Ethylene

Cover image from Science Vol. 241, no. 4869, 26 August 1988, reprinted with permission from AAAS; photo by Kurt Stepnitz, Michigan State
University
 Ethylene (C2H4) is a gaseous
hormone with diverse actions
Ethylene regulates: Air Ethylene
Air (control) 7 days ethylene
•fruit ripening
•organ expansion
•senescence
•gene expression
•stress responses

Cotton plants

Arabidopsis

Beyer, Jr., E.M. (1976) A potent inhibitor of ethylene action in plants. Plant Physiol. 58: 268-271.
Early fruit-ripening practices
Ethylene in smoke has long been
used to ripen fruit; this practice
has included ripening pears in the
smoke from incense. Gashing of
unpollinated figs has also been
practiced; the ethylene produced
upon wounding induces ripening

Image sources: British Museum; Kurt Stüber

 Ethylene synthesis and
homeostasis

Illuminating gas distilled from

tar contains very high levels
Increasing ethylene of ethylene

In 1901, Dimitry Neljubow traced the InIn1901,

1901,ethylene
ethylenewaswas
source of the strange growth patterns of his identified as a
identified as a
dark-grown pea seedlings to the ethylene compound
compoundthat thataffects
affects
produced by gas-burning lamps plant growth
plant growth
Neljubov, D.N. (1901) Uber die horizontale Nutation der Stengel von Pisum sativum und einiger anderen Pflanzen. Beih. Bot. Centralbh. 10: 129–
139.
In 1959 gas chromatography (GC)
was used to measure ethylene
levels
This new method was a million-fold more
sensitive than earlier methods. Using GC,
Burg and Thimann showed that ethylene
production is temperature dependent

Burg, S.P., and Thimann, K.V. (1959). The physiology of ethylene formation in apples. Proc. Natl. Acad. Sci. USA 45: 335-344.
 GC revealed that ethylene is a
cause, not consequence, of ripening
Ethylene production
precedes ripening
and its associated
CO2 production

Burg, S.P., and Burg, E.A. (1962). Role of ethylene in fruit ripening. Plant Physiol. 37: 179-189.
Burg and Thimann made a key
discovery about ethylene
production
When an apple deprived of
oxygen for four hours is
returned to an aerobic
Return to air after
environment, there is a
4 hours oxygen
dramatic burst of ethylene
deprivation
production

This suggests that an

ethylene precursor
accumulates in
Controls oxygen-deprived cells!

Burg, S.P., and Thimann, K.V. (1959). The physiology of ethylene formation in apples. Proc. Natl. Acad. Sci. USA 45: 335-344.
 Compound X is
aminocyclopropane-carboxylic
acid (ACC)

N2

O2
Air
 Ethylene synthesis

Ethylene is produced from methionine (Met) via

S-adenosylmethionine (AdoMet) by the action of
ACC synthase (ACS) and ACC oxidase (ACO)

Reprinted from Chae, H.S., and Kieber, J.J. (2005). Eto Brute? Role of ACS turnover in regulating ethylene
biosynthesis. Trends Plant Sci.10: 291-296 with permission from Elsevier.
Ethylene synthesis

Methionine
Methionineisis
regenerated
regeneratedviaviathe
the
Yang cycle, elucidated
Yang cycle, elucidated
by
byShang
ShangFaFaYang
Yang
Shang Fa Yang
1932 – 2007
Reprinted from Chae, H.S., and Kieber, J.J. (2005). Eto Brute? Role of ACS turnover in regulating ethylene biosynthesis. Trends
Plant Sci.10: 291-296 with permission from Elsevier.; Image sources: University of California; Crenim
 The two key enzymes, ACS and
ACO, are rare and unstable

