Download ETHYLENE

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
Air (control)
7 days ethylene
Ethylene regulates:
•fruit ripening
•organ expansion
•senescence
•gene expression
•stress responses
Air
Ethylene
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 responses in Arabidopsis
Ethylene-induced gene expression
Inhibition of root
elongation
Inhibition of leaf
cell expansion
Acceleration of leaf senescence
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; Rüžička, K., Ljung, K., Vanneste, S., Podhorská, R., Beeckman, T., Friml, J., and Benková, E. (2007). Ethylene regulates root
growth through effects on auxin biosynthesis and transport-dependent auxin eistribution. Plant Cell 19: 2197-2212.
When germinating in the dark, impeded
seedlings produce ethylene which confers a
characteristic “triple response”
Ethylene induces the triple
response:
•reduced elongation,
•hypocotyl swelling,
•apical hook exaggeration.
C2H4
C2H4
It’s thought that this response
helps the seedling push past
the impediment.
By treating dark-grown seedlings with exogenous ethylene,
ethylene-response mutants could be identified quickly and
easily based on the triple response phenotype.
The response to ethylene is very
rapid
A single dark-grown
Arabidopsis seedling
photographed every 30
minutes over seven hours.
The rapid elongation that
preceded ethylene addition
stopped immediately, and
resumed rapidly after
ethylene was removed.
Binder, B.M., O’Malley, R.C., Wang, W., Moore, J.M., Parks, B.M., Spalding, E.P., and Bleecker, A.B. (2004). Arabidopsis seedling growth response and recovery to
ethylene. A kinetic analysis. Plant Physiol. 136: 2913–2920.
Ethylene synthesis and homeostasis
In 1901, ethylene was identified as a
compound that affects plant growth
Increasing ethylene
Illuminating gas distilled from
tar contains very high levels
of ethylene.
In 1901, Dimitry Neljubow traced the
source of the strange growth patterns of
his dark-grown pea seedlings to the
ethylene produced by gas-burning lamps.
Neljubov, D.N. (1901) Uber die horizontale Nutation der Stengel von Pisum sativum und einiger anderen Pflanzen. Beih. Bot. Centralbh. 10: 129–139.
In 1934, Gane purified ethylene from
ripening apples, demonstrating that
it is an endogenous hormone
How to measure
ethylene circa 1943
The relative insensitivity of the
early methods made it difficult
to detect small changes in
ethylene production.
Avocados were an
early model for
studying fruit ripening
Pratt, H.K., Young, R.E., and Biale, J.B. (1948). The identification of ethylene as a
volatile product of ripening avocados. Plant Physiol. 23: 526-531.
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
Return to air after
4 hours oxygen
deprivation
Controls
When an apple deprived of
oxygen for four hours is
returned to an aerobic
environment, there is a
dramatic burst of ethylene
production.
This suggests that an
ethylene precursor
accumulates in
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.
This ethylene precursor was called
“Compound X”
N2
Compound X
Air
O2
Radiolabeled methionine was used
to identify Compound X
Adams and Yang incubated
apple slices in 14C-Met to
see what compound
accumulated when oxygen
was withheld.
Air
14C-Ethylene
N2
to
Air
N2
Adams, D.O., and Yang, S.F. (1979). Ethylene biosynthesis: Identification of 1-aminocyclopropane-1-carboxylic acid
as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. USA 76: 170-174.
They identified Compound X!
Compound X
14C-Met
14C-Ethylene
Air
N2
to
Air
Air
N2
N2
N2 to Air
Adams, D.O., and Yang, S.F. (1979). Ethylene biosynthesis: Identification of 1-aminocyclopropane-1-carboxylic acid
as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. USA 76: 170-174.
Compound X is aminocyclopropanecarboxylic acid (ACC)
N2
Air
O2
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 is
regenerated via the
Yang cycle, elucidated
by Shang Fa 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
(1950 – 2005)
Hans Kende
(1937 - 2006)
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).
