History of Research on the Plant Hormone Ethylene

Abstract

Ethylene is the simplest of the olefin gasses and was the first known gaseous biological signaling molecule. It is synthesized by plants during certain stages of development and in response to abiotic and biotic stresses. Ethylene affects many aspects of plant growth, development as well as responses to environmental cues. Research leading to the discovery of ethylene as a plant hormone started in the 1800s with scientists examining the effects of illuminating gas on plants. In 1901, Dimitry Neljubow determined that ethylene is the active component of illuminating gas that affects plants and thus launched this important field of research. It is generally accepted that in 1934 Richard Gane provided the conclusive evidence that plants biosynthesize ethylene. This early research showed that ethylene is both biosynthesized and sensed by plants. From the 1930s to the 1960s, there was scant research on ethylene as a hormone because many researchers did not believe that ethylene was indeed a plant hormone and because that the detection of ethylene was difficult. However, in the late 1950s, the application of gas chromatography led to an increased interest in ethylene research. From the 1960s through the early 1980s, the biochemical pathway for ethylene biosynthesis in plants was elucidated and membrane-bound ethylene-binding sites were discovered and characterized. The use of Arabidopsis thaliana as a model plant system and the widespread use of molecular biological techniques starting in the 1980s correlates with a second and larger increase in ethylene research productivity. Information gleaned from this model plant is now being applied to many plant species. In recent years, detailed models for the regulation of ethylene biosynthesis and ethylene signal transduction have emerged. This article provides an overview of the key historical discoveries regarding ethylene as a plant hormone.

Full text

History of Research on the Plant Hormone Ethylene
Arkadipta Bakshi
1
•Jennifer M. Shemansky
2
•Caren Chang
2
•Brad M. Binder
3
Received: 28 January 2015 / Accepted: 30 April 2015 / Published online: 4 July 2015
ÓSpringer Science+Business Media New York 2015
Abstract Ethylene is the simplest of the olefin gasses and
was the first known gaseous biological signaling molecule.
It is synthesized by plants during certain stages of devel-
opment and in response to abiotic and biotic stresses.
Ethylene affects many aspects of plant growth, develop-
ment as well as responses to environmental cues. Research
leading to the discovery of ethylene as a plant hormone
started in the 1800s with scientists examining the effects of
illuminating gas on plants. In 1901, Dimitry Neljubow
determined that ethylene is the active component of illu-
minating gas that affects plants and thus launched this
important field of research. It is generally accepted that in
1934 Richard Gane provided the conclusive evidence that
plants biosynthesize ethylene. This early research showed
that ethylene is both biosynthesized and sensed by plants.
From the 1930s to the 1960s, there was scant research on
ethylene as a hormone because many researchers did not
believe that ethylene was indeed a plant hormone and
because that the detection of ethylene was difficult. How-
ever, in the late 1950s, the application of gas chromatog-
raphy led to an increased interest in ethylene research.
From the 1960s through the early 1980s, the biochemical
pathway for ethylene biosynthesis in plants was elucidated
and membrane-bound ethylene-binding sites were discov-
ered and characterized. The use of Arabidopsis thaliana as
a model plant system and the widespread use of molecular
biological techniques starting in the 1980s correlates with a
second and larger increase in ethylene research produc-
tivity. Information gleaned from this model plant is now
being applied to many plant species. In recent years,
detailed models for the regulation of ethylene biosynthesis
and ethylene signal transduction have emerged. This article
provides an overview of the key historical discoveries
regarding ethylene as a plant hormone.
Keywords Ethylene Biosynthesis Illuminating gas 
Signal transduction Triple response Mutant 
Arabidopsis Hormone
Historical Overview
The term hormone was first used in 1905 by Ernest Starling
who posited that a hormone is a chemical biosynthesized at
low levels by a multicellular animal that has a physiolog-
ical effect at a distance from the site of synthesis within the
organism (Starling 1905). Even though initially applied to
animals, this term is also used for plants (So
¨ding 1923).
Research in the late 19th and early 20th centuries showed
that ethylene, the simplest of olefin gasses, fulfilled both of
Arkadipta Bakshi and Jennifer M. Shemansky have contributed
equally to this work.
&Brad M. Binder
bbinder@utk.edu
Arkadipta Bakshi
abakshi@utk.edu
Jennifer M. Shemansky
jshemans@umd.edu
Caren Chang
carenc@umd.edu
1
Genome Science and Technology Program, University
of Tennessee, Knoxville, TN 37996, USA
2
Department of Cell Biology and Molecular Genetics,
University of Maryland, College Park, MD 20742, USA
3
Department of Biochemistry, Cellular and Molecular Biology
and the Genome Science and Technology Program,
University of Tennessee, Knoxville, TN 37996-0840, USA
123
J Plant Growth Regul (2015) 34:809–827
DOI 10.1007/s00344-015-9522-9

the criteria for a hormone, making it the first gaseous
hormone to be recognized. Because ethylene can diffuse to
and affect surrounding plants, it also has characteristics of
a pheromone. Unfortunately, ground-breaking discoveries
like this in plants are often overlooked. In 1998, the Nobel
Prize in Physiology or Medicine was given for the finding
that nitric oxide is a signaling molecule. The Nobel com-
mittee claimed that this represented the first discovery of a
gas that can act as a biological signaling molecule. As
documented in this review and noted in a letter to Science
by prominent plant biologist Hans Kende (Kende 1998),
the Nobel committee was wrong about nitric oxide being
the first gaseous signaling molecule discovered, because
plant biologists were on the path to showing that ethylene
gas acts as a signaling molecule over a century before this
award was given.
The discovery of ethylene having effects on plants can
be traced back to observations on the effects of smoke and
illuminating gas on plants in the 1800s. Illuminating gas
was first used in the late 1700s (Sedgwick and Schneider
1911) and by the late 1800s it was used in many commu-
nities around the world for lighting in homes and busi-
nesses, as well as for streetlights. This gas was initially
manufactured by the destructive distillation of bituminous
coal and was sometimes referred to as coal gas. Later,
carbureted water gas was also used. In both cases, illumi-
nating gas companies produced the gas and then distributed
it via underground pipes. The first published case of illu-
minating gas affecting plants was in 1858 by George
Fahnestock who noted that illuminating gas leaking from
pipes caused damage to plants in a greenhouse in
Philadelphia (Fahnestock 1858). This observation was
substantiated by numerous reports throughout the late
1800s and into the 1900s that illuminating gas and smoke
affected various plant species (Giardin 1864; Kny 1871;
Spa
¨th and Meyer 1873; Lackner 1873; Molisch 1884;
Eulenberg 1876; Deuber 1932; Wehmer 1900; Sorauer
1916; Stone 1907; Wallace 1926; Woffenden and Priestley
1924; Priestley 1922). Damage to plants from leaking
illuminating gas lines even resulted in lawsuits against gas
companies (Stiness 1909; Thompson 1878; Frey 1918; Hun
1897). This clearly was of interest to both growers and
producers of illuminating gas, as this issue was reported in
magazines such as The Florist’s Review,American Gar-
dening,Gas World, and American Gas Light Journal.
Because of the widespread use of illuminating gas, there
was broad interest in determining the cause of plant death
and damage near illuminating gas lines.
