers and
ripening of climacteric fruits (Kende and Baumgartner, 1974; Kende and Hanson, 1976; Beyer
and Sundin, 1978; Woodson et al., 1985; Halevy and Mayak, 1979). In carnation flowers, an
increase in ethylene production rate, similar to the climacteric associated with fruit ripening,
coincides with the first visible signs of senescence by petal in-rolling (Peiser, 1986; Mayak and
Tirosh, 1993). During the aging of petals, the transition to autocatalytic ethylene production
results from a change in tissue responsiveness to ethylene and an increase in their capacity to
respond to exogenous ethylene by the induction of autocatalytic ethylene production, indicating a
gradual release of the ethylene biosynthetic pathway from restriction (Wang and Woodson,
1989). Several studies have been shown that rose flower senescence is regulated by ethylene.
Many cultivars of miniature roses also show a climacteric rise in ethylene production during
flower senescence, even though large differences are found among cultivars. In some cultivars,
flower senescence is accompanied by a clear climacteric rise in ethylene production, and by a
moderate or low ethylene production in others. Longevity of miniature roses in the absence of
exogenous ethylene is presumably a function of endogenous ethylene production. For example,
the short life of individual flowers in ‘Bronze’ is associated with a clear climacteric peak in
ethylene production, similar to that found in typical climacteric flowers. The excellent longevity
of ‘Charming Parade’ in an ethylene-free environment, however, is related to a very low
ethylene production of flowers (Müller and Stummann, 2003). 1.4 Pollination-induced senescence
Pollination in many flowers causes ethylene biosynthesis and developmental changes such as
ovary growth and petal wilting and abscission. These processes are induced by a translocated
signal that precedes the growing pollen tube signal, a compatible pollination, to ovary and petals.
In addition, styles are sources of high ethylene production, which can be activated by pollination
(Jones and Woodson, 1999a; Nichols, 1977). Two or three phases of pollination-induced ethylene
3 General Introduction
production occur in petunia, tobacco, and carnation. The first peak of ethylene is detectable
within a few minutes of pollination, then sustains climacteric of ethylene production and follows
in response to a compatible pollination and hastens senescence of the style and petals. An
unidentified senescence signal appears to be translocated to other flower parts, which become
active sites of autocatalytic ethylene production within a few hours of pollination (Holden et al.,
2003). Application of amino-oxyacetic acid (AOA), an inhibitor in ethylene biosynthesis, blocks
pollination-induced ethylene production in orchid flowers, decreases ovary growth and delays
senescence (Ketsa and Rugkong, 2000). 1.5 Non-ethylene-mediated senescence
Senescence of the petals of many cut flowers such as Compositae, Iridaceae, Liliaceae and also
most of the important geophytes appears not to be related to ethylene (Reid, 1989; Woltering and
Van Doorn, 1988).
Flower senescence in Hemerocallis, daylily, is ethylene-independent. The senescence of these
flowers is not accelerated by exposure to exogenous ethylene, nor delayed by inhibitors of
ethylene biosynthesis or by ethylene antagonists (Lukaszewski and Reid, 1989). The major events
that occurred in the ethylene-unresponsive daylily are an early decline in phospholipid synthesis,
an increase in cell permeability that leads to an increase efflux of sugars and ions, a respiration
climacteric, early wilting and then autolysis of petal tissue. Moreover, flower senescence in
daylilies is accompanied by both the decline in protein content and the breakdown of specific
proteins in the petals. The inhibition of flower senescence by cyclohexamide (CHI) could
maintain protein content and protein population of the petals. These effects of CHI may require
de novo protein synthesis in these species during senescence and are key factors controlling petal
senescence in a species where the regulation of this event appears to be independent of ethylene
(Bieleski and Reid, 1992; Lay-Yee et al., 1992).
Gladiolus, like other geophytes in Liliaceace, is an ethylene-insensitive flower (Lay-Yee et al.,
1992). Ethylene is not a factor in floret senescence since wilting of florets is not accelerated by
exposure to ethylene, nor delayed by ethylene antagonists (Serek et al., 1994a). 4 General Introduction
In Cyclamen persicum Mill flowers, senescence is not associated with an increase in ethylene
production. Flowers do not respond to exogenous ethylene even when exposed to very high
concentrations of the gas. Pollination, however, induces a dramatic increase in ethylene
evolution. This presumably indicates that the promotion of senescence (corolla drop) in
pollinated cyclamen flowers is mediated by ethylene (Halevy et al., 1984). 1.6 Ethylene biosynthesis
The pathway of ethylene biosynthesis in plants has been explained (Yang, 1985; Van Alvorst and
Bovy, 1995). (1) SAM synthase
(2) ACC synthase
(3) ACC oxidase
(4) ACC malonyl-transferase
recycling (1) Senescence
AOA ------------------AVG SAM
Triton X-100 -------------------Ethanol
Allocoronamic acids (3) (4)
Malonyl CoA Ethylene _
NBD --------------------Irradiated DACP MACC + + Receptor(s) Signal transduction Gene expression Protein synthesis Response: Flower Fig. 1 Ethylene biosynthesis pathway by Van Alvorst and Bovy (1995). 5 General Introduction
In higher plants, ethylene is synthesized from methionine via a pathway involving the conversion
of S-adenosylmethionine (SAM) to 1-amino cyclopropane-1-caboxylic acid (ACC) and the
oxidation of ACC to ethylene. The enzyme ACC synthase converts SAM to ACC while ACC
oxidase catalyzes the conversion of ACC to ethylene, HCN and CO2. In addition, methionine is
regenerated in the Yang cycle (Adam and Yang, 1979) (Fig. 1). 1.6.1 ACC
The intermediate precursor of ethylene in higher plants is 1-aminocyclopropene-1-carboxylic acid
(ACC) (Adam and Yang, 1979). The endogenous ACC level in various flower parts increases
during senescence (Veen and Kwakkenbos, 1982; Nichols et al., 1983; Hsieh and Sacalis, 1986).
Application of ACC stimulates wilting in whole carnation flowers (Veen and Kwakkenbos,
1982). Exposure to ethylene of isolated carnation petals, separated into upper and basal parts,
shows that the majority of ethylene production is evolved from the basal part of the petals.
Endogenous ACC content in the basal portions of senescing carnation petal is 3 to 5 times higher
than in the upper parts. The upper portions respond to ethylene by delaying wilting and much
lower ethylene production. Application of ACC to the upper portion of senescing petals increases
their ethylene production (Mor et al., 1985). During flower senescence, ACC is translocated from
the basal part where it is synthesized to the upper part where it is converted into ethylene
(Overbeek and Woltering, 1990). In addition, the endogenous ACC content of pollen correlates
with the amount of ethylene that is produced by petunia styles immediately after pollination
(Singh et al., 1992). The detection of ACC in petunia pollen indicates that ACC diffusing from
pollen might be responsible for early ethylene production, initiating autocatalytic ethylene
production (Stead, 1985). 1.6.2 ACC synthase and ACC oxidase
The biosynthesis of ethylene in flower tissues is under strict metabolic regulation and is subject to
induction by a variety of signals including emasculation, pollination, wounding, auxin, abscissic
acid and environment stress (Borochov and Woodson, 1989). The conversion of SAM to ACC is
catalyzed by the peridoxal phosphate-requiring enzyme ACC synthase, which represents the ratelimiting step in ethylene biosynthesis in many plant tissues (Kende, 1989, 1993). All evidence
6 General Introduction
indicates that ACC synthase is a cytoplasmic enzyme (Kende, 1993). Boller et al. (1979) found
that ACC synthase is a soluble enzyme in tomato fruits. The final step in the ethylene
biosynthetic pathway is catalyzed by ACC oxidase, formerly referred to as the ethylene-forming
enzyme (EFE) (Kende, 1989, 1993). ACC oxidase is located in the cell walls of apple and tomato
fruit cells. In apple, the enzyme is actually located at the external face of the plasma membrane
(Ramassamy et al., 1998; Rombaldi et al., 1994). ACC oxidase requires ascorbate, Fe2+, and CO2
which act as essential cofactors (Smith and John, 1993a, b). Both ACS and ACO enzymes from a
number of different species are encoded by multigene families whose members are differentially
regulated in a tissue-specific manner by a variety of developmental cues, mechanical and
environmental stresses, and by ethylene, which stimulates its own biosynthesis (Kende, 1993;
Fluhr and Mattoo, 1996). 1.7 Strategies to breed ethylene-insensitive flowers
The development of ethylene-resistant cultivars that are mutant with impaired ethylene
biosynthesis or sensitivity has been studied in carnations as a model system leading to a more
focused breeding effort in the genetic improvement of postharvest longevity of these flowers. The
extended vaselife genotypes are responsive to genetic differences by synthesis and response to
ethylene. For example, genotype 799 is impaired in its ability to respond to ethylene by
increasing ethylene production or premature petal senescence. The second genotype, 87-37G-2,
appears to represent a mutation affecting the synthesis of ethylene but not ethylene
responsiveness. The third genotype, 81-2, is able to synthesize ethylene in response to ethylene
treatment but failed to produce ethylene during normal aging. This pattern shows that these
flowers fail to produce elevated ethylene during aging but respond to exogenous ethylene by
increased ethylene synthesis and premature senescence (Brant and Woodson, 1992). Similar to
Sandrosa carnations, these flowers do not exhibit an autocatalytic increase in ethylene production
nor do they develop petal in-rolling, but the sensitivity to ethylene diminishes with age, whereas
other flowers show increased sensitivity to ethylene with age (Mayak and Tirosh, 1993). In
‘killer’ carnations that show non-climacteric behavior, the lack of ethylene production is due to a
limited-available ACC, likely resulting from low ACC synthase activity and failure to convert
ACC into ethylene because of restricted ACC oxidase activity (Serrano et al., 1991). 7 General Introduction
‘Sandra’ and ‘Chinera’ carnation flowers last about twice as long as those of ‘White Sim’ whose
senescence closely correlates with the normal climacteric of respiration and ethylene production
and is accompanied by a marked increase in ACC content and ACC oxidase activity. In contrast,
the long vase life of Sandra flowers is due to inhibition, under normal conditions, of the pathway
for ethylene biosynthesis (Wu et al., 1989, 1991a). Although behavior of non-climacteric carnation
‘Sandra’ fails to produce elevated ethylene during aging, it responds to exogenous ethylene by
increasing ethylene synthesis and premature senescence. ‘Chinera’ flowers not only produce less
ethylene during natural senescence, but are also much less sensitive to ethylene (Wu et al., 1991b). 1.8 Ethylene sensitivity
Various processes during plant development are determined both by ethylene production and by
the tissue responsiveness to ethylene. Therefore, differences in sensitivity to hormones are the
main factor controlling plant response (Trewavas, 1982). During senescence, changes in a
climacteric rise in ethylene production and a gradual increase in ethylene sensitivity occur in the
corollas of climacteric flowers such as carnation and petunia (Nichols, 1968; Halevy and Mayak,
1981; Whitehead and Halevy, 1989). In addition, pollination of these flowers leads to an
acceleration of senescence involved in a marked stimulation of ethylene synthesis and a sudden
increase in sensitivity of the corolla to ethylene (Whitehead and Halevy, 1989; Whitehead and
Vasijevic, 1993; Porat et al., 1994; Halevy et al., 1996).
The increase in ethylene sensitivity following pollination is independent of endogenous ethylene
production since it occurs in flowers treated with AOA, which prevents the increase in ethylene
synthesis. An increase in ethylene sensitivity following pollination is thought to render the tissue
to respond to low basal ethylene, thus inducing the later autocatalytic increase in ethylene
production. This is also related to its ability to increase ethylene binding by altering certain
membrane properties which could lead to an increase in the availability of ethylene binding sites
(Porat et al., 1993, 1994, 1995b; Whitehead and Vasijevic, 1993). It is clear that ethylene cannot
be the pollination signal inducing sensitivity to ethylene (Porat et al., 1993, 1994, 1995b).
The possibility that the sensitivity factor is short-chain saturated fatty acids (C7-C10) has been
postulated (Whitehead and Halevy, 1989). Application of these acids to the stigmas of petunia
8 General Introduction
and carnation flower results in a sudden increase in ethylene sensitivity and a marked acceleration
of senescence (Whitehead and Vasijevic, 1993). Treatment of Dutch iris (Iris) ‘Sapphire Beauty’
bulb with the short-chain saturated fatty acid octanoic acid (C8) increases ethylene sensitivity by
stimulating flowering (Botha et al., 1998).
However, in carnation, petunia and cymbidium (Cymbidium), short-chain saturated fatty acid
does not play an important role in flower senescence and ethylene sensitivity (Wotering et al., 1993). 1.9 Statement of the problem
Miniature potted roses are popular greenhouse crops in many parts of the world. In the 1980s,
there was an introduction of new varieties that have had a dramatic impact on the European and
North American markets. Current annual world production is estimated at 60-80 million pots in
Europe and 26 million pots in the United States. Major centers of production include Denmark,
The Netherlands, the United States, Canada and Japan with production also in France, Germany
and Italy (Pemberton et al., 2003; USDA, 2003: http://www.ars.usda.gov). The acceptability and
trade value of miniature potted roses can be influenced by postharvest longevity, where in
Denmark, extensive efforts have been taken to improve the quality and increase the production of
these plants, for example with a breeding program (Serek and Andersen, 1993, Müller et al.,
1998). Loss of quality during postharvest and marketing in miniature potted roses are important
problems caused by ethylene action (Serek, 1993; Serek et al., 1994c), such as accelerated flower
wilting, leaf yellowing, bud and leaf abscission and infection by grey mould (Botrytis cinerea). It
is assumed that ethylene is not the primary cause of leaf yellowing in miniature potted roses, but
that it accelerates flower senescence as well as bud, flower and leaf drop (Müller and Stummann,
2003). Variation in postharvest life of miniature roses is partly the result of differences in
endogenous ethylene during flower senescence, stress-induced ethylene production and
sensitivity to exogenous ethylene. Ethylene sensitivity has important implications during the
transport and handling of potted roses in supermarkets and other areas where the air is commonly
contaminated with ethylene (Müller et al., 1998, 2000a). In ethylene-contaminated air, rose
cultivars with high sensitivity to exogenous ethylene exhibit a short flower life (Müller et al.,
2000a). Ethylene action involves binding to a specific receptor (Schaller and Breecker, 1995;
Sisler and Serek, 1997). Since ethylene inhibitors can block the plant tissues from endogenous
9 General Introduction
and exogenous ethylene, they are considered very potent for horticultural use (Sisler and Serek,
1997; Feng et al., 2000). 1-MCP and some new putative inhibitors of ethylene action, which are
structurally analogous to 1-MCP containing a longer side chain at 1-position (1-OCP and 1-DCP),
have been found to be effective in protecting cut flowers against ethylene (Kebene et al., 2003a,
b) and delaying the sensitivity of bananas to ethylene (Sisler et al., 2003). In addition, the
expression patterns of the genes for the ethylene biosynthetic enzyme ACC synthase, and for
ethylene receptors and signal transduction pathway during flower development and senescence
processes seem essential to understanding ethylene response. Understanding the roles of the
various ethylene-relevant genes in flower development may lead to an ability to selectively block
processes in flower senescence that are economically detrimental, and to improve display life
using both conventional breeding and biotechnological engineering. 1.10 Objectives
1. To investigate the effect of extended chain length of the activity of inhibitors of ethylene
receptors 1-OCP and 1-DCP as compared with 1-MCP, in preventing the effects of
ethylene on display quality of miniature potted roses. 2. To determine the optimum concentrations, exposure time and temperature at which 1-OCP
and 1-DCP effectively improve the longevity of miniature potted roses. 3. To determine the effectiveness of 1-OCP, 1-DCP and 1-MCP between miniature potted
rose cultivar ‘Lavender’ with sensitivity to ethylene and ‘Vanilla’ with long-lasting vase
life. 4. To investigate the effect of 1-OCP, 1-DCP and 1-MCP on the expression of the genes for
the ethylene biosynthetic enzymes, ethylene perception and signal transduction pathway
in miniature potted roses. 10 Efficacy of new inhibitors of ethylene perception 2. Efficacy of new inhibitors of ethylene perception in improvement of display
quality of miniature potted roses (Rosa hybrida L.)
1-Octylcyclopropene (1-OCP) and 1-Decylcyclopropene (1-DCP), which act as ethylene
receptor inhibitors and are analogues to 1-MCP, but are substituted with longer carbon chains
in the 1-position, were investigated in miniature potted rose cultivar ‘Lavender’. All tested levels
of both chemicals were protected as compared to untreated plants. 1-OCP and 1-DCP were the
most effective at concentrations of 1000 and 1500 nl l-1, which was five times higher than the
concentration of 1-methylcyclopropene (1-MCP) (200 nl l-1) used as a standard. The
effectiveness of 1-OCP and 1-DCP was a function of time and temperature. At short (2 h)
exposure times, the plants were highly sensitive to ethylene. Exposure time of 4 h for both 1-OCP
and 1-DCP was sufficient to improve display life of miniature roses and longer exposures did not
have any additional beneficial effect. Apparently, exposing miniature potted roses to various
temperatures did not have an influence on the performance of either 1-OCP or 1-DCP. They were
effective at temperatures between 5 to 20oC. The effectiveness of this group of compounds on the
display quality of miniature roses is discussed.
Key words: 1-DCP, Ethylene receptor inhibitors, 1-MCP, 1-OCP, Postharvest performance 11 Efficacy of new inhibitors of ethylene perception
Ethylene has been shown to play a central role in physiological and developmental processes
such as seed germination, growth, flower initiation, organ abscission, fruit ripening, senescence
of leaf and flowers and response to pathogen attack (Abeles et al., 1992). Ethylene binding would
be mediated by a transition metal cofactor (Burg and Burg, 1967). The isotopic competition
technique can explain the presence of a specific ethylene binding site in carnation petals exposed
to 14C-C2H4. A substantial proportion of the bound radioactive ethylene could be displaced by the
addition of unlabelled ethylene, identifying a specific binding site. Treatment with ethylene action
inhibitors silver or 2,5-NBD to flowers reduces binding activity (Sisler et al., 1983). In addition,
the binding of ethylene in petal tissues is significantly higher for young carnation petals than for
older flowers. The peak in ethylene binding precedes the climacteric-like rise in ethylene
production. Ethylene binding changes in senescent flowers because the number of binding sites
and the affinity for ethylene in the older tissue decreases (Brown, 1986).
