Biotechnology Advances 24 (2006) 410 – 419

www.elsevier.com/locate/biotechadv

Research review paper

Genetic modulation of ethylene biosynthesis and signaling in plants
Jennifer C. Czarny a , Varvara P. Grichko b , Bernard R. Glick a,⁎
a
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
b
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA
Available online 9 March 2006

Abstract

With the isolation and characterization of the key enzymes and proteins, and the corresponding genes, involved in ethylene
biosynthesis and sensing it has become possible to manipulate plant ethylene levels and thereby alter a wide range of physiological
processes. The phytohormone ethylene is an essential signaling molecule that affects a large number of physiological processes;
plants deprived of ethylene do not grow and develop normally. In a search for flexible on–off ethylene control, scientists have used
inducible organ- and tissue-specific promoters to drive expression of different transgenes. Here, the various strategies that have
been used to genetically engineer plants with decreased ethylene biosynthesis and sensitivity are reviewed and discussed.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Ethylene; Signal transduction; ACC deaminase; ACC oxidase; ACC synthase; Transgenic plants

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
2. Key targets for genetic manipulation of ethylene levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
3. Modulating plant development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
3.1. Flower senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
3.2. Ripening of climacteric fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
4. Stress tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
4.1. Engineering plants with increased resistance to pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . 414
4.2. Improving ozone tolerance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
4.3. Increasing resistance to other abiotic stresses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416

1. Introduction

The hormone ethylene is essential for proper plant

⁎ Corresponding author. Tel.: +1 519 888 4567x5208; fax: +1 519
746 0614.
E-mail address: glick@sciborg.uwaterloo.ca (B.R. Glick).
0734-9750/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2006.01.003
development, growth and survival. It is responsible for
signaling changes during germination, flower and fruit
development, the onset of plant defense responses, and
 J.C. Czarny et al. / Biotechnology Advances 24 (2006) 410–419
cross-talk with other plant hormones (Mattoo and
Shuttle, 1991; Abeles et al., 1992; Arshad and Franken-
berger, 2002; Stepanova and Alonso, 2005). Transcrip-
tional profiling by cDNA-AFLP (amplified fragment
length polymorphism) and microarray analysis revealed
that ethylene regulates transcription of a large number of
genes governing cell wall and lipid metabolism, protein
degradation through the ubiquitin–proteasome-depen-
dent proteolysis, water transport, peptide and ion cellular
localization, signalling, and many other basic cellular
processes (De Paepe et al., 2004). In Arabidopsis, 7% of
the investigated 6000 genes are ethylene-regulated
(Zhong and Burns, 2003). Ethylene has the ability to
trigger exaggerated disease symptoms and exacerbate an
environmental pressure. Except for fruit ripening and
lateral root initiation, high levels of ethylene are usually
deleterious to plant growth and health.
2. Key targets for genetic manipulation of ethylene
levels
Biosynthesis of ethylene requires S-adenosylmethio-
nine (SAM), which is a precursor in several other path-
ways and is abundant within plant tissues. The rate-
limiting step in the ethylene biosynthetic pathway is
generally considered to be the conversion of SAM to 1-
aminocyclopropane-1-carboxylic acid (ACC) and 5′-
methylthioadenosine (MTA) in the reaction catalyzed by
ACC synthase. MTA is then recycled to L-methionine to
allow for levels of L-methionine to remain relatively
unchanged even during high rates of ethylene production
(Abeles et al., 1992).
ACC is oxidized to ethylene and other products by
oxygen in a reaction catalyzed by ACC oxidase, which
is present in most tissues at very low levels (so that the
induction of ACC oxidase may also be considered to be
rate limiting). Both ACC synthase and ACC oxidase are
encoded by multi-gene families, and the transcription of
different genes is induced under different environmental
and physiological conditions (Abeles et al., 1992;
Arshad and Frankenberger, 2002; Andersson-Gunneras
et al., 2003; Wang et al., 2005).
The essential elements of ethylene perception and
signal transduction are conserved among the plants
where they have been examined, suggesting their im-
portance to plant development and survival. Ethylene
action occurs through a chain of events involving pro-
teins that were first identified in Arabidopsis (Chang et
al., 1993; Guo and Ecker, 2004). There are five known
ethylene receptor proteins in Arabidopsis: ETR1 and
ERS1 (subfamily I), and ETR2, EIN4, and ERS2
(subfamily II), for ETHYLENE RESPONSE 1 and 2,
411
ETHYLENE INSENSITIVE 4, and ETHYLENE RE-
SPONSE SENSORS 1 and 2, respectively (reviewed in
Guo and Ecker, 2004; Stepanova and Alonso, 2005;
Chen et al., 2005). Expression of genes coding ERS1,
ERS2 and ETR2 is induced by ethylene (Hua et al.,
1998). Ethylene receptors form dimers and bind ethylene
with the help of a copper (I) cofactor (Rodriguez et al.,
1999; Woeste and Kieber, 2000) that is located in the
transmembrane domain. ETR1 is an intracellular recep-
tor located on the endoplasmic reticulum (ER) (Chen et
al., 2002). Expression in yeast of ethylene-binding do-
mains from the five receptor isoforms from Arabidopsis
thaliana and five receptor isoforms from tomato
demonstrated that all receptors bind ethylene with a
high affinity and release it very slowly (O'Malley et al.,
2005). Ethylene receptors are related to the two-com-
ponent hybrid sensor kinases, first found in bacteria,
which are characterized as having both a sensor and
response regulator domain (Chang et al., 1993). In two-
component sensors, a conserved histidine residue
autophosphorylates following ligand binding, and a
phosphate is then transferred to an aspartic acid residue,
which is located within the receiver domain (Pirrung,
1999). Only ETR1, EIN4 and ETR2 contain a receiver
domain, whereas the others do not.
Ethylene receptors of subfamily I have three hydro-
phobic transmembrane regions at the N terminal end and
a conserved histidine kinase domain (Stepanova and
Alonso, 2005). However, the role of histidine kinase in
ethylene receptor activity remains uncertain (Gamble et
al., 2002; Wang et al., 2003). The other three ethylene
receptors, ETR2, ERS2 and EIN4, contain four trans-
membrane domains, lack key features of the histidine
kinase domain (Hua and Meyerowitz, 1998), yet have
serine kinase activity (Moussatche and Klee, 2004).
They might form heterodimers with other receptors in
order to assemble functional receptors (Wang et al.,
2002). It is also of interest to note that ETR1 has recently
been shown to mediate cellular responses to hydrogen
peroxide (Desikan et al., 2005).