In 1896, a plant physiologist at the Botanical Institute of
St. Petersburg University, Dimitry Neljubow, identified
ethylene as the active component in illuminating gas that
affects plants (Neljubow 1901). He showed that ethylene
altered the growth of etiolated pea seedlings and caused
them to have shorter, thicker epicotyls that displayed dia-
geotropism. This was the first time that ethylene was shown
to have a biological effect. This response became known as
the etiolated seedling triple response (Knight and Crocker
1913) and was used as a sensitive bioassay for ethylene
(Crocker and others 1913). Neljubow’s results were later
verified and expanded by several scientists (Crocker and
Knight 1908; Knight and Crocker 1913; Harvey 1915;
Harvey and Rose 1915; Knight and others 1910a).
It took several decades after Neljubow’s discovery
before it was shown that plants also could biosynthesize
ethylene. The first indication that plants biosynthesize
ethylene came from research by Herbert Cousins who
reported that fungus-damaged oranges produced a gas that
accelerated banana fruit ripening (Cousins 1910). With
hindsight, it is now unclear whether the ethylene was pro-
duced by the damaged oranges or by the fungus infecting
them. In 1934, Richard Gane used a quantitative chemical
method to show that apples release ethylene, and was thus,
the first person to chemically show that plants biosynthesize
ethylene (Gane 1934). A year later, he provided evidence
that other fruits also produce ethylene (Gane 1935). Even
though not initially accepted by all scientists (Michener
1938; Went and Thimann 1937; Nord 1936), these early
studies demonstrated that ethylene has the two key char-
acteristics of a hormone and led to the suggestion in 1935 by
William Crocker, Alfred Hitchcock, and Percy Zimmer-
man, at the Boyce Thompson Institute, that ethylene is a
plant hormone (Crocker and others 1935). This conclusion
has now been substantiated by numerous studies making it
the first gaseous hormone to be discovered.
Research on ethylene was initially hindered by two
factors. One was the above-mentioned reticence of some
researchers to accept ethylene as a signaling molecule. The
other was the difficulty of accurately measuring ethylene.
Until the 1950s, a variety of bioassays and analytical
chemical techniques were used. However, they all had the
drawback of low sensitivity and difficulty of use. For
instance, it required the accumulation of gas from 60
pounds of apples over a period of 4 weeks for Gane to
detect ethylene in his 1934 study. This changed with the
invention of gas chromatography and its application to
ethylene research (Burg and Stolwijk 1959; Huelin and
Kennett 1959), which was easier than prior methods, and
the introduction of flame ionization detectors (Meigh 1959)
made sensitivity much higher than other methods. This
likely led to an increase in the number of papers published
about ethylene as a plant hormone (Fig. 1). During the
1960s and 1970s, the biochemical pathway for ethylene
biosynthesis in plants was determined and ethylene-binding
sites were identified. However, it was the advent of
molecular biology techniques and the development of
Arabidopsis thaliana as a genetic model system for plants
810 J Plant Growth Regul (2015) 34:809–827
123

that led to major advances in our understanding of ethylene
signaling and the control of biosynthesis. Arabidopsis has
many advantages including a small size, small compact
genome, rapid generation time, and ease of manipulation
(Meyerowitz and Pruitt 1985). Using the sensitive bioassay
originally developed with pea seedlings (Crocker and
others 1913), it was found that gassing dark-grown Ara-
bidopsis seedlings with ethylene leads to a triple response
phenotype characterized by a short root and hypocotyl,
thicker hypocotyl, and exaggerated apical hook (Fig. 2).
This rapid and simple assay provided a powerful method to
screen for and identify signaling and biosynthesis mutants
(Bleecker and others 1988; Guzma
´n and Ecker 1990). The
first paper using this approach was published in 1988 and
identified an ethylene-insensitive mutant that was later
shown to be an ethylene receptor mutant (Bleecker and
others 1988). This heralded a new era of plant molecular
biology that correlates with a large increase in the number
of papers in ethylene biology published in the last two
decades (Fig. 1).
This rapid increase in information has led to more
refined models for ethylene biosynthesis and signaling that
continue to be honed. This article reviews the history of
research on this plant hormone, highlighting key early
discoveries that have led to our current understanding
regarding the biosynthesis and perception of this gaseous
molecule by plants.
Early Studies on the Physiological Effects
of Ethylene on Plants
After Neljubow’s report in 1901, a large number of studies
documented various responses to ethylene (Abeles and
others 1992; Reviewed by: Mattoo and Suttle 1991).
Initially, there was a great interest in determining the range
of effects and the sensitivity of various plant species so that
greenhouse managers could detect illuminating gas leaks or
the accumulation of ethylene from other sources (Doubt
1917; Knight and others 1910b). There was also interest in
determining whether or not ethylene was the only active
component of the gas that was affecting plants (Knight and
others 1910a; Richards and MacDougal 1904; Crocker and
others 1932). As it became clear that ethylene was the main
causative agent, researchers began to focus on further
characterizing the effects of ethylene and trying to under-
stand the underlying mechanism for its actions. From these
studies, we now know that ethylene affects many aspects of
plant growth, development, and responses to the environ-
ment including fruit ripening, leaf and flower senescence
and abscission, adventitious root formation, seed germi-
nation, interactions with microbes, and responses to stress,
to name a few. Here we will provide an overview of some
of these early discoveries. Because of space constraints, we
will only briefly cover early research about the effects of
ethylene on selected responses. For more information,
readers are referred to the book Ethylene in Plant Biology
by Abeles and others (1992) who have exhaustively sum-
marized ethylene research up to the early 1990s.
Fig. 1 Number of papers on ethylene as a plant hormone from 1908
through 2013. Based on an online database search for the term
ethylene in the Web of Science (http://webofknowledge.com/)on5
January 2014. The search was limited to those citations within the
research areas of plant sciences and agriculture
Fig. 2 Photo of wild-type seedlings and one ethylene-insensitive
mutant of Arabidopsis thaliana grown in an atmosphere containing
ethylene. From the cover of Science Vol. 241, No. 4869 (Aug. 26,
1988). Photo by Kurt Stepnitz, Michigan State University, E. Lansing,
MI 48824. Reprinted with permission of the AAAS and the
photographer
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Abscission and Senescence
As mentioned earlier, historically it was the observation
that illuminating gas caused yellowing and death of leaves
that eventually led to the discovery of ethylene as a plant
hormone. It is now clear that ethylene stimulates floral
organ and leaf senescence and abscission in many plant
species. The first paper documenting an effect of ethylene
on floral organs was by William Crocker and Lee Knight
(Crocker and Knight 1908), who reported that ethylene
prevented young carnation flowers from opening and
caused open flowers to close and become discolored and
withered. Several early papers also documented that ethy-
lene-stimulated senescence and abscission of leaves (Doubt
1917; Wehmer 1917). Even though ethylene levels rise in
aging leaves (Aharoni and others 1979), this is not required
for senescence (Mayak and Halevy 1972; McAfee and
Morgan 1971) and some plants have very little response to
ethylene for one or another of these processes (Woltering
1987). Research starting in the 1960s focused on the bio-
chemical and cellular changes that lead to senescence and
abscission (Abeles and Holm 1967; Holm and Abeles
1967; Horton and Osborne 1967; Jackson and others 1972),
both of which continue to be topics of interest today.