Copper is the metal required for the high affinity ethylene binding that receptors display
(Rodriguez et al, 1999). Therefore, the binding of ethylene to its membrane associated with
receptor sites is a crucial step in its action (Goren et al., 1984). Additionally, the number of
receptors could have an essential role in determining the sensitivity to ethylene and the type of
response that is elicited. When ethylene binds to the receptors, it induces a series of events that
initiate a response to the hormone (Tian et al., 1997).
2.1.1 Compounds interacting with the ethylene receptor in plants
126.96.36.199 Ethylene and ethylene analogues
Compounds such as ethylene, some olefins, propylene, acetylene, carbon monoxide and
isocyanides, which bind to the receptor, are capable of triggering ethylene-induced changes in the
growth and development of plants. Ethylene and its analogues are thought to interact with a
metal-containing receptor (Sisler and Wood, 1988; Sisler et al., 1990; Sisler and Serek, 2003). Of
the group, ethylene is considerably more effective than the others, which are not used for some
experimental research, but give some insight into how ethylene behaves (Sisler and Serek, 2001).
12 Efficacy of new inhibitors of ethylene perception
188.8.131.52 Competitive ethylene antagonists
Compounds preventing an ethylene response interact with the receptor and compete with ethylene
for binding. A single exposure of plant tissue to these compounds is enough to prevent binding of
ethylene because they remain bound for a long period of time, with even high levels of ethylene
not inducing any action (Sisler and Serek, 2003).
2.1.2 Mode of action of compounds blocking the receptor
Competitive ethylene antagonist compounds presumably bind to a metal in the ethylene receptor.
They compete with ethylene for the receptor and prevent the ethylene receptor from binding in
treated tissues. While they are bound, ethylene cannot bind (Sisler and Serek, 1997).
Both those compounds inducing a response and those blocking the receptor should bind to the
supposed metal (M) on the receptor and withdraw electrons from the metal, which causes a
rearrangement or change in ligand (L1-L5) on the metal. Then, ethylene (E) is likely to come off
of the metal in a few minutes while other inhibitory compounds that block receptors appear to
take at least several hours or, in some, many days to leave (Sisler and Serek, 1999) (Fig. 2).
Ethylene leaves the complex and causes it to become active probably by ligand arrangement.
Ethylene then would not be a part of the active complex, but the initiator of its formation. While
the inhibitory compounds act in a similar manner to ethylene, they do not move away from the
complex, thus an active complex is not formed (Sisler and Serek, 1997), which accounts for the
mode of action of ethylene and 1-MCP that blocks ethylene responses (Sisler and Serek, 1997). 13 Efficacy of new inhibitors of ethylene perception Fig. 2. Proposed model for action of ethylene and 1-methylcyclopropene (1-MCP) on the
ethylene receptor (Sisler and Serek, 1997).
It is believed that the ligands could be in a protein group such as histidine, methionine, thiol
groups, tyrosine groups and in a hydrophobic environment such as a membrane. Perhaps the
double bonds in unsaturated fatty acids, on a single protein forming a cross link between two
proteins, could be all ligands (Sisler and Serek, 1999).
2.1.3 Ethylene antagonists inactivating the receptor for an extended period of time
Silver ion, applied as Silver thiosulfate (STS), is a potent anti-ethylene compound in various
plants. Silver acts through the irreversible interaction with ethylene binding sites (Sisler et al.,
1986; Veen, 1986; Rodriguez et al, 1999). STS is in widespread commercial used to inhibit
effects of ethylene and prolong vase life in many ornamentals including orchid (Cattleya) ‘Loiuse
Georgeianna’ (Beyer, 1976), carnation (Veen, 1979), sweet pea (Lathyrus odoratus) (Mor et al.,
1984), potted Christmas cactus (Schlumbergera truncate) (Serek and Reid, 1993) and potted rose
(Rosa hybrida) (Serek, 1993). However, as Silver is a heavy metal, it cannot be used on food and
feed, and many countries prohibit its use (Veen, 1986).
Cyclic olefins appear to block ethylene responses rather than to induce a response. All of these
compounds are released from the receptor and diffuse from the binding site over a period of
14 Efficacy of new inhibitors of ethylene perception
several hours. 2,5-norbonadiene (2,5-NBD) is found to compete with ethylene for binding sites in
carnation petals, but it requires continuous exposure to be effective (Wang and Woodson, 1989;
Sisler and Serek, 1999). Another compound in this group is trans-cyclooctene, which is much
more effective, in terms of concentration, than 2,5-NBD. However, the practical use of this
compound is limited by a very pungent and objectionable odor (Sisler et al., 1990).
Diazocyclopentadiene (DACP) is a putative photoaffinity label for ethylene binding sites. These
compounds form highly reactive carbons under UV light, called diazo-compounds. Under UV
light, nitrogen gas is split from the molecule leaving a carbine, which almost immediately reacts
with many other compounds (Sisler and Serek, 2003). It binds irreversibly to the ethylene
receptor or at least remains bound for many days to reduce tissue responses to ethylene in mung
bean sprouts and tobacco leaves (Sisler and Blankenship, 1993), potted roses (Rosa hybrida)
(Serek et al., 1994b) and tomato fruit (Tian et al., 1997). A major problem with DACP is that it is
explosive in high concentrations, which limits its commercial usefulness (Sisler and Serek, 1997).
A series of cyclopropenes, cyclopropene (CP), 1-methylcyclopropene (1-MCP), 3-methylcyclopropene
(3-MCP) and 3,3-dimethylcyclopropene (3,3-DMCP), have been prepared and tested by using
banana fruit as an assay system. Based on the substitution of the cyclopropene ring in positions 1,
2 and 3, all compounds are effective in preventing banana from exogenous ethylene but none are
more effective, concentration-wise, than 1-MCP (Sisler et al, 1996a, b, 1999, 2001). CP and
1-MCP act at low concentrations. In banana, they are effective around 0.5 nl l-1. The required
concentration of 3,3-DMCP is about 1000 times as high as CP and 1-MCP. It is effective in the μl l-1
range and also protects banana for only 7 days (Sisler et al., 1996a, b). 3-MCP is also effective at
higher concentrations than for 1-MCP. In banana, about 2.5 times as much 3-MCP is needed
(Sisler et al., 1999).
In the substitution on the cyclopropene ring, both the concentration required for inactivation and
duration of binding appear to be influenced by steric and inductive effects (Sisler et al., 1996b,
1999, 2001). The presence of the methyl groups in these compounds is associated with steric
effect. Flat molecules (cyclopropene, 1-MCP and 3-MCP) are more active than compounds with
the methyl groups isolated from the double bond. Inductive effect, however, is caused by methyl. 15 Efficacy of new inhibitors of ethylene perception
A methyl group releases electrons into the cyclopropene ring and is capable of relieving the strain
related to anti-ethylene effects of compounds in plants (Sisler et al., 2001).
The position of the methyl group is more important. In 1-MCP the methyl group is adjacent to the
double bond, thus allowing an allylic-type of arrangement. This may interact with the ethylene
receptor more rapidly than isolating a double bond in 3-MCP and result in a low concentration
requirement for a given time exposure (Sisler, 1999).
Stability is an important factor in these compounds in considering commercial use. CP is unstable
in the liquid phase even at -78oC, and in a dilute gas phase seems to polymerize at room
temperature. 1-MCP, 3-MCP and 3,3-DMCP are relatively stable (Sisler et al., 1996a, b; Sisler et al.,
1-MCP, one of the most useful compounds among substituted CPs, is a non-toxic compound, stable
at room temperature, active at relatively low concentration that provides protection for a longer
period of time up to 12 days after a single exposure, without any detectable odor. Moreover, in the
floriculture industry 1-MCP can be used as a replacement of silver thiosulfate (STS), which is
considered to be toxic (Sisler and Serek, 2001, 2003). Other CPs substituted with the methyl group
in the 1-position have been developed and tested as ethylene antagonists (Sisler et al., 2001).
1-MCP prevents damage from exogenous ethylene in many potted plant species including
heimalis begonia ‘Najada and ‘Rosa’ and tuberous begonia (Begonia x tuberhybrida), rose
‘Victory Parade’ (Rosa hybrida) (Serek et al., 1994c), kalanchoe ‘Tropicana’ (Kalanchoe
blossfeldiana) (Serek and Reid, 2000b), Christmas cactus (Schlumbergera truncate) and
bellflower (Campanula carpatica) (Serek and Sisler, 2001) and in some countries has already
been approved for use in edible crops (Blankership and Dole, 2003). However, the effect of 1-MCP
on ethylene-induced petal abscission in ivy geranium ‘Pink Blizzard’ (Pelargonium peltalum) is
transient (Cameron and Reid, 2001).
Recently, a series of cyclopropenes substituted with a methyl group in the 1-position, analogues
of 1-MCP, protected for longer periods of time than those substituted in other positions (Sisler et
al., 2003; Sisler and Serek, 2003). When the chain length is extended to more than four carbons,
16 Efficacy of new inhibitors of ethylene perception
the minimum concentration requirement declines (Sisler et al., 2001), however, 2 or 3 carbon
side chains substituted at the 1-position, 1-ethylcyclopropene (1-ECP) and 1propylcyclopropene (1-PCP), are still effective blockers of ethylene receptors and are able to
inhibit ethylene action in a wide scope of systems. These include climacteric fruits like avocado
and tomato, ‘the triple response’ in etiolated peas and abscission of citrus leaf explants
(Feng e t al ., 2004). A concentration as low as 0.3 nl l -1 i s needed for both 8-carbon
chain, 1-octylcyclopropene (1-OCP), and 10-carbon, 1-decylcyclopropene (1-DCP), to protect
against ethylene response in banana fruits. Furthermore, the time of protection for 1-substituted
CPs at ambient temperature (22-23oC) is significantly longer for 1-DCP (36 days) than that of
1-MCP (12 days) (Sisler et al., 2003). Further 1-substituted CP tests have been done on
kalanchöe and sweet pea flowers, but their inhibiting effects are lower than in bananas (Kebenei
et al., 2003a, b).
The effects of these compounds as ethylene antagonists are attributed to their structural molecular
strain that permits a very tight binding to electron donor compounds such as low valency, in the
receptor. They compete with ethylene for the binding sites and remain bound to the receptor for a
long time, thus preventing ethylene from binding (Sisler et al., 1999). The activity of CPs
depends on the concentration required for inactivation of the receptor, with the duration of
binding possibly due to steric and inductive effects, regarding the position of a methyl group with
respect to double bonds (Sisler et al., 1996a, b, 1999, 2001). Ethylene receptor inhibitors, such as
1-MCP, act by binding to a metal in the receptor. They compete with ethylene for the receptor,
but do not induce ethylene responses (Sisler et al., 1990).
Ethylene mediated flower senescence is a significant problem in horticulture, most obvious in the
floriculture industry. The regulation of flower senescence is of great interest to horticulturists in
search of methods to improve the postharvest quality of ornamentals. The need for chemical
protection from ethylene action has been recommended in many potted flowering plants in which
low concentrations of ethylene causes rapid loss in display quality (Serek, 1993; Serek et al.,
1994b, c). Therefore, much research has been conducted in order to develop anti-ethylene agents
that block the synthesis and action of ethylene, as mentioned before. It is proposed that the
analogues inhibit ethylene action by competing for the binding sites on the ethylene receptor,
similar to the mode of action suggested for 1-MCP (Feng et al., 2004). It is hypothesized that
17 Efficacy of new inhibitors of ethylene perception
1-OCP and 1-DCP, CPs with 8 and 10-carbon chain length, respectively, could significantly
improve the display quality of miniature roses, important commercial potted plants, in protecting
them against ethylene. This study reported the difference in the display quality between the two
cultivars ‘Lavender’ with short flower life and ‘Vanilla’ with long-lasting flower, after
pretreatment with 1-OCP, 1-DCP and 1-MCP, and afterwards continuous exposure to ethylene.
The influence of treatment conditions, such as concentration, exposure time and temperature, on
the postharvest quality of miniature rose plants was then investigated. Fig. 3. Chemical structures of compounds interacting with the ethylene receptor (Sisler and
Serek, 2003). 18 Efficacy of new inhibitors of ethylene perception
2.2 Materials and methods
1-OCP and 1-DCP were synthesized at the Department of Molecular and Structural
Biochemistry, North Carolina State University, Raleigh, USA, from 2-bromoalkenes and
bromoform using 50% NaOH to produce a carbine and form a 1,1,2-cyclopropane, which was
then reacted with methyllithium at dry ice temperature, to form cyclopropenes (Al Dulayymi et al.,
1996, 1997). These compounds were then sent on dry ice to the University of Hannover,
Germany. The compounds were subsequently divided into smaller samples of 0.5 ml and kept at
-80oC until needed for the experiments. Each sample was then diluted with ether to a volume of
50 ml before being used for experiments. 1-MCP was obtained from AgroFresh Inc. (Rohm and
Haas, AgroFresh Inc., Philadelphia, USA) in a commercially-available form.
2.2.2 Plant material
Rosa hybrida L. cultivar ‘Lavender’, which is sensitive to ethylene, was used to investigate the
effects of extended chain length on the activity of inhibitors of ethylene receptors, 1-OCP and
1-DCP as compared with 1-MCP. In addition, miniature potted rose cultivar ‘Vanilla’, with
excellent postharvest performance and low sensitivity to exogenous ethylene (Müller et al.,
2001), was compared to ‘Lavender’ in the effects of exogenous ethylene after pretreatment with
inhibitors of ethylene receptors (1-OCP and 1-DCP). ‘Lavender’, obtained from Kordana
breeding line of Rosen Kordes, W. Kordes’ Söhne Rosenschulen GmbH & Co KG, Sparrishoop,
Germany, was used to investigate the effect of 1-OCP concentration on display quality of
miniature potted roses. The other miniature potted roses were produced in 10 cm-diameter pots (2
cuttings per pot) in a greenhouse at the University of Hannover during January-December, under
the following conditions: 19_20oC/20oC (day/night temperature) and 60-85% relative humidity
(RH), natural daylight was supplemented with 60 µmol m-2 s-1 from SON-T lamps (Osram,
400W, Philips, Eindhoven, The Netherlands) over a 16 h (7.00-23.00) photoperiod. Paclobutrazol
was used as a growth retardant at a concentration of 0.5%. Watering, fertilizer application and
pest and disease control were carried out until plants were required for experiments. Miniature
potted roses were used in the experiments when two to three flowers per pot had opened.
19 Efficacy of new inhibitors of ethylene perception
2.2.3 Effects of concentration of 1-OCP and 1-DCP on display quality
‘Lavender’ plants were placed in 54 l glass chambers. The plants were exposed to the desired
concentrations of 1-OCP or 1-DCP (200, 500, 1000 and 1500 nl l-1), respectively, and 200 nl l-1
of 1-MCP (Serek and Reid, 2000), sealed in glass chambers for 6 h at 20oC. The calculated
volumes of the 1-OCP and 1-DCP were pipetted on filter paper in the glass chambers to increase
the surface area and facilitate evaporation. 1-MCP was released from a commercial powdered
formulation (SmartFresh™, 0.14% a.i.) via addition of a few drops of water (≈0.5 ml). Control
plants were sealed in identical containers containing air for 6 h at 20oC. After the treatments, the
chambers were vented for 30 min and the plants were transferred and placed randomly in a glass
chamber, sealed and kept in an interior environment room, which was maintained at 20oC, 6070% RH. The plants were exposed to 12 h light from cool white fluorescent tubular lamps
providing 20 µmol m-2 s-1. The glass chambers were ventilated (200 l h-1) with air containing a
range of ethylene concentration of 1.25 (+/- 0.25) µl l-1 provided by a simple diffusion system
(Saltveit, 1978) throughout the experimental period. The concentration of ethylene was
determined daily using a Perkin-Elmer portable digital gas chromatograph (GC Voyager
FFKG312, Ontario, Canada) equipped with a photoionisation detector. The carrier gas was N2 at
40 ml min-1, and the injection temperature was 60oC with a column temperature of 60oC. The
display quality was determined after every 3 days by recording the percentage of leaf drop (80%
leaf drop was considered unacceptable), and the number of days to onset of total bud drop
(number of abscised or dead buds) and flower drop (individual flower longevity from the day of
anthesis to petal drying or abscission).
2.2.4 Effects of treatment time on 1-OCP and 1-DCP on display quality
The optimum concentrations from section 2.2.3 were used. Potted ‘Lavender’ plants were
selected and placed in 54 l glass chambers. They were treated with 1000 nl l-1 1-OCP or 1-DCP in
sealed glass chambers at 20oC but at different exposure times (2, 4, 6 and 12 h). The 1-MCP
treatments (200 nl l-1) were sealed for 6 h (Serek et al., 1994b, c; Serek and Reid, 2000b). Control
plants were sealed in identical containers containing air for 6 h at 20oC. After treatments, the
plants were exposed to the same procedure as previously described in section 2.2.3. 20 Efficacy of new inhibitors of ethylene perception
2.2.5 Effects of temperature on efficacy of 1-OCP and 1-DCP on display quality
The temperature inside the growth chambers was set at 5, 10, 15 and 20oC and allowed to
stabilize overnight. Potted ‘Lavender’ plants were selected and placed in 54 l glass chambers.
They were treated with 1000 nl l-1 1-OCP and 1-DCP in sealed glass chambers at 20oC but at
different exposure times (2, 4, 6 and 12 h). 1-MCP treatments (200 nl l-1) were sealed for 6 h
(Serek et al., 1994b, c; Serek and Reid, 2000b). Control plants were sealed in identical containers
containing air for 6 h at 20oC. After treatments, the plants were handled as previously described
in section 2.2.3.
2.2.6 Comparison of effectiveness of 1-MCP, 1-OCP and 1-DCP between ‘Lavender’ and
Potted ‘Lavender’ and ‘Vanilla’ plants were selected and placed in 54 l glass chambers. They
were treated with 1-OCP and 1-DCP at the best condition from the previous experiments in
sealed glass chambers at 20oC. 1-MCP treatments (200 nl l-1) were sealed for 6 h (Serek et al.,
1994b, c; Serek and Reid, 2000b). Control plants were sealed in identical containers containing
air for 6 h at 20oC. After treatments, the plants were exposed to the same procedure as previously
described in section 2.2.3. During the experimental period, 1-OCP and 1-DCP experiments were
conducted separately and 1-MCP was used in both experiments as the standard.