The ethylene signal pathway is negatively regulated
(Hua and Meyerowitz, 1998; Tieman et al., 2000). When
ethylene occupies a receptor, the receptor is thought to
undergo a conformational change and interacts with the
Raf-like kinase CTR1 (CONSTITUTIVE TRIPLE
RESPONSE 1) which is a negative regulator of the
signal transduction pathway and is controlled at the post-
transcriptional level by ethylene (Gao et al., 2003).
Inactivation of CTR1 releases suppression of the MAPK
cascade and results in activation of EIN2 (ETHYLENE
INSENSITIVE 2) protein, a key positive regulator in the
ethylene signal transduction pathway. Activation of
412 J.C. Czarny et al. / Biotechnology Advances 24 (2006) 410–419
EIN2 is followed with the initiation of the transcriptional
cascade and the establishment of ethylene responses. In
Arabidopsis, loss-of-function mutations in EIN2 result
in ethylene insensitivity, indicating its central role in
ethylene signaling (Stepanova and Alonso, 2005).
3. Modulating plant development
3.1. Flower senescence
The central role of ethylene in flower senescence,
fruit ripening and spoilage has caused it to be of interest
to the horticulture, agriculture and food industry. Ethyl-
ene biosynthesis, perception and signal transduction
have become the targets of extensive genetic manipula-
tion in order to extend the shelf life and appearance of
ornamental flowers.
Flower wilting is caused by the death of cells as a
result of increased membrane permeability, activation of
reactive oxygen species and the decreased expression of
protective enzymes (Rubinstein, 2000). These changes
are triggered in many cases by ethylene and the up-
regulation of ethylene biosynthesis genes can be seen
prior to the senescence of many flower species (Rottman
et al., 1991; Woodson, 1994; Tang et al., 1994).
Two main approaches have emerged as a result of
numerous efforts to engineer transgenic plants with
longer-lived flowers. The first is based on the antisense
ACC synthase and oxidase gene strategies and the
second employs heterologous expression of mutated
ethylene receptor genes.
Transgenic plants with lowered ethylene biosynthesis,
including tomato (Hamilton et al., 1990), broccoli (Henzi
et al., 1999) and tobacco (De Martinis and Mariani,
1999), have been produced primarily to study reproduc-
tion and senescence, whereas carnation, begonia and
torenia have been produced for commercial reasons. All
of the above mentioned plants were transformed with an
antisense version of an ACC oxidase gene that is up-
regulated during senescence. With tobacco, the pistil
specific S3 promoter from Petunia inflata was used to
direct expression of the antisense construct to the floral
parts. In all cases, there was a dramatic reduction in the
levels of ethylene produced, and in turn increased flower
longevity. For instance, transgenic carnations produced
90% less ethylene than non-transformed plants (Savin et
al., 1995), transgenic begonia flowers had longevity
similar to non-transformed flowers treated with silver
thiosulfate (Einset and Kopperud, 1995), and antisense
ACC oxidase transgenic torenia had a vase life up to 3.5
times that of non-transformed plants (Aida et al., 1998).
Transgenic torenia with an antisense ACC oxidase gene
also produced more flowers per stem. Kosugi et al. (2002)
reported that cut flowers of a transgenic carnation with an
ACC oxidase cDNA in the sense orientation (sACO
transgene) had a longer vase life than flowers of non-
transformed carnation plants, and produced negligible
amount of ethylene. The sACO transgene is thought to
inhibit expression of ACO1, probably by cosuppression
in the gynoecium, which then suppresses ethylene pro-
duction in all flowers of sACO-1 plants.
Mutated ethylene receptor genes have frequently been
used to convey resistance to ethylene in transgenic floral
species. Bleecker et al. (1988) showed that etr1-1 is a
dominant mutation that confers ethylene insensitivity in
other plants. Ethylene-insensitive petunia were created
by Wilkinson et al. (1997) when they introduced etr1-1
under the control of the constitutive CaMV 35S promoter.
The main disadvantage of this strategy is that plants
deprived of ethylene perception do not develop normally.
Therefore, the use of organ- and tissue-specific promoters
that are inducible either developmentally or by a selective
external stimulus is indicated. Transfection of petunia
with etr1-1 placed under the control of floral-specific
promoters FBP1 (floral binding protein) and AP3
(involved in floral organ development) resulted in a
vase life of up to five times that of non-transformed
flowers (Cobb et al., 2002). Carnation has also been
transformed with etr1-1 under the control of these same
floral specific promoters and a CMB2 (carnation MADS
box) containing promoter (Baudinette et al., 2000);
MADS domain proteins are important in fruit ripening
(Vrebalov et al., 2002). Transgenic carnation cut flowers
had three times the vase life of non-transformed flowers
and lasted up to 16days, which is longer than flowers
treated with either inhibitors of ethylene biosynthesis or
ethylene antagonists (Bovy et al., 1999; Baudinette et al.,
2000).
Shaw et al. (2002) introduced the boers gene, a
mutated ers gene from Brassica oleracea, into petunia.
Similar to etr1-1, boers codes for an ethylene receptor
with a non-functional sensor domain that is not able to
bind ethylene. The transgenic petunia plants were insen-
sitive to ethylene, and produced flowers that were larger
and had a longer vase life than those from non-trans-
formed plants. They found, however, that disease re-
sistance was compromised. Earlier, Clark et al. (1999)
observed reduced adventitious root formation in ethyl-
ene-insensitive transgenic petunia and concluded that
ethylene plays an important role in the response of roots
to environmental stimuli. More recently, Clevenger et al.
(2004) reported that transgenic ethylene-insensitive
petunias carrying the etr1-1 transgene had a decrease
in pollen viability, root mass, seed weight, and seed
 J.C. Czarny et al. / Biotechnology Advances 24 (2006) 410–419
germination. While seed germination and weight are
maternally regulated, the transgene was found to be
completely dominant in its effect on flower senescence.
A mutated ethylene receptor from tomato was also re-
ported to delay senescence in transgenic flowers, despite
the fact that transgenic plants showed a triple response
(Wilkinson et al., 1997).
Shibuya et al. (2004) have recently isolated PhEIN2,
a petunia homolog of the Arabidopsis EIN2 gene, and
constructed transgenic petunia plants with reduced
PhEIN2 expression, and significantly delayed flower
senescence. Some undesirable traits similar to those
seen in transgenic etr1-1 plants were observed including
inhibition of adventitious and seedling root hair
formation, increased hypocotyl length, and premature
death. A more dramatic reduction in ethylene sensitivity
was achieved with the expression of both the etr1-1 and
PhEIN2 transgenes (Shibuya et al., 2004).
3.2. Ripening of climacteric fruit
Climacteric fruits including apple, avocado, banana,
guava, mango, melon, passion fruit, pawpaw fruit, peach,
and pear differ from non-climacteric fruits in their ability
to continue to ripen after harvest (Abeles et al., 1992;
Giovannoni, 2004). The ripening of climacteric fruit
features an autocatalytic ethylene synthesis stage, which
is preceded by a rapid increase in respiration followed by
a series of developmental processes that allow plants to
produce edible fruit and disperse their seeds.