Growth
The modulation of seedling growth by ethylene also
became a topic of study. In addition to examining the
effects of long-term ethylene treatment, several laborato-
ries examined the timing of growth inhibition by ethylene.
This was first done by P.A. van der Laan who used manual
time-lapse photography to determine the latent time for
growth inhibition of dark-grown pea and oat seedlings in
response to ethylene (van der Laan 1934). Subsequent
studies by several laboratories using diverse techniques to
examine the growth inhibition kinetics when ethylene was
applied generally found latent times of approximately
10 min for ethylene to cause a measurable effect (Warner
and Leopold 1971; Rauser and Horton 1975; Goeschl and
Kays 1975; Jackson 1983; Burg 1973). Some also inves-
tigated the time for growth recovery after removal of
ethylene and found 20-min latent times (Warner and Leo-
pold 1971; Burg 1973; Jackson 1983). The timing of these
effects was correlated with cellular and biochemical
changes caused by ethylene, such as cell division and
expansion, DNA and RNA synthesis, and the reorientation
of cellulose microfibrils (Burg 1973).
Application of ethylene can also promote growth in
various organs (Ku and others 1970; Walter and Osborne
1979; Konings and Jackson 1979). Of particular interest is
that ethylene stimulates the growth of aquatic plants, pre-
dominantly by increasing cell expansion (Musgrave and
others 1972; Musgrave and Walters 1974a,b). A related
response is ethylene-stimulated leaf epinasty which is
observed in some species including aquatic plants (Crocker
and others 1932; Woltering 1987; Doubt 1917). Probably
the best studied aquatic plant is deep-water rice, where
submergence causes a buildup of ethylene that stimulates
internode expansion (Me
´traux and Kende 1983). The latent
time for promotion of growth in deep-water rice by ethy-
lene is slower than the times noted above for growth
inhibition of eudicots (Satler and Kende 1985; Rose-John
and Kende 1985). The molecular mechanism for this dif-
ference in response and timing is not yet completely
understood but likely involves differential regulation of
gibberellic acid by ethylene in different plant species (Kim
and others 2012). With the advent of molecular tools and
automated time-lapse imaging, a great deal of information
has recently been discovered concerning the complexity of
ethylene signaling and cross-talk with other hormones
(Binder and others 2004a,b,2006; Vandenbussche and
others 2010).
Fruit Ripening
Due to its obvious economic importance, fruit ripening is
another area that received early interest. There are pre-
modern examples of ethylene being used to stimulate fruit
ripening including the scraping of figs mentioned in
Egyptian texts (Galil 1968), which we now know causes
wound-induced production of ethylene. Additionally, the
ancient Chinese used smoke, which we now know contains
ethylene, to ripen pears (Miller 1947). In many ways, sci-
entific research on the effects of ethylene on fruit ripening
paralleled the research on the regulation of growth. In the
early 1900s, kerosene stoves were in widespread use. When
citrus packers and shippers switched to other heating
methods, they noted that the citrus fruits failed to develop
good color (Reviewed in: Chace 1934). Two USDA sci-
entists, Arthur Sievers and Rodney True, showed that heat
was not the cause of improved fruit color (Sievers and True
1912). A few years later, Frank Denny, at the Boyce
Thompson Institute, concluded that ethylene was the active
component from kerosene heaters that stimulated color
change in citrus fruit and soon afterwards took out a patent
for the use of ethylene to improve citrus fruit color (Denny
1923,1924). Since these early observations, many, but not
all, fruits have been shown to be sensitive to ethylene. As
noted above, apples were the first fruit to be shown to
produce ethylene, and they too respond to ethylene with
accelerated ripening (Kidd and West 1933).
The above summary highlights some of the diverse
effects of ethylene that were identified early in the history
of this field. Because of these effects, there is intense sci-
entific and commercial interest in understanding how
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plants respond to ethylene. This is especially important
because ethylene has long been recognized as an air pol-
lutant that can adversely affect plants (Scott and others
1957; Crocker 1913). A major source of this pollution is
exhaust from automobiles and manufacturing plants. In
fact, average daytime ethylene levels around urban areas
have been found to be well above the threshold for
affecting plants (Abeles and Heggestad 1973; Abeles and
others 1971; Gordon and others 1968).
More recent studies have made substantial progress in
unraveling the mechanisms of ethylene biosynthesis and
perception in plants. The remainder of this article will
highlight key discoveries leading to our current under-
standing of these processes.
Ethylene Biosynthesis and Regulation in Plants
Pathway for Biosynthesis
After recognizing that ethylene is a plant hormone that
impacts diverse aspects of plant growth and development,
researchers became interested in how this simple two-car-
bon molecule is synthesized in plants and how this
biosynthesis is regulated. One direction of research was to
identify the metabolic intermediates in this process. Some
of the proposed precursor molecules of ethylene were
linolenic acid, ethanol, fumaric acid, acetic acid, glucose,
sucrose, organic acids, propanal, b-alanine, acrylic acid,
and methionine (Yang and Hoffman 1984). To investigate
these candidates, researchers conducted in vitro studies
using these and other molecules to determine which, if any,
of the proposed intermediates could be converted to ethy-
lene. For instance, Morris Lieberman and L.W. Mapson
showed that ethylene can be formed from linolenic acid in
the presence of a metal catalyst (Lieberman and Mapson
1964). To decipher the role of free radicals in this process,
methionine was used as a free radical scavenger. Surpris-
ingly, more ethylene was produced by the addition of
methionine, which identified methionine as a possible
precursor for the production of ethylene by plants (Fig. 3).
In the same year, Frederick Abeles and Bernard Rubin-
stein, at the US Army Biological Laboratories, stated in a
government report that addition of flavin mononucleotide
to pea extracts led to ethylene production (Abeles and
Rubinstein 1964). Soon after, Shang Fa Yang and col-
leagues at the University of California-Davis showed that
methionine was indeed the substrate for this reaction (Yang
and others 1966). In parallel with the above in vitro studies,
in vivo studies were also pointing to methionine as the
ethylene precursor molecule (Lieberman and Kunishi
1965). Using isotope-labeled methionine, it was shown that
apple fruits produce ethylene from the C3 and C4 carbons
of methionine (Lieberman and others 1966; Burg and
Clagett 1967; Baur and others 1971).
The next intriguing puzzle was to determine whether
methionine was converted to ethylene directly or via
intermediates. Stanley Burg was the first to show that
S-adenosylmethionine (SAM) was an intermediate during
the synthesis of ethylene (Burg 1973). Later studies with
apple tissues confirmed that SAM was formed from
methionine in the course of ethylene biosynthesis (Adams
and Yang 1977). The Yang group also noted that apple
tissue given
14
C-SAM under anaerobic conditions accu-
mulated another compound (Adams and Yang 1977). This
discovery correlated with earlier findings showing that
ethylene production was inhibited in apple tissues main-
tained in an anaerobic environment; this inhibition was
reversed upon re-exposure to aerobic conditions. These
observations resulted in several laboratory groups
attempting to identify this metabolic intermediate between
SAM and ethylene, which was eventually identified as
1-aminocyclopropane-1-carboxylic acid (ACC) (Adams
and Yang 1979). In this study, Adams and Yang also
discovered that the conversion of ACC to ethylene is
oxygen dependent. In the same year, Lu
¨rssen and col-
leagues in Germany reported that ACC is an ethylene
precursor that induced the biosynthesis of ethylene in
different plant tissues (Lu
¨rssen and others 1979a,b).