2.2.7 Experimental design and statistics
The experiments were conducted in a completely randomized design using 3 pots per treatment
and two replications. Each pot contained 2 miniature rose cuttings. The data obtained was
subjected to analysis of variance (ANOVA) using the general linear models (Proc GLM)
procedure of the Statistical Analysis System (SAS, 2002) program package. Multiple
comparisons among means was done using the Least Significant Difference (LSD) at P = 0.05. 21 Efficacy of new inhibitors of ethylene perception
2.3.1 Effect of concentrations of 1-OCP and 1-DCP on display quality
Display quality was characterized by leaf flower and bud drop. In ‘Lavender’ the most obvious
symptom of ethylene sensitivity was accelerated leaf drop. Miniature rose plants treated with a
concentration of 200 nl l-1 1-OCP and 1-DCP reached 80% leaf drop after 9 days of continuous
exposure to ethylene and were not different from untreated (control) plants (Fig. 4A, B). Plants
pretreated with 500-1500 nl l-1 of 1-OCP or 1-DCP exhibited reduced leaf drop and therefore
improved display quality up to 15 days as compared to the control (Fig. 4A, B). Pretreatment
with 1-MCP, 500-1500 nl l-1 1-OCP, and all used concentrations of 1-DCP significantly
(P<0.001) increased the number of days to the total bud and flower drop compared to control
plants. There were no differences between 1-MCP and long chains CPs (Fig. 4C, D). The best
display quality was achieved by pretreating miniature roses with 1000, 1500 nl l-1 for both 1-OCP
and 1-DCP (Fig. 4A, B, C, D). Increasing the concentration of 1-OCP and 1-DCP from 1000 to
1500 nl l-1 did not show further improvement in the display quality of the plants. Also, no toxicity
was observed with a high concentration of 1500 nl l-1. Additionally, ether used to dissolve 1-OCP
and 1-DCP had no effect on the display quality of ‘Lavender’ (data not shown). In terms of
effectiveness of different concentrations, 1-MCP was more effective than 1-OCP and 1-DCP. 22 Efficacy of new inhibitors of ethylene perception aa aa a A a 80
b b d
c bc d
d bc c control 200 500 1000 ab 10
c control 200 a a 500 1000 1500 a bc 8
2 1500 1-MCP Concentrations of 1-OCP (nl l )
-1 aa a
a B b b b
c b 60 c cd
a b c cd cd b 12
10 ab ab ab ab 200 500 1000 1500 a 8
6 c 4
2 c c c D 14 a
bb 1-MCP Concentrations of 1-OCP (nl l-1) aa b 3
15 12 0 bb Leaf drop (%) d cd 80 day
day d c c 20 100 c
c cd 40 30
bc b a day
day b Days until total bud and flower drop Leaf drop (%) a
60 C 14
Days until total bud and flower drop 100 0 0
control 200 500 1000 1500 1-MCP control 1-MCP Concentrations of 1-DCP (nl l-1) Concentrations of 1-DCP (nl l-1) Fig. 4. Mean of percent leaf drop (A, B) and total bud and flower drop (C, D) of miniature potted
rose cultivar ‘Lavender’ pretreated with 1-OCP and 1-DCP at concentrations of 0, 200, 500,
1000, 1500 nl l-1 and untreated controls, respectively. These were compared to treatment with
1-MCP (200 nl l-1) for 6 h at 20oC. After the treatments, the plants were exposed to 1.25 (+/- 0.25)
µl l-1 continuous ethylene throughout the experiments. Bars marked with the same letter (for
figures A, B, C, D bars representing the same day) are not statistically different at P<0.05. Means
were separated by LSD.
23 Efficacy of new inhibitors of ethylene perception
2.3.2 Effect of exposure time of 1-OCP and 1-DCP on display quality
Exposing miniature potted roses to 1000 nl l-1 1-OCP or 1-DCP for 4 to 12 h significantly
(P<0.001) delayed leaf drop (Fig. 5A, B) and increased the number of days to the onset of total
bud and flower drop (Fig. 5C, D). However, an exposure time of 2 h had no effect on leaf drop or
total bud and flower drop. Plants pretreated for a short time (2 h) showed a fast response to
ethylene by reaching 80% leaf drop after day 9 (Fig. 5A, B) and took about 7 days to reach the
total bud and flower drop for both 1-OCP and 1-DCP (Fig. 5C, D). The untreated (control) plants
reached 80% leaf drop within 6 days as well as the total bud and flower drop, respectively (Fig.
5A, B, C, D). Within the time range (4-12 h), 1-OCP treatments were less effective than 1MCP, with respect to leaf drop. It also took 1-OCP treated plants 8 days to attain the total bud
and flower drop while 1-MCP-treated plants took 12 days. The 1-DCP treatments were not
different from 1-MCP with respect to percent leaf drop. Exposing plants to 1000 nl l-1 1-DCP for
2 to 12 h significantly increased the number of days to total bud and flower drop (Fig. 5D).
Additionally, after 12 h exposure to 1-DCP, it took 10 days to attain total bud and flower drop.
However, 1-MCP was the most effective in preventing bud and flower drop. 24 Efficacy of new inhibitors of ethylene perception aaa aa a A 80 d
e 40 30
15 80 b b control 2 d
d de c e
4 6 c
12 10 b 8
6 d c 2
control 2 bc bb bc b b
d cd d
a a control ab
2 e c c
6 6 12 1-MCP d b
12 D 14 b c c 4 Time of 1-OCP (h) B b a b b 4 1-MCP aa aa a 60 30
15 a 12 Time of 1-OCP (h) 100 Leaf drop (%) d c b a day
day c d 60 20
day cb b D ays until total bud and flower drop Laef drop (%) b C 14 b
Days until total bud and flower drop 100 d a 12
10 c c 4 6 d 8
control 1-MCP Time of 1-DCP (h) 2 12 1-MCP Time of 1-DCP (h) Fig. 5. Mean of percent leaf drop (A, B) and total bud and flower drop (C, D) of miniature potted
rose cultivar ‘Lavender’ pretreated with 1-OCP and 1-DCP (1000 nl l-1) at exposure time of 0, 2,
4, 6, 12 h and untreated controls, respectively. These were compared to treatment with 1-MCP
(200 nl l-1) for 6 h at 20oC. After the treatments, the plants were exposed to 1.25 (+/- 0.25) µl l-1
continuous ethylene throughout the experiments. Bars marked with the same letter (for figures A,
B, C, D bars representing the same day) are not statistically different at P<0.05. Means were
separated by LSD.
25 Efficacy of new inhibitors of ethylene perception
2.3.3 Effect of temperature of 1-OCP and 1-DCP on display quality
Treatment of miniature roses with 1-OCP and 1-DCP (1000 nl l-1) for 4 h at all temperature
regimes (5, 10, 15 and 20oC) significantly improved their display quality as showed by reduced
leaf drop, total bud and flower drop compared with the control (Fig. 6A, B, C, D). All
temperature levels significantly (P<0.001) decreased leaf drop and increased number of days to
the onset of total bud and flower drop as compared to the untreated (control) plants (Fig. 6A, B,
C, D). No significant difference was observed in display life among all the temperature levels (5
to 20oC) in flowers treated with 1-OCP and 1-DCP. Plants treated with 1-MCP gave better results
than that of 1-OCP and 1-DCP. 26 Efficacy of new inhibitors of ethylene perception aaa A a 80 b b b b b b bc 60 Leaf drop (%) b b bc bc bb c
d 40 20 3
15 b bcd
day b b 0 bc b control 5 cd 15 20 bc b bc 5 10 15 20 8
6 d 4
2 1-MCP control B
bb b bc b b b bc b c b 60 cc b
40 c c d
20 e a
control bc bcd
5 10 cd
20 D 14
D ays until total bud and flower drop bb 1-MCP Temperature of 1-OCP (oC) a aa 80 Leaf drop (%) bc 0 10 a 3
15 a 10 Temperature of 1-OCP (oC) 100 day
day 12 b b b C 14
Days until total bud and flower drop 100 12
6 b b b 5 10 15 20 c 4
0 1-MCP control 1-MCP Temperature of 1-DCP (oC) Temperature of 1-DCP (oC) Fig. 6. Mean of percent leaf drop (A, B) and total bud and flower drop (C, D) of miniature potted
rose cultivar ‘Lavender’ pretreated with 1-OCP and 1-DCP at temperatures of 5oC, 10oC, 15oC,
20oC, and untreated controls, respectively. These were compared to treatment with 1-MCP (200 nl l-1)
for 6 h at 20oC and untreated controls. After the treatments, the plants were exposed to 1.25 (+/- 0.25)
µl l-1 continuous ethylene throughout the experiments. Bars marked with the same letter (for
figures A, B, C, D bars representing the same day) are not statistically different at P<0.05. Means
were separated by LSD. 27 Efficacy of new inhibitors of ethylene perception
2.3.4 Efficacy of 1-MCP, 1-OCP and 1-DCP in cultivars ‘Lavender’ and ‘Vanilla’
Efficacy of ethylene action inhibitors 1-MCP, 1-OCP and 1-DCP between ‘Vanilla’ (long lasting
flower) and ‘Lavender’ (short flower life) was investigated. In both cultivars, ethylene
accelerated flower senescence. However, there were distinct differences between ‘Vanilla’ and
‘Lavender’ when they were continuously exposed to 1.25 µl-1 ethylene (Fig. 7A, B, C, D).
Untreated ‘Vanilla’ plants attained 40% leaf drop after 6 days of continuous exposure to ethylene,
while the display quality of ‘Lavender’ was clearly reduced to 80% leaf drop in the same period
(Fig. 7A, B). Additionally, it took 6 days to attain total bud and flower drop in ‘Lavender’ (Fig. 7D).
Pretreatment with ethylene receptor inhibitors 1-OCP, 1-DCP and 1-MCP, improved the display
quality in both cultivars. In ‘Vanilla’, the trend of percent leaf drop in flowers treated with 1-OCP
and 1-DCP was similar to that of ‘Lavender’. Also, 1-OCP-treated plants reached 100% leaf drop
after day 15 and were less effective than 1-DCP (Fig. 7A, B). There was no significant difference
between the display quality of 1-DCP and 1-MCP. However, it took 9 and 10 days for ‘Vanilla’
plants to attain total bud and flower drop for 1-OCP and untreated (control) plants, respectively.
It took 11 and 12 days for ‘Vanilla’ plants treated with 1-DCP and 1-MCP to attain the total bud
and flower drop (Fig. 7C), respectively. In ‘Lavender’, 1-OCP compared favorably with 1-DCP
with respect to percent leaf drop, while pretreatment with 1-MCP was the most effective in
improving the display quality of miniature roses. The untreated ‘Lavender’ plants took 6 days to
attain total bud and flower drop while it took 8, 9 and 10 days for 1-OCP, 1-DCP and 1-MCP,
respectively (Fig. 7D). 28 Efficacy of new inhibitors of ethylene perception aa 100 a
b Leaf drop (%) 80 b bb b 10 a a a a ab 2 a 0 1-OCP 1-DCP control 1-MCP aa a B a Leaf drop (%) D 14
12 bb b a 10
b 60 c c 40 8 c a a 6 d 4 a bd 20 ab b b 30
15 1-OCP Treatments c day
day b Treatments 80 1-MCP b 4 b
a a control 100 1-DCP C 6 20 30
15 a 8 c 40 a 12 bb 60 b day
day 14 A a c
a 2 ad
0 control 1-OCP 1-DCP 1-MCP control 1-OCP 1-DCP 1-MCP Treatments Treatments Fig. 7. Mean of percent leaf drop and days of total bud and flower drop of miniature potted rose
cultivars ‘Vanilla’ (A, C) and ‘Lavender’ (B, D) pretreated with 1-OCP, 1-DCP (1000 nl l-1),
respectively, 1-MCP (200 nl l-1) for 6 h at 20oC and the control. After the treatments, the plants
were exposed to 1.25 (+/- 0.25) µl l-1 continuous ethylene throughout the experiments. Bars
marked with the same letter (for figures A, B, C, D bars representing the same day) are not
statistically different at P<0.05. Means were separated by LSD. 29 Efficacy of new inhibitors of ethylene perception
The duration of quality improvement was dependent on the concentrations. 1-OCP and 1-DCP,
two structural analogues of 1-MCP with substitution in 1-position, were considered to be putative
inhibitors of ethylene action. Their potency was evaluated by their ability to counteract ethyleneinduced responses (leaf, bud and flower abscission). These two analogues exerted their effect in a
similar manner to 1-MCP by blocking the binding site of ethylene in the receptor (Sisler and
Serek, 1997; Feng et al., 2004). In this study, all of these compounds were effective in blocking
the ethylene receptor and improving the display quality of miniature potted roses when compared
to untreated control plants. Based on the substitution with 8 and 10 carbon chains at 1-position of
the cyclopropene, both compounds were effective in improving display quality from exogenous
ethylene, but in terms of concentration, they were less effective than 1-MCP. Moreover, the
concentration required for 1-OCP and 1-DCP was about 5 times higher than that of 1-MCP. This
is in contrast to the previous studies of Sisler et al., (2003) and Kebenei et al., (2003a, b), where
1-OCP was effective at lower concentrations than 1-MCP in delay ripening in bananas and
improving display life and quality of kalanchöe and sweet pea, respectively. Sisler and Serek
(1997) suggested that for the maximum response in growing vegetative tissue, a higher
concentration of an inhibitor is needed. Besides, flowers on intact plants, as used by Müller et al.
(2000a, 2002b), may react differently to exogenous ethylene treatments than detached kalanchöe
flowers (Kebenei et al., 2003a, b). Probably the presence of many leaves in whole plants could
have absorbed higher amounts ethylene than excised flowers (Müller and Stummann, 2003). In
miniature roses, pretreatment with 1-MCP obviously reduced the rise in autocatalytic ethylene
production in flowers (Müller and Stummann, 2003), whereas in vegetative tissue, such as
tobacco leaves, ethylene production was autoinhibitited by a negative feedback control
(Philosoph-Hadas et al., 1985). Furthermore, another factor that should be taken into
consideration is the time it takes ethylene inhibitors to bind to the receptors and regain activity.
This suggests that the same receptor is becoming active again or that new receptors are being
produced (Sisler et al., 1990). The fact that some time after the treatment with 1-OCP or 1-DCP
the tissue became sensitive to ethylene again, assumes that free binding sites on the ethylene
receptor are present in the tissue at the point of recovery from the inhibition. However, the
analogue 1-OCP was found to be a more potent inhibitor than 1-DCP, when pretreating ethylene- 30 Efficacy of new inhibitors of ethylene perception
sensitive cultivars like ‘Lavender’. In contrast, pre-treating ‘Vanilla’, which has long-lasting
flowers, with 1-DCP was more effective than 1-OCP.
Both 1-OCP and 1-DCP are gases at room temperature (Closs, 1966). They need to be dispersed
as liquids that need time to evaporate and diffuse to the binding sites in order to bind to them.
Display quality was dependent on exposure time, such that longer exposure times improved the
display quality better than shorter exposure times. Similar observations were reported on other
cyclopropenes (Dupille and Sisler, 1995; Sisler et al., 1996a, 1999). Molecular weight has no
influence on evaporation, in that higher molecular weight compounds bind as rapidly as the lower
ones, suggesting evaporation time into the gaseous phase is not a big factor (Sisler et al., 2003).
Continuous exposure for longer periods results in saturation of the receptor and further treatment
does not give any additional effect. For example, exposure of both chemicals for 12 h gave the
same effect as 4 h exposure. Therefore, a 4-h exposure is sufficient to give better improvement
against ethylene. Shorter exposure time (2 h) with the same concentration was not satisfactory.
This may require a higher concentration of the respective cyclopropenes (Sisler et al., 1999,
2003) for the same beneficial effect to be realized.
Temperature has a direct influence on the exposure time and concentration of the treatment. At
lower temperatures, a higher concentration and longer time of treatment is required (Sisler et al.,
1996a, 1999; Sisler and Serek, 1997). In this study, the temperature range 5-20oC had no effect
on the activity of both chemicals, implying that pre-treatment of potted roses with these
compounds can be done within temperatures that range from 5-20oC without any affects on their
effectiveness. Contrary to kalanchöe (Kebenei et al., 2003a, b), the activity of 1-OCP was
influenced by temperature, and it was more potent at 15 and 20oC than at lower (5 and 10oC)
temperatures. In carnations and Penstemon, a higher concentration of 1-MCP was required at low
temperatures in order to give protection against ethylene (Serek et al., 1995a; Sisler et al., 1996a).
Differences in ethylene production and/or sensitivity are a vital factor regulating flower life. For
this reason ethylene partly resulted in differential display quality (Müller et al., 1998, 2001).
Ethylene caused leaf, bud and flower drop, but there were differences among cultivars.
‘Lavender’, which is sensitive to ethylene, significantly exhibited 80% leaf drop as well as a total
bud and flower drop within 6 days as compared to ‘Vanilla’, which took 9 days. It could be
31 Efficacy of new inhibitors of ethylene perception
assumed that ‘Lavender’ was similar to cultivar ‘Bronze’, in which exposure to ethylene induces
an autocatalytic rise in ethylene with constant increase in endogenous ethylene levels (Müller et al.,
2001). Although ‘Vanilla’ had excellent longevity and seemed to be almost completely
insensitive to exogenous ethylene (Müller et al., 2001), continuous exposure to ethylene caused
flower, bud and leaf drop. In the current study, ‘Vanilla’ had about 80% leaf and total bud drop
within 9 days, which was retarded by ethylene inhibitors. In the miniature rose ‘Victory Parade’, 1MCP protected against exogenous ethylene, increased display life and reduced abscission of
buds, flowers and leaves (Serek et al., 1994c). Therefore, for ethylene-sensitive cultivars like
‘Lavender’, ethylene inhibitor treatment will be an important practical tool in increasing the
postharvest life after ethylene exposure. It would also be more effective in improving the display
quality of the ethylene-sensitive cultivar ‘Lavender’ than ethylene-insensitive ‘Vanilla’.
In conclusion, these results demonstrated the potency of 1-MCP and long chain CPs, 1-OCP and
1-DCP, as ethylene antagonists in improving display quality of miniature potted roses.
Additionally, 1-MCP was the most potent ethylene inhibitor in terms of concentration and
duration of exposure in improving display quality as compared to 1-OCP and 1-DCP. Therefore,
1-MCP is more suitable for use in potted plants while 1-OCP is more effective in prolonging the
display life of individual flowers like kalanchöe. 32 Expression analysis of genes for ethylene biosynthesis enzyme 3. Expression analysis of genes for ethylene biosynthesis enzyme, ethylene
perception and signal transduction pathway after pretreatment with ethylene
receptor inhibitors of miniature potted roses (Rosa hybrida L.)