Characterization of ACC synthase
Proteins extracted
from ripening
tomatoes were used
to make monoclonal
antibodies.
Bleecker, A.B., Kenyon, W.H., Somerville, S.C., and Kende, H. (1986). Use of monoclonal antibodies in the purification and characterization of 1-aminocyclopropane-1carboxylate synthase, an enzyme in ethylene biosynthesis. Proc. Natl. Acad. Sci. USA 83: 7755-7759.
Characterization of ACC synthase
The antibodies were
screened for selectivity
to ACC synthase and
then used to
immunoprecipitate the
enzyme.
ACC
synthase
The other two
proteins are
derived from the
antibody.
Bleecker, A.B., Kenyon, W.H., Somerville, S.C., and Kende, H. (1986). Use of monoclonal antibodies in the purification and characterization of 1-aminocyclopropane-1carboxylate synthase, an enzyme in ethylene biosynthesis. Proc. Natl. Acad. Sci. USA 83: 7755-7759.
An antibody purification scheme was
used to clone an ACC synthase cDNA
Proteins were purified
from ripening zucchini
ACC synthase expression levels were
induced to enrich the protein extract
Auxin
cytokinin,
ACC
Synthase
inhibitors
Uninduced
protein
extraction
Induced
protein
extraction
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.
Y
The partially purified inducedprotein extract was used to produce
antiserum
Induced
protein
extract
Rabbit by Danko
Y
The antiserum was passed over a
column containing uninduced
extract
The contaminating antibodies
from the antiserum were
removed by absorption onto
the uninduced zucchini
extract, which contains very
little ACC synthase. The
resulting antiserum was highly
enriched for anti-ACC
synthase antibodies.
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.
The anti-ACC antibody was used to
screen a cDNA expression library
Induced
extract
A cDNA expression
library made from
induced zucchini mRNA
was screened using the
purified antiserum to
obtain an ACC synthase
cDNA.
Y
Uninduced
extract
Y
Blot probed with
purified
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.
Yeast or E. coli cells expressing ACS
cDNA make ACC
This study provided proof
that the cloned cDNA
encodes ACS
Cloning an ACO cDNA was similarly
challenging….
Control
ACO
antisense
Ethylene
production in
ripening fruit
•A cDNA whose kinetics matched that of ethylene accumulation was cloned
•introduction of an antisense construct into tomato reduced or eliminated
ethylene production after wounding and during fruit ripening
Reprinted by permission from Macmillan Publishers Ltd (Nature) Hamilton, A.J., Lycett, G.W., and Grierson, D. (1990).
Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346: 284-287 Copyright 1990.
Yeast expressing the ACO cDNA can
make ethylene
C2H4
After these key genes
were cloned, it was
possible to examine how
their expression was
regulated.
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
Type I
Type III
The ACS gene family products
can potentially form 45 homoand 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-aminocyclopropane-1-carboxylate synthase isozymes encoded by the arabidopsis gene family. J. Biol. Chem. 278: 49102-49112.
Different ACS dimers have different
catalytic properties
The subset of genes
are that are
expressed in any cell
determines the types
of ACS dimers that
can form, and affects
the rate of ethylene
synthesis.
The ACC synthase genes are
differentially regulated and induced
Tsuchisaka, A., and Theologis, A. (2004). Unique and overlapping expression patterns among the Arabidopsis 1-amino-cyclopropane-1-carboxylate
synthase gene family members. Plant Physiol. 136: 2982-3000.
ACS genes have unique and
common functions
Single mutant analysis shows that each gene has a unique and specific function
Higher order mutants show that there are common essential functions including
effects on flowering time.....
Higher order ACS mutants flower
earlier: ethylene delays flowering
The pentuple
mutant lacks
activity of 5
genes, hexuple
lacks 6, etc.
