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:•fruit ripening•organ expansion •senescence •gene expression•stress responses

Cotton plants

7 days ethyleneAir (control)Air Ethylene

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

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.

Inhibition of leaf cell expansion

Acceleration of leaf senescence

Ethylene-induced gene expression

Inhibition of root elongation

When germinating in the dark, impeded seedlings produce ethylene which confers a

characteristic “triple response”

C2H4

C2H4

Ethylene induces the triple response: •reduced elongation,•hypocotyl swelling,•apical hook exaggeration.

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.

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

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.

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.

Ethylene synthesis and homeostasis

In 1901, ethylene was identified as a compound that affects 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.

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.

Increasing ethylene

In 1934, Gane purified ethylene from ripening apples, demonstrating that

it is an endogenous hormone

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.

How to measure ethylene circa 1943

Avocados were an early model for

studying fruit ripening

The relative insensitivity of the early methods made it difficult to detect small changes in ethylene production.

In 1959 gas chromatography (GC) was used to measure ethylene levels

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

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 Burg, E.A. (1962). Role of ethylene in fruit ripening. Plant Physiol. 37: 179-189.

GC revealed that ethylene is a cause, not consequence, of ripening

Ethylene production precedes ripening and its associated CO2 production.

Burg and Thimann made a key discovery about ethylene production

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

Controls

Return to air after 4 hours oxygen deprivation

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!

This ethylene precursor was called “Compound X”

Compound X

O2

N2

Air

Radiolabeled methionine was used to identify Compound X

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.

Air

N2

N2 to Air

Adams and Yang incubated apple slices in 14C-Met to see what compound accumulated when oxygen was withheld.

14C-Ethylene

They identified Compound X!

Air

N2

N2 to Air

14C-Met14C-Ethylene

Compound X

Air

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 aminocyclopropane-carboxylic acid (ACC)

O2

N2

Air

Ethylene synthesis

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 is produced from methionine (Met) via S-adenosylmethionine (AdoMet) by the action of ACC synthase (ACS) and ACC oxidase (ACO).

Ethylene synthesis

Shang Fa Yang1932 – 2007

Methionine is regenerated via the Yang cycle, elucidated by Shang Fa Yang.

Methionine is regenerated via the Yang cycle, elucidated by Shang Fa Yang.

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

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).

Isolating these proteins and the genes that encode them was a significant effort.

Tony Bleecker (1950 – 2005)

Hans Kende (1937 - 2006)

Tony Bleecker and Hans Kende made major contributions to the study of ethylene synthesis and responses.

ACS is ACC synthase ACO is ACC oxidase

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-1-carboxylate synthase, an enzyme in ethylene biosynthesis. Proc. Natl. Acad. Sci. USA 83: 7755-7759.

Characterization of ACC synthase

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-1-carboxylate synthase, an enzyme in ethylene biosynthesis. Proc. Natl. Acad. Sci. USA 83: 7755-7759.

ACC synthase

The antibodies were screened for selectivity to ACC synthase and then used to immunoprecipitate the enzyme.

The other two proteins are derived from the antibody.

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

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.

Uninduced protein extraction

Induced protein extraction

Auxincytokinin,

ACC Synthaseinhibitors

The partially purified induced- protein extract was used to produce

antiserum

Induced protein extractY

YY

Y

YY

Y

Y

Y

YRabbit by Danko

The antiserum was passed over a column containing uninduced

extract

Y

YY

Y

YY

Y

Y

Y

Y

Y

YY

YY

Y

Y

Y

Y

Y

Y

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

YA cDNA expression library made from induced zucchini mRNA was screened using the purified antiserum to obtain an ACC synthase cDNA.

Y

YY

Y YYY

Y

Y

Y

Y

Uninduced extract

Induced extract

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….

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.

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

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

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.

Type III

Type I

Type II

S S S S

S

Type I

Type III

Type II

The ACS gene family products can potentially form 45 homo- and heterodimers of which 25 are functional.

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

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.

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.

ACS genes have unique and common functions

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.

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.