In order to investigate transcript abundance of genes for ethylene biosynthesis enzyme (RhACS1-5),
ethylene perception (RhETR1-4) and signal transduction pathway (RhCTR1-2, RhEIN3, RhEIL),
expression of these transcripts in petal and leaf tissues was examined in two rose cultivars
‘Vanilla’ and ‘Lavender’ during continuous exposure to exogenous ethylene and ethylene-free air
after pretreatment with inhibitors of ethylene receptors 1-MCP, 1-OCP and 1-DCP. Additionally,
one of the ethylene-responsive element-binding factor (ERFs) genes was isolated from miniature
potted roses (RhERF1) which was 92 bp long, encoding a predicted polypeptide for an Ap2 DNA
binding domain and sharing high homology with other reported ERF domains. Northern blot
hybridization analysis was used to compare with results of RT-PCR. However, it was difficult to
distinguish the hybridization signal of ethylene receptors between RhETR1 and RhETR2, which
were highly homologue. RT-PCR analysis revealed that ethylene induced the expression of all
genes investigated in control ‘Vanilla’ and ‘Lavender’ petals except RhACS1-2 and RhETR4,
respectively, while pretreatments of 1-MCP and 1-OCP led to the supression of all investigated
mRNAs in the presence or absence of ethylene. However, strong expression of RhETR3, RhEIN3
and RhEIL transcripts was detectable in ‘Lavender’ petals pretreated with 1-DCP. In the absence
of ethylene, strong expression of all genes was detectable in control ‘Vanilla’ leaves but there
was no expression of RhACS1, while the accumulation of all genes was eliminated after
pretreatments of ‘Vanilla’ leaves with ethylene receptor inhibitors. The level of RhEIN3 mRNA
was upregulated by pretreatment of 1-MCP, 1-OCP and 1-DCP in ‘Vanilla’ leaves in the
presence of ethylene, while the accumulation of RhEIN3 transcript was suppressed in ‘Lavender’
leaves after pretreatment of 1-MCP and 1-OCP. RhEIN3 transcript is probably rate-limiting for
ethylene perception and signal transduction pathway, which is regulated in leaves during flower
senescence at the transcriptional level. In the absence of ethylene, 1-MCP suppressed the
expression of RhACS1, RhETR3 and RhERF1 transcripts. Similarly, 1-OCP inhibited the
expression of RhACS1, RhCTR2 and RhERF1 transcripts. 1-DCP inhibited the expression of
33 Expression analysis of genes for ethylene biosynthesis enzyme
RhEIN3, RhEIL genes. However, in the presence of ethylene, pretreatment of ‘Lavender’ leaves
with 1-MCP suppressed the expression of RhACS1, RhETR4, RhCTR1-2, RhEIL and RhERF1
transcripts. Similarly, 1-OCP treatment inhibited the expression of RhACS1, RhETR2, RhETR4,
RhCTR1-2, RhEIN3 and RhERF1 transcripts. Pretreating ‘Lavender’ leaves with 1-DCP
suppressed the expression of RhACS1 and RhETR4 transcripts. These results indicated that the
expression of genes for the ethylene biosynthesis enzyme, ethylene perception and signal
transduction pathway was regulated by both positive and negative feedback regulation
mechanisms in ‘Vanilla’ and Lavender’ pretreated with ethylene receptor inhibitors in the
presence or absence of ethylene. This might depend on plant species and tissues under
investigation. Physiologically and molecularly, these results suggest that pretreatments with
inhibitors of ethylene receptors improved the display life of miniature roses by delaying bud,
flower and leaf drop. However, 1-MCP is more effective than 1-OCP and 1-DCP by suppressing
the expression of ethylene signal transduction genes or the feedback regulations of genes for
ethylene biosynthesis and ethylene perception.
Key words: ACC synthase, display quality, ethylene, ethylene perception and signal transduction,
gene expression, inhibitors of ethylene receptor, Rosa hybrida, senescence 34 Expression analysis of genes for ethylene biosynthesis enzyme
3.1.1 Expression regulation of genes for ethylene biosynthesis enzyme
Ethylene biosynthesis and perception are modulated during development of plant tissues
(Abeles et al., 1992) and responsible for inducing many biochemical processes which lead to
programmed cell death, including the expression of senescence-related genes (Woodsons et al.,
1992; Kim et al., 1997). Ethylene biosynthesis in higher plants has been well characterized and
1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), an enzyme of ethylene
biosynthesis, has been recognized as the rate-limiting step (Yang and Hoffman, 1984; Kende,
In all plants, ACC synthase is encoded by a medium-size gene family, which is differentially
regulated in a tissue-specific manner by a variety of developmental cues, mechanical and
environmental stresses and by ethylene itself (Kende, 1993; Zarembinski and Theologis, 1994). In
tomato fruits, ACC synthase is a soluble enzyme (Boller et al., 1979). This indicates that ACC
synthase is a cytoplasmic enzyme (Kende, 1993).
Sequence analysis of the Arabidopsis genome reveals that ACS genes are putatively encoded by
twelve genes (Yamagami et al., 2003). All genes, except ACS3, are transcriptionally active and
differentially expressed in Arabidopsis during growth and development. Additionally, IAA
induces all genes, except ACS7 and ACS9; cyclohexamide (CHI) enhances the expression of all
functional ACS genes. These ACS genes, which have a biochemically-diverse function in unique
cellular environments for the biosynthesis of ethylene, permit the signaling molecule to exert
their unique effects in a tissue- or cell-specific manner (Yamagami et al., 2003).
In Rosa hybrida, ACS genes consist of a multigene family (Mibus and Serek, 2004; Wang et al.,
2004). Analysis of the expression of ACS genes shows that RhACS1 is specifically expressed in
rose petals, the ovary and sepals. This expression increases dramatically as the flower matures to
senescence and also correlates positively with ethylene levels (Wang et al., 2004). 35 Expression analysis of genes for ethylene biosynthesis enzyme
3.1.2 Ethylene receptor gene expression
Ethylene actions are involved with binding to a receptor, followed by activation of one or more
signal transduction pathways that lead to the cellular response. Ultimately, ethylene exerts its
effects primarily by changing the pattern of gene expression (Taiz and Zeiger, 2002).
Furthermore, the development of molecular genetic approaches in the model plant Arabidopsis
has established the linear signal transduction pathway for the response to ethylene (Hongwei and
Ecker, 2004). Genetic screens based on the triple-response phenotype, inhibition of hypocotyls
and root cell elongation, radial swelling of hypocotyls and exaggerated curvature of the apical
hook, have been extensively conducted on etiolated Arabidopsis seedlings to isolate mutants that
are affected in their response to ethylene (Bleecker et al., 1988; Guzman and Ecker, 1990). Two
classes of mutants have been identified:
1. Mutants that fail to respond to exogenous ethylene (ethylene-resistant or ethyleneinsensitive mutants).
2. Mutants that display the response even in the absence of ethylene (constitutive mutants).
Ethylene-insensitive mutants are identified as tall seedlings extending above the lawn of short,
triple-responding seedlings when grown in the presence of ethylene. Conversely, constitutive
ethylene response mutants are identified as seedlings displaying the triple response in the absence
of exogenous ethylene (Guzman and Ecker, 1990).
The ETR1 gene is the first member of the receptor gene family to be cloned from Arabidopsis
(Chang et al., 1993). Expression of ETR1 protein, which is obtained from plants and transgenic
yeast, is not only membrane-associated but also exists as a covalently-linked dimer. Two
cycteines at the N-terminus (Cys4 and Cys6) are the sites of disulphide cross-linkage between
ETR1 monomers. This N-terminal sensor domain also consists of all the elements essential and
sufficient to bind ethylene with high affinity when expressed in yeast (Schaller et al., 1995;
Schaller and Bleecker, 1995). The C-terminal half of the ETR1 protein contains all of the
conserved sequence elements found in the histidine kinase domains of bacterial two-component
regulators. These are typically composed of a sensor protein with an input domain receiving
signals and a catalytic transmitter domain which autophosphorylates on an internal histidine
36 Expression analysis of genes for ethylene biosynthesis enzyme
residue. The second component is a response regulator protein consisting of a receiver domain,
which receives phosphate from the transmitter and an output domain that mediates responses
depending on the phosphorylation state of the receiver. The ETR1 protein has a receiver domain
fused to the C-terminus of the histidine kinase domain (Parkinson, 1993; Maeda et al., 1994). In
addition, the two-component system is a signaling mechanism primarily used by bacteria to
respond to a broad array of environmental signals (Stock et al., 2000). However, the region that is
located between the hydrophobic sensor domain and the histidine kinase transmitter is of
unknown function. Besides, there are two blocks of sequence within this region, which shows
homology to sequences that flank the chromophore-binding domain in the light-sensing
phytochromes from higher plants and cyanobacteria (Kehoe and Grossman, 1996). It has been
reported that the region between these blocks of homology in ETR1 contains a GAF domain
associated with cyclic GMP binding and light regulation in a number of proteins, but the function
of the GAF domain in ETR1 is unknown (Aravind and Ponting, 1997). Studies of ETR1 indicate
that localization of transmembrane domains on the receptor also serve to the endoplasmic
reticulum (ER), an unusual location for a hormone receptor, but compatible with the ready
diffusion of ethylene in aqueous and lipid environments (Chen et al., 2002). Fig. 8. Basic scheme of the two-component system (Chang and Meyerowitz, 1995).
Ethylene-binding domain of ETR1 lies within the first 165 amino acids of the protein containing
the sequences that are necessary and sufficient to bind ethylene. Further characterization of
ethylene binding to ETR1 occurs in a hydrophobic pocket, located at the N terminus of the
receptor, and requires a transition metal, copper, as a cofactor (Schaller and Bleecker, 1995;
Rodriguez et al., 1999). The identification of a copper ion is associated with the ethylene-binding
domain of ETR1 with theoretical considerations dating back to Stanley Burg’s original hypothesis
37 Expression analysis of genes for ethylene biosynthesis enzyme
that ethylene binding would be mediated by a transition metal cofactor (Burg and Burg, 1967).
Interestingly, silver (Ag(I)), which has long been known to inhibit ethylene-responses, may also
occupy the binding site and interact with ethylene but fail to induce the changes that would
normally occur following copper-mediated ethylene binding (Rodriguez et al., 1999). Cloning of
Arabidopsis RAN1 resembles copper transporting P-type ATPase and rescues a copper transport
defect in yeast (Hirayama et al., 1998). Within the plant, RAN1 potentially serves to produce
functional ethylene receptor via intracellular delivery of copper. The loss-of-function of ran1
mutants displays a rosette-lethal phenotype, which is thought to be caused either by general
effects due to the reduced copper availability for other copper-utilizing enzymes, or by disruption
of ethylene receptor function (Hirayama et al., 1998; Woeste and Kieber, 2000). Fig. 9. The Arabidopsis ethylene receptor family (Chang and Shockey, 1999).
Five ETR1-like genes have been identified in Arabidopsis so far. These five genes can be divided
into two main subfamilies based on sequence similarity and structure features of the protein.
Members of type-I subfamily ETR1 and ERS1 (Hua et al., 1995, 1998), are most closely related
in sequence and have three hydrophobic subdomains at the N-terminus. The type-II subfamily
receptors, ETR2 (Sakai et al., 1998), ERS2 and EIN4 (Hua et al., 1998), share close sequence
similarity and all have four hydrophobic subdomains at the N-terminus.
Like ETR1, ERS1 binds ethylene when expressed in yeast (Schaller et al., 1995), and its
transmitter domain containing all the conserved subdomains (H, N, G1, F and G2) is thought to
be required for histidine kinase activity. ERS1 differs from ETR1 in that it lacks the fused
38 Expression analysis of genes for ethylene biosynthesis enzyme
receiver domain. Conversely, members of the ETR2-like subfamily appear to be degenerate in the
histidine kinase domain. Each member of this subfamily lacks one or more elements that are
thought to be essential for histidine kinase activity; therefore it is unlikely that they are functional
kinases (Schaller et al., 1995).
In miniature potted roses, fragments of four ethylene receptor genes have been isolated and
cloned, termed RhETR1-4 (Müller et al., 2000a, b). They are an ethylene receptor gene family
with 2 subfamilies, as in Arabidopsis. RhETR1 and RhETR4 exhibit high amino acid similarity to
AtERS1, while RhETR2 is more similar to AtETR1. Three of the receptors belong to subfamily 1,
whereas RhETR3 belongs to subfamily 2. The northern blot analysis is conducted in miniature
roses for RhETR1-3 during flower senescence after treatment with ethylene and ABA to
investigate the differences in flower longevity and ethylene sensitivity in miniature roses. The
relative transcript abundance in the flowers varies significantly during development and after
hormone treatment, but the expression of all three genes is detectable at all flower stages.
Exposure to low ethylene concentrations results in an upregulation of RhETR1 and RhETR3 in
flowers of ‘Bronze’, a short-life flower, and are distinctly higher than long-lasting ‘Vanilla’. It is
assumed that differences of cultivars in flower sensitivity to ethylene may partly be due to
differences in receptor expression levels during flower development (Müller et al., 2000a, b).
While RhETR2 expression varies slightly during flower development and in response to ethylene
and ABA treatment, the expression of RhETR1 and RhETR3 exhibits differently during flower
development and appears to be rate-limiting for ethylene perception and the determinants of
flower longevity. It is found that the expression of RhETR1 in ‘Bronze’ is distinctly higher than
long-lasting ‘Vanilla’. Although the expression of RhETR1 precedes the ethylene production by
the flowers, abundance of the RhETR3 transcript increases during flower senescence in ‘Bronze’,
indicating that the ethylene response system in rose flowers consists of multiple receptor genes
with an overlapping pattern of expression. In ‘Vanilla’, a cultivar with excellent flower longevity
despite moderate ethylene production (Müller et al., 2000b), expression of RhETR1 and RhETR3
is reduced. These results indicate that differences in flower life among rose cultivars – in an
ethylene free environment and in response to exogenous ethylene – may be due to differences in
receptor expression levels. These results do not conform to the standard model of ethylene signal
transduction (Bleecker, 1999), which predicts that a reduction in the level of receptors would
39 Expression analysis of genes for ethylene biosynthesis enzyme
result in increased ethylene sensitivity, while an increase in receptor numbers would result in
3.1.3 Ethylene-insensitive mutant of ethylene receptor
Mutations in the ethylene receptors cause ethylene insensitivity or constitutive ethylene
responses, dependent on the nature of the mutation. Ethylene insensitivity is a result of single
amino acid changes within the region of the receptor involved in ethylene binding (Chang et al.,
1993; Hua et al, 1995, 1998; Sakai et al, 1998). The ethylene-insensitive mutations are dominant.
Little or no effect of single loss-of-function mutations upon ethylene signaling transduction is
investigated by experiments using combinations of receptor knock-out (Hua and Meyerowitz,
1998), indicating that there is functional overlap among the receptor family members and the
receptors serve as negative regulators of the ethylene response pathway, because elimination of
receptors activates ethylene responses. According to this model for negative regulation, wild-type
ethylene receptors actively repress ethylene responses in the air but express in the presence of
ethylene. Wild type receptors switch to a signaling inactive state that allow for induction of
ethylene responses (Bleecker, 1999). In contrast to the single and double loss-of-function, triple
and quadruple receptor mutants show constitutive-ethylene response phenotypes because the
basal levels of ethylene, which are constitutively produced in these plants, are sufficient to
inactivate the remaining receptor ‘repressive’ activity (Cancel and Larsen, 2002). Therefore, an
ethylene-insensitive mutation in one member of the five-member ethylene insensitivity suggests
that signaling by one family member is enough to repress ethylene responses. Conversely, lossof-function mutations in three receptors are simultaneously sufficient to induce ethylene
responses (Hua and Meyerowitz, 1998). Ethylene bind to the receptor alters the activity of the His
kinase domain, which in turn influences downstream components CTR1 and EIN2 (Guzman and
Ecker, 1990; Kieber et al., 1993; Ecker, 1995).
Mutant, etr1-1, which no longer binds copper, is incapable of binding ethylene (Rodriguez et al.,
1999), and the receptor is unable to turn CTR1 “on” even when ethylene is present, resulting in
the ethylene-insensitive phenotype. The expression of the Arabidopsis etr1-1 gene in transgenic
carnations delays flower senescence, which leads to a significant increase in vase life. About half
of the transgenic plants obtain flower senescence, which delays a 3-fold increase in vase life by at
40 Expression analysis of genes for ethylene biosynthesis enzyme
least 6 days relative to control flowers, with a maximum delay of 16 days. These flowers do not
show the phenotype typical of ethylene-dependent carnation flower senescence (Bovy et al.,
1999). The expression of the ACO1 gene in etr1-1 transgenic carnation flowers is down
regulated, suggesting that the autocatalytic induction of ethylene biosynthesis is absent due to
dominant ethylene insensitivity. The delay in senescence observed in etr1-1 transgenic flowers is
longer than in flowers pretreated with inhibitors of either ethylene biosynthesis (AOA) or
ethylene receptor (STS) (Van altvorst et al., 1995, 1997; Bovy et al., 1999). Agrobacteriummediated transformation of tomato, tobacco and petunia plants with Arabidopsis etr1-1 gene
under control of the constitutive CaMV 35S promoter is successfully conferred ethylene
insensitivity. An ethylene-insensitive phenotype delays flower senescence after pollination and
after exogenous ethylene treatment (Wilkinson et al., 1997). Ciardi and Klee (2001) elucidated
mode of the dominant etr1-1 mutation, which causes a substitution of tyrosine (Y65) for cysteine
at amino acid position 65, eliminating both ethylene and copper binding (Fig. 10). Since ethylene
does not bind, phosphorylation cannot be inactivated and the ethylene response is constitutively
suppressed. Additionally, ethylene inhibitor action, STS and 1-MCP, associated with theoretical
considerations (Sisler and Serek, 1997), can be explained at the molecular level that the receptor
(ETR1) exhibits strong affinity for another metal, silver, and mediates ethylene binding. Silver
probably acts by displacing copper in the active site of the receptor complex that still binds to
ethylene (Ciardi and Klee, 2001). Therefore it must prevent a conformational change that would
normally occur following copper-mediated ethylene binding. The compound 1-MCP, can also be
bound by ethylene, but binding does not increase ethylene sensitivity and serves to block ethylene
perception instead (Fig. 10). Although ethylene receptors are capable of binding several different
molecules, the interaction required to induce an ethylene response is extremely specific (Ciardi
and Klee, 2001). 41 Expression analysis of genes for ethylene biosynthesis enzyme Fig. 10. A model for the role of kinase activity in ethylene signalling. A, The Arabidopsis ETR1
receptor. B, Ethylene binding to wild-type receptor. C, Loss-of-function mutations in multiple
ethylene receptors. D, The dominant etr1-1 mutation. E, Silver ion competes with copper for
receptor binding sites. F, The ethylene action inhibitor 1-methylcyclopropene (1-MCP) competes
with ethylene to bind to the receptor. (Ciardi and Klee, 2001).