ACS is ACC synthase

ACO is ACC oxidase

Isolating these proteins and the

genes that encode them was a
significant effort

Tony Bleecker and Hans Kende

made major contributions to the
study of ethylene synthesis and
responses

Tony Bleecker Hans Kende

(1937 - 2006)
(1950 – 2005)
Reprinted from Chae, H.S., and Kieber, J.J. (2005). Eto Brute? Role of ACS turnover in regulating ethylene biosynthesis. Trends Plant Sci.10: 291-296 with permission
from Elsevier.; Photos courtesy of Alan Jones (University of North Carolina) and Kurt Stepnitz (Michigan State University).
 The anti-ACC antibody was used
to screen a cDNA expression
library
Uninduced Induced
extract extract
A cDNA expression
library made from induced
zucchini mRNA was
Y screened using the
purified antiserum to
obtain an ACC synthase
cDNA
Y Y
Y

Y Y
YY
Blot probed with Y
purified
Y

Y
Y

antiserum

Blot probed with

crude antiserum

Sato, T., and Theologis, A. (1989). Cloning the mRNA encoding 1-aminocyclopropane-1-carboxylate
synthase, the key enzyme for ethylene biosynthesis in plants. Proc.Natl. Acad. Sci. USA 86: 6621-6625.
Ethylene production is primarily
regulated by ACS accumulation

•ACS is encoded by 9 genes with diverse

functions and expression patterns

•Some ACS proteins are strongly

regulated post-translationally
 ACS is encoded by 9 genes and
functions as a dimer
The ACS gene family products
Type I
can potentially form 45 homo-
Type III and heterodimers of which 25
are functional

Type II

Type I S SSS
Type II S
Type III

Yamagami, T., Tsuchisaka, A., Yamada, K., Haddon, W.F., Harden, L.A., and Theologis, A. (2003). Biochemical diversity among the 1-
amino-cyclopropane-1-carboxylate synthase isozymes encoded by the arabidopsis gene family. J. Biol. Chem. 278: 49102-49112.
 Genetic studies identified
ethylene-overproducer (eto)
mutants
Wild Type eto1

AIR ETHYLENE AIR

eto mutants
show a triple-
response in air
and
overproduce
ethylene

Guzman, P., and Ecker, J.R. (1990). Exploiting the triple response of Arabidopsis to
identify ethylene-related mutants. Plant Cell 2: 513-523.
ETO1 is a component of a ubiquitin-
ligase complex

ETO1 targets ACS proteins for ETO1

ACS
ubiquitination and proteolysis by the
26S proteosome

CUL3

WT eto1
ACS5 is selectively
stabilized in loss-of- ACS5 26S proteasome
function eto1 mutants
-tubulin

Reprinted by permission from Macmillan Publishers Ltd: Wang, K.L.C., Yoshida, H., Lurin, C., and Ecker, J.R. (2004). Regulation
of ethylene gas biosynthesis by the Arabidopsis ETO1 protein. Nature 428: 945-950, copyright 2004.
Regulation by proteolysis allows for
rapid responses
2 1. Transcription
1. Transcription
2. RNA processing A process regulated by
2. RNA processing
1 3.
3.
Translation
Translation de novo transcription
3 4. Enzyme action
4. Enzyme action has a considerable lag
before beginning

A process regulated by proteolysis

can respond very rapidly

This method however requires a

constant influx of energy to
X maintain
 Ethylene synthesis and
homeostasis - summary

SAM
ACS proteins
ACS stabilized by
Ethylene ACC wounding, other
Biosynthesis ACO hormones
C2H4

•Simple biosynthetic pathway regulated by expression

and stability of ACS and ACO

•ACS and ACO activities are tightly regulated

transcriptionally and post-transcriptionally and sensitive
to developmental cues, wounding and pathogen attack
 Ethylene response – receptors and
downstream signaling
In the 1980s, a genetic screen
was carried out by Tony
Bleecker, Hans Kende and
colleagues to dissect the
ethylene signaling pathway at
the molecular level