Tomato is the model of choice to study climacteric
fruit ripening due to its economic importance as well as
the relative ease with which it can be studied genetically.
In tomato, ripening begins with the transcription of two
ACC synthase genes (LeACS1A and LeACS4), which
are thought to be activated by the LeMADS-RIN
transcription factor (Vrebalov et al., 2002). Ripening-
specific ACC oxidase transcripts also begin to appear at
this time and help to sustain autocatalytic ethylene
synthesis. Increased ethylene production results in
changes in chlorophyll, carotenoid and flavonoid
concentrations and, thus, in fruit color change; changes
in cell wall turgor, that alters fruit texture; and
modifications to sugar, acid and volatile compound
profiles, affecting fruit flavor and aroma (Giovannoni,
2004).
Biotechnological strategies that have been employed
to delay fruit ripening in tomato plants have included the
development of transgenic lines with altered S-adeno-
sylmethionine (SAM) levels, gene silencing achieved
with the antisense expression of ACC synthase and
ACC oxidase (Theologis and Sato, 1998; Oeller et al.,
413
1991; Hamilton et al., 1990), RNAi (RNA interference)
silencing of the ACC oxidase gene (Xiong et al., 2005),
and heterologous expression of ACC deaminase (Klee et
al., 1991; Robison et al., 2001a).
SAM is a universal methylating agent and involved in
the methylation of nucleic acids, proteins, carbohydrates,
lipids, and many other biomolecules. Its level in fruit can
be altered by expressing transgenes that code for SAM
degrading enzymes. For example, SAM hydrolase from
bacteriophage T3 can convert SAM to 5′-methylthioa-
denosine and homoserine (Good et al., 1994) which then
may re-enter the L-methionine cycle. Genetic control of
ethylene biosynthesis in plants using SAM hydrolase
was patented in 1995 by Ferro et al. Over-expression of
the SAM hydrolase gene under the control of the
ripening-specific E8 promoter in tomato reduced
ethylene levels in transgenic fruit compared to control
fruit (Good et al., 1994). Another enzyme that can be
used to control SAM levels is SAM decarboxylase that
converts SAM to its decarboxylated form. Decarboxy-
lated SAM acts as a propylamine group donor in the
biosynthesis of polyamines (Kumar et al., 1996).
Expression of the SAM decarboxylase gene under
control of either the CaMV 35S promoter or the
tetracycline promoter, in both the sense and antisense
orientation, has been utilized in potato plants (Kumar et
al., 1996). A number of transgenic lines, however,
displayed unusual phenotypes that could be a conse-
quence of the central role of SAM in a number of
physiological processes.
Prominent among the strategies that have been
employed to delay fruit ripening in tomato are the use
of antisense constructs of ACC synthase or ACC oxidase
(Oeller et al., 1991; Hamilton et al., 1990). Plants con-
taining antisense ACC synthase showed a 99.5% de-
crease in ethylene production and fruits did not ripen
without the addition of exogenous ethylene. However, in
other studies, there has been only limited success in
using antisense ACC synthase to lower plant ethylene
levels (Knoester et al., 1997; Oeller et al., 1991). This
may be because of the many different isoforms of ACC
synthase within tomato. Use of an antisense ACC oxi-
dase gene was more successful perhaps due to higher
conservation among ACC oxidase genes. When a ripen-
ing specific ACC oxidase gene was used in the antisense
direction, tomato plants were found to produce less eth-
ylene than their non-transformed counterparts in flood-
ing conditions as well as during senescence (English et
al., 1995; John et al., 1995).
The use of small antisense RNA has been effective in
decreasing transcript levels in plants. Han and Grierson
(2002) showed that small interfering RNAs (siRNAs,
414 J.C. Czarny et al. / Biotechnology Advances 24 (2006) 410–419
21–28 nucleotides) could effectively lower expression
of ACC oxidase transcripts in tomato. They also found
that the addition of inverted repeats of the 5′ UTR region
of the transgene increased silencing of ACC oxidase
mRNA even though the siRNAs were produced from the
3′ end of the transgene. Although ethylene levels were
not measured, this work holds promise for attenuation of
ethylene biosynthesis in a stable and specific way.
In a similar strategy, Xiong et al. (2005) produced
double-stranded interfering RNAs complementary to
ACC oxidase and placed them under the control of a
modified CaMV 35S promoter. These transgenic tomato
plants produced fruit that released only traces of ethylene
and had a shelf life of more than 120days.
Strategies similar to those employed with tomato
have been used in melon and apple. Auyb et al. (1996)
produced a transgenic cantaloupe line with lower levels
of ethylene synthesis due to the over-expression of an
antisense ACC oxidase gene called Mel1. Ethylene
production in ripening fruit and wounded leaves was
reduced by 99% and 68%, respectively (Auyb et al.,
1996). With these melons, the pulp ripened at the same
rate as control plants, but the rind stayed greener due to
retention of chlorophyll and had an increased concen-
tration of sucrose and citric acid (Flores et al., 2001;
Flores et al., 2002). Dandekar et al. (2004) used antisense
versions of the apple ACC synthase and ACC oxidase to
transform the Greensleeves variety of apple with the aim
of studying the role of ethylene in apple ripening. They
observed very low levels of ethylene synthesis in three-
year old apple trees grown in the field, and hence fruit
that ripened more slowly. One side effect of this strategy
was a decrease in the concentration of enzymes res-
ponsible for the formation of flavour esters, an effect that
may adversely affect consumer acceptance.
A dioxygenase gene from tomato, E8, is similar to the
ACC oxidase family of genes and has been found to
increase ethylene levels when expressed in the antisense
orientation in tomato (Penarrubia et al., 1992), and it
lowers ethylene levels when it is over-expressed (Kneissl
and Deikman, 1996). E8 does not convert ACC to
ethylene, but may be involved in a pathway that has an
indirect effect on ethylene biosynthesis (Penarrubia et
al., 1992).
The microbial enzyme ACC deaminase, first discov-
ered by Honma and Shimomura (1978), converts ACC to
ammonia and α-ketobutyrate, both of which may be
further metabolized by the microorganism. ACC deam-
inase genes have been isolated from a number of soil
bacteria (Klee et al., 1991; Klee and Kishore, 1996;
Honma, 1993; Jacobson et al., 1994; Glick et al., 1995;
Campbell and Thomson, 1996; Burd et al., 1998; Mina-
mi et al., 1998; Belimov et al., 2001; Ghosh et al., 2003;
Ma et al., 2003; Mayak et al., 2004a; Hontzeas et al.,
2005). By inserting the ACC deaminase gene into to-
mato plants under the control of the CaMV 35S pro-
moter, fruits with delayed ripening were readily
produced (Klee et al., 1991). No other significant phe-
notypic differences were observed between plants with
high levels of ACC deaminase activity and non-
transformed plants. Other transgenic tomato lines have
been produced using the same strategy with similar
results (Reed et al., 1995; Grichko et al., 2005).