Tsuchisaka, A., Yu, G., Jin, H., Alonso, J.M., Ecker, J.R., Zhang, X., Gao, S., and Theologis, A. (2009). A combinatorial interplay among the 1aminocyclopropane-1-carboxylate isoforms regulates ethylene biosynthesis in Arabidopsis thaliana. Genetics 183: 979-1003.
ACS genes have unique and
common functions
Higher order ACS mutants are more susceptible to
pathogens; ethylene contributes to pathogen resistance
A mutant lacking all 9 ACS genes is not viable –
ethylene is necessary for plant survival.
Tsuchisaka, A., Yu, G., Jin, H., Alonso, J.M., Ecker, J.R., Zhang, X., Gao, S., and Theologis, A. (2009). A combinatorial interplay among the 1aminocyclopropane-1-carboxylate isoforms regulates ethylene biosynthesis in Arabidopsis thaliana. Genetics 183: 979-1003.
Post-translational control of ACS
activity
Genetic studies identified ethyleneoverproducer (eto) mutants
Wild Type
AIR
eto1
ETHYLENE
AIR
eto mutants
show a tripleresponse 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 ubiquitinligase complex
ETO1 targets ACS proteins for
ubiquitination and proteolysis by the
26S proteosome.
ETO1
ACS
CUL3
WT
ACS5 is selectively
stabilized in loss-ofACS5
function eto1
mutants.
-tubulin
eto1
26S proteasome
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.
The eto2 and eto3 mutations affect
stability of ACS5 and ACS9
ACS5
eto2
ACS9
eto3
The mutations in eto2 and eto3 are due to
changes in the C-terminal region of ACS5 or
ACS9. The mutant proteins are stabilized,
enhancing ethylene synthesis.
Chae, H.S., Faure, F., and Kieber, J.J. (2003). The eto1, eto2, and eto3 mutations and cytokinin treatment increase ethylene
biosynthesis in Arabidopsis by increasing the stability of ACS protein. Plant Cell 15: 545-559.
ACS proteins are normally subject to
rapid proteolysis
Degradation by
the 26S
proteasome
Translation
Normally ACS is continually
synthesized and continually
degraded, maintaining a
very low level of ethylene
ACS
ETO1
CUL3
Liu, Y., and Zhang, S. (2004). Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive
mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis. Plant Cell 16: 3386-3399.
C-terminal phosphorylation
stabilizes ACSs by interfering with
ETO1 action
C-terminal serines are
targets for regulated
phosphorylation
Target of
MAP
Kinase
P P P
Type I
S SSS
P
Type II
Type III
S
Target of
CDP
Kinase
The kinase activities are regulated
by wounding and other hormones
WOUNDING,
PATHOGEN
ATTACK
ATP
MAP
kinase
P P P
Type I
S SSS
P
Type II
Type III
S
CDP
kinase
ATP
ABIOTIC
STRESS,
OTHER
HORMONES
Regulation by proteolysis allows for
rapid responses
2
1
3
1.
2.
3.
4.
Transcription
RNA processing
Translation
Enzyme action
A process regulated by
de novo transcription
has a considerable lag
before beginning.
4
A process regulated by proteolysis
can respond very rapidly.
X
This method however requires a
constant influx of energy to
maintain.
Ethylene synthesis and homeostasis
- summary
SAM
Ethylene
Biosynthesis
ACS
ACC
ACO
ACS proteins
stabilized by
wounding, other
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
ETR1
GAF
histidine kinase
receiver
The etr1-1 mutation is dominant
WT
WT
WT
WT
ETR1
etr1-1
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 bound to
ethylene, the
receptor does
not shut off the
ethylene
response.
When not
bound to
ethylene, the
receptor shuts
off the ethylene
response.
Responses
OFF
Responses
ON
A receptor that always shuts off
signaling is dominant
Ethylene
Responses
OFF
Responses
ON
Responses
OFF
The dominant
negative effect of
etr1-1 and some
other receptor
mutants is
because they
always shut off
responses,
whether or not
ethylene is
present.
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.