3.1.4 Expression regulation of ethylene signal transduction pathway
Genetic analysis indicates that CTR1, which encodes a protein consisting of 821 amino acids, acts
downstream of the receptor in the ethylene signal pathway and localizes at the ER (Kieber et al.,
1993; Roman et al., 1995; Sakai et al., 1998; Guo et al., 2003). Because CTR1 has no obvious
trans-membrane domain or membrane motifs, its ER localization probably results from direct
interaction with the ER-associated receptor (Guo et al., 2003). CTR1 is a negative regulator of the
42 Expression analysis of genes for ethylene biosynthesis enzyme
ethylene-response pathway because ctr1 null mutants exhibit constitutive ethylene responses even
in the absence of ethylene (Kieber et al., 1993). The deduced CTR1 protein sequence is most
similar to the Raf family of protein kinase, which initiates mitogen-activated protein (MAP)kinase signaling cascades in mammals (Fig. 11) (Kyriakis et al., 1992; Pelech and Sanghera,
1992). The similarity of CTR1 known MAPKKKs implies that ethylene signaling may operate
through a MAP-kinase cascade. Although many genes with homology to MAPKKs and MAPKs
have been identified in the Arabidopsis genome sequence, currently none have been involved
with ethylene signaling. Thus, no intermediate components have been identified genetically or
biochemically to act between the receptors and the CTR1 kinase. The two-hybrid yeast assay
shows that the amino-terminal domain of CTR1 can interact with the His kinase domains of ETR1
and ERS1 (Clark et al., 1998). The function of CTR1 is dependent on both its carboxy-terminal
Ser/Thr kinase activity and the association of its amino-terminal domain with ER-bound ethylene
receptors. Interestingly, CTR1 is also able to interact weakly with carboxy-terminal domain ETR2
(type-II). Thus, it is hypothesized that all five ethylene receptors are able to interact with CTR1
via their carboxy-terminal kinase domains. However, type-I receptors (ETR1 and ERS1) have a
high affinity for CTR1, whereas type-II (at least ETR2) possess a low binding affinity for CTR1
(Cancel and Larsen, 2002). Loss of function in the ethylene receptors or CTR1 results in the
induction of ethylene responses. In air, the ethylene receptors maintain CTR1 in an “on” state that
serves to repress the ethylene responses. Binding of ethylene to the receptors turns CTR1 “off”
and leads to derepression of the pathway. Specifically, inactivation of CTR1 would result in the
activation of EIN2, and consequently activation of the transcriptional cascade that involves the
EIN3/EIL and the ERFs (Ecker, 1995). 43 Expression analysis of genes for ethylene biosynthesis enzyme Fig. 11. A current view of the ethylene signal transduction pathway formulated on the basis of
cloned Arabidopsis genes (Chang and Shockey, 1999).
In Arabidopsis, signal propagated from CTR1 to the nucleus requires EIN2 to encode an integral
membrane protein of 1294 amino acids. Null mutations in EIN2 result in the complete loss of
ethylene responsiveness throughout plant development, suggesting that EIN2 is an essential
positive regulator in the ethylene signal pathway (Fig. 11). EIN2 comprises 12 predicted
transmembrane domains in the third of the polypeptide, a region that resembles members of the
NRAMP family, including metal-ion transporters. The carboxy-terminal portion of EIN2 is a
novel sequence predicted to be soluble and cytosolic. EIN2 is also membrane-associated, but
lacks detectable metal transport activity. The amino-terminal domain may serve as a sensor of
divalent cations or as a membrane anchor. Overexpression of hydrophobic carboxy-terminal
domain in an EIN2 null background results in constitutive activation of some, but not all, of the
ethylene response pathway. These results suggest that ethylene regulation of this activity requires
the EIN2 amino-terminal domain. It is hypothesized that the amino-terminal region of EIN2
44 Expression analysis of genes for ethylene biosynthesis enzyme
represents an input domain, interacting with upstream signaling factors, while the carboxyterminal region represents an output domain, interacting with downstream signaling factors
(Alonso et al., 1999).
Based on genetic evidence, EIN3 is a novel nuclear-localized DNA binding protein that encodes a
protein of 628 amino acids and acts downstream of EIN2. EIN3, which contains acidic, pralinerich and glutamine-rich domains found in transcriptional activation domains, is nuclear-localized.
Loss of function of EIN3 results in an ethylene insensitive phenotype, indicating that EIN3 is a
positive regulator of ethylene signal transduction (Fig. 11). In Arabidopsis, EIN3 is a multigene
family, in which EIN3 and EIN3-like (EIL1) are the most closely related proteins. Loss of
function mutation in the EIL1 gene shows incomplete ethylene insensitivity like ein3 mutants, but
eil1 mutants are more sensitive to ethylene than ein3 mutants. In addition, overexpression of
EIN3 and EIL1 genes confers a constitutive ethylene response phenotype on dark-grown
seedlings as well as those grown in the light. Moreover, EIN3 and EIL proteins comprise a novel
family of sequence-specific DNA-binding proteins and define a primary ethylene response
element (PERE) present in the promoters of several ethylene-regulated genes (Chao et al., 1997).
EIN3 is both necessary and sufficient for the activation of ethylene responsive target genes,
particularly for ERF1 (Solano et al., 1998).
ERF1, which is induced by the EIN3/EIL1, is itself a transcription factor, indicating that ethylene
signal transduction involves a transcription cascade (Fig. 11) (Solano et al., 1998), and is
formerly known as EREBP (ethylene-responsive element-binding protein). This gene belongs to a
family of ethylene-responsive element-binding factor (ERF) proteins (Ohme-Takagi and Sinshi,
1995; Fujimoto et al., 2000). ERF1, known to be a trans-acting factor responsible for ethyleneregulated expression of genes, induces ripening and senescence and interacts specifically with
sequences consisting of AGCCGCC motifs (GCC box) that are found in the promoters of several
pathogenesis genes, such those encoding β-1, 3-glucanase, basic chitinase, and defensin (PDF1.2)
(Ohme-Takagi and Sinshi, 1995; Hao et al., 1998; Solano et al., 1998). Some ERFs contain GCC
boxes, a cis-acting promoter element, which is both necessary and sufficient to confer ethylene
responsiveness in their promoters indicating that they are targets for other members of the ERF
family, and suggesting the existence of a tertiary level of transcriptional regulation in ethylene
signal transduction (Solano et al., 1998). Moreover, ERFs share a well-conserved 58-59 amino
45 Expression analysis of genes for ethylene biosynthesis enzyme
acid domain, ERF domain, which has a novel structure containing three β-sheets and an α-helix
binding to DNA as a monomer, with high affinity (Allen et al., 1998; Hao et al., 1998). This
domain is related to the AP2 domain in the Arabidopsis gene APATALA2 (Jokofu et al., 1994),
although ERF proteins contain a single DNA-binding domain whereas the APATALA2-type
proteins typically contain two (Riechmann and Meyerowitz, 1998). Overexpression of ERF1
induces a partial triple-response phenotype in dark-grown seedlings, with the seedlings lacking
the exaggerated apical hook normally associated with the triple response (Solano et al., 1998).
This result indicates that the roles of ERF1 are not confined to the pathogenesis response and
other factors. ERF1 is required for initiating a full ethylene response. Analysis of the ERF family
in Arabidopsis reveals that some members, such as ERF1, function as transcriptional activators
while other members function as repressors (Fujimoto et al., 2000).
ERFs can be divided into three classes based on their amino acid sequences. All classes consist of
the ERF domain, but the acidic domain in class I has been found in the N-terminal region. In
addition, members of this group possess a region rich in basic amino acids (P/L-K-K/R-R-R) that
could serve as a putative nuclear localization signal (Raikhel, 1992). In class II, ERF domain is
located close to the N-terminus of the protein. It comprises 58 amino acids, with one residue
shorter than that of class I ERFs. Class III has the longest coding region among members of ERF
protein. Acidity domains are located in both the N- and C-terminal region flanking the ERF
domain. Additionally, a putative mitogen-activated protein (MAP) kinase phosphorylation site is
conserved in the C-terminal region. Class I and III are activators, whereas class II has active
repressors that can suppress the activity of other transcriptional activators without competing for
DNA binding sites (Fujimoto et al., 2000; Ohta et al., 2001). Class IV is the new ERF class that
displays a novel and highly conservative N-terminal motif (MCGGAII/L). Though the function of
MCGGAII/L motif has not been established, deletion studies indicate that it is required neither for
nucleus localization nor for binding to the GCC box (Tournier et al., 2003). In Arabidopsis,
AtERF1 and AtERF2 belong to class I, AtERF3 and AtERF4 possess class II and AtERF5 is the
only identified member of class III (Fujimoto et al., 2000). ERF proteins play important roles in
plant responses to signals that are induced by extracellular stress. They are involved in the
induction of gene expression by stress factors, such as pathogens and cold, and by components of
stress signal transduction pathways, such as ethylene, abscisic acid and jasmonic acid (OhmeTakagi and Shinshi, 1995; Büttner and Singh, 1997; Stockinger et al., 1997; Zhou et al., 1997;
46 Expression analysis of genes for ethylene biosynthesis enzyme
Finkelstein et al., 1998; Solano et al., 1998; Menke et al., 1999). Most of the members of this
family have been characterized and shown to participate in stress and/or hormonal responses
(Solano et al., 1998; Fujimoto et al., 2000). The ERF domain-containing Pti4/5/6 gene products
bind to the pathogenesis-related Pto protein kinase in tomato (Zhou et al., 1997) and over
expression of Pti4 in Arabidopsis induces the expression of GCC box-containing genes (Wu et
al., 2002), thereby enhancing resistance to pathogen attack (Gu et al., 2002).
In Rosa hybrida, the expression of RhCTR1 (66% amino acid identity to Arabidopsis CTR) and
RhCTR2 (87% amino acid identity to AtEDR1 and 90% identity to LeCTR2) during flower
senescence and in response to ethylene has been examined. RhCTR1 levels have been upregulated
in senescing flowers, while RhCTR2 is constant during flower senescence. The expression of both
genes increases in response to ethylene, indicating the role of these genes in ethylene sensitivity
and postharvest performance (Müller et al., 2002a). However, it can be assumed that flowers
become more sensitive to ethylene during senescence. CTR expression will decrease in the
developmental stage of flowers and in response to exogenous ethylene as the prediction of the
negative regulator model.
RhEIN3, a cDNA encoding part of an EIN3 transcription factor homologue, has been isolated in
Rosa hybrida. The deduced protein has 83% and 88% identity to the corresponding regions of
Arabidopsis EIN3 and EIL1, respectively. The expression of RhEIN3 transcript during flower
development has been investigated in the miniature rose cultivars ‘Bronze’, of short flower life
and high ethylene production, and ‘Vanilla’, long-lasting and moderate ethylene production, at
three stages of flower development: the bud, the opening flower and the incipient senescence
stage. For the opening flower stage, changes in expression of RhEIN3 after ethylene and ABA
treatment have been investigated. The gene RhEIN3 is constitutively and stably expressed during
flower development in both cultivars and in response to ethylene and ABA. The constitutive
pattern of RhEIN3 expression could not be correlated with the previous observed cultivar
differences in flower life or ethylene sensitivity in flower or leaves of miniature roses (Müller et al.,
2002b). These observations are in accordance with previous studies in Arabidopsis (Chao et al.,
1997) and tobacco (Kosugi and Ohasi, 2000), in that ethylene does not affect the level of EIN3
mRNA, and suggests that control of ethylene sensitivity occurs upstream of EIN3 and its
47 Expression analysis of genes for ethylene biosynthesis enzyme
Therefore, the biochemical investigation of inhibitors of ethylene responses, and studies of the
ethylene signal transduction pathway using a molecular genetic approach can determine the
manner in which ethylene influences these processes. From the previous study of physiological
investigation, it is assumed that the difference in the display quality of miniature potted roses
after pretreatment with air, 1-OCP, 1-DCP, 1-MCP and afterwards continuous exposure to
ethylene, might have an influence on the expression of genes for ethylene biosynthesis enzymes,
ethylene perception and the signal transduction pathway. Two selected cultivars, ‘Vanilla’ (longlasting flower) and ‘Lavender’ (short flower life) were selected in order to understand the
mechanisms that regulate ethylene induced flower senescence and genetic control of the ethylene
response pathway. This could lead to a possibility of selective block processes in flower
development that are economically detrimental, and to improve display quality using new
chemicals for ethylene receptor inhibition. However, there is no report about the ERF domaincontaining genes in miniature roses so far. In order to identify and characterize differentiallyexpressed genes that might provide more insight into explaining the genetic basis of the ethylene
response pathway, the cloning and initial characterization of a novel ERF gene, RhERF1, from
Rosa hybrida was reported. 48 Expression analysis of genes for ethylene biosynthesis enzyme
3.2 Materials and Methods
3.2.1 Plant material
The plants were grown as previously described in chapter 2.2.6. They were harvested after
continuous exposure to exogenous ethylene and ethylene-free air for 9 days and then put in 15 ml
polyethylene tubes (Sarstedt, Germany) and immediately frozen in liquid nitrogen. They were
then ground in liquid nitrogen and stored at -80oC in a deep freezer until extraction of DNA or
3.2.2 Database analyses and primer design
A sequence comparison between known ERF sequences of different plant species was
done according to Mibus (2003). The ERF primer pairs were designed using program Primer 3
(Steve and Skaletsky, 2000) (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and was
commercially synthesized by MWG Biotech AG, as shown in Table 1.
Table1. Primer pair for amplification of the Ap2 (DNA binding Domain) coding sequence of
Name Sense Primer Name Antisense Primer Annealing
Temp ERF1s 5’TACAGAGGCGTTAGGAAGCG3’ ERF1as 5’CGAAAGTGCCAAGCCAGA3’ 55 Size (bp) (oC)
92 3.2.3 DNA isolation and PCR
Genomic DNA was isolated from 80 mg of ground petals of miniature roses ‘Vanilla’ and
‘Lavender’ using DNeasyR Plant Mini Kit (Qiagen GmbH, Hilden, Germany), according to the
manufacturer’s instructions. The concentration of ‘Vanilla’ and ‘Lavender’ genomic DNA was
determined by comparing it with standard concentrations (5, 10, 25, 50, 100 and 200 µg ml-1) of
λDNA (Fermentas GmbH, St, Leon-Rot, Germany) in 1% agarose flatted gel electrophoresis
visualised by staining with 40 µg ethidium bromide. A temperature gradient PCR was performed
to optimize the annealing temperatures for the ERF primer (Table 1) and various gene-specific
primers (table 2) to minimize the number of incorrect base pairings (mismatches). This
49 Expression analysis of genes for ethylene biosynthesis enzyme
phenomenon is enhanced by low annealing temperature which is responsible for mismatching
(Rychlick et al., 1990). For PCR analysis, 15 ng genomic DNA was used as a template in a 20 µl
volume reaction that contained 0.03 µM of ERF1 primer pair: ERFs and ERFas (Table 1) or 1.2 µM
gene-specific primer pairs (table 2), 150 µM of each dNTP and 0.2 units of DyNAzymeTM II
DNA polymerase (Finnzymes Oy, Espoo, Finland) in the 10x (100 mM Tris-HCL, pH 8.3, 500
mM KCl, 20 mM MgCl2 and 0.01% gelatin) Williams buffer. The reaction mixture was incubated
in a Thermocycler (Biometra, Göttingen, Germany) and the PCR conditions were: 2 min at 94oC
(initial denaturation), followed by 40 cycles lasting 1 min at 94oC (denaturation), 1 min at 49oC to
56oC (40 cycles) (annealing), and 2 min at 72oC (extension) and a cooling step at 4oC.
3.2.4 Cloning, DNA sequencing and sequence analysis
Orange G (30% Glycerin, 20 mM EDTA-pH 8, 1.25% Orange G and 10 ml deionised
water) loading buffer was added to the PCR products and centrifuged for 5 seconds. This mixture
was then loaded into 1% agarose gels and separated in a flatted gel electrophoresis using 1xTAE
(40 mM Tris-Acetate, 1mM EDTA, pH 8.0) Buffer. The gels had a capacity of either 50 ml or
150 ml and were run at 120V or 80V, respectively. They were also visualised by staining with 0.3
µg/ml ethidium bromide and observed using a BioDocAnalyze UV trans-illuminator (Biometra,
Göttingen, Germany). The sizes of amplicons were estimated by comparing them to a 100 base
pair-ladder DNA marker. The desirable fragments were cloned by ligation into a TA plasmid
cloning vector using pCRR4-TOPOR TA Kit (Invitrogen, Carlsbad, CA; Appendix. 5) and
transformed into Escherichia coli.
The plasmid vector (pCRR4-TOPOR) was supplied linearised, also called “activated vector”. The
Vaccinia virus Topoisomerase I binds to duplex DNA at specific sites and cleaves phosphodiester
backbone after 5’-(T/C)CCTT in one strand (Shuman, 1991) and the energy released is conserved
through formation of a covalent bond between 3’ phosphate of the cleaved strand and a tyrosyl
residue (Tyr-274) of topoisomerase I. The phosphate-tyrosyl bond between DNA and enzyme can
subsequently be attacked only by a 5’ hydroxyl of the original cleaved strand, reversing the
reaction and releasing topoisomerase (Shuman, 1994). The plasmid vector has two adjacent
cutting surfaces and since both DNA fragments are blocked by Topoisomerase, this prevents self
ligation. Moreover, the vector allows direct selection of positive recombinants via disruption of
50 Expression analysis of genes for ethylene biosynthesis enzyme
the lethal Escherichia coli, ccdB gene (Bernard et al., 1994) since the cells that contain a nonrecombinant vector are killed upon plating, which eliminates the need for blue/white screening.
After cloning (50 ng of the PCR product) and transformation into Escherichia coli according to the
manufacturer’s instructions (TOPO TA Cloning kit; Invitrogen), 125 µl S.O.C. medium (2%
Tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM
glucose) was added to the plasmid–E. coli mixture and shaken horizontally using a shaker at 240
rpm for 1.5 hours at 37oC in order for bacteria to multiply. The transformants were then cultured
in LB (1% Tryptone, 0.5% Yeast extract, 1% NaCl, 950 ml deionised water, pH 7.0) medium
containing 150 µg µl-1 Ampicillin at 37oC. Plasmid DNA from transformed E. coli was recovered
using the NucleoSpinR Plasmid Kit (Macherey-Nagel GmbH, Düren, Germany). Positive
transformants were directly analysed using 0.2 µg T3 and 0.2 µg T7 primer pairs in a PCR
reaction. The nucleotide sequences for T3 5’-ATTAACCCTCACTAAAGGGA-3’ and T7 5’TAATACGACTCAACTATAGGG-3’. Sequencing by dideoxynucleotide method was performed
commercially by MWG Biotech AG (Ebersberg-Munich, Germany). The isolated sequences were
analysed using CLUSTAL W program, European Bioinformatics Institute (EMBL; Higgens,
1994), and the homology search was done using the BLUSTN programme, National Center for
Biotechnology Information (NCBI; Altschul et al., 1997).