Another strategy that has been utilized to decrease the
rate of fruit ripening includes the insertion of the etr1-1
mutant gene from A. thaliana into tomato plants
resulting in decreased ethylene perception and little to
no ethylene action (Chang et al., 1993). In this case, fruits
either ripened more slowly than controls or did not ripen
at all, and were not susceptible to exogenous ethylene
application (Wilkinson et al., 1997).
There are a number of different strategies that can be
used to efficaciously lower ethylene levels in ripening
fruit and thereby decrease the rate at which these fruits
ripen. In fact, it would appear that this technology is
essentially ready for commercialization.
4. Stress tolerance
4.1. Engineering plants with increased resistance to
pathogens
Plants respond to pathogen invasion in a complex
way. The plant defense system against pathogen attack
includes the hypersensitive response (HR), a component
of systemic acquired resistance (SAR) responsible for
the rapid death of infected cells and formation of local
and necrotic lesions to restrict the growth of pathogens.
HR causes the production of reactive oxygen species
(ROS) followed by the induction of SAR genes such as
1,3-glucanase, chitinases and other pathogenesis related
(PR) proteins.
Plants often respond to an infection with an
accelerated rate of ethylene biosynthesis (Van Loon,
1984; Abeles et al., 1992). A small initial peak of ethyl-
ene is produced close in time to the onset of the stress and
is followed by a second much larger peak some time
later. The first peak of ethylene consumes the existing
pool of ACC within plant tissues, after which ACC
synthase genes are expressed to fuel the second wave of
ethylene production (Ciardi et al., 2000; Robison et al.,
2001a). The second peak triggers senescence, chlorosis
and abscission, and is deleterious to the plant. The
second peak of ethylene is thought to act as a modulator
 J.C. Czarny et al. / Biotechnology Advances 24 (2006) 410–419
in apoptosis and stress-induced cell death (Rubinstein,
2000) and amplifies damage to a stressed plant (Van
Loon, 1984; Hyodo, 1991).
Inhibition of ethylene biosynthesis significantly de-
creases the severity of fungal infections (Cronshaw and
Pegg, 1976; Elad, 1988; Biles et al., 1990; Hyodo,
1991; Bashan, 1994). Both Lund et al. (1998) and
Robison et al. (2001b) observed that transgenic tomato
plants that expressed ACC deaminase and had a
lowered level of stress ethylene were significantly
protected from damage by various phytopathogens.
Tobacco plants containing sense and antisense ACC-
synthase and/or ACC-oxidase had altered ethylene
levels and resisted infection with tobacco mosaic virus
(TMV) (Knoester et al., 1997). In contrast, both
ethylene-insensitive (etr1-1) tobacco and Arabidopsis
plants were both impaired in their resistance to some
necrotrophic pathogens. These transgenic tobacco
plants developed wilting and stem rot when grown in
non-autoclaved soil, and were more sensitive to
infection by Fusarium, Thielaviopsis, and Pythium
than were non-transformed plants. On the other hand,
these plants were less susceptible to the biotrophic fun-
gus Oidium neolycopersici, the oomycete Peronospora
tabacina and TMV (Geraats et al., 2003). Earlier,
Knoester et al. (1998) observed that tobacco plants
transformed with the etr1-1 gene expressed almost no
defense-related PR proteins and developed spontaneous
stem browning, a consequence of the loss of resistance
to normally non-pathogenic fungi. The ethylene-insen-
sitive Never Ripe tomato mutant, however, showed in-
creased tolerance to pathogens, and plants over-
expressing the Never Ripe (NR) cDNA displayed
greatly reduced necrosis in response to pathogens
(Ciardi et al., 2000).
Due to the complex relationship between plants and
their pathogens, and the role of plant growth regulators
in infection, it is extremely difficult to accurately predict
how lowering ethylene levels in the plant will affect
tolerance to pathogens. It also appears that plants with
lowered ethylene signaling may become tolerant to one
pathogen only to lose tolerance to another.
4.2. Improving ozone tolerance
Ozone, a component of photochemical smog, causes
necrotic lesions that are visible on the surface of the
leaves of plants. The oxidative burst that occurs after
ozone treatment acts to initiate ethylene biosynthetic
pathways that cause plant tissue damage, and the amount
of damage correlates with the level of ethylene biosyn-
thesis (Moeder et al., 2002). Thus, damage to plants
415
exposed to ozone may occur via the activation of defense
processes and not necessarily through the direct action of
reactive oxygen species.
Most attempts to produce ozone-tolerant plants have
focused on ethylene biosynthesis. ACC synthase genes
from tomato (Le-ACS2 and Le-ACS6) and potato were
found to be rapidly induced by ozone (Tuomainen et
al., 1997; Moeder et al., 2002; Sinn et al., 2004).
Antisense expression of one of these ACC synthase
genes resulted in 82% and 66% less ozone-induced
ethylene in tobacco and potato plants, respectively
(Nakajima et al., 2002; Sinn et al., 2004). The
transgenic potatoes, however, were not as tolerant to
ozone as the non-transformed plants when grown in the
field. Tobacco plants with either an antisense ACC
synthase or senescence-associated ACC oxidase gene
from carnation showed a 55% and 37% decrease in
ethylene production following the treatment with H2O2,
respectively (Wi and Park, 2002).
The detrimental effects of ozone can be also
attenuated by lowering the ability of the plant to sense
ethylene. Transgenic birch trees are unable to perceive
ethylene, since they express the etr1-1 gene from Arabi-
dopsis; they show significantly reduced stress symp-
toms when treated with ozone even though the level of
ethylene produced in the transgenic tree was the same as
in the non-transformed plants (Vahala et al., 2003).
Except in ethylene-insensitive plants, the amount of
damage induced by oxidative stress always correlates
with the level of endogenous ethylene.