Cytokinin
receptor
AHK4
CHASE
domain
histidine kinase
receiver
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
Subfamily II
58%
54%
38%
52%
55%
40%
ETR2
ERS2
53%
Loss-of-function mutations in
ethylene receptors show
constitutive ethylene responses
Wild-type
Responses
OFF
ers1 etr1
double loss-offunction mutant
Responses
ON
Wang, W., Hall, A.E., O'Malley, R., and Bleecker, A.B. (2003). Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from
Arabidopsis is not required for signal transmission. Proc. Natl. Acad. Sci. USA 100: 352-357, copyright National Academy of Sciences USA.
But different receptors have
different signaling strengths
ers1 etr1
(Loss of
Subfamily 1)
Subfamily I
ETR1 ERS1
Subfamily II
etr1 etr2 ein4
EIN4 ETR2 ERS2
Hall, A.E., and Bleecker, A.B. (2003). Analysis of combinatorial loss-of-function mutants in the Arabidopsis ethylene receptors
reveals that the ers1 etr1 double mutant has severe developmental defects that are EIN2 dependent. Plant Cell 15: 2032-2041.
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
Wild type
Never
ripe
Never
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 Neverripe. 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.
The ethylene-binding domain
ethylene
binding
GAF
histidine kinase
receiver
ETR1
NH2
There are three transmembrane
segments in the ethylene binding domain
of ETR1 (four in subfamily II receptors)
From Rodríguez, F.I., Esch, J.J., Hall, A.E., Binder, B.M., Schaller, G.E., and Bleecker, A.B. (1999). A copper cofactor for the ethylene receptor ETR1 from
Arabidopsis. Science 283: 996-998, Reprinted with permssion from AAAS
Mutations in the transmembrane
domain abolish ethylene binding
NH2
Abolishing ethylene binding
causes a dominant ethyleneinsensitive phenotype.
From Rodríguez, F.I., Esch, J.J., Hall, A.E., Binder, B.M., Schaller, G.E., and Bleecker, A.B. (1999). A copper cofactor for the ethylene receptor ETR1 from Arabidopsis.
Science 283: 996-998, Reprinted with permssion from AAAS; Hall, A.E., Grace Chen, Q., Findell, J.L., Eric Schaller, G., and Bleecker, A.B. (1999). The relationship
between ethylene binding and dominant insensitivity conferred by mutant forms of the ETR1 ethylene receptor. Plant Physiol. 121: 291-300.
Control of receptor activity
by interaction with RTE/GR
RTE/GR
WT
etr1-2
ETR1
rte
REVERSION-TOETHYLENE SENSITIVITY
Loss-of-function of
RTE suppresses
ethylene insensitive
etr1-2 phenotype.
These studies suggest
that RTE/GR is a
negative regulator of
ethylene signaling.
Green ripe gain-offunction alleles confer
a dominant, ethyleneinsensitive phenotype
in tomato fruit.
Resnick, J.S., Wen, C.-K., Shockey, J.A., and Chang, C. (2006). REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene that regulates ethylene receptor
function in Arabidopsis. Proc. Natl. Acad. Sci. USA 103: 7917-7922; Barry, C.S. and Giovannoni, J.J. (2006) Ripening in the tomato Green-ripe mutant is inhibited by
ectopic expression of a protein that disrupts ethylene signaling. Proc. Natl. Acad. Sci. USA 103: 7923-7928; copyright National Academy of Sciences USA.
Signaling downstream of the
receptors
Genetic epistasis studies determined the
order of action of the genes
ethylene
+
etr1
=
ctr1
etr1 ctr1
The double mutant has
the same phenotype as
ctr1, indicating that it
acts downstream from
ETR1.
ETR1
CTR1
responses
The genetic pathway of ethylene
signaling
C 2H 4
Receptor
family
ETR1 ERS1 ETR2 EIN4 ERS2
(insensitive - dominant)
CTR1
(constitutive)
EIN2 EIN3 EIN5 EIN6
(insensitive - recessive)
responses to ethylene
CTR1 is a negative regulator of
ethylene signaling
ctr1
Wild type
The ctr1 mutant has a
constitutive triple response.