Normal triple
response

Bleecker, A.B., Estelle, M.A., Somerville, C., and Kende, H. (1988). Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis
thaliana. Science 241: 1086-1089 reprinted with permission from AAAS; photo by Kurt Stepnitz, Michigan State University.
 Many signaling components were
identified genetically
Ethylene-insensitive mutants
Ethylene-insensitive –
no triple response in ethylene etr1 etr2 ein4
ein2 ein3 ein5 ein6

C2H4
Constitutive response –
triple response in air Constitutive-response mutants

ctr1

air
ETHYLENE RESPONSE1 (ETR1)
encodes an ethylene receptor
ETR1 was the first protein to be unambiguously
identified as a phytohormone receptor (1993)
•ETR1 binds ethylene
•ETR1 is similar in sequence to known-receptors in animal cells
•ETR1 is membrane localized

ethylene
binding GAF histidine kinase receiver
ETR1
 The etr1-1 mutation is dominant
WT WT WT WT etr1-1
ETR1 etr1-1 etr1-1
Introduction of the mutant
etr1-1 allele into a wild-type
plant causes an ethylene
insensitive phenotype

From Chang, C., Kwok, S., Bleecker, A., and Meyerowitz, E. (1993). Arabidopsis ethylene-response gene ETR1:
similarity of product to two-component regulators. Science 262: 539 – 544; reprinted with permission from AAAS.
How can a mutant receptor have a
dominant phenotype???
The receptors negatively regulate the responses

No Ethylene Ethylene

When not When bound to

bound to ethylene, the ( ) (H
ethylene, the receptor does
receptor shuts not shut off the
off the ethylene ethylene
response response

Responses Responses
OFF ON
 A receptor that always shuts off
signaling is dominant
Ethylene
The dominant
negative effect of
etr1-1 and some
( )
other receptor
(H
mutants is
because they
always shut off
responses,
whether or not
ethylene is
Responses Responses Responses present
OFF ON OFF
 Arabidopsis ethylene receptors
resemble hybrid histidine kinases
ethylene
binding GAF histidine kinase receiver
ETR1

The ethylene receptors structurally

resemble the cytokinin receptors.
However, unlike the cytokinin
receptors, the histidine kinase domain
has little role in signaling in vivo
CHASE
Cytokinin domain histidine kinase receiver
receptor
AHK4
 Arabidopsis ethylene receptor family
ethylene
binding GAF histidine kinase receiver
ETR1
Subfamily I
83% 64% 64%
ERS1

44-54% 38-41% 16-29% 32%

EIN4
58% 54% 38% 53%
Subfamily II
ETR2

52% 55% 40%

ERS2
Ethylene receptor mutants have also
been identified in other plants
The tomato Never ripe mutant has
a dominant, ethylene-insensitive
phenotype, like etr1-1

Wild type Never

ripe

Never
Wild type ripe

From Wilkinson, J.Q., Lanahan, M.B., Yen, H.-C., Giovannoni, J.J., and Klee, H.J. (1995). An ethylene-inducible component of signal transduction encoded by
Never-ripe. Science 270: 1807-1809, reprinted with permisison from AAAS; Lanahan, M.B., Yen, H.C., Giovannoni, J.J., and Klee, H.J. (1994). The Never ripe
mutation blocks ethylene perception in tomato. Plant Cell 6: 521-530.
Genetic epistasis studies determined the
order of action of the genes

ethylene
+ =
ETR1
ctr1 etr1 ctr1
etr1 The double mutant has
CTR1
the same phenotype as
ctr1, indicating that it
acts downstream from
ETR1
responses
 The genetic pathway of ethylene
signaling
C2H4
Receptor
family ETR1 ERS1 ETR2 EIN4 ERS2
(insensitive - dominant)

CTR1
(constitutive)

EIN2 EIN3 EIN5 EIN6

(insensitive - recessive)

responses to ethylene
CTR1 acts (somehow) through EIN2,
a positive regulator of ET signaling

( ) (H

Genetic studies show that EIN2 acts

? downstream of CTR1, but how the signal

is transduced remains a mystery!