3.2.5 RNA Isolation and RT-PCR
Total RNA was isolated from 30 mg of ground petals and leaves of miniature rose
cultivars ‘Vanilla’ and ‘Lavender’ after treatment with continuous ethylene for 9 days or
ethylene-free air as described in section 2.2.6, using InvisorbR Spin Plant RNA Mini Kit (Invitek
GmbH, Berlin, Germany) following the protocols by the manufacture. Total RNA was
determined by use of a spectrophotometer (Biorad, California). To check for the presence of
contaminating genomic DNA, total RNA was compared with standard concentrations (5, 10, 25,
100 and 200 µg ml-1) of λDNA (Fermentas GmbH, St. Leon-Rot, Germany) in a flatted gel
electrophoresis visualized by staining with 0.3 µg/ml ethidium bromide then stored at -20oC for
short-term or -80oC deep freezer for long-term storage, until further use. Under these conditions,
no degradation of RNA is detectable for at least 1 year. RNA was handled using latex hand
gloves and was kept under ice when taking aliquots. Additionally, the laboratory working
51 Expression analysis of genes for ethylene biosynthesis enzyme
surfaces and equipments were cleaned with RNase degrading chemicals (RNase Exitus;
cDNA was synthesized and used as a template for PCR. Before RT-PCR, the residual genomic
DNA in the total RNA preparation was removed through a RNase-free DNase treatment as
follows: 0.5 µg RNA was treated with 1 µl 10x buffer, 0.5 units Deoxyribonuclease I (Rnase-free)
(Fermentas GmbH, Heidelberg, Germany), 0.5 units RiboLockTM Ribonuclease Inhibitor
(Fermentas GmbH, Heidelberg, Germany), then the mixture was incubated in a Thermocycler
(Biometra, Göttingen, Germany) for 30 min at 37oC. After incubation, 40 µM EDTA was added
to the mixture to deactivate DNase. This was done at 65oC for 10 min. 0.5 µg of total RNA was
used as a template for cDNA synthesis using M-MLV RT, RNase H Minus: a point Mutant
(Promega Corporation, Manheim, Germany). First strand cDNA synthesis was carried out using
first strand oligo(dT)23 for 10 min at 40°C, then 2 h at 50° C and a final deactivation for 10 min
at 70°C. The resulting cDNA was diluted to a concentration of 20 pg µl-1 for the PCR reaction.
The PCR reaction mixture contained 2 µl of cDNA, 2 µl of 10x Williams Buffer (see section
3.2.3) recommended by the supplier (Biometra, Göttingen, Germany), 150 µM dNTPs, 1.2 µM
gene-specific primer pairs and 0.5 units Taq DNA polymerase (Invitek GmbH, Berlin, Germany)
in a total volume of 20 µl. Primers (0.25 µM of each) used for the amplification of β-Actin (used
as a reference) were: ACC synthase; RhACS1, RhACS2, RhACS3, RhACS4, RhACS5, ETR genes;
RhETR1, RhETR2, RhETR3, RhETR4, CTR genes; RhCTR1, RhCTR2, EIN3 and EIN-like genes;
RhEIN3, RhEIL and ERF gene; RhERF1 were designed from sequences available in the databases
(Rosa hybrida) (http://www.ncbi.nlm.nih.gov) (Table 2). For the PCR reaction, between 35-45
cycles, were observed with the clear differences of amplification detected at 40 cycles. The
reaction mixture was incubated in a Thermocycler (Biometra, Göttingen, Germany). PCR
conditions were: 5 min at 94oC (initial denaturation), followed by 40 cycles consisting of 30 s at
94oC (denaturation), 30 s at 49oC to 55oC (annealing) (see Table.2), and 30 s at 72oC (extension)
and a cooling step at 4oC. The products of PCR were separated as described for the normal PCR
above. PCR and the experiments themselves were repeated three times for each sample. A
representative result is presented in chapter 3.3.2. 52 Expression analysis of genes for ethylene biosynthesis enzyme
Table 2. Gene specific primer pairs for β-Actin (Accession No. X55751), ACC synthase; RhACS1
(Accession No. AY061946), RhACS2 (Accession No. AY525066), RhACS3 (Accession No.
AY525067), RhACS4 (Accession No. AY525068), RhACS5 (Accession No. AY525069), ETR
genes; RhETR1 (Accession No. AF394914), RhETR2 (Accession No. AF127220), RhETR3
(Accession No. AF154119), RhETR4 (Accession No. AF159172), CTR genes; RhCTR1
(Accession No. AY032953), RhCTR2 (Accession No. AY029067), EIN3 and EIN-like genes;
RhEIN3 (Accession No. AF443783), RhEIL (Accession No. AY052825) and ERF gene; RhERF1
designed using the program Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).
Name Name Antisense Primer Annealing Size Temp (oC) Actin_for Sense Primer (bp) 5’AGATCTTTATGGAAACATTGTGCTC3’ Actin_Rev 5’ATCCAGACACTGTATTTCCTCTCT3’ 49 150 RhACS1s 5’AAAACCCAGAAGCCTCCATT3’ RhACS1as 5’ATTTCTGGTTCCCGTTCCTT3’ 55 205 RhACS2s 5’AAAACCCAGAAGCCTCCATT3’ RhACS2as 5’ATTTCTGGTTCCCGTTCCTT3’ 55 249 RhACS3s 5’CCATGGCCTTTTGTCCTTTA3’ RhACS3as 5’ACTGCAGCCAATGAGCTTTT3’ 55 126 RhACS4s 5’GCTTCCAACTTGGGATCAAA3’ RhACS4as 5’TTCCAACCCCATACTACCCA3’ 55 237 RhACS5s 5’CAGCCGGATTCAAGAGAAAC3’ RhACS5as 5’CTCTTATGTTTTGCCTCGCC3’ 55 203 RhETR1s 5’TGGTATGAACCTTCAACTTTCTCAT3’ RhETR1as 5’TGCTATTCTTGAAGAGTCTATGCGA3’ 55 393 RhETR2s 5’CTCAAACTTCCAAATCAATGACTG3’ RhETR2as 5’GGATCTGCTAATGGAGCAGAATAT3’ 55 213 RhETR3s 5’CACTGCTATAACGCTCATCACTCT3’ RhETR3as 5’ATCCTTGAAGAGTCCCAACTAATG3’ 55 661 5’TTTGAATCTGCAACTTTCTCACAC3’ RhETR4as 5’TGTCATGAACCACGAAATGC3’ 55 500 5’TCAATGGCCTCAAAGATTCC3’ 52 706 5’CCCACTCCAAGCCAATTTTA3’ 55 357 5’ACCCTGATTTCATCCACCAA3’ 56 236 5’GAGGCCACCATTCCTCATTA3’ 56 192 5’GCTTCCTAACGCCTCTGTAA3’ 55 64 RhETR4s
RhERF1as The β-Actin primer used as a reference gene is in boldface.
3.2.6 Northern blot hybridization
Polysomal RNA was extracted as described in section 3.2.5. Northern blotting was performed
according to Terefe (2005). Equal amounts (8 µg per sample) of total RNA were denatured in
freshly prepared loading buffer (2.2 M formaldehyde, 50% foramide, 0.5x MOPS and 0.05%
bromophenol blue (0.25% (w/v) bromophenol blue, 30% (v/v) Glycerin) in a final volume of 30
µl for 15 min at 65oC. The denatured RNA was then fractioned by electrophoresis in 1% agarose
gel containing 0.7 M formaldehyde in 1x MOPS-buffer (200 mM 3-(N-Morpholino)53 Expression analysis of genes for ethylene biosynthesis enzyme
propanesulfonic acid (MOPS), 80 mM Sodium acetate 3-hydrate, 12.5 mM EDTA, pH 7.0) for
4-5 h (at the constant voltage of 5V/cm gel length). The gels were stained with 1,5 µg/ml
ethidium bromide to check the intactness of the RNA under UV light, then washed first in water
for 15 min and twice in 10x SSC (1.5 M NaCl, 0.15 M Na-citrate, pH 7.0) for 15 min each. RNA
was then transferred to positively-charged nylon membranes (Hybond-N+, Amersham Biosciences UK
Ltd, Buckinghamshire, England) in 10x SSC using a vaccuum-system (Pharmacia; 60 mbar, 90
min). After crosslinking by irradiation with UV light, the membranes were rinsed with water and
stored until hybridization.
3.2.7. DIG labeling of the probes for northern blot hybridization
The ethylene receptor and signal transduction pathway, RhETR1-2, RhEIN3 and RhEIL were
labeled with DIG (digoxigenin) (Roche Diagnostics GmbH, Mannheim, Germany) for nonradioactive hybridization. DIG labeling was performed by PCR using 10 pg plasmid, containing
each gene of interest: 2.5 µM of each standard T3 5’-ATTAACCCTCACTAAAGGGA-3’ and T7
5’-TAATACGACTCAACTATAGGG-3’ primers, 10x reaction William Buffer (GeneCraft
GmbH, Lüdinghausen, Germany) as recommended by the supplier (Biometra, Göttingen,
Germany), 10x DIG DNA labeling mix and 1 unit DyNAzymeTM II DNA polymerase
(Finnzymes Oy, Espoo, Finland) filled to a final volume of 20 µl with ddH2O. The reaction
mixture was incubated in a Thermocycler (Biometra, Göttingen, Germany). PCR conditions
were: 2 min at 94oC (initial denaturation), followed by 40 cycles lasting 30 s at 94oC
(denaturation), 1 min at 50oC (annealing), 2 min at 72oC (extension) and 5 min at 72oC (final
extension). As a control, additional PCR reactions were set for every probe, labeled by using
normal dNTPs without DIG. 5 µl DIG-labeled PCR product and the control were checked by
flatted electrophoresis in 1% agarose gel visualised by staining with 40 µg ethidium bromide. The
size of fragments was estimated using 100 base pair-ladder DNA marker. Due to the presence of
DIG, the DIG-labeled probes migrate slower and appear to be larger than PCR fragments
obtained using the normal dNTPs. The DIG-labeled probes were denaturated at 95oC for 10 min
and immediately chilled on ice for denaturation. The denaturated probe was then used for
northern blot hybridization. 54 Expression analysis of genes for ethylene biosynthesis enzyme
3.2.8. Hybridization and washing conditions for northern blots
The membranes were prehybridized in prehybridization solution containing Dig Easy Hyb
(Roche Diagnostics GmbH, Mannheim, Germany) at 50oC for 30 min. Hybridization was carried
out overnight at the same condition with 100 ng of the denatured DIG-labeled probe in 7.5 ml
Dig Easy Hyb. After hybridization, membranes were washed twice for 5 min each in 2x
SSC/0.1% SDS at 50oC. The membranes were then agitated in 1x blocking solution (Roche
Diagnostics GmbH, Mannheim, Germany) for 30 min to eliminate nonspecific background and
for another 30 min in an Anti-Dig-AP solution (Roche Diagnostics GmbH, Mannheim, Germany)
(2.5 µl (0.75 unit/µl) Anti-Dif-AP in 50 ml 1x blocking solution). Detection of the DIG-labeled
probes was performed according to the instructions of the supplier, using CDP-star (Roche
Diagnostics GmbH, Mannheim, Germany) as substrate. X-ray films (Hyperfilm, Amersham
Biosciences UK Ltd, Buckinghamshire, England) were exposed to the membranes for about 1 h.
The transcript sizes were estimated by comparison with the RNA Molecular Weight Marker I
(Roche Diagnostics GmbH, Mannheim, Germany) covering the range 0.3-6.9 kb. Blots were used
for multiple hybridizations after stripping in 0.1x SDS boiling. 55 Expression analysis of genes for ethylene biosynthesis enzyme
3.3.1 Cloning and sequence analysis of RhERF1
Using PCR with primer pairs deduced from the conserved region (Ap2 Domain) of
Arabidopsis ERF homologues, a fragment encoding a partial ERF homologue was amplified from
genomic DNA from ‘Vanilla’ and ‘Lavender’. A database search revealed that the deduced amino
acid sequence of RhERF1 contained a highly conserved DNA binding domain. This consisted of
30 amino acids (92 bp for partial-length of DNA sequence) and a shared high identity with other
reported ERF domain-containing proteins (Fig. 12).
β-sheet-1 β-turn-1 β-sheet-2 β-turn-2 β-sheet-3 ** * * * ** Position RhERF1 : YRGVRKRPWGRYAAEIRDPWKK-TRVWLGTF----------------------------- 1-30 AtERF1 : YRGVRQRPWGKFAAEIRDPAKNGARVWLGTFETAEDAALAYDRAAFRMRGSRALLNFPLR 147-201 AtERF2
: YRGVRQRPWGKFAAEIRDPAKNGARVWLGTFETAEDAALAYDIAAFRMRGSRALLNFPLR 116-176
YRGVR RPWGKFAAEIRDP K G RVWLGTY Fig. 12. Alignment of the DNA-binding domains of RhERF1 in comparison with other ERF
proteins: AtERF1 (GeneBank Accession No. AAL25588), AtERF2 (GeneBank Accession No.
NM_124093), NtERF1 (GeneBank Accession No. AF057373), LeERF1 (GeneBank Accession
No. AY192367), GmERF1 (GeneBank Accession No. AAM45475). The identical amino acids
among all the aligned ERF proteins are marked in grey. The amino acids with asterisks in the
ERF consensus indicate residues that interact with nucleotides with one word in the GCC box.
The numbers at right indicate the position of the amino acids in the ERF domain for each protein. 56 Expression analysis of genes for ethylene biosynthesis enzyme
3.3.2 Expression patterns of genes for the ethylene biosynthesis enzyme, ethylene perception and
signal transduction pathway after pretreatment with ethylene receptor inhibitors
Ethylene caused leaf, bud and flower drop, but there were differences among cultivars. In both
cultivars, ethylene accelerated flower senescence. Continuous exposure to exogenous ethylene at
1.25 nl l-1 hastened leaf drop (as expressed in percentage) for ‘Vanilla’ and ‘Lavender’ (Fig 13A,
B). ‘Vanilla’ control plants attained 90% leaf drop after 9 days of continuous exposure to
ethylene, as opposed to ‘Lavender’, which attained 100% leaf drop after the same period.
Pretreatment with ethylene receptor inhibitors, 1-OCP, 1-DCP and 1-MCP, significantly
improved the display quality in both cultivars (Fig 13A, B). 1-MCP was the most effective of all
ethylene receptor inhibitors tested in improving the display quality in both cultivars. However, 1OCP and 1-DCP were not different from each other with respect to leaf drop in both cultivars (see
chapter 2) (Fig. 13A, B). Vanilla a b 80
60 Lav ender B 100 a Percent leaf drop after 9 days Percent leaf drop after 9 days 100 A b c 40
b 60 b 40
control 1-MCP 1-OCP control 1-DCP 1-MCP 1-OCP 1-DCP Treatments Treatments Fig. 13. Percent leaf drop of miniature potted rose cultivars ‘Vanilla’ (A) and ‘Lavender’ (B),
after day 9 pretreated with 200nl l-1 1-MCP for 6 h, and 1000 nl l-1 1-OCP and 1-DCP for 4 h at
20oC, respectively. After treatments, the plants were exposed to 1.25 (+/- 0.25) µl l-1 continuous
ethylene throughout the experiments. Bars marked with the same letter are not statistically
different at P<0.05. Means were separated by LSD (selected data from Fig. 7; p. 28). 57 Expression analysis of genes for ethylene biosynthesis enzyme
To investigate transcript expression, abundance of genes for the ethylene biosynthesis enzyme,
ethylene perception and other ethylene-related genes (RhACS1, RhACS2, RhACS3, RhACS4,
RhACS5, RhETR1, RhETR2, RhETR3, RhETR4, RhCTR1, RhCTR2, RhEIN3, RhEIL a nd
RhERF1) in petal and leaf tissues was examined in the two rose cultivars ‘Vanilla’ and
‘Lavender’. This was done after pretreatment with air, 200 nl l-1 1-MCP for 6 h, 1000 nl l-1
1-OCP and 1-DCP for 4 h at 20oC, followed by either continuous exposure to exogenous ethylene
or ethylene-free air for 9 days (Fig. 15-16, A, B). Two ACS genes, RhACS1 and RhACS2, were
expressed in rose tissues while RhACS3-5 was undetectable in the investigated tissues (leaves and
petals) (method previously described). Northern blotting with RhETR2 probes from miniature
rose was performed to compare the results of RT-PCR. The position was consistent with the
expected size of RhETR2, but transcription signals of ethylene perception could not be clearly
seen on the autoradiogram (Fig. 14). RT-PCR was utilized to compare the expression level of
ethylene receptors in miniature rose tissues. 1049 bp
575 bp CM O
Petals D CM O
C Leaves -C2H4 D C MO
Petals D C M OD
Leaves +C2H4 Fig. 14. Northern blot hybridization analysis of ethylene receptor, RhETR2, on 8 µg total RNA
obtained from petals and leaves of miniature rose cultivar ‘Vanilla’ in untreated (control) plant
(C) and pretreatment with 200 nl l-1 1-MCP (M) for 6 h and 1000 nl l-1 1-OCP (O) and 1-DCP (D)
for 4 h at 20oC after 9 days of continuous exposure of exogenous ethylene (+C2H4) and without
ethylene treatment (–C2H4). 58 Expression analysis of genes for ethylene biosynthesis enzyme
Pretreating ‘Vanilla’ petals with 1-MCP, 1-OCP and 1-DCP led to the suppression of the mRNA
level of genes for ethylene biosynthesis, ethylene perception and ethylene signal transduction in
both the presence and absence of ethylene (Fig. 15A). In the untreated petals (control), ethylene
induced the expression of genes for ethylene perceptions (RhETR1, RhETR2, RhETR3 and
RhETR4) and other ethylene-related genes (RhCTR1, RhCTR2, RhEIN3, RhEIL, RhERF1).
However, RhACS1 and RhACS2 were not affected by ethylene treatment, and in the absence of
ethylene they had no effect on the expression of any of the genes investigated. (Fig. 15A).