Post-translational control of ACS activity

Genetic studies identified ethylene-overproducer (eto) mutants

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

Wild Type eto1

AIR AIRETHYLENE

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

ETO1 is a component of a ubiquitin-ligase complex

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.

CUL3

ETO1

WT eto1

ACS5

-tubulin

ACS5 is selectively stabilized in loss-of-function eto1 mutants.

26S proteasome

ETO1 targets ACS proteins for ubiquitination and proteolysis by the 26S proteosome.

ACS

The eto2 and eto3 mutations affect stability of ACS5 and ACS9

ACS5

ACS9

eto2

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

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.

ACS

Translation

Normally ACS is continually synthesized and continually degraded, maintaining a very low level of ethylene

Degradation by the 26S proteasome

CUL3

ETO1

C-terminal phosphorylation stabilizes ACSs by interfering with

ETO1 action

S S S S

S

P PP

P

C-terminal serines are targets for regulated phosphorylation

Target of MAP

Kinase

Target of CDP

Kinase

Type I

Type III

Type II

The kinase activities are regulated by wounding and other hormones

S S S S

S

P PP

P

MAP kinase

ATPWOUNDING, PATHOGEN ATTACK

CDP kinase

ATP

ABIOTIC STRESS, OTHER HORMONES

Type I

Type III

Type II

Regulation by proteolysis allows for rapid responses

1

2

3

4

A process regulated by de novo transcription has a considerable lag before beginning.

1. Transcription2. RNA processing3. Translation4. Enzyme action

1. Transcription2. RNA processing3. Translation4. Enzyme action

A process regulated by proteolysis can respond very rapidly.

This method however requires a constant influx of energy to maintain. X

Ethylene synthesis and homeostasis - summary

•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 Biosynthesis

SAM

ACC

C2H4

ACS

ACO

ACS proteins stabilized by wounding, other hormones

Normal triple response

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.

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.

Ethylene response – receptors and downstream signaling

Many signaling components were identified genetically

ctr1

ein2 ein3 ein5 ein6

Constitutive-response mutants

Ethylene-insensitive mutants

etr1 etr2 ein4

air

C2H4

Ethylene-insensitive – no triple response in ethylene

Constitutive response –triple response in air

ETHYLENE RESPONSE1 (ETR1) encodes an ethylene receptor

ETR1 was the first protein to be unambiguouslyidentified as a phytohormone receptor (1993) •ETR1 binds ethylene•ETR1 is similar in sequence to known-receptors in animal cells•ETR1 is membrane localized

ETR1

histidine kinase receiverGAFethylenebinding

The etr1-1 mutation is dominant

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.

WT etr1-1WT WT WT

etr1-1etr1-1 ETR1Introduction of the mutant etr1-1 allele into a wild-type plant causes an ethylene insensitive phenotype.

How can a mutant receptor have a dominant phenotype???

Responses ON

)( (H

Ethylene

ResponsesOFF

The receptors negatively regulate the responses

No Ethylene

When not bound to ethylene, the receptor shuts off the ethylene response.

When bound to ethylene, the

receptor does not shut off the

ethylene response.

A receptor that always shuts off signaling is dominant

Responses ON

ResponsesOFF

Responses OFF

)( (H

Ethylene

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

ETR1

histidine kinase receiverGAFethylenebinding

histidine kinase receiverCHASE domainCytokinin

receptor AHK4

The ethylene receptors structurally resemble the cytokinin receptors. However, unlike the cytokinin receptors, the histidine kinase domain has little role in signaling in vivo.

ERS1

EIN4

ETR2

ERS2

53%

32%16-29%

58% 38%

52%

83%

44-54%

40%

64%

38-41%

64%

54%

55%

Subfamily I

Subfamily II

ETR1

histidine kinase receiverGAFethylenebinding

Arabidopsis ethylene receptor family

Loss-of-function mutations in ethylene receptors show

constitutive ethylene responses

ResponsesOFF

ResponsesON

Wild-typeers1 etr1 double loss-of-function

mutant

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

ERS1EIN4 ETR2 ERS2

Subfamily I Subfamily II

ETR1

ers1 etr1(Loss of

Subfamily 1)

etr1 etr2 ein4

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

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.