These studies led to our current understanding that
Fig. 3 Biochemical pathway for ethylene biosynthesis in plants. In
plants, ethylene is biosynthesized from the amino acid methionine.
Three enzymes are involved: SAM synthetase converts methionine to
SAM, ACC synthase converts SAM to ACC, and ACC oxidase
converts ACC to ethylene. SAM synthetase and ACC synthase are
part of the Yang or methionine cycle. The reaction catalyzed by ACC
synthase is the rate-limiting reaction for ethylene biosynthesis
J Plant Growth Regul (2015) 34:809–827 813
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methionine is converted sequentially to SAM, ACC, and
finally to ethylene (Fig. 3).
Even though this is a simple biosynthetic pathway, a
major challenge was to identify the enzymes involved in
this process. We now know that these enzymes are SAM
synthetase (which converts methionine to SAM), ACC
synthase (which converts SAM to ACC), and ACC oxidase
(which converts ACC to ethylene) (Fig. 3). Both SAM
synthetase and ACC synthase are part of what is called the
Yang or methionine cycle. This cycle is part of a salvage
pathway for the methylthio group (Adams and Yang 1979;
Miyazaki and Yang 1987; Yang and Hoffman 1984; Baur
and Yang 1972). A bifurcation between the Yang cycle and
ethylene production occurs at ACC synthase, which pro-
duces 50-methylthioadenosine (which enters the cycle) and
ACC (which is converted to ethylene by ACC oxidase). In
early studies, ACC oxidase was referred to as ethylene-
forming enzyme. Here we will focus on the steps leading to
the biosynthesis of ethylene.
Various studies identified SAM as a major methyl group
donor in transmethylation reactions of proteins, nucleic
acids, fatty acids, and polysaccharides (Tabor and Tabor
1984). The formation of SAM is a major step in methionine
metabolism (Giovanelli and others 1985). Early studies
using baker’s yeast showed that SAM synthetase could
convert methionine to SAM, releasing pyrophosphate and
phosphate in the process (Chou and Talalay 1972). Later,
several laboratories successfully purified, cloned, and
characterized SAM synthetase from several sources (Lar-
sen and Woodson 1991; Peleman and others 1989a,b;
Tabor and Tabor 1984). Cross-species comparisons showed
that this enzyme was highly conserved (Horikawa and
others 1989,1990; Horikawa and Tsukada 1992).
In 1979, research from the Yang and Kende laboratories
using tomato pericarp tissues identified ACC synthase as
part of the ethylene biosynthesis pathway (Boller and
others 1979; Adams and Yang 1979). This turned out to
belong to the pyridoxal-50-phosphate-dependent enzyme
family and was the rate-limiting step of ethylene biosyn-
thesis (Argueso and others 2007; Alexander and others
1994). When it was obvious that ACC synthase played a
vital role in ethylene biosynthesis, there was an active
competition between different laboratories to purify,
characterize, and clone the enzyme in order to understand
how it was regulated. However, purification was chal-
lenging due to its low abundance and instability. In the
meantime, other sources of the ACC synthase were being
identified and it was discovered that wounding and treat-
ment with LiCl increased enzyme levels (Ramalingam and
others 1985; Nakagawa and others 1988; Boller 1984).
Eventually, several laboratories did manage to purify,
sequence, and characterize ACC synthase from several
plant sources (Dong and others 1991b,1992; Nakajima and
Imaseki 1986; Yip and others 1991; Van der Straeten and
others 1989; Sato and others 1991; Bleecker and others
1986). Five independent research groups cloned ACC
synthase around this time period as well (Sato and Theol-
ogis 1989; Olson and others 1991; Nakajima and others
1990; Nakagawa and others 1991; Dong and others 1991a;
Van der Straeten and others 1990). Due to the high vari-
ability in the predicted amino acid sequences obtained by
these groups, it was discovered that ACC synthase is in a
multigene family. Structural studies have contributed more
insights as to how ACC synthase functions (Yip and others
1990; White and others 1994; Tarun and others 1998;
Tarun and Theologis 1998; Capitani and others 1999).
The final step in ethylene biosynthesis is catalyzed by
ACC oxidase. The isolation and characterization of ACC
oxidase was very challenging, in part because the enzyme
was widely believed to be membrane associated (John
1983; Abeles and others 1992). This proved to be wrong
(John and others 1985), however, and led to delays in this
area of research. One advance came with the identification
of a ripening-related cDNA from tomato called pTOM13,a
gene that is required in ethylene biosynthesis and encodes
for ACC oxidase (Davies and Grierson 1989; Hamilton and
others 1990). The deduced amino acid sequence of
pTOM13 has homology to flavanone-3-hydroxylase and
other hydroxylases from different plant sources (Hamilton
and others 1991). This information allowed others to
develop methods to extract and characterize ACC oxidase
enzymatic activity in vitro (Ververidis and John 1991).
Although significant progress has been made on ACC
oxidase, many questions still remain including a good
understanding of its subcellular localization, reaction
mechanism, and regulation.
Regulation of Ethylene Biosynthesis
Ethylene is produced throughout the plant but levels are
generally low. The level of ethylene increases in response
to both developmental cues and environmental signals
(Reviewed by: De Paepe and Van Der Straeten 2005; Chae
and Kieber 2005). Probably the best known case of this is
the accumulation of ethylene in many fruits during ripening
during which the biosynthesis of ethylene increases dra-
matically; however, other events and cues also stimulate
ethylene biosynthesis including many biotic and abiotic
stressors. Because of this, ethylene is often called the
‘‘stress’’ hormone. Under some conditions, ACC oxidase is
a site of regulation. However, the major site for the regu-
lation of ethylene biosynthesis occurs at ACC synthase. A
major focus of research has been to determine the mech-
anism(s) for the regulation of ACC synthase.
One of the first screens for ethylene mutants in Ara-
bidopsis identified eto1 which has a constitutive ethylene
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response phenotype (Guzma
´n and Ecker 1990). Subsequent
studies showed that eto1, along with two other mutants
with similar traits, eto2 and eto3, have higher ACC syn-
thase activities (Woeste and others 1999; Vogel and others
1998). Subsequently, several laboratories showed a corre-
lation between ACC synthase protein levels and ethylene
levels (Chae and others 2003; Joo and others 2008;
Christians and others 2009). These studies pointed to a
central role for ACC synthase, of which there are three
types. Type 1 ACC synthases contain target motifs for
mitogen-activated protein kinases (MAPKs) and calcium-
dependent protein kinases (CDPKs), whereas type 2 ACC
synthases only contain target motifs for CDPKs, and type 3
enzymes carry neither motif (Liu and Zhang 2004;
Kamiyoshihara and others 2010). Stress-activated MAPKs
cause increased phosphorylation of specific ACC synthase
isoforms resulting in protein stabilization and increased
ethylene production (Han and others 2010; Joo and others
2008; Liu and Zhang 2004; Sebastia
`and others 2004). Not
surprisingly, dephosphorylation also has an important role
(Skottke and others 2011). Research from several labora-
tories has now linked the phosphorylation state of specific
ACC synthase isoforms to protein stability. In some cases,
the phosphorylation state was shown to be regulated by
environmental signals and involves ubiquitin-dependent
proteolysis (Skottke and others 2011; Christians and others
2009; Tan and Xue 2014; Chae and others 2003; Wang and
others 2004; Yoon and Kieber 2013; Liu and Zhang 2004;
Han and others 2010; Lyzenga and others 2012; Prasad and
others 2010; Prasad and Stone 2010). Although more
research is needed to fully understand how ethylene levels
are regulated, these studies provide important mechanistic
insights into how environmental signals and developmental
cues affect ethylene biosynthesis.