4.3. Increasing resistance to other abiotic stresses
A wide range of abiotic stresses including flooding,
drought, high and low temperature, metals and salt can
induce the synthesis of stress ethylene. The most suc-
cessful strategy for reducing stress ethylene in response
to abiotic stresses has been with the use of the ACC
deaminase gene. When plants are grown in association
with plant growth-promoting bacteria that express the
enzyme ACC deaminase, the bacteria act as a sink for
ACC and thereby lower plant ethylene levels (Glick et
al., 1998). In this way, ACC deaminase-containing plant
growth-promoting bacteria can protect plants against
some of the deleterious effects of a wide variety of
ethylene-inducing stresses including flooding (Grichko
and Glick, 2001b), drought (Mayak et al., 2004a), salt
(Mayak et al., 2004b), the presence of metals or organic
contaminants (Burd et al., 1998, 2000; Huang et al.,
2004, 2005; Reed and Glick, 2005), and pathogens
(Wang et al., 2000). Similarly, transgenic tomato plants
that expressed ACC deaminase were protected from
416 J.C. Czarny et al. / Biotechnology Advances 24 (2006) 410–419
heavy metals and flooding (Grichko et al., 2000; Grichko
and Glick, 2001a), respectively.
As part of a phytoremediation strategy, transgenic
tobacco and canola plants expressing a bacterial ACC
deaminase gene were constructed. These transgenic
plants were tested for their resistance to growth inhi-
bition by nickel, arsenic and high salt. Similar to what had
previously been found with tomatoes, the transgenic
plants, especially those in which the ACC deaminase
gene was under the control of the rolD promoter, were
significantly protected from the growth inhibition that
often occurs as a consequence of these stresses (Nie et al.,
2002; Stearns et al., 2005; Sergeeva et al., in press).
5. Conclusion
There is considerable commercial interest in devel-
oping fruit that will not ripen until a specific external cue
is provided, or having flowers with an extended shelf
life, or plants that are able to withstand a range of envi-
ronmental stresses. Ethylene is a pivotal signaling
molecule, and by modulating aspects of its synthesis
and perception the bioengineering of plants that are more
robust than their non-transformed counterparts in the
face of pathogen infection, flooding, drought, high sali-
nity, organic and inorganic pollutants is already attain-
able for a variety of plants. Successful strategies for
creating transgenic plants with attenuated ethylene
signaling have included the use of antisense genes cor-
responding to important ethylene biosynthesis enzymes,
RNA interference, the insertion of bacterial and other
foreign genes, and the over-expression of native and
mutant ethylene receptor genes. In all cases the use of
tissue specific and inducible promoters has improved the
results significantly.
Acknowledgements
We thank the Natural Science and Engineering
Research Council of Canada for its ongoing support of
research in our laboratory.
References
Abeles FB, Morgan PW, Saltveit Jr ME. Ethylene in plant biology.
New York: Academic Press, 1992.
Aida R, Yoshida T, Ichimura K, Goto R, Shibata M. Extension of
flower longevity in transgenic torenia plants incorporating ACC
oxidase transgene. Plant Sci 1998;138:91-101.
Andersson-Gunneras S, Hellgren JM, Björklund S, Regan S, Moritz T,
Sundberg B. Asymmetric expression of a poplar ACC oxidase
controls ethylene production during gravitational induction of
tension wood. Plant J 2003;34:339–49.
Arshad M, Frankenberger Jr WT. Ethylene: agricultural sources and
applications. New York: Kluwer Academic/Plenum Publishers,
2002.
Auyb R, Guis M, Ben Amor M, Gillot L, Roustan J, Latche A, et al.
Expression of ACC oxidase antisense gene inhibits ripening of
cantaloupe melon fruits. Nat Biotechnol 1996;14:862–5.
Bashan Y. Symptom expression and ethylene production in leaf blight
of cotton caused by Alternaria macrospora and Alternaria
alternata alone and combined. Can J Bot 1994;72:1574–9.
Baudinette SC, Stevenson TW, Savin KW. Isolation and characterisa-
tion of the carnation floral-specific MADS box gene, CMB2. Plant
Sci 2000;155:123–31.
Belimov AA, Safronova VI, Sergeyeva TA, Egorova TN, Matveyeva
VA, Tsyganov VE, et al. Characterization of plant growth
promoting rhizobacteria isolated from polluted soils and contain-
ing 1-aminocyclopropane-1-carboxylate deaminase. Can J Micro-
biol 2001;47:642–52.
Biles CL, Abeles FB, Wilson CL. The role of ethylene in anthracnose
of cucumber, Sucumis sativus, caused by Colletotrichum lagenar-
ium. Phytopathology 1990;80:732–6.
Bleecker AB, Estelle MA, Somerville C, Kende H. Insensitivity to
ethylene conferred by a dominant mutation in Arabidopsis
thaliana. Science 1988;241:1086–9.
Bovy AG, Angenet GC, Dons HJM, van Altvorst A-C. Heterologous
expression of the Arabidopsis etr1-1 allele inhibits the senescence
of carnation flowers. Mol Breed 1999;5:301–8.
Burd GI, Dixon DG, Glick BR. A plant growth promoting bacterium
that decreases nickel toxicity in plant seedlings. Appl Environ
Microbiol 1998;64:3663–8.
Burd GI, Dixon DG, Glick BR. Plant growth-promoting bacteria
that decrease heavy metal toxicity in plants. Can J Microbiol
2000;46:237–45.
Campbell BG, Thomson JA. 1-Aminocyclopropane-1-carboxylate
deaminase genes from Pseudomonas strains. FEMS Microbiol Lett
1996;138:207–10.
Chang C, Kwok SF, Bleecker AB, Meyerowitz EM. Arabidopsis
ethylene-response gene ETR1: similarity of product to two-
component regulators. Science 1993;262:539–44.
Chen YF, Randlett MD, Findell JL, Schaller GE. Localization of the
ethylene receptor ETR1 to the endoplasmic reticulum of Arabi-
dopsis. J Biol Chem 2002;277:19861–6.
Chen YF, Etheridge N, Schaller GE. Ethylene signal transduction. Ann
Bot–London 2005;95:901–15.
Ciardi JA, Tieman DM, Lund ST, Jones JB, Stall RE, Klee HJ.
Response to Xanthomonas campestris pv. vesicatoria in tomato
involves regulation of ethylene receptor gene expression. Plant
Physiol 2000;123:81–92.
Clark DG, Gubrium EK, Barrett JE, Nell TA, Klee HJ. Root formation
in ethylene-insensitive plants. Plant Physiol 1999;121:53–9.
Clevenger DJ, Barrett JE, Klee HJ, Clark DG. Factors affecting seed
production in transgenic ethylene-insensitive petunias. J Am Soc
Hortic Sci 2004;129:401–6.
Cobb D, Schneiter N, Guo S, Humiston GA, Harriman B, Bolar J.
Flower specific expression of an ethylene receptor (ETR1-1)
confers ethylene insensitivity in transgenic petunia. XXVIth
International Horticultural Congress, Toronto, Canada. Book of
abstracts; 2002. p. 83.
Cronshaw DK, Pegg GF. Ethylene as a toxin synergist in Verticillium
wilt of tomato. Physiol Plant Pathol 1976;9:33–8.