EIN2

Responses
ON
Reprinted from Kendrick, M.D., and Chang, C. (2008). Ethylene signaling: new levels of complexity and regulation. Curr.
Opin. Plant Biol. 11: 479-485 with permission from Elsevier.
 EIN2 is subject to proteolysis in the
absence of ethylene
Ethylene
EIN2
ETP1 and ETP2 are
ETP1, 2 components of the ubiquitin
ligase complex that targets
proteins for proteolysis
Responses
ON

Ethylene destabilizes ETP1 and

ETP2, stabilizing EIN2 and
promoting downstream effects

From Qiao, H., Chang, K.N., Yazaki, J., and Ecker, J.R. (2009). Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation
of EIN2 triggers ethylene responses in Arabidopsis. Genes Devel. 23: 512-521.
 Downstream of EIN2 a transcriptional
cascade controls gene expression
EIN3 and EIL1 are
transcription factors that bind EIN2
an ethylene binding site (EBS)
in the promoter of ERF1.
ERF1 encodes another TF
that targets ethylene-
Nucleus
responsive genes

EIN3/EIL1

EBS
ERF1

GCC C2H4 Responsive Gene

Reprinted from Chao, Q., Rothenberg, M., Solano, R., Roman, G., Terzaghi, W., and Ecker, J. (1997). Activation of the ethylene gas response pathway in Arabidopsis
by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins Cell 89: 1133 – 1144 with permission from Elsevier; Kendrick, M.D., and Chang, C. (2008).
Ethylene signaling: new levels of complexity and regulation. Curr. Opin. Plant Biol. 11: 479-485 with permission from Elsevier.
Transcription factors mediate many
of ethylene’s functions

Fruit ripening genes

FLS2, encoding a receptor
that recognizes pathogens
EIN3/EIL1

EBS Defense response genes

ERF1

GCC C2H4 Responsive Gene

Salicylic acid
biosynthesis genes

Reprinted from Kendrick, M.D., and Chang, C. (2008). Ethylene signaling: new levels of complexity and regulation. Curr.
Opin. Plant Biol. 11: 479-485 with permission from Elsevier.
Summary of ethylene synthesis
and signaling
SAM
ACS
Ethylene ACC
Biosynthesis ACO
C2H4

ETR1 and others RTE/GR

CTR1
Ethylene EIN2 ETP1 and ETP2
Signaling
EIN3, EILs EBF1 and EBF2

ERF1 and ERFs

Ethylene perception and signaling
- summary
• Arabidopsis genetics, and especially the
easy-to-score triple response, were
instrumental in identifiying the genes
encoding the signaling pathway
• The pathway has a novel combination of
proteins acting in a mainly linear pathway
• Negative regulation plays an important role!
• Protein turnover is an important regulatory
mechanism
Ethylene’s roles in whole-plant
processes
• Shoot and Root elongation
• Reproductive development
• Sex determination
• Petal senescence
• Fruit ripening
• Flooding responses –
• Aerenchyma formation, leaf epinasty
• Deepwater rice
• Pathogen responses
Ethylene restricts elongation of
the shoot and root in the dark

C2H4 C2H4
C2H4
C2H4
 Ethylene contributes to apical
hook formation through auxin
effects
Ethylene
AIR

HOOKLESS

ARF2
ETHYLENE

Differential growth

Reprinted from Lehman, A., Black, R., and Ecker, J.R. (1996). HOOKLESS1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis
hypocotyl. Cell 85: 183-194 with permission from Elsevier.
Sex determination in Cucumis

Hermaphrodite Male Female

Imperfect (non-
hermaphroditic) flowers
can lead to increased
outcrossing and
increased fitness.