Ethylene Signal Transduction
Early Studies on Ethylene Signaling
In addition to studies on ethylene biosynthesis, there were
investigations into how ethylene is perceived and how the
signal is transduced in the cell. Initial studies on ethylene
signaling were focused on two areas: the characterization
of ethylene-binding sites and the analysis of genes/proteins
that are expressed in response to ethylene. Early on, Burg
and Burg (1967) astutely proposed that the binding of
ethylene to a protein would require a transition metal.
Additional clues regarding the properties and characteris-
tics of ethylene-binding sites came from the identification
of ethylene response inhibitors, such as 2,5-norbornadiene
(a competitive inhibitor, Sisler and Pian 1973) and silver
nitrate (a noncompetitive inhibitor, Beyer 1976; Sisler
1982). Using various plant tissues and extracts, two high
affinity and saturable binding sites (with a short and long
half-life of ethylene binding, respectively) were detected
(Sanders and others 1991; Sisler 1991). However, efforts to
purify these ethylene-binding proteins were unsuccessful
for a variety of reasons, including the difficulty of main-
taining the binding activity in a detergent environment
(Sisler 1980; Thomas and others 1984,1985; Smith and
others 1987; Williams and others 1987). Based on the
amount of ethylene bound to tobacco leaves, Edward Sisler
(1979) calculated that there are approximately 4000 bind-
ing sites per cell. Cell fractionation/autoradiography data
suggested that binding likely occurs at the ER membrane
(Evans and others 1982a,b). Despite these studies, there
was no evidence to link the observed binding with ethylene
signaling, and thus it was unclear whether the partially
purified binding sites represented an actual ethylene
receptor.
At the opposite end of the pathway, ethylene-induced
proteins were identified, such as endochitinase (Boller and
others 1983), and with the availability of molecular biology
tools in the 1980s, ethylene-responsive genes began to be
studied. Many of the genes were induced in response to
pathogen attack and included genes such as basic chitinases,
defensins, and b-1-3-glucanases. Analysis of the promoter
regions of such genes led to the identification of a cis-acting
element called the GCC box, which is necessary and suf-
ficient for control by ethylene (for example, Eyal and others
1993; Hart and others 1993). A search for trans-acting
factors that bind to the GCC box led to the isolation of
ethylene-responsive element-binding-proteins (EREBPs) in
tobacco (Ohme-Takagi and Shinshi 1995). These EREBPs
are members of the family of AP2-like DNA-binding
transcription factors. The big remaining question was,
however, what were the identities of the proteins that sig-
naled the presence of ethylene leading to the expression or
activation of the EREBPs?
Genetic Dissection of the Central Ethylene Signaling
Pathway in Arabidopsis
Major breakthroughs in our understanding of the ethylene
signaling pathway took place in the late 1980s and 1990s
with the emergence of Arabidopsis as a genetic model
system for plants and have led to models for ethylene
signaling (Fig. 4). The use of Arabidopsis allowed for the
genetic dissection of numerous signaling pathways,
developmental processes, and metabolic pathways (Som-
erville and Koorneef 2002; Koorneef and Meinke 2010).
The genetic dissection approach, involving the isolation of
mutants specific to the process of interest followed by
cloning of the corresponding genes, was impractical with
the crop and horticultural models used at that time
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(Meyerowitz and Pruitt 1985). As a result, much of what is
known regarding the ethylene signaling pathway has come
from pioneering studies in Arabidopsis. There were several
‘‘firsts’’ in the genetic dissection of ethylene signaling in
Arabidopsis, and thus advances in ethylene signaling were
at the forefront of Arabidopsis research for a number of
years.
As a graduate student in Hans Kende’s laboratory at
Michigan State University, Tony Bleecker devised a simple
but elegant genetic screen for isolating Arabidopsis ethy-
lene response (etr) mutants. The screen was based on the
triple response of etiolated pea seedlings that Neljubow had
observed 100 years earlier. Bleecker’s screen yielded a
dominant ethylene-insensitive mutant, etr1-1, the first
Fig. 4 Model of the ethylene signal transduction pathway in
Arabidopsis.aEthylene signaling pathway, circa 1998, based entirely
on mutants and their genetic epistatic relationships. The current view
of the ethylene signaling pathway without ethylene band with
ethylene c. Ethylene (white circle) is perceived by a family of five
receptors represented here by the ETR1 dimer. Copper (orange oval),
transported by RAN1, serves as a cofactor for ethylene binding and is
required for proper biogenesis of the receptors. Interaction of RTE1
with ETR1 (but not the other ethylene receptors) is believed to
promote the signaling conformation of ETR1. In b, in the absence of
ethylene binding, ETR1 activates the associated CTR1 protein kinase,
which phosphorylates the C-terminus (C-term) of EIN2, potentially
leading EIN2 to be targeted for degradation by two F-box proteins
(ETP1 and ETP2) via the 26S proteasome. Consequently, two F-box
proteins, EBF1 and EBF2 (EBF1/2), target the key transcription
factors EIN3 and EIL1 for degradation by the 26S proteasome
preventing downstream ethylene signaling from occurring. In c, when
ethylene is bound, the receptors no longer activate CTR1, allowing
the unphosphorylated EIN2 C-term to be cleaved (by an unknown
protease) and translocated into the nucleus where it plays a role in
activating downstream responses. The EBF1/2 F-box proteins are
somehow repressed, involving the general exoribonuclease XRN4
(also called EIN5/EIN7), and thus the EIN3/EIL1 proteins are now
stabilized. EIN3/EIL1 homodimers bind to the PERE element of
target genes, such as ERF1, activating their expression. In addition,
the expressed ERF1 transcription factor binds to the GCC box in the
promoters of additional ethylene-responsive genes leading to ethylene
responses
816 J Plant Growth Regul (2015) 34:809–827
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ethylene response mutant in Arabidopsis (Bleecker and
others 1988). The now iconic image of Bleecker’s tall,
ethylene-insensitive etr1-1mutant surrounded by short,
responsive wild-type Arabidopsis seedlings in a Petri dish
was featured on the cover of Science in 1988 (Fig. 2). The
mutant was insensitive to ethylene not only at the seedling
stage, but also in all other aspects measured in the mature
plant. Because the triple response assay is highly specific to
ethylene, as well as relatively simple and quick to carry
out, it has remained the definitive assay for measuring
ethylene response.