Wild typeNever ripe

The tomato Never ripe mutant has a dominant, ethylene-insensitive phenotype, like etr1-1.

Never ripe

Wild type

The ethylene-binding domain

NH2

ETR1

histidine kinase receiverGAFethylenebinding

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 ethylene-insensitive 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

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.

ETR1

etr1-2

rte

REVERSION-TO-ETHYLENE SENSITIVITY

RTE/GR

WT

Loss-of-function of RTE suppresses ethylene insensitive etr1-2 phenotype.

Green ripe gain-of-function alleles confer a dominant, ethylene-insensitive phenotype in tomato fruit.

These studies suggest that RTE/GR is a negative regulator of ethylene signaling.

Signaling downstream of the receptors

Genetic epistasis studies determined the order of action of the genes

+ =

etr1ctr1 etr1 ctr1

ETR1

CTR1

responses

ethylene

The double mutant has the same phenotype as ctr1, indicating that it acts downstream from ETR1.

The genetic pathway of ethylene signaling

CTR1

ETR1 ERS1 ETR2 EIN4 ERS2

EIN2 EIN3 EIN5 EIN6

responses to ethylene

C2H4

(insensitive - dominant)

(insensitive - recessive)

(constitutive)

Receptor family

CTR1 is a negative regulator of ethylene signaling

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.

Air

Ethylene

Wild typectr1The ctr1 mutant has a constitutive triple response.

CTR1 is a serine/threonine protein kinase that

resembles animal Raf kinases and is predicted to

act in a MAPK cascade

No substrates have been identified yet

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 receptors directly interact with CTR1 and affect its activity

In the absence of ethylene, CTR1 is active and inhibits the ethylene responses.

Ethylene

Ethylene

)( (H

ResponsesOFF

CTR1 (active)

CTR1 (inactive)

In the presence of ethylene, CTR1 is inactive.

ResponsesON

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.

The ethylene receptors directly interact with CTR1

ETR1ERS

CTR1

A yeast two-hybrid assay revealed a specific interaction between the C-terminal region of the ethylene receptors and the N-terminal region of CTR1.

Colony growth and lacZ expression means the two proteins interact.

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

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

Responses ON

)( (H

?Genetic studies show that EIN2 acts downstream of CTR1, but how the signal is transduced remains a mystery!

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

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

EIN2 has 12 membrane spanning domains but its function is unknown.

Responses ON

)( (H

Loss-of-function mutants are ethylene insensitive –EIN2 has a positive role.

?

EIN2 is subject to proteolysis in the absence of ethylene

EIN2

ETP1, 2

Responses ON

Ethylene

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

C2H4 Responsive Gene

Nucleus

GCC

EBS ERF1

EIN3/EIL1

EIN3 and EIL1 are transcription factors that bind an ethylene binding site (EBS) in the promoter of ERF1. ERF1 encodes another TF that targets ethylene-responsive genes.

EIN2

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

EBF1/2

C2H4 Responsive Gene

Nucleus

GCC

EBS ERF1

EIN3/EIL1

Degradation by the 26S proteasome via

SCFEBF1/2

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

EBF1/2

C2H4 Responsive Gene

Nucleus

GCC

EBS ERF1

EIN3/EIL1

Degradation by the 26S proteasome via

SCFEBF1/2

EIN5/XRN4

EIN5 encodes a RNA exoribonuclease that affects the stability of EBF mRNA and so affects ethylene signaling.

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

Ethylene Biosynthesis

SAM

ACC

C2H4

ACS

ACO

ETR1 and others

Ethylene Signaling

CTR1

EIN2

EIN3, EILs

ERF1 and ERFs

ETP1 and ETP2

RTE/GR

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

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.

Auxin-signaling is required for ethylene-induced gene expression in the elongating region of the root.