Air
CTR1 is a serine/threonine
protein kinase that
resembles animal Raf
kinases and is predicted to
act in a MAPK cascade
Ethylene
No substrates have
been identified yet
Reprinted from Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A., and Ecker, J.R. (1993). CTR1, a negative regulator of the ethylene
response pathway in arabidopsis, encodes a member of the Raf family of protein kinases. Cell 72: 427-441 with permission from Elsevier.
The receptors directly interact with
CTR1 and affect its activity
Ethylene
Ethylene
In the presence of
ethylene, CTR1 is
inactive.
CTR1
(active)
Responses
OFF
In the absence of
ethylene, CTR1 is
active and inhibits
the ethylene
responses.
CTR1
(inactive)
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.
The ethylene receptors directly
interact with CTR1
ETR1
ERS
A yeast two-hybrid assay revealed a
specific interaction between the Cterminal region of the ethylene receptors
and the N-terminal region of CTR1.
CTR1
Colony growth and
lacZ expression
means the two
proteins interact.
Clark, K.L., Larsen, P.B., Wang, X., and Chang, C. (1998). Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene
receptors. Proc. Natl. Acad. Sci. USA 95: 5401-5406, copyright National Academy of Sciences USA.
CTR1 acts (somehow) through EIN2,
a positive regulator of ET signaling
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.
CTR1 acts (somehow) through EIN2,
a positive regulator of ET signaling
EIN2 has 12
membrane spanning
domains but its
function is unknown.
?
EIN2
Responses
ON
Loss-of-function
mutants are ethylene
insensitive –EIN2 has a
positive role.
From Alonso, J., Hirayama, T., Roman, G., Nourizadeh, S., and Ecker, J. (1999). EIN2, a bifunctional transducer of ethylene and stress responses in
Arabidopsis. Science 284: 2148 – 2152 reprinted with permission from AAAS; 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, 2
Responses
ON
ETP1 and ETP2 are
components of the ubiquitin
ligase complex that targets
proteins for proteolysis.
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
an ethylene binding site (EBS)
in the promoter of ERF1.
ERF1 encodes another TF
that targets ethyleneresponsive genes.
EIN2
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.
In the absence of ethylene, EIN3 and
EIL1 are targeted for proteolysis
EBF1 and EBF2 are
F-box proteins that
target EIN3 and
EIL1 for proteolysis
EBF1/2
Degradation by the 26S
proteasome via
SCFEBF1/2
EIN3/EIL1
EBS
ERF1
GCC
C2H4 Responsive Gene
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.
Accumulation of EBF1 and EBF2 is
regulated in part at the RNA level
EIN5 encodes a RNA
exoribonuclease that affects
the stability of EBF mRNA and
so affects ethylene signaling.
EIN5/XRN4
EBF1/2
Degradation by the 26S
proteasome via
SCFEBF1/2
EIN3/EIL1
EBS
ERF1
GCC
C2H4 Responsive Gene
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
Ethylene
Biosynthesis
ACS
ACC
ACO
C2H4
ETR1 and others
RTE/GR
CTR1
Ethylene
Signaling
EIN2
EIN3, EILs
ERF1 and ERFs
ETP1 and ETP2
EBF1 and EBF2
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 role 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
Auxin is required for ethylene
effects in the root
Auxin-signaling is
required for
ethylene-induced
gene expression
in the elongating
region of the root.
EBS
GUS
A reporter construct for
ethylene-induced gene
expression
Stepanova, A.N., Yun, J., Likhacheva, A.V., and Alonso, J.M. (2007). Multilevel interactions between
ethylene and auxin in Arabidopsis roots. Plant Cell 19: 2169-2185.