As a postdoc, Bleecker sought to clone the ETR1 gene by
chromosome walking in Elliot Meyerowitz’s laboratory at
Caltech. The chromosome walking approach was made
possible by the genetic map of DNA markers for Arabidopsis
produced by the Meyerowitz laboratory through efforts led
by then graduate student, Caren Chang (Chang and others
1988). With such a map, one could clone the gene for any
mapped phenotype via chromosome walking from the
nearest physically linked marker. This was a very powerful
approach, as it required no prior knowledge of a gene’s
properties or its encoded protein. In the early 1990s, Chang
(by then a postdoc in the Meyerowitz lab) together with
Bleecker (by then an assistant professor at University of
Wisconsin at Madison) used chromosome walking to iden-
tify the ETR1 gene (Chang and others 1993). ETR1 was
among the first genes to be isolated by map-based cloning in
Arabidopsis and the first plant hormone receptor gene to be
cloned in any plant. The ETR1 sequence showed similarity to
the two-component family of histidine kinase receptors
widely found in prokaryotes (Chang and others 1993); ETR1
was the first two-component homolog identified in a higher
eukaryote (Koshland 1993). Because of the similarity to this
prokaryotic receptor family, it was speculated that ETR1
might encode an ethylene receptor. Subsequently, Eric
Schaller, who at that time was a postdoc in Bleecker’s lab,
demonstrated that the ETR1 protein is a homo-dimer capable
of binding ethylene and that binding was disrupted by the
etr1-1mutation (Schaller and Bleecker 1995; Schaller and
others 1995). This was a major breakthrough, as ethylene
binding by ETR1 was directly linked to ethylene responses
through the etr1 mutant phenotype.
Soon after the isolation of the etr1-1mutant, Joseph
Ecker’s laboratory (currently at The Salk Institute) isolated
many additional mutants that have been critical in eluci-
dating the ethylene signaling pathway. Ecker’s laboratory
screened for two different kinds of mutants based on the
triple response assay: ethylene-insensitive (ein) mutants
that lack the triple response phenotype and constitutive
triple response (ctr) mutants that display the triple response
even in the absence of ethylene treatment (Guzma
´n and
Ecker 1990; Kieber and others 1993; Roman and others
1995). In addition to new dominant alleles of etr1 (ein1),
they isolated mutants of ein2,ein3,ein4,ein5,ein6,ein7,
and ctr1 (all recessive).
On the basis of genetic epistatic relationships, the Ecker
laboratory could place the ethylene response mutants into a
pathway that suggested the order of action of these com-
ponents (for example, Roman and others 1995; Kieber and
others 1993); the recessive ein mutants acted at or down-
stream of ctr1, which acted at or downstream of the
dominant mutants (Fig. 4a). Many molecular details have
since been added to this framework (for example, see
reviews by Merchante and others 2013; Shakeel and others
2013) (Fig. 4b, c), based on cloning of the corresponding
genes followed by functional studies ranging from genetic
analyses of mutants to cellular and biochemical analyses of
the encoded proteins.
Importantly, the ethylene signaling pathway in Ara-
bidopsis is essentially conserved in other plant species (for
example, Wilkinson and others 1997; Klee 2004). For
example, cloning of the Arabidopsis ETR1 gene paved the
way for the isolation of ethylene receptor genes from
numerous other plants on the basis of sequence similarity
(for example, Sato-Nara and others 1999; Mita and others
1998; Terajima and others 2001; Ma and Wang 2003;
Vriezen and others 1997; Shibuya and others 2002). It
turned out that the gene responsible for the semi-dominant
Never-ripe tomato mutant, first identified in 1956 (Rick and
Butler 1956), was an ethylene receptor homolog (Wilkin-
son and others 1995).
The following sections of this article cover some high-
lights regarding the central ethylene signaling pathway and
how the signaling mechanisms were discovered.
The Ethylene Receptors
Soon after the cloning of ETR1, it was discovered that
ETR1 belongs to a family of homologous ethylene recep-
tors in plants. Arabidopsis has five members (Fig. 4a),
whereas tomato has six (Klee 2004). The most closely
related ETR1 homolog, ETHYLENE RESPONSE SENSOR
(ERS1), was discovered in the Meyerowitz laboratory by
screening an Arabidopsis DNA library for clones that
hybridized with an ETR1 probe (Hua and others 1995). In
the absence of a mutant phenotype for ERS1, a mutation
analogous to the dominant mutation of etr1-4was intro-
duced into an ERS1 transgene, creating a dominant mutant
version of ERS1 that conferred ethylene insensitivity sim-
ilar to that of ETR1. It was confirmed a decade later that
ERS1 and the other Arabidopsis ethylene receptors (ETR2,
EIN4, and ERS2) bind ethylene (O’Malley and others
2005).
The ETR2 ethylene receptor gene was discovered
serendipitously in the Meyerowitz laboratory in a chro-
mosome walk to clone an unrelated gene (Sakai and others
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1998). Upon realizing the sequence similarity between an
unidentified gene within the walk and the ETR1 gene,
Sakai and others (1998) undertook experiments that
revealed that the gene corresponded to an unpublished,
dominant ethylene-insensitive mutant, etr2-1, which had
been isolated by Bleecker. Using ETR2 as a probe, the
Meyerowitz laboratory isolated the EIN4 and ERS2 ethy-
lene receptor genes via hybridization screening of an
Arabidopsis DNA library (Hua and others 1998). EIN4
corresponded to the dominant ethylene-insensitive ein4-1
mutant isolated in the Ecker laboratory (Roman and others
1995). For ERS2, a dominant ethylene-insensitive pheno-
type was obtained using ERS2 transgenes carrying domi-
nant mutations analogous to that of etr2-1and ein4-1(Hua
and others 1998).
Identification of the ethylene receptors was followed by
more than a decade of intense characterization. As a result,
we know that all of the receptors have a hydrophobic
N-terminal domain comprising the ethylene-binding
domain (Schaller and Bleecker 1995; Rodriguez and others
1999; O’Malley and others 2005; Hall and others 1999),
followed by a cytosolic GAF domain, which plays a role in
protein–protein interactions between the receptors (Xie and
others 2006; Grefen and others 2008; Gao and others
2008). Following, the GAF domain is a histidine kinase
domain, and in some of the ethylene receptors, a C-ter-
minal receiver domain. (Receptors named ERS lack a
receiver domain.) The ethylene receptors are disulfide-
linked homodimers that exist in clusters (Schaller and
others 1995; Hall and others 2000; Takahashi and others
2002; Gao and others 2008; Chen and others 2010) local-
ized at the ER membrane (Chen and others 2002) and the
Golgi apparatus membrane (Dong and others 2008). It is
thought that one ethylene molecule is bound per receptor
dimer (Schaller and others 1995; Rodriguez and others
1999).