Image courtesy of Abdelhafid Bendahmane, URGV - Plant Genomics Research INRA

Female flowers arise when stamen
primordia abort

Pistil

Stamen

Petals

Sepals

Image courtesy of Abdelhafid Bendahmane, URGV - Plant Genomics Research INRA

Genes affecting sex determination
encode ACS genes

Elevated levels of ethylene

production are correlated
with developmental arrest
of the stamen primordia
Ethylene promotes petal
senescence

Azad, A.K., Ishikawa, T., Ishikawa, T., Sawa, Y., and Shibata, H. (2008). Intracellular energy depletion triggers programmed cell
death during petal senescence in tulip. J. Exp. Bot. 59: 2085-2095, by permission of Oxford University Press.
 Chemical and genetic approaches
can prolong petal longevity
STS and CACP interfere with
ethylene binding to receptor

Wild-
type

Expression of etr1-1 mutant

etr1-1 allele represses petal
responses to ethylene

0 3 8
DAYS AFTER POLLINATION
Reprinted from Serek, M., Woltering, E.J., Sisler, E.C., Frello, S., and Sriskandarajah, S. (2006) Controlling ethylene responses in flowers at the receptor level.
Biotech. Adv. 24: 368-381 with permission from Elsevier; Wilkinson, J.Q., Lanahan, M.B., Clark, D.G., Bleecker, A.B., Chang, C., Meyerowitz, E.M., and Klee,
H.J. (1997). A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants. Nat Biotech 15: 444-447.
 Fruit ripening is induced by
ethylene

Ethylene

Ripening includes:
Changes in cell wall structure
Pigment accumulation
Flavor and aromatic volatile production
Conversions of starches to sugars
Fruit ripening can be controlled by
controlling ethylene synthesis
ACC ACC
synthase oxidase
S-adenosyl H H
methionine C C
H H
ACC Ethylene

Antisense
ACC synthase

Control

Theologis, A., Zarembinski, T.I., Oeller, P.W., Liang, X., and Abel, S. (1992). Modification of fruit ripening by
suppressing gene expression. Plant Physiol. 100: 549-551.
 Ethylene/ JA responses are
mediated by ERF1 and other TFs

Lorenzo, O., Piqueras, R., Sanchez-Serrano, J.J., and Solano, R. (2003). ETHYLENE RESPONSE FACTOR1 integrates
signals from ethylene and jasmonate pathways in plant defense. Plant Cell 15: 165-178.
 ETHYLENE- SUMMARY

SAM
ACS
Ethylene ACC
Biosynthesis ACO
C2H4
Ethylene
ETR1 and others Responses

CTR1 •Cell elongation

Ethylene •Auxin synthesis and
EIN2
Signaling transport
•Fruit ripening
EIN3, EILs •Senescence
•Pathogen defense
ERF1 and ERFs
 Ongoing research - 1
Does ACC itself What signals contribute to the post-
function as a growth translational regulation of ACS
regulator? accumulation?

SAM
ACS
How can ethylene ACC
production be ACO What is the mechanism of
optimized to enhance C2H4 ethylene production by ACO?
fruit quality?

What are the transcriptional

regulators of ACS and ACO genes?
 Ongoing research - 2
CTR1 What are the roles of How does EIN2
MAP kinases in synthesis EIN2 work?
EIN3/EIL1 and signaling?
What role if any is played by the
ACS P P P
S SSS histidine kinase domain in the
receptors? What do the different
receptor isoforms do?
ETR1
Many other ethylene-
response mutants are
being characterized How can we
and integrated into best use this
the pathway – what knowledge to
do they do? improve access
to fresh food?
enhanced ethylene Robles, L.M., Wampole, J.S., Christians, M.J., and Larsen, P.B. (2007). Arabidopsis enhanced ethylene response 4
response 4 encodes an EIN3-interacting TFIID transcription factor required for proper ethylene response, including ERF1
induction. J. Exp. Bot. 58: 2627-2639, by permission of Oxford University Press.