Dandekar AM, Teo G, Defilippi BG, Uratsu SL, Passey AJ, Kader AA,
et al. Effect of down-regulation of ethylene biosynthesis on fruit
flavor complex in apple fruit. Transgenic Res 2004;13:373–84.
 J.C. Czarny et al. / Biotechnology Advances 24 (2006) 410–419
De Martinis D, Mariani C. Silencing gene expression of the ethylene-
forming enzyme results in a reversible inhibition of ovule
development in transgenic tobacco plants. Plant Cell 1999;11:
1061–71.
De Paepe A, Vuylsteke M, Van Hummelen P, Zabeau M, Van Der
Straeten D. Transcriptional profiling by cDNA-AFLP and
microarray analysis reveals novel insights into the early
response to ethylene in Arabidopsis. Plant J 2004;39:537–59.
Desikan R, Hancock JT, Bright J, Harrison J, Weir I, Hooley R, et al. A
role for ETR1 in hydrogen peroxide signaling in stomatal guard
cells. Plant Physiol 2005;31:913–20.
Einset JW, Kopperud C. Antisense ethylene genes for Begonia
flowers. Acta Hort 1995;405:190–6.
Elad Y. Involvement of ethylene in the disease caused by Botrytis
cinerea on rose and carnation flowers and the possibility of control.
Ann Appl Biol 1988;113:589–98.
English PJ, Lycett GW, Roberts JA, Jackson MB. Increased 1-
aminocyclopropane-1-carboxylic acid oxidase activity in shoots of
flooded tomato plants raises ethylene production to physiologi-
cally active levels. Plant Physiol 1995;109:1435–40.
Ferro AJ, Bestwick RK, Brown LR, Inventors; Agrotope, assignee.
1995/05/16. Genetic control of ethylene biosynthesis in plants using
S-adenosylmethionine hydrolase. US Patent # 5416250; 1995.
Flores FB, Martinez-Madrid MC, Sanchez-Hidalgo FJ, Romojaro F.
Differential rind and pulp ripening of transgenic antisense ACC
oxidase melon. Plant Physiol Biochem 2001;39:37–43.
Flores F, Yahyaoui FE, de Billerbeck G, Romojaro F, Latché A,
Bouzayen M, et al. Role of ethylene in the biosynthetic pathway of
aliphatic ester aroma volatiles in Charentais cantaloupe melons.
J Exp Bot 2002;53:201–6.
Gamble RL, Qu X, Schaller EG. Mutational analysis of the ethylene
receptor ETR1. Role of the histidine kinase domain in dominant
ethylene insensitivity. Plant Physiol 2002;128:1428–38.
Gao ZY, Chen YF, Randlett MD, Zhao XC, Findell JL, Kieber JJ,
et al. Localization of the Raf-like kinase CTR1 to the endoplas-
mic reticulum of Arabidopsis through participation in ethylene
receptor signaling complexes. J Biol Chem 2003;278:34725–32.
Geraats BPJ, Bakker PAHM, Lawrence CB, Achuo EA, Hofte M,
Van Loon LC. Ethylene-insensitive tobacco shows differentially
altered susceptibility to different pathogens. Phytopathology
2003;93:813–21.
Ghosh S, Penterman JN, Little RD, Chavez R, Glick BR. Three newly
isolated plant growth-promoting bacilli facilitate the growth of
canola seedlings. Plant Physiol Biochem 2003;41:277–81.
Giovannoni JJ. Genetic regulation of fruit development and ripening.
Plant Cell 2004;16:S170–80.
Glick BR, Karaturovíc D, Newell P. A novel procedure for rapid
isolation of plant growth-promoting rhizobacteria. Can J Microbiol
1995;41:533–6.
Glick BR, Penrose DM, Li J. A model for the lowering of plant
ethylene concentrations by plant growth promoting bacteria.
J Theor Biol 1998;190:63–8.
Good X, Kellogg JA, Wagoner W, Langoff D, Matsumura W,
Bestwick RK. Reduced ethylene synthesis by transgenic tomatoes
expressing S-adenosylmethionine hydrolase. Plant Mol Biol
1994;26:781–90.
Grichko VP, Glick BR. Flooding tolerance of transgenic tomato plants
expressing the bacterial enzyme ACC deaminase controlled by the 35S,
rolD or PRB-1b promoter. Plant Physiol Biochem 2001a;39:19–25.
Grichko VP, Glick BR. Amelioration of flooding stress by ACC
deaminase-containing plant growth-promoting bacteria. Plant
Physiol Biochem 2001b;39:11–7.
417
Grichko VP, Filby B, Glick BR. Increased ability of transgenic plants
expressing the bacterial enzyme ACC deaminase to accumulate
Cd, Co, Cu, Ni, Pb and Zn. J Biotechnol 2000;81:45–53.
Grichko VP, Glick BR, Grishko VI, Pauls KP. Evaluation of tomato
plants with the constitutive, root-specific and stress-induced ACC
deaminase gene expression. Russ J Plant Physiol
2005;52:406–11.
Guo HW, Ecker JR. The ethylene signaling pathway: new insights.
Curr Opin Plant Biol 2004;7:40–9.
Hamilton AJ, Lycett GW, Grierson D. Antisense gene that inhibits
synthesis of the hormone ethylene in transgenic plants. Nature
1990;346:284–7.
Han Y, Grierson D. The influence of inverted repeats on the production
of small antisense RNAs involved in gene silencing. Mol Genet
Genomics 2002;267:629–35.
Henzi MX, McNeil DL, Christey MC, Lill RE. A tomato antisense 1-
aminocyclopropane-1-carboxylic acid oxidase gene causes re-
duced ethylene production in transgenic broccoli. Aust J Plant
Physiol 1999;26:179–83.
Honma M. Stereospecific reaction of 1-aminocyclopropane-1-carbox-
ylate deaminase. In: Pech JC, Latché A, Balagué C, editors. Cellular
and molecular aspects of the plant hormone ethylene. Dordrecht,
The Netherlands: Kluwer Academic Publishers; 1993. p. 111–6.
Honma M, Shimomura T. Metabolism of 1-aminocyclopropane-1-
carboxylic acid. Agric Biol Chem 1978;43:1825–31.
Hontzeas N, Richardson AO, Belimov AA, Safranova VI, Abu-
Omar MM, Glick BB. Evidence for horizontal gene transfer
(HGT) of ACC deaminase genes. Appl Environ Microbiol
2005;71:7556–8.
Hua J, Meyerowitz EM. Ethylene responses are negatively regu-
lated by a receptor gene family in Arabidopsis thaliana. Cell
1998;94:261–71.
Hua J, Sakai H, Nourizadeh S, Chen QG, Bleecker AB, Ecker JR, et al.