The Arabidopsis ethylene receptors fall into two sub-
families. Subfamily I receptors (ETR1 and ERS1) are
predicted to contain three N-terminal transmembrane
domains and histidine autokinase activity (ERS1 also has
serine/threonine kinase activity) (Gamble and others 1998;
Moussatche and Klee 2004; Chen and others 2009). Sub-
family II receptors (ETR2, ERS2, and EIN4) are predicted
to have four N-terminal transmembrane domains and a less
conserved histidine kinase domain that displays serine/
threonine kinase activity (Gamble and others 1998;
Moussatche and Klee 2004; Chen and others 2009). There
is debate regarding the extent to which the kinase domain
plays a role in ethylene signaling (Gamble and others 2002;
Wang and others 2003; Hall and Bleecker 2003; Qu and
Schaller 2004; Hall and others 2012). Autophosphorylation
of the receiver domain is believed to play a minor role in
ethylene response (Wang and others 2003) but the receiver
domain appears to play a role in recovery following
exposure to ethylene (Binder and others 2004b; Kim and
others 2011). The exact signaling mechanism of the
receptors is still unknown.
Although the mechanisms of ethylene receptor signaling
are not entirely understood, genetic analyses have revealed
that the ethylene receptors are negative regulators of
ethylene signaling. This conclusion is based on the isola-
tion of recessive loss-of-function mutations for the ethy-
lene receptor genes in Arabidopsis by Hua and Meyerowitz
(1998) and later Qu and others (2007). These studies
showed that the receptors have some functional redun-
dancy, which explains why no recessive mutations and
only dominant gain-of-function mutations were obtained
from genetic screens. When Hua and Meyerowitz (1998)
created a quadruple mutant of four of the receptor genes,
they found that the mutant confers constitutive ethylene
responses. Therefore, by deduction, functional ethylene
receptors must repress ethylene responses (in the absence
of ethylene binding), whereas ethylene binding inactivates
the receptors resulting in ethylene responses. This is known
as the inverse agonist model (Hall and others 1999).
Soon after demonstrating that ETR1 binds ethylene, the
Bleecker laboratory showed that the binding requires a
copper cofactor (Rodriguez and others 1999), confirming
the 1967 prediction that a transition metal would be
required for ethylene binding. Around the same time,
RESPONSIVE-TO-ANTAGONIST1 (RAN1), a copper-
transporting P-type ATPase homologous to the human
Menkes/Wilson proteins, was found to be required for the
biogenesis of the ethylene receptors (Hirayama and others
1999; Woeste and Kieber 2000; Binder and others 2010)
(Fig. 4b, c). The first ran1 mutants were isolated in the
Ecker laboratory in a genetic screen originally aimed at
identifying ethylene receptor mutants that respond to a
competitive inhibitor of ethylene (before ETR1 was found
to encode the ethylene receptor). The screen instead yiel-
ded mutants that alter the ethylene receptor indirectly as a
result of reduced availability of copper ions. The strongest
ran1 mutant allele confers constitutive ethylene responses,
which are thought to result from the receptors being
improperly folded and non-functional without the copper
cofactor (Woeste and Kieber 2000).
Another ethylene receptor modifier, REVERSION-TO-
ETHYLENE-SENSITIVITY1 (RTE1), was identified
using a genetic screen for suppression of ethylene insen-
sitivity of an etr1 mutant (Resnick and others 2006)
(Fig. 4b, c). Although its biochemical function is unknown,
RTE1 appears to specifically target the ETR1 receptor
(Resnick and others 2006; Zhou and others 2007; Rivarola
and others 2009; Dong and others 2010) and may play a
role in ETR1 folding, keeping ETR1 functionally active
(Resnick and others 2008). RTE1 is homologous to tomato
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Green-ripe, found through a dominant ethylene-insensitive
mutant in which Green-ripe is overexpressed (Barry and
Giovannoni 2006).
The CTR1 Protein Kinase in the Ethylene Receptor
Complex
The CTR1 gene encoding a serine/threonine protein kinase
was the first gene reported in the ethylene signaling path-
way (8 months prior to the publication of the ETR1 gene)
(Kieber and others 1993). As a postdoc in the Ecker lab,
Joseph Kieber cloned CTR1 by virtue of a T-DNA insertion
tag combined with genetic mapping of ctr1. The constitu-
tive response phenotype of ctr1 mutants had indicated that
CTR1 is a negative regulator of ethylene responses (that is,
CTR1 kinase activity represses ethylene responses), and
epistasis analysis indicated that CTR1 acts at or down-
stream of the ethylene receptors (Roman and others 1995)
(Fig. 4a). We now know that the CTR1 N-terminal regu-
latory domain physically associates with the ethylene
receptors (Clark and others 1998; Cancel and Larsen 2002;
Gao and others 2003), and that this interaction is required
for CTR1 kinase activity (Huang and others 2003).
Although this was an important finding, the mechanism of
how the receptors activate CTR1 is still not understood, but
ETR1 histidine kinase activity does not seem to be
involved (Gao and others 2003).
Until recently, the substrate of CTR1 phosphorylation
has remained a mystery. The CTR1 sequence is most
similar to that of the Raf family of protein kinases, which
are MAPKKKs (Kieber and others 1993). Therefore,
CTR1, which is often referred to as a ‘‘Raf-like kinase,’’
has been presumed to act in a canonical MAPK pathway.
Candidate MAPKKs/MAPKs have been proposed (Ouaked
and others 2003; Yoo and others 2008; Novikova and
others 2000); however, no CTR1-containing MAPK path-
way has been conclusively identified to date. The discovery
that CTR1 phosphorylates EIN2 (described below) sug-
gests that the primary ethylene signaling pathway does not
require a MAPKK substrate for CTR1.
The EIN2 Protein Bridges the ER Membrane
and the Nucleus
The EIN2 gene, which was the last of the central ethylene
pathway genes to be identified, was cloned by map-based
cloning by Jose
´Alonso when he was a postdoc in the Ecker
laboratory (Alonso and others 1999). The biochemical
function(s) of EIN2 have remained elusive. The N-termi-
nus of EIN2 consists of twelve predicted transmembrane
domains that show sequence similarity to Nramp metal ion
transporters (Alonso and others 1999), but it is unclear
whether EIN2 is capable of transporting metals. The EIN2
C-terminal portion consists of a plant-specific domain that
activates downstream ethylene responses by an unknown
mechanism (Alonso and others 1999).
The epistatic relationship between ein2 and ctr1 indi-
cated that EIN2 acts at or downstream of CTR1 (Roman
and others 1995) (Fig. 4a), and until recently, the ethylene
signaling pathway has been depicted with EIN2 being
controlled by a putative CTR1-containing MAPK pathway
(for example, Gray 2004). Additionally, EIN2 was pre-
dicted to localize to the nuclear membrane so that it would
be able to signal to the transcription factor EIN3, the next
known downstream component of the pathway (described
in the next section). Consequently, models of the ethylene
signaling pathway often showed EIN2 residing at the
nuclear membrane (for example, Gray 2004). It was not
until 2009, a decade after the EIN2 gene was cloned, that
EIN2 was localized to the ER membrane by Georg Groth’s
laboratory at Du
¨sseldorf University (Bisson and others
2009). Given this finding, there was now a physical gap
that needed to be bridged between the ER membrane-lo-
calized EIN2 and EIN3 in the nucleus.