The absence of ethylene in ‘Vanilla’ control leaves led to a strong expression of RhCTR2,
moderate expression of RhETR1, RhETR2, RhETR4, RhEIN3, RhEIL, weak expression of
RhACS1, RhETR3, RhERF1 and no expression of RhACS1 (Fig. 15B). Moreover, pretreating
‘Vanilla’ leaves with 1-MCP, 1-OCP and 1-DCP, in the absence of ethylene, eliminated the
abundance of mRNAs for all investigated genes (Fig. 15B). In ‘Vanilla’ control leaves, the
ethylene treatment resulted in strong expression of RhETR1, RhETR2, RhETR4, RhCTR2 and
RhEIL, moderate expression of RhACS2, RhCTR1 and RhEIN3, weak expression of RhETR3 and
RhERF1 and no expression of RhACS1 (Fig. 15B). Furthermore, RhEIN3 was strongly and
constitutively expressed after pretreating ‘Vanilla’ leaves with 1-MCP, 1-OCP and 1-DCP (Fig.
15B), while RhEIL was also expressed in ‘Vanilla’ leaves in response to 1-OCP and 1-DCP.
Pretreating ‘Vanilla’ leaves with 1-DCP in the presence of ethylene led to a weak accumulation
of RhETR1-2, RhCTR1-2 transcripts (Fig. 15B). 59 Expression analysis of genes for ethylene biosynthesis enzyme
-C2H4 RhERF Leaves
+C2H4 -C2H4 A +C2H4 B RhEIL
C M O D C M O D C M O D C M O D Fig. 15. Expression of genes for ethylene biosynthesis, ethylene perception and ethylene signal
transduction in miniature potted rose cultivar ‘Vanilla’ (A, B) in untreated (control) plant and
pretreatment with 1-MCP, 1-OCP and 1-DCP after 9 days of continuous exposure of exogenous
ethylene (+ C2H4) and without ethylene treatment (- C2H4). Lane 1, control (C); lane 2, 1-MCP
(M); lane 3, 1-OCP (O) and lane 4, 1-DCP (D). PCR reaction contained 20 pg µl-1 of cDNA. βActin was used as an internal control to normalize the amount of cDNA.
In the absence of ethylene, none of the genes were detectable at 40 cycles of PCR reaction in the
petals of ‘Lavender’, irrespective of the treatment applied (Fig. 16A). Exposing ‘Lavender’
control petals to ethylene resulted in strong expression of RhETR2, RhCTR1, RhCTR2, RhEIN3
and RhEIL, moderate expression of RhETR1 and RhETR3, weak expression of RhACS1, RhACS2
and RhERF1 and no expression of RhETR4 (Fig. 16A). However, pretreating ‘Lavender’ petals
with 1-MCP and 1-OCP inhibited the expression of all genes while strong accumulation of
RhETR3, RhEIN3 and RhEIL transcripts was detectable in ‘Lavender’ petals pretreated with
1-DCP (Fig. 16A). 60 Expression analysis of genes for ethylene biosynthesis enzyme
In the absence of ethylene, pretreating ‘Lavender’ leaves with 1-MCP upregulated the expression
of RhACS2, RhCTR1 and RhEIL as compared to the control leaves (Fig. 16B), while downregulating
the expression of RhETR1, RhETR2 and RhETR3 as opposed to the control. Also, 1-MCP
suppressed the expression of RhACS1, RhETR3 and RhERF1, whereas it had no effect on the
expression of RhETR4 and RhCTR2 (Fig. 16B). Similarly, pretreatment of ‘Lavender’ leaves with
1-OCP upregulated the expression of RhACS2, RhETR4, RhCTR1, RhEIN3, but downregulated
the expression of RhETR2, RhETR3 as compared to the control leaves (Fig. 16B). 1-OCP
inhibited the expression of RhACS1, RhCTR2 and RhERF1 and had no effect on the expression of
RhETR1 and RhEIL as compared to the control. Likewise, pretreatment of 1-DCP upregulated the
expression of all RhACS and RhETR genes, but downregulated the expression of RhCTR2 as
opposed to control leaves. Moreover, 1-DCP inhibited the expression of RhEIN3, RhEIL and had
no effect on the expression of RhCTR1 and RhERF1 as compared to the control (Fig. 16B).
In the presence of ethylene, pretreating ‘Lavender’ leaves with 1-MCP suppressed the expression
of RhACS1, RhETR4, RhCTR-2, RhEIL and RhERF1, while it downregulated the expression of
RhETR1-2 and RhEIL, and had no effect on the expression of RhACS2, RhETR3 when compared
to control ‘Lavender’ leaves. Similarly, pretreatment of 1-OCP inhibited the expression of
RhACS1, RhETR2, RhETR4, RhCTR1-2, RhEIN3 and RhERF1 but upregulated the expression
RhEIL. 1-OCP downregulated the expression of RhETR1 whereas it did not affect the expression
of RhACS2, RhETR3 when compared to the control (Fig 16B). Pretreating ‘Lavender’ leaves with
1-DCP suppressed the expression of RhACS1 and RhETR4. However, 1-DCP upregulated
RhETR3 and RhCTR2 while it downregulated the expression of RhETR2, RhEIN3, RhEIL and
RhERF1, but had no effect on the expression of RhACS2, RhETR1 and RhETR3 when compared
to the control (Fig. 16B). 61 Expression analysis of genes for ethylene biosynthesis enzyme
-C2H4 RhERF Leaves
+C2H4 -C2H4 A +C2H4 B RhEIL
C M O D C M O D C M O D C M O D Fig. 16. Expression of genes for ethylene biosynthesis enzyme, ethylene perception and ethylene
signal transduction in miniature potted rose cultivar ‘Lavender’ (A, B) in untreated (control) plant
and pretreatment with 1-MCP, 1-OCP and 1-DCP after 9 days of continuous exposure of
exogenous ethylene (+ C2H4) and without ethylene treatment (- C2H4). Lane 1, control (C); lane
2, 1-MCP (M); lane 3, 1-OCP (O) and lane 4, 1-DCP (D). PCR reaction contained 20 pg µl-1 of
cDNA. β-Actin was used as an internal control to normalize the amount of cDNA loaded. 62 Expression analysis of genes for ethylene biosynthesis enzyme
Differences in flower display quality are partly due to differences in endogenous ethylene
production and/or sensitivity to exogenous ethylene (Müller et al., 1998, 2001). ‘Vanilla’ and
‘Lavender’ showed differences in ethylene sensitivity for their leaf abscission. After 9 days of
continuous exposure to ethylene, ‘Vanilla’ control plants showed about 80% leaf drop, while
display quality of ‘Lavender’ control plants was clearly reduced by 100% leaf drop (see chapter 2).
In rose, 1-MCP protects against exogenous ethylene, increasing display life and reducing
abscission of buds, flowers and leaves (Serek et al., 1994c). To investigate the role of ethylene
response in miniature roses pretreated with ethylene action inhibitors, 1-MCP, 1-OCP and 1-DCP,
the differential regulation of genes for ethylene biosynthesis enzyme, ethylene perception and
signal transduction pathway in petals and leaves following ethylene treatment and ethylene freeair was assessed by reverse transcription-PCR (RT-PCR) analysis. Northern blot hybridization
analysis was also used to compare the results from RT-PCR, but transcription signals of the
ethylene receptor, RhETR2, on the gel made it difficult to distinguish the clear bands. It could be
due to a cross hybridization between other members of the ethylene receptor family in miniature
rose that are highly homologous. Nucleotide sequence for probe of RhETR2 is 77% identical to
RhETR4 and 63% identical to RhETR1, indicating that RhETR2 is the most homolog with
RhETR4 and RhETR1. Northern blotting depends on minimal RNA degradation, efficient transfer
out of the gel, and ability to reliably detect the sequence of interest (Sobel et al., 2002). This
method, however, is time-consuming and requires a large quantity of RNA (Chelly and Kahn,
1994). Northern blot analysis is effective for quantifying gene expression, while RT-PCR
converts RNA into first strand cDNA used as a template for PCR. Moreover, RT-PCR is more
rapid and sensitive and can be more specific than northern blot analysis (Gause and Adamovicz, 1995).
Gene-specific primer pairs were used since they are able to detect lower levels of gene
expression. RT-PCR has a disadvantage in that, quantification is difficult because many sources
of various exist, including template concentration and amplification efficiency. For RT-PCR to be
accurate and quantitative, it must be analyzed in the linear range of amplification before reaction
components become limiting, which occurs after 20 cycles. RT-PCR, however, with 35 cycles
was shown to closely resemble northern blot analysis, indicating relatively low template amount
or amplification efficiency (Dean et al., 2002). In this study, 40 cycles were used for RT-PCR
analysis, which was considered to be effective in amplifying some genes, such as RhACS1 and
63 Expression analysis of genes for ethylene biosynthesis enzyme
RhACS2. The number of cycles to reach saturation greatly depends on the amplification
efficiency (Diaco, 1995). However, qualitative RT-PCR in this study was difficult to explain the
variations in RNA levels. Marone et al. (2001) suggests that in most cases, a qualitative study is
not sufficient to deliver a satisfactory answer for the detection of any variation in the expression
levels under different experimental conditions. RT-PCR analysis showed that the expression of
all genes investigated was undetectable in both ‘Vanilla’ and ‘Lavender’ petals in the absence of
In carnation (Wang and Woodson, 1991; Woodson et al., 1992) and Orchid flowers (O’Neill et al.,
1993), senescence is associated with increased ethylene production arising from concomitant
increases in the activities of both ACC synthase and ACC oxidase enzymes. ACS gene is encoded
by gene families, which are responsible for the differences in the regulation of expression among
the different family members. Transcription of the ACS family members in Arabidopsis is
differentially regulated during development, in different stimuli (Liang et al., 1992; Vahala et al.,
1998). In this study, RhACS1 was upregulated in ‘Vanilla’ control leaves by treatment of
ethylene, while expression of both RhACS1 and RhACS2 were downregulated in ‘Lavender’
control leaves. The expression of RhACS1 and RhACS2 was also induced by ethylene treatment
in ‘Lavender’ control petals (Fig. 16A), indicating that these genes were specifically expressed in
different organs and cultivars of rose. Müller et al. (2000a) has shown that the ACC synthase
transcript increases during flower senescence in ‘Vanilla’, with long-lasting flower longevity but
remains constant at a low level in ‘Bronze’, a sensitive flower. However, RhACS1 in rose leaves
and buds is not detectable but is expressed in rose floral organs, such as ovary and sepals, and at
very low level in anthers (Wang et al., 2004). Functionally, ACS genes can be classified in terms
of their primary stimulus such as wounding, auxin or ripening that induces expression. The
primary stimulus-induced expression of ACS genes is further positively or negatively modulated
by secondary stimuli (Imaseki, 1999). In tomato fruit, a massive ethylene production is responsible
for increases in LE-ACS2 and LE-ACS4 transcripts (Van Der Streten et al., 1990; Olson et al., 1991;
Rottmann et al., 1991; Yip et al., 1992; Lincoln et al., 1993; Barry et al., 1996). Expression of
these genes in preclimacteric tomato fruit is rapidly induced and/or enhanced by ethylene
(Maunder et al., 1987; Rottmann et al., 1991; Lincoln et al., 1993). Additionally, expression of
ACC synthase gene is proposed to be the rate-limiting step that controls the burst of ethylene
occurring during fruit ripening in Prunus mume (Mita et al., 1999). In carnation flower,
64 Expression analysis of genes for ethylene biosynthesis enzyme
expression of CARAS1 and CARACC3 transcripts in ovary is rapidly induced by ethylene (Have
and Woltering, 1997). There might be several mechanisms that regulate the expression of
ethylene biosynthesis genes during fruit ripening and flower senescence, and these mechanisms
might depend, in turn, on plant species and tissue (Mita et al., 1999).
Ethylene perception requires specific receptors and a signal transduction pathway to coordinate
downstream response (Müller et al., 2000a). Hua et al., (1998) showed in Arabidopsis leaves that
the receptor genes are differentially regulated by ethylene. The different receptors have tissue- or
stage-specific functions (Chang and Stewart, 1998). Exposure to ethylene may also increase
tissue sensitivity of ethylene receptors, possibly providing a mechanism to react to low ethylene
concentration (Müller et al., 2000a). The present results indicate that the putative ethylene
receptor genes were upregulated in both ‘Vanilla’ and ‘Lavender’ petals in the absence of
ethylene, while RhETR4 transcript was not detectable in ‘Lavender’ control petals (Fig. 15-16A).
The expression pattern of the ethylene receptor in both cultivar petals was similar to that of
leaves, in which RhETR1, RhETR2 and RhETR4 transcripts were upregulated by treatment of
ethylene. However, RhETR3 was constitutively expressed in ‘Vanilla’ control leaves as opposed
to ‘Lavender’ control leaves, whereby RhETR3 was downregulated by ethylene treatment (Fig.
15-16B). This was consistent with the findings of Müller et al. (2000a, b) with miniature rose
cultivars ‘Bronze’ and ‘Vanilla’ in which, exposure to low ethylene concentrations results in
upregulation of RhETR1 in ‘Vanilla’ flowers. RhETR2 was constitutively expressed during flower
senescence, while the RhETR3 transcript in ‘Vanilla’ flowers appeared to be constitutively
expressed at a very low level. Tieman and Klee (1999) found that expression levels of LeETR4
and LeETR5 transcripts in tomato are highly regulated among plant tissue with high levels of
expression in productive tissues such as flower buds and mature flowers. Additionally, Nr gene
expression can be induced by treatment with exogenous ethylene, or with treatments that induce
in vivo ethylene production such as fruit ripening (Wilkinson et al., 1995; Payton et al., 1996),
while other ETR homologues, LeETR1, LeETR2, LeETR4 and LeETR5, are constant throughout
the ripening process (Lashbrook et al., 1998). In Arabidopsis leaves, transcript levels of ERS1,
ETR2 and ERS2 genes are upregulated, while ETR1 and EIN4 are not affected by ethylene
treatment (Hua et al., 1998). In mung bean seedlings, ethylene positively modulates the
expression of its receptor genes (Kim et al., 1999). By contrast, exogenous ethylene does not
induce PhETR1 or PhETR2 mRNA accumulation in geranium, suggesting control of ethylene65 Expression analysis of genes for ethylene biosynthesis enzyme
induced petal abscission in geranium florets may lie in another member of the PhETR gene
family or at a post-transcriptional point (Dervinis et al., 2000).
Regulation of ethylene response can possibly occur downstream of the ethylene receptor. RhCTR1-2
transcripts were upregulated after ethylene treatment in both ‘Vanilla’ and ‘Lavender’ control
petals (Fig. 15A and 16A). This result was consistent with Müller et al. (2002). In ‘Bronze’ petals
exogenous ethylene increases expression of RhCTR1 and 2 and results in accelerated flower
senescence (Müller et al. 2002). RhCTR1-2 were constitutively expressed in ‘Vanilla’ leaves after
ethylene treatment and RhCTR1 was also ethylene-independent expressed in ‘Lavender’ control
leaves. These results were consistent with the Arabidopsis CTR1 gene that was not ethylene
inducible (Kieber et al., 1993; Guo et al., 2003). However, RhCTR2 was upregulated in ethylenetreated ‘Lavender’ leaves.
EIN3 and its homologues have been shown to act as a positive regulator of ethylene response
(Chao et al., 1997). The present study showed that expression of RhEIN3 and RhEIL transcripts
in petals and leaves of both cultivars were upregulated by treatment of ethylene (Fig. 15-16A, B).
However, Müller et al. (2003) found that the RhEIN3 gene was constitutively and stably
expressed in petals during flower development in cultivar ‘Bronze’ and ‘Vanilla’ and in response
to ethylene and ABA. Additionally, ethylene does not affect the level of EIN3 and EIL1 genes in
Arabidopsis (Chao et al., 1997; De Paepe et al., 2004) and in tobacco (Kosugi and Ohashi, 2000).
In contrast to carnation flowers, the expression pattern of EIL1 (a homologue to AtEIN3)
decreases during senescence, suggesting EIN3 and its homologue may possibly be involved in the
regulation of ethylene sensitivity (Waki et al., 2001). However, EIN3/EIL transcription factors
are not primarily regulated by ethylene at the transcriptional level (Chao et al., 1997; Tieman et al.,
2001; Lee and Kim, 2003). Post-transcriptional regulation in ethylene signal transduction
pathway is under regulation of a key transcription factor, EIN3. In the absence of ethylene, EIN3
is continuously degraded through the proteasome-mediated pathway, and prevents activation of
its transcriptional targets. In the presence of ethylene, degradation of EIN3 is suppressed, and
allows EIN3 protein levels to increase and thus promotes the ethylene response (Guo and Ecker,
2003; Potuschak et al., 2003). 66 Expression analysis of genes for ethylene biosynthesis enzyme
The primary ethylene response element, ERF1, is considered as an immediate target of EIN3
(Solano et al., 1998).
The protein of the newly-isolated gene RhERF1 contained a highly-conserved Ap2 DNA binding
domain. This domain was the only part of the protein that exhibited significant sequence
homology with ERF proteins from other species. The binding of some ERF proteins to the GCC
box in the ethylene-responsive element suggests a role for proteins in the regulation of expression
of the ethylene-responsive gene. As a transcription factor initiating the expression of related
target genes, the ERF protein plays an important role in physiological plant processes (Qin et al.,
Both ‘Vanilla’ and ‘Lavender’ miniature rose cultivars showed that RhERF1 was positively
regulated when exposed to exogenous ethylene (Fig. 15-16A, B). In Arabidopsis, AtERF1,
AtERF2 and AtERF5 are rapidly induced by ethylene, 12 h after treatment and their roles are to
regulate the ethylene-inducible gene (Solano et al., 1998). The transcripts of GbERF accumulate
rapidly to a high level of G. barbadense in the leaf, when treated with exogenous ethylene (Oin et
al., 2004). This study indicated that all genes are specifically expressed in miniature roses and the
levels of mRNAs are regulated in a tissue-specific manner (Shibiya et al., 2002).