EIN4 and ERS2 are members of the putative ethylene receptor
family in Arabidopsis. Plant Cell 1998;10:1321–32.
Huang X-D, El-Alawi Y, Penrose DM, Glick BR, Greenberg BM.
Responses of plants to creosote during phytoremediation and their
significance for remediation processes. Environ Pollut
2004;130:453–63.
Huang X-D, El-Alawai Y, Gurska J, Glick BR, Greenberg BM. A
multi-process phytoremediation system for decontamination of
persistent total petroleum hydrocarbons (TPHs) from soils.
Microchem J 2005;81:139–47.
Hyodo H. Stress/wound ethylene. In: Mattoo AK, Shuttle JC, editors. The
plant hormone ethylene. Boca Raton: CRC Press; 1991. p. 43–64.
Jacobson CB, Pasternak JJ, Glick BR. Partial purification and
characterization of 1-aminocyclopropane-1-carboxylate deaminase
from the plant growth promoting rhizobacterium Pseudomonas
putida GR12-2. Can J Microbiol 1994;40:1019–25.
John I, Drake R, Farrell A, Cooper W, Lee P, Horton P, et al. Delayed
leaf senescence in ethylene-deficient ACC-oxidase antisense
tomato plants: molecular and physiological analysis. Plant J
1995;7:483–90.
Klee HJ, Kishore GM, Inventors; Monsanto Company, assignee. 1996/
04/30. Control of fruit ripening and senescence in plants. US Patent
# 5702933; 1996.
Klee HJ, Hayford MB, Kretzmer KA, Barry GF, Kishmore GM.
Control of ethylene synthesis by expression of a bacterial enzyme
in transgenic tomato plants. Plant Cell 1991;3:1187–93.
Kneissl ML, Deikman J. The tomato E8 gene influences ethylene
biosynthesis in fruit but not in flowers. Plant Physiol
1996;112:537–47.
418 J.C. Czarny et al. / Biotechnology Advances 24 (2006) 410–419
Knoester M, Linthorst HJM, Bol JF, Van Loon LC. Modulation of
stress-inducible ethylene biosynthesis by sense and antisense gene
expression in tobacco. Plant Sci 1997;126:173–83.
Knoester M, Van Loon LC, Van den Heuvel J, Hennig J, Bol JF,
Linthorst HJM. Ethylene-insensitive tobacco lacks nonhost
resistance against soil-borne fungi. Proc Natl Acad Sci USA
1998;95:1933–7.
Kosugi Y, Waki K, Iwazaki Y, Tsuruno N, Mochizuki A, Yoshioka T,
et al. Senescence and gene expression of transgenic non-ethylene-
producing carnation flowers. J Jpn Soc Hort Sci 2002;71:638–42.
Kumar A, Taylor MA, Arif SAM, Davies HV. Potato plants expressing
antisense and sense S-adenosylmethionine decarboxylase (SAMDC)
transgenes show altered levels of polyamines and ethylene: anti-
sense plants display abnormal phenotypes. Plant J 1996;9:147–58.
Lund ST, Stall RE, Klee HJ. Ethylene regulates the susceptible res-
ponse to pathogen infection in tomato. Plant Cell 1998;10:371–82.
Ma W, Sebestianova S, Sebestian J, Burd GI, Guinel F, Glick BR.
Prevalence of 1-aminocyclopropane-1-carboxylate deaminase in
Rhizobia spp. Anton Leeuw Int J G 2003;83:285–91.
Mattoo AK, Shuttle JC, editors. The plant hormone ethylene. Boca
Raton: CRC Press, Inc.; 1991.
Mayak S, Tirosh T, Glick BR. Plant growth-promoting bacteria that
confer resistance to water stress in tomato and pepper. Plant Sci
2004a;166:525–30.
Mayak S, Tirosh T, Glick BR. Plant growth-promoting bacteria that
confer resistance in tomato to salt stress. Plant Physiol Biochem
2004b;42:565–72.
Minami R, Uchiyama K, Murakami T, Kawai J, Mikami K, Yamada T,
et al. Properties, sequence, and synthesis in Escherichia coli of
1-aminocyclopropane-1-carboxylate deaminase from Hansenula
saturnus. J Biochem 1998;123:1112–8.
Moeder W, Barry CS, Tauriainen AA, Betz C, Tuomainen J, Utriainen
M, et al. Ethylene synthesis regulated by biphasic induction of
1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocy-
clopropane-1-carboxylic acid oxidase genes is required for hy-
drogen peroxide accumulation and cell death in ozone-exposed
tomato. Plant Phys 2002;130:1918–26.
Moussatche P, Klee HJ. Autophosphorylation activity of the
Arabidopsis ethylene receptor multigene family. J Biol Chem
2004;279:48734–41.
Nakajima N, Itoh T, Takikawa S, Asai N, Tamaoki M, Aono M, et al.
Improvement in ozone tolerance of tobacco plants with an
antisense DNA for 1-aminocyclopropane-1-carboxylate synthase.
Plant Cell Environ 2002;25:727–35.
Nie L, Shah S, Rashid A, Burd GI, Dixon DG, Glick BR. Phyto-
remediation of arsenate contaminated soil by transgenic canola and
the plant growth-promoting bacterium Enterobacter cloacae
CAL2. Plant Physiol Biochem 2002;40:355–61.
Oeller PW, Lu MW, Taylor LP, Pike DA, Theologis A. Reversible
inhibition of tomato fruit senescence by antisense RNA. Science
1991;254:437–9.
O'Malley RC, Rodriguez FI, Esch JJ, Binder BM, O'Donnell P, Klee
HJ, et al. Ethylene-binding activity, gene expression levels, and
receptor system output for ethylene receptor family members from
Arabidopsis and tomato. Plant J 2005;41:651–9.
Penarrubia L, Aguilar M, Margossian L, Fischer R. An antisense gene
stimulates ethylene hormone production during tomato fruit
ripening. Plant Cell 1992;4:681–7.
Pirrung MC. Histidine kinases and two-component signal transduction
systems. Chem Biol 1999;6:167–75.
Reed MLE, Glick BR. Use of plant growth-promoting bacteria to
facilitate phytoremediation of copper and polycyclic aromatic
hydrocarbons by canola (Brassica napus). Can J Microbiol
2005;51:1061–9.
Reed AJ, Magin KM, Anderson JS, Austin GD, Rangwala T, Linde
DC, et al. Delayed ripening in tomato plants expressing the
enzyme 1-aminocyclopropane-1-carboxylic acid deaminase: 1.
Molecular characterization, enzyme expression, and fruit rip-
ening traits. J Agric Food Chem 1995;43:1954–62.
Robison MM, Griffith M, Pauls KP, Glick BR. Dual role of ethylene in
susceptibility of tomato to Verticillium wilt. J Phytopathol
2001a;2:385–8.