A proteomic study of ethylene-treated and -untreated
Arabidopsis seedlings using mass spectrometry provided a
critical breakthrough in understanding how EIN2 is regu-
lated (Chen and others 2011). A goal of the study was to
identify missing pathway components that could not be
obtained through genetic screens or other means, but it
instead offered hints that the EIN2 C-terminal domain
might be differentially phosphorylated in response to
ethylene (Chen and others 2011). In the ethylene-treated
samples, no phosphorylation of EIN2 was detected, but in
the untreated samples, phosphorylation of conserved resi-
dues of EIN2 was detected. Given that CTR1 was known to
be an active protein kinase in the absence of ethylene,
CTR1 was a logical candidate for directly phosphorylating
EIN2. A collaboration between the Chang and Kieber
laboratories (at University of Maryland College Park and
University of North Carolina Chapel Hill, respectively)
showed that CTR1 can interact with and directly phos-
phorylate EIN2 (Ju and others 2012) (Fig. 4b, c). They
additionally showed that preventing phosphorylation on a
particular serine residue results in constitutive ethylene
responses. A similar result was shown for a different EIN2
serine residue by Qiao and others (2012) in the Ecker lab.
This indicated that the lack of phosphorylation of EIN2
activates EIN2 signaling, whereas phosphorylation serves
to keep EIN2 inactive.
Another breakthrough was the discovery by three dif-
ferent groups that in the presence of ethylene, the C-ter-
minus of EIN2 is proteolytically cleaved and moves into
the nucleus (Qiao and others 2012; Wen and others 2012;
Ju and others 2012) (Fig. 4b, c). The cleavage appears to be
controlled by phosphorylation of EIN2 by CTR1, because
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preventing phosphorylation with alanine substitutions
results in constitutive cleavage and nuclear translocation of
the C-terminus of EIN2, concomitant with constitutive
ethylene responses (Qiao and others 2012; Wen and others
2012; Ju and others 2012). This cleavage and translocation
provides a mechanism by which the ethylene signal is
transmitted from the site of perception at the ER membrane
into the nucleus, filling an important gap in the pathway.
The phosphorylation of EIN2 by CTR1, together with
cleavage of EIN2 and translocation of the EIN2 C-terminal
domain into the nucleus, represented significant advances
in understanding the ethylene signaling pathway shown in
Fig. 4.
Additional progress has come from protein–protein
interaction studies. For example, by screening for proteins
that physically interact with the C-terminus of EIN2, Qiao
and others (2009) discovered two F-box proteins, ETP1 and
ETP2 (EIN2-TARGETING PROTEIN1/2), which target
EIN2 for degradation in the absence of ethylene. In the
Groth lab, the C-terminal domain of EIN2 was also found to
physically interact with the kinase domain of all five Ara-
bidopsis ethylene receptors (Bisson and Groth 2010). The
ER-localized ethylene receptor complex therefore contains
both CTR1 and EIN2. The biological relevance of the
EIN2-receptor association is unknown and may be one of
the next pieces of the puzzle to be solved, in addition to
understanding the function of the EIN2 C-terminus in the
nucleus and its connection to the regulation of EIN3.
The EIN3 Transcription Factor and its Regulation
The next known downstream component in the ethylene
pathway is ETHYLENE-INSENSITIVE3 (EIN3), a plant-
specific transcription factor and a positive regulator of
ethylene responses. EIN3 was cloned in the Ecker labora-
tory by plasmid rescue based on a T-DNA insertion mutant
(Chao and others 1997). Genetic epistasis had placed EIN3
(as well as EIN2 and EIN5) at or downstream of CTR1
(Roman and others 1995), and further analyses placed
EIN3 downstream of EIN2 and EIN5 (Guo and Ecker
2003). Using EIN3 as a hybridization probe, Chao and
others (1997) isolated the homolog EIN3-LIKE (EIL1),
which is partially redundant with EIN3 (An and others
2010; Binder and others 2007).
As a postdoc in the Ecker lab, Hongwei Guo found that
EIN3 protein levels are highly responsive to ethylene;
EIN3 starts accumulating within 15 min of ethylene treat-
ment, and is turned over within 30 min after removal of
ethylene treatment (Guo and Ecker 2003). The key regu-
latory mechanism underlying this response was discovered
simultaneously by three groups (Guo and Ecker 2003;
Potuschak and others 2003; Gagne and others 2004). Using
protein–protein interaction screens, EIN3-BINDING
F-BOX PROTEIN1 and 2 (EBF1/2) were found to interact
with and target EIN3 for degradation by the 26S protea-
some. This post-translational regulation of EIN3and the
similar regulation of EIL1 (An and others 2010) were the
major findings that explained how ethylene responses could
be so rapid. Regulation by protein turnover is now known
to be a common feature in many phytohormone signaling
pathways (McSteen and Zhao 2008).
Regulation of EIN3 and EIL1 is more complex than
outlined above and there are regulators whose functions are
not yet clearly understood. For example, EIN5, encodes a
50to 30exoribonuclease known as EXORIBONUCLEASE4
(XRN4) (also allelic with ein7) (Potuschak and others 2006;
Olmedo and others 2006). EIN5 appears to indirectly reg-
ulate EBF1/2 expression, consequently affecting EIN3
protein levels (Potuschak and others 2006; Olmedo and
others 2006). However, the mechanism for this regulation
is not yet understood.
Downstream of EIN3
The research summarized above has resulted in a largely
linear model for the primary signaling events for ethylene,
although, non-linear models that invoke feedback are now
being explored. Downstream of EIN3 and EIL1, a second
level of transcriptional regulation was found to occur that
results in a complex web of transcriptional regulation to
modulate diverse responses to ethylene. For instance, in the
tobacco EREBPs mentioned above, which had been iden-
tified on the basis of binding to the GCC promoter element,
it was discovered that EIN3 and EIL1 are responsible for
activating Arabidopsis EREBP genes. In particular, the
Arabidopsis ETHYLENE RESPONSE FACTOR1 (ERF1)
is a GCC-box-binding transcription factor that was found in
the Ecker laboratory (Solano and others 1998) on the basis
of homology to tobacco EREBP1, which had been purified
by Ohme-Takagi and Shinshi (1995). Solano and others
(1998) found that dimerized EIN3 binds directly to a
conserved ethylene response element in the promoter
region of ERF1 (PERE element), activating expression of
the ERF1 gene. In turn, the ERF1 protein binds to the GCC
box in the promoters of secondary target genes, such as
chitinase and PDF1.2. This finding linked ethylene per-
ception and the entire signaling pathway to a transcrip-
tional cascade that controls downstream responses to
ethylene (Fig. 4c).
Concluding Remarks
The historical summary above highlights many of the key
discoveries that have led to our current understanding of
the biosynthesis and perception of the plant hormone
820 J Plant Growth Regul (2015) 34:809–827
123

ethylene. This increased understanding is leading to better
ways to control ethylene for agricultural and horticultural
applications. Despite these major advances, questions still
remain. Recent studies have identified proteins that affect
ethylene biosynthesis or signaling, but the underlying
mechanisms and how they fit into current models remain to
be determined. Given the increasingly fast pace of scien-
tific research and the development of new research tools,
we are likely to continue to see major changes in our
understanding of ethylene as a signaling molecule.
Acknowledgments This work was supported by National Science
Foundation Grants (IOS-1254423) to BMB and (MCB-1244303) to
CC, and a University of Maryland Ann G. Wylie Dissertation Fel-
lowship to JMS. The authors thank Roxane Bouten, John Clay, Randy
Lacey, and Jaden Lee for comments on the manuscript.
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