Ethylene-receptor inhibitors such as 1-MCP and CPs long-chain, are thought to act by binding to
a metal in the receptor (Sisler and Serek, 2003). 1-MCP suppresses the ethylene response
pathway by permanently binding to a sufficient number of ethylene receptors that are bound to
the copper cofactor of the ethylene receptor. Due most likely to steric hindrance, the binding is
insufficient to convert the receptors to the inactive (off) state, which keeps CTR1 in its active
(inhibiting) state (Binder and Bleecker, 2003). By analogy to 1-MCP, CPs long-chain binds to the
receptor in the same way (Sisler et al., 2003). Lelievre et al. (1997) showed that treatment with
1-MCP results in reduced accumulation of ACC oxidase and ACC synthase transcripts, and
ethylene production during chilling of pear fruits. In addition, 1-MCP antagonizes not only
ethylene responses but also ethylene biosynthesis, by downregulating CsACS1 and CsACO1 in
citrus fruitlets (Katz et al., 2004). Moreover, 1-MCP results in the elimination of mRNA levels
for receptor genes of pears during the cold treatment (El-Sharkawy et al., 2003). In miniature
roses, pretreatment with 1-MCP obviously reduces the rise in autocatalytic ethylene production in
67 Expression analysis of genes for ethylene biosynthesis enzyme
flowers (Müller and Stummann, 2003). In the present study, pretreatment with 1-MCP and 1-OCP
showed strong suppression of the expression of all genes investigated in both ‘Vanilla’ and
‘Lavender’ petals in the absence and presence of ethylene, while RhETR3, RhEIN3 and RhEIL
were strongly expressed in ‘Lavender’ petals pretreated with 1-DCP in the presence of ethylene.
1-MCP, 1-OCP and 1-DCP eliminated the expression of all genes in ‘Vanilla’ leaves in the
absence of ethylene, while RhEIN3 transcript was upregulated after pretreatments of 1-MCP and
1-OCP in the presence of ethylene. In contrast to ‘Lavender’ leaves, 1-MCP and 1-OCP
pretreatments prevented the accumulation of RhEIN3 mRNA (Fig. 15-16B). The results provided
evidence that at least some signal transduction genes may play an important role in ethylene
perception and signal transduction pathway, and are involved in ethylene-induced flower
senescence. RhEIN3 transcript is probably rate-limiting for ethylene perception and signal
transduction pathway, which is regulated during flower senescence. The expression of RhEIN3
transcript in ‘Vanilla’ and ‘Lavender’ leaves pretreated with 1-MCP and 1-OCP may also be
regulated at the transcriptional level. A physiological investigation can explain a large difference
of display qualities in both miniature rose cultivars, when pretreated with 1-OCP and 1-MCP,
caused the increased number of leaf drop in ‘Vanilla’ more than in ‘Lavender’ (Fig. 7A, B, C, D).
In accordance with 1-MCP, pretreatment of 1-OCP of ‘Lavender’ leaves reduced the expression
of all genes investigated except for RhEIL, which was upregulated in the presence of ethylene,
while 1-DCP treatment upregulated transcripts of RhETR1, RhCTR2 and RhEIN3. This may result
in plants being more sensitive to ethylene. Additionally, the abundance of the individual
transcripts varied with different tissues (Lashbrook et al., 1998; Tieman and Klee, 1999).
‘Vanilla’ was characterized by excellent longevity (Müller et al., 1998), while ‘Lavender’
exhibited short postharvest life. Therefore, exposure to ethylene may increase the sensitivity of
tissues in ‘Lavender’ than that of ‘Vanilla’, thus resulting in reduction in postharvest longevity.
It seems possible that ‘Vanilla’ differs from other miniature rose flowers in its ethylene-binding
site or signal transduction pathway (Müller et al., 1998). Even though ‘Vanilla’ seemed
insensitive to ethylene, exogenous ethylene positively modulates the expression of ethylene
receptor gene in flowers (Müller et al., 1998, 2000). 68 Expression analysis of genes for ethylene biosynthesis enzyme
Based on the genetic evidence, these results correlated with physiological investigation that 1-MCP
is an ethylene-action inhibitor that binds to the receptor site competitively, thereby preventing
tissue response to ethylene (Sisler and Serek, 1997). 1-MCP was more effective than any other
long chain CPs in preventing flowers from exogenous ethylene. Differences between 1-MCP and
CPs long chain may be due to different chemical groups attached to the cyclopropene molecule or
to the side chain(s), and some of them may have interesting physiological properties (Sisler et al., 2003).
In conclusion, it is known that ethylene biosynthesis, ethylene perception and the signal
transduction pathway contribute to the regulation of ethylene responses in plant tissues. These
results indicated both positive and negative feedback regulation mechanism exists in miniature
rose cultivars ‘Vanilla’ and Lavender’ pretreated with ethylene receptor inhibitors, 1-MCP, 1OCP and 1-DCP, in the presence or absence of ethylene. The mechanisms that regulate the
expression of genes for ethylene biosynthesis, ethylene perception and the signal transduction
pathway during flower senescence might depend on plant species and tissues under investigation.
1-MCP, 1-OCP and 1-DCP treatments suppressed the expression of these genes. Therefore, an
explanation of the role of the ethylene receptor inhibitors treatment in miniature roses is required
to understand the mechanism of gene expression in the same response but under different
conditions. 69 Summary 4. Summary
Ethylene-mediated flower senescence, especially in ornamental crops, is a significant problem in
the horticulture industry (Müller and Stummann, 2003). Exogenous ethylene reduces longevity of
commercial miniature rose cultivars (Serek, 1993; Serek et al., 1994c), which showed great
variation in their display life in an ethylene-free environment. Sensitivity to ethylene seems to be
an important natural regulator of rose flower senescence and varies greatly among cultivars
(Müller et al., 2000). Therefore, developmental regulation of ethylene response in flowers may
occur at the level of hormone sensitivity (Tieman and Klee, 1999).
Ethylene action involves binding to a specific receptor (Schaller and Bleecker, 1995). In the present
study, the potency of two structural analogues of 1-MCP, 1-OCP and 1-DCP, containing a side
chain at 1-position, was assessed. These chemicals are new putative inhibitors of ethylene action.
Their potency was evaluated by testing their ability to counteract ethylene-induced responses
(display life, bud, flower and leaf drop). 1-OCP and 1-DCP protected plant tissue against the
detrimental effect of ethylene for a longer time as compared to untreated (control) plant,
suggesting these analogues were similar to 1-MCP (Sisler and Wood, 1988; Sisler and Serek,
1997). These two analogues exerted their effect by blocking the site of ethylene binding at the
receptor. The best response with 1-OCP and 1-DCP was achieved at a concentration 5 times
higher than that with 1-MCP. The results of parallel experiments with 1-MCP showed that the
two analogues were less potent than 1-MCP. 1-MCP improved display life by delaying bud,
flower and leaf abscission for an extended period at 200 nl l-1, whereas two analogues improved
display life at 1000 nl l-1. However, no phytotoxicity was observed at higher concentrations of 1-OCP
and 1-DCP. The time of exposure had an influence on the binding of two chemicals to the
ethylene receptor, thus affecting the display life of the plants. 4-h exposure time to 1-OCP and
1-DCP treatments was sufficient to improve the display life for about 9 days. This was
significantly different from untreated plants, which showed 80% leaf drop after 6 days.
Continuous exposure of two analogues for a longer period of time possibly resulted in saturation
of the receptor and did not give any additional benefit. However, after 9 days of pretreatment
with 1-OCP and 1-DCP, the tissue became sensitive to ethylene again, indicating that free binding
sites on the ethylene receptor were present in the tissue at the point of recovery from the
inhibition, or these binding sites were newly formed, or the sites became dissociated from the
inhibitors (Feng et al., 2004). Miniature roses pretreated with 1-OCP and 1-DCP at the range of
5-10oC were not significantly different but were different from untreated (control) plants in
improving display life. However, 1-MCP was more effective than 1-OCP and 1-DCP. Based on
the concentrations required and the duration of protection, ‘Lavender’ flowers, which are
sensitive to ethylene pretreated with 1-OCP, was more effective in protecting detrimental effects
of ethylene than pretreated with 1-DCP. Conversely, 1-DCP pretreatment was more effective in
‘Vanilla’, with long-lasting flowers, than with a 1-OCP pretreatment. We also showed that 1-MCP
analogues, with substitution at the 1-position and bearing eight and ten carbon side chains, are
effective as blockers of the ethylene receptor.
The molecular study explained the expression of genes for ethylene biosynthesis, ethylene
perception and signal transduction after pretreatment with 1-MCP, 1-OCP and 1-DCP, and
afterwards continuous exposure to exogenous ethylene was performed in petals and leaves of
both ‘Vanilla’ and ‘Lavender’. It is widely recognized that ethylene biosynthesis, ethylene
perception and the signal transduction pathway involve the regulation of ethylene response in
plants (Wilkinson et al., 1995). To gain a better understanding of the ethylene response pathway,
therefore, new partially-putative ethylene-responsive element-binding factors (ERF) were
isolated from Rosa hybrida. RhERF1 showed a similar, highly-conserved DNA-binding domain
consisting of 30 amino acids and shared homology with other reported ERF-domain containing
Northern blot hybridization analysis was used to compare the results of RT-PCR. However, it
was difficult to distinguish transcriptional signals of ethylene receptor, RhETR2, which is a high
homologue to other members of the ethylene receptor family in miniature roses, such as RhETR1
and RhETR4. Thus RT-PCR was utilized to compare the expression level of genes investigated in
miniature potted roses. RT-PCR analysis revealed that the expression of mRNAs for many
ethylene-related genes in both rose cultivars was regulated in a tissue-specific manner. Also,
differential cultivars suggest they have different roles in ethylene biosynthesis enzyme, ethylene
perception and signal transduction pathway. In the petals of the two miniature rose cultivars in
the absence of ethylene, no expression of mRNAs for any genes in any treatments was detectable.
However, the accumulation of mRNA transcripts was upregulated by ethylene in control petals.
In vegetative tissues like leaves, the level of mRNAs was detectable in both untreated cultivars in
the absence and presence of ethylene indicating the ethylene signaling pathway contains both
positive and negative regulator genes. This implied that some proteins serve to induce the
ethylene response while others suppress it (Chen et al., 2005). In ‘Vanilla’ control leaves,
RhACS1, RhACS2, RhETR1, RhETR2, RhETR4, RhEIN3, RhEIL and RhERF1 transcripts were
upregulated by ethylene, while RhETR3 and RhCTR1-2 were constitutively expressed. In
‘Lavender’ untreated leaves, RhACS1, RhACS2 and RhETR3 transcripts were downregulated,
while RhETR1, RhETR2, RhETR4, RhCTR1-2, RhEIN3, RhEIL and RhERF1 transcripts were
upregulated by ethylene. Pretreatment with ethylene receptor inhibitors, 1-MCP, 1-OCP and 1-DCP,
suppressed the expression of all genes investigated in ‘Vanilla’ petals, in the absence or presence
of ethylene, while pretreatment with 1-DCP upregulated the expression of RhETR3, RhEIN3 and
RhEIL transcripts in ‘Lavender’ petals in the presence of ethylene. In ‘Vanilla’ leaves, in the
absence of ethylene, all chemicals suppressed the expression of all genes investigated, whereas
the level of RhEIN3 mRNA was upregulated by pretreatment of ethylene receptor inhibitors in
the presence of ethylene, while the accumulation of RhEIN3 mRNA was suppressed in
‘Lavender’ leaves. These results suggest that the expression of RhEIN3 transcript in ‘Vanilla’,
which is insensitive to ethylene, and ‘Lavender’, with short-flower life, may be rate-limiting for
ethylene perception and the signal transduction pathway that is involved in ethylene-induced
flower senescence. With physiological investigation, this can explain a great difference of display
quality between ‘Vanilla’ and ‘Lavender’ when pretreated with 1-OCP and 1-MCP. Pretreating
‘Lavender’ leaves with 1-MCP and 1-OCP in the presence of ethylene suppressed the expression
of other genes but upregulated the accumulation of RhETR1, RhETR2 and RhEIL transcripts,
respectively, while pretreatment of ‘Lavender’ leaves with 1-DCP upregulated RhETR1, RhETR4,
RhCTR2, RhEIN3 and RHEIL transcripts.
The regulation of genes for the ethylene biosynthesis enzyme, ethylene perception and signal
transduction pathway in miniature rose is partly due to the differences in sensitivity of cultivars
and tissues to ethylene. Exposure to ethylene may increase the sensitivity of tissues in ‘Lavender’
than that of ‘Vanilla’, thus resulting in reduction in postharvest longevity. This might depend on
plant species and tissue. However, 1-MCP was more potent in protecting plants against ethylene
than 1-OCP and 1-DCP, probably by blocking the binding site of ethylene on the ethylene
receptor in plant tissues, thus controlling ethylene responses (Sisler and Wood, 1988; Sisler and
Serek, 1997). 1-MCP is a highly strained olefin, which binds in an apparently irreversible manner
to the ethylene receptor. Also, it seems that 1-OCP and 1-DCP inhibited ethylene action in a
similar mode to that suggested for 1-MCP. These results were consistent with physiological
investigation except after a certain period, when the plant tissues resumed sensitivity to ethylene
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Appendix 1. gene-specific primer pairs for RhERF1 using partial DNA fragment of Arabidopsis
CTTGGCACTTTCG Appendix 2. Sequence analysis of Rosa hybrida 92 bp partial DNA fragment. (a) nucleotide
sequence, (b) mRNA sequence, (c) deduced amino acid sequence (d) homology comparison with
LeERF1 (GeneBank Accession No. AY192367), LeERF2 (GeneBank Accession No.
AY192368), AtERF1 (GeneBank Accession No. AAL25588), AtERF2 (GeneBank Accession No.
NM_124093) and AtERF5 (GeneBank Accession No. NM_124094) using CLUSTAL W
program. *= identical amino acid, : = two nucleotides out of the triplicate amino acid code are
identified, . = one nucleotide out of the triplicate amino acid code are identified.
YRGVRKRPWGRYAAEIRDPWKK-TRVWLGTF----------------------------- ***:*:****::****** :. ******: 91 166
Appendix 3. Sequence analysis for specific primers of Rosa hybrida (a) nucleotide sequence, (b)
mRNA sequence. Border between intron and exon sequence was detected with splicing specific
sequence GT and AG, respectively.
β-Actin: X55751 373 bp mRNA (b)
mRNA size 150 bp
RhACS1: AY061946 1089 bp mRNA (b)
mRNA size: 205 bp
ATCAACGAAGTGAAGCTCAACGTTTCGCCGGGGTCTTCCTTCCGTTGCGTGGAGCCAGGCTGGTTCCGGGTTTGT 92 Appendices
RhACS2: AY525066 1346 bp DNA (a)
Genomic size 369 bp
mRNA size 249 bp
RhETR1: AF394914 1479 bp mRNA (b)
mRNA size: 393 bp
AGTGATCAGCTCGTAGAGCAGAATGGTGCTTTAGATTTGGCCCGGAGAGAGGCAGAGCTGGCAATCCATGCTCGCAAC 93 Appendices
RhETR2: AF127220 534 bp DNA RhETR2s CTCAAACTTCCAAATCAATGACTG
RhETR2as ATATTCTGCTCCATTAGCAGATCC (a)
Genomic size: 978 bp
mRNA size: 213 bp
AGGAGGGAAGCAGAAACGGCAATTTGTGCTCGCAATGATTTCTTGGCCGTGATGAACCACGAGAT 94 Appendices
RhETR3: AF154119 798 bp mRNA (b)
mRNA size: 661 bp
RhETR4: AF159172 534 bp mRNA (b)
mRNA size: 500 bp
RhCTR1: AY032953 3121 bp mRNA (b)
mRNA size:706 bp
CCGGATGAATCTTCGTCGAGGTTGTCCAGCTCTGCCGATGCAGTTTCGCATCGATTTTGGGTGAATGGCTGTCTGTCA 95 Appendices
RhCTR2: AY029067 681 bp mRNA (b)
mRNA size: 375 bp
AAAGAAGTTGATCCCCCTGTTGCAAGGATAATTTGGCAATGCTGGCAAAACGACCCC 96 Appendices
RhEIN3: AF443783 553 bp mRNA (b)
mRNA size: 236 bp
RhEIN3-like: AY052825 407 bp mRNA (b)
RhERF: 92 bp DNA (a, b)
TAACGCCTCTGTAA 97 Appendices
Appendix 4. Computer programs used for analysing gene sequences (a) NCBI Blast program, (b)
ClustalW program, (c) Chromas and construction of primers (d) Primer 3 Input program.
(a). NCBI Blast program
TER=L&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&NCBI_G). 98 Appendices
(b). ClustalW program (http://www.ebi.ac.uk/clustalw/). (c). Chromas (http://www.technelysium.com.au/chromas.html). 99 Appendices
(d). Primer 3 Input program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). 100 Appendices
Appendix 5. The principle of ligation into a Topo TA Cloning Kit (Invitrogen, Carlsbad, CA,
http://www.invitrogen.com/content/sfs/vectors/pcr4topo_map.pdf). Appendix 15. The principle of ligation into a Topo TA Cloning Kit (Invitrogen, Carlsbad, CA,
http://www.invitrogen.com/content/sfs/vectors/pcr4topo_map.pdf). 101 Eidesstattliche Erklärung
Hiermit erkläre ich an Eides Statt, das ich die vorliegende Arbeit selbständig angefertigt habe und
keine anderen als die angegebenen Quellen und Hilfsmittel benutzt habe sowie dass diese Arbeit
noch nicht als Dissertation oder andere Prüfungsarbeit vorgelegt worden ist. Hannover, den 18.10.2005 Mantana BUANONG Publication
Buanong, M., Mibus, H., Sisler, E.C. and Serek, M. (2005). Efficacy of new inhibitors of ethylene
perception in improvement of display quality of miniature potted roses (Rosa hybrida L.). Plant
Growth Regul. 47:29-38. Curriculum Vitae
Name: Mantana BUANONG Address: Zim. 491, Dorotheen Str. 5A, 30419, Hannover Date and Place of Birth: 08.11.1973, Khonkaen-Thailand Nationality: Thai School and Higher Education
1992-1996 Horticulture at King Mongkut's Institute of Technology
Ladkrabang, Bangkok, Thailand.
Bachelor Degree of Science (Agriculture). 1997-2000 Postharvest Technology at King Mongkut's University of
Technology Thonburi, Bangkok, Thailand.
Master Degree of Science (Postharvest Technology). Since 09/2002 Ph.D. Student at University of Hannover, Faculty of Natural
Science, Department of Horticulture. Work Experience
2001-2002 Researcher at Division of Postharvest Technology, School of
Bioresources and Technology, King Mongkut's University of
Technology Thonburi, Bangkok, Thailand. Research Interests
Physiology and Molecular effects after pretreatment of inhibitors
of ethylene receptor in improving postharvest quality of ornamental
Honours and Awards
Since 09/2002: Scholarship by the Asian Development Bank (ADB) for
Doctoral studies at University of Hannover. 2000 Scholarship by University Mobility in Asia and the Pacific
(UMAP), as an exchange student at Faculty of Science and
Technology, University of Western Sydney, Hawkesbury, New
South Wales, Australia. 1998 Scholarship by King Mongkut’s University of Technology
Thonburi, as assistant researcher. ...
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