Robison MM, Shah S, Tamot B, Pauls KP, Moffatt BA, Glick BR.
Reduced symptoms of Verticillium wilt in transgenic tomato
expressing a bacterial ACC deaminase. Mol Plant Pathol
2001b;2:135–45.
Rodriguez FI, Esch JJ, Hall AE, Binder BM, Schaller GE, Bleecker
AB. A copper cofactor for the ethylene receptor ETR1 from
Arabidopsis. Science 1999;283:996–8.
Rottman WH, Peter GF, Oeller PW, Keller JA, Shen NF, Nagy BP,
et al. 1-aminocyclopropane-1-carboxylate synthase in tomato is
encoded by a multigene family whose transcription is induced
during fruit and floral senescence. J Mol Biol 1991;222:937–61.
Rubinstein B. Regulation of cell death in flower petals. Plant Mol Biol
2000;44:303–18.
Savin KW, Baudinette SC, Graham MW, Michael MZ, Nugent GD, Lu
C, et al. Antisense ACC oxidase RNA delays carnation petal
senescence. HortScience 1995;30:970–2.
Sergeeva, E, Shah, S, Glick, BR. Tolerance of transgenic canola
expressing a bacterial ACC deaminase gene to high concentrations
of salt. World J Microbiol Biotechnol in press.
Shaw J-F, Chen H-H, Tsai M-F, Kuo C-I, Huan LC. Extended flower
longevity for Petunia hybrida plants transformed with boers, a
mutated ERS gene of Brassica oleracea. Mol Breed 2002;9:211–6.
Shibuya K, Barry KG, Ciardi JA, Loucas HM, Underwood BA,
Nourizadeh S, et al. The central role of PhEIN2 in ethylene
responses throughout plant development in petunia. Plant Physiol
2004;136:2900–12.
Sinn JP, Schlagnhaufer CD, Arteca RN, Pell EJ. Ozone-induced
ethylene and foliar injury responses are altered in 1-aminocyclo-
propane-1-carboxylate synthase antisense potato plants. New
Phytol 2004;164:267–77.
Stearns JC, Shah S, Greenberg BM, Dixon DG, Glick BR.
Tolerance of transgenic canola expressing 1-aminocyclopro-
pane-1-carboxylic acid deaminase to growth inhibition by
nickel. Plant Phys Biochem 2005;43:701–8.
Stepanova AN, Alonso JM. Ethylene signaling and response pathway:
a unique signaling cascade with a multitude of inputs and outputs.
Physiol Plant 2005;123:195–206.
Tang X, Gomes AMTR, Bhatia A, Woodson WR. Pistil-specific and
ethylene-regulated expression of 1-aminocyclopropane-1-carbox-
ylate oxidase genes in petunia flowers. Plant Cell 1994;6:1227–39.
Theologis A, Sato T. Inventors; The United States of America as re-
presented by the Secretary of the Agriculture, assignee. 1998/03/03.
Control of fruit ripening through genetic control of ACC synthase
synthesis. US Patent # 5723766; 1998.
Tieman DE, Taylor MG, Ciardi JA, Klee H. The tomato ethylene
receptors NR and LeETR4 are negative regulators of ethylene
response and exhibit functional compensation within a multigene
family. Proc Natl Acad Sci USA 2000;97:5663–8.
Tuomainen J, Betz C, Kangasjarvi J, Ernst D, Yin ZH, Langebartels C,
et al. Ozone induction of ethylene emission in tomato plants:
regulation by differential accumulation of transcripts for the
biosynthetic enzymes. Plant J 1997;12:1151–62.
 J.C. Czarny et al. / Biotechnology Advances 24 (2006) 410–419
Vahala J, Ruonala R, Keinanen M, Tuominen H, Kangasjarvi J.
Ethylene insensitivity modulates ozone-induced cell death in birch.
Plant Phys 2003;132:185–95.
Van Loon LC. Regulation of pathogenesis and symptom expression in
diseased plants by ethylene. In: Fuchs Y, Chalutz E, editors.
Ethylene: biochemical, physiological and applied aspects. The
Hague: Martinus Nijhoff/Dr. W. Junk; 1984. p. 171–80.
Vrebalov J, Ruezinsky D, Padmanabhan V, White R, Medrano D,
Drake R, et al. A MADS-box gene necessary for fruit ripening at
the tomato ripening-inhibitor (rin) locus. Science 2002;296:343–6.
Wang C, Knill E, Glick BR, Défago G. Effect of transferring 1-
aminocyclopropane-1-carboxylic acid (ACC) deaminase genes
into Pseudomonas fluorescens strain CHA0 and its gacA derivative
CHA96 on their growth-promoting and disease-suppressive
capacities. Can J Microbiol 2000;46:898–907.
Wang KL, Li H, Ecker JR. Ethylene biosynthesis and signaling
networks. Plant Cell Suppl 2002;14:S131–51.
Wang W, Hall AE, O'Malley R, Bleecker AB. Canonical histidine
kinase activity of the transmitter domain of the ETR1 ethylene
receptor from Arabidopsis is not required for signal transduction.
Proc Natl Acad Sci USA 2003;100:352–7.
Wang NN, Shih MC, Li N. The GUS reporter-aided analysis of the
promoter activities of Arabidopsis ACC synthase genes AtACS4,
AtACS5, and AtACS7 induced by hormones and stresses. J Exp Bot
2005;56:909–20.
419
Wi SJ, Park KY. Antisense expression of carnation cDNA encoding
ACC synthase or ACC oxidase enhances polyamine content and
abiotic stress tolerance in transgenic tobacco plants. Mol Cells
2002;13:209–20.
Wilkinson JQ, Lanahan MB, Clark DG, Bleeker AB, Chang C,
Meyerowitz EM, et al. A dominant mutant receptor for
Arabidopsis confers ethylene insensitivity in heterologous plants.
Nat Biotechnol 1997;15:444–7.
Woeste KE, Kieber JJ. A strong loss-of-function mutation in RAN1
results in constitutive activation of the ethylene response pathway
as well as a rosette-lethal phenotype. Plant Cell 2000;12:443–55.
Woodson WR. Molecular biology of flower senescence in carnation.
In: Scott RJ, Stead AD, editors. Molecular and cellular aspects of
plant reproduction. Cambridge, UK: Cambridge University Press;
1994. p. 255–67.
Xiong AS, Yao QH, Peng RH, Li X, Han PL, Fan HQ. Different effects
on ACC oxidase gene silencing triggered by RNA interference in
transgenic tomato. Plant Cell Rep 2005;23:639–46.
Zhong GV, Burns JK. Profiling ethylene-regulated gene expression in
Arabidopsis thaliana by microarray analysis. Plant Mol Biol
2003;53:117–31.