GUSEBSA reporter construct for ethylene-induced gene expression

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

AIR

ETHYLENE

Ethylene

HOOKLESS

ARF2

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

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

Hermaphrodite Male Female

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

Female flowers arise when stamen primordia abort

Sepals

Petals

Pistil

Stamen

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

Klee, H.J. (2004). Ethylene signal transduction. Moving beyond Arabidopsis. Plant Physiol. 135: 660-667.

Downregulation of the ethylene receptor in stamen primordia makes these tissue more sensitive to ethylene

Cell in developing pistil

Cell in developing stamen

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

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.

DAYS AFTER POLLINATION0 3 8

Wild-type

etr1-1

STS and CACP interfere with ethylene binding to receptor

Expression of etr1-1 mutant allele represses petal responses to ethylene

Fruit ripening is induced by ethylene

Ethylene

Ripening includes: Changes in cell wall structurePigment accumulationFlavor and aromatic volatile productionConversions 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

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.

SAM ACC C2H4 PerceptionACOACS

LEACS4

LEACS6

LEACS1A

LEACS2

Developmentally regulated

Positive regulation – steep increase in ethylene production

Fruit ripening can be controlled by controlling ethylene synthesis

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.

ACC synthase

ACC oxidase

EthyleneACC

S-adenosyl methionine C C

H

HH

H

Antisense ACC synthase

Control

Ethylene synthesis increases upon hypoxia caused by flooding

C2H4

C2H4

O2O2

Normally, soil has air pockets from which plant roots can take up oxygen.

Normally, soil has air pockets from which plant roots can take up oxygen.

When flooded, roots cannot take up oxygen, and become hypoxic – oxygen deprived.

When flooded, roots cannot take up oxygen, and become hypoxic – oxygen deprived.

Hypoxia induces ACC synthase and ethylene production.

Ethylene synthesis increases upon hypoxia caused by flooding

C2H4

C2H4

O2O2

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

C2H4

ACC

C2H4

In some plants ACC moves through the xylem into the shoot where it is converted to ethylene by ACC oxidase.

C2H4

Leaf epinasty, caused by differential growth of the petiole, reduces light absorption by the leaves.

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.

Ethylene

Gibberellin

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.

These plants can grow as much as 15m high when subjected to flooding.

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.

Deepwater

The elongation response is encoded by two ethylene-responsive transcription factors (ERFs)

Deepwater rice

Non-deepwater rice

Transcriptional response

No transcriptional response

SNORKEL1 & 2

Flooding

Flooding Non-deepwater rice does not have these genes

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

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.

Higher order ACS mutants are more susceptible to pathogens

Plants expressing a dominant ETR1 mutant gene lack resistance to

normally harmless soil-borne fungi.

Ethylene is required for wound or pathogen 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.

Plants that do not produce or respond to ethylene fail to induce expression of proteinase inhibitor 2 (pin2).

No treatment

Wounding Wounding + silver thiosulfate, an inhibitor of ethylene responses.

ACO antisense plantHours after wounding

In fact, both ethylene and jasmonate are needed for the defense response

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.

PDF1.2 is a defense gene that requires BOTH ethylene and jasmonate for induction (coi1 is a jasmonate-insensitive mutant).

Ethylene works with jasmonate in defense-related gene expression.

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

Ethylene Biosynthesis

SAM

ACC

C2H4

ACS

ACO

ETR1 and others

Ethylene Signaling

CTR1

EIN2

EIN3, EILs

ERF1 and ERFs

•Cell elongation•Auxin synthesis and transport•Fruit ripening•Senescence•Pathogen defense

Ethylene Responses

Ongoing research - 1

SAM

ACC

C2H4

ACS

ACO

What signals contribute to the post-translational regulation of ACS

accumulation?

Does ACC itself function as a growth

regulator?

How can ethylene production be

optimized to enhance fruit quality?

What are the transcriptional regulators of ACS and ACO genes?

What is the mechanism of ethylene production by ACO?

Ongoing research - 2

EIN2How does EIN2 work?

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?

Many other ethylene-response mutants are being characterized and integrated into the pathway – what

do they do?

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.

EIN3/EIL1

S S S SP PP

What are the roles of MAP kinases in synthesis

and signaling?

ACS

CTR1

ETR1