Ethylene’s effects are mediated by
auxin in the root
Stepanova, A.N., Yun, J., Likhacheva, A.V., and Alonso, J.M. (2007). Multilevel interactions between
ethylene and auxin in Arabidopsis roots. Plant Cell 19: 2169-2185.
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 (nonhermaphroditic) 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
Another sex determination gene
affects receptor expression
Downregulation of the
ethylene receptor in
stamen primordia makes
these tissue more
sensitive to ethylene
Cell in developing pistil
Cell in developing stamen
Klee, H.J. (2004). Ethylene signal transduction. Moving beyond Arabidopsis. Plant Physiol. 135: 660-667.
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
Wildtype
Expression of etr1-1 mutant
allele represses petal
responses to ethylene
etr1-1
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
Ethylene synthesis increases
dramatically during fruit ripening
Ethylene
accumulation
Giovannoni, J.J. (2004). Genetic regulation of fruit development and ripening. Plant Cell 16: S170-180.
Ethylene induces expression of ACS
genes during ripening
ACS
Positive regulation –
steep increase in
ethylene production
ACO
SAM
ACC
LEACS6
C2H4
Perception
LEACS1A
LEACS4
LEACS2
Developmentally
regulated
Adapted from Barry, C.S., Llop-Tous, M.I., and Grierson, D. (2000). The regulation of 1-aminocyclopropane-1-carboxylic acid synthase
gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato. Plant Physiol. 123: 979-986.
Fruit ripening can be controlled by
controlling ethylene synthesis
ACC
synthase
ACC
oxidase
H
S-adenosyl
methionine
H
C
ACC
C
H
H
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 synthesis increases upon
hypoxia caused by flooding
Normally, soil has
air pockets from
which plant roots
can take up
oxygen.
O2
When flooded, roots
cannot take up
oxygen, and become
hypoxic – oxygen
deprived.
O2
C2H4
C2H4
Hypoxia induces ACC
synthase and
ethylene production.
Ethylene synthesis increases upon
hypoxia caused by flooding
O2
O2
C2H4
C2H4
Ethylene induces
cell death or cell
separation and
formation of
aerenchyma – air
channels through
which oxygen can
move into roots.
Photo Author: Gordon Beakes©University of Newcastle upon Tyne Image courtesy LTSN Bioscience. A darkfield
micrograph of a transverse section of a stem of Hippuris spp., showing aerenchyma.
ACC moving from root to shoot induces
ethylene formation and epinasty
C2H4
In some plants ACC
moves through the
xylem into the shoot
where it is converted
to ethylene by ACC
oxidase.
C2H4
ACC
C2H4
Leaf epinasty, caused
by differential growth
of the petiole, reduces
light absorption by the
Cleaves.
2H4
Rice is grown in regions subject to
flooding
After prolonged flooding,
many strains of rice die,
but submergence
tolerant lines survive
using either an escape
or quiescence strategy.
Reprinted by permission from Macmillan Publishers Ltd. NATURE from Voesenek, L.A.C.J., and Bailey-Serres, J. (2009). Genetics of
high-rise rice. Nature 460: 959-960 copyright 2009
Rice is grown in regions subject to
flooding
The escape strategy
involves an ethylene
response.
The quiescence
strategy involves a
gibberellin response.
Gibberellin
Ethylene
Reprinted by permission from Macmillan Publishers Ltd. NATURE from Voesenek, L.A.C.J., and Bailey-Serres, J. (2009). Genetics of
high-rise rice. Nature 460: 959-960 copyright 2009
In deepwater rice, ethylene induces
internode elongation
Preserved
deepwater
rice specimen
These plants
can grow as
much as 15m
high when
subjected to
flooding.
Deepwater
Reprinted by permission from Macmillan Publishers Ltd. From Hattori, Y., et al. (2009). The ethylene response factors SNORKEL1 and
SNORKEL2 allow rice to adapt to deep water. Nature 460: 1026-1030, copyright 2009. Photo credit Moto Ashikari, Nagoya University.
The elongation response is encoded
by two ethylene-responsive
transcription factors (ERFs)
Deepwater rice
Flooding
SNORKEL1 & 2
Transcriptional
response
Non-deepwater rice
Flooding
Non-deepwater
rice does not have
these genes
No transcriptional
response
Reprinted by permission from Macmillan Publishers Ltd. From Hattori, Y., et al. (2009). The ethylene response factors SNORKEL1 and
SNORKEL2 allow rice to adapt to deep water. Nature 460: 1026-1030, c opyright 2009.
Ethylene-insensitive tobacco has an
impaired immune system
Plants expressing a dominant ETR1
mutant gene lack resistance to
normally harmless soil-borne fungi.
Higher order ACS mutants are more susceptible to
pathogens
Knoester, M., van Loon, L.C., van den Heuvel, J., Hennig, J., Bol, J.F., and Linthorst, H.J.M. (1998). Ethylene-insensitive tobacco lacks nonhost resistance against soil-borne
fungi. Proc. Natl. Acad. Sci. USA 95: 1933–1937, copyright National Academy of Sciences USA.; Tsuchisaka, A., Yu, G., Jin, H., Alonso, J.M., Ecker, J.R., Zhang, X., Gao, S.,
and Theologis, A. (2009). A combinatorial interplay among the 1-aminocyclopropane-1-carboxylate isoforms regulates ethylene biosynthesis in Arabidopsis thaliana. Genetics
183: 979-1003.
Ethylene is required for wound or
pathogen responses
Plants that do not produce or
respond to ethylene fail to
induce expression of
proteinase inhibitor 2 (pin2).
Wounding
ACO antisense plant
Hours after wounding
No treatment
Wounding + silver
thiosulfate, an inhibitor
of ethylene responses.
From O'Donnell, P.J., Calvert, C., Atzorn, R., Wasternack, C., Leyser, H.M.O., and Bowles, D.J. (1996). Ethylene as a signal mediating
the wound response of tomato plants. Science 274: 1914-1917. Reprinted with permission from AAAS.
In fact, both ethylene and jasmonate
are needed for the defense response
PDF1.2 is a defense gene
that requires BOTH ethylene
and jasmonate for induction
(coi1 is a jasmonateinsensitive mutant).
Ethylene works with jasmonate in
defense-related gene expression.
Penninckx, I.A.M.A., Thomma, B.P.H.J., Buchala, A., Metraux, J.-P., and Broekaert, W.F. (1998). Concomitant activation of jasmonate and ethylene
response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell 10: 2103-2114.
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
Ethylene
Biosynthesis
ACS
ACC
ACO
C2H4
ETR1 and others
CTR1
Ethylene
Signaling
EIN2
EIN3, EILs
ERF1 and ERFs
Ethylene
Responses
•Cell elongation
•Auxin synthesis and
transport
•Fruit ripening
•Senescence
•Pathogen defense
Ongoing research - 1
What signals contribute to the posttranslational regulation of ACS
accumulation?
Does ACC itself
function as a growth
regulator?
SAM
How can ethylene
production be
optimized to enhance
fruit quality?
ACS
ACC
ACO
C2H4
What is the mechanism of
ethylene production by ACO?
What are the transcriptional
regulators of ACS and ACO genes?
Ongoing research - 2
What are the roles of
MAP kinases in synthesis
EIN3/EIL1
and signaling?
CTR1
ACS
P P P
S SSS
ETR1
Many other ethyleneresponse mutants are
being characterized
and integrated into
the pathway – what
do they do?
How does
EIN2 work?
EIN2
What role if any is played by the
histidine kinase domain in the
receptors? What do the different
receptor isoforms do?
How can we
best use this
knowledge to
improve access
to fresh food?
enhanced ethylene
response 4
Robles, L.M., Wampole, J.S., Christians, M.J., and Larsen, P.B. (2007). Arabidopsis enhanced ethylene 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.