1. Ethylene (C2H4) is a gaseous hormone with diverse actions
Ethylene regulates:
fruit ripening
organ expansion
senescence
gene expression
stress responses
Cotton plants
7 days ethylene
Air (control)
Air
Ethylene
Arabidopsis
Beyer, Jr., E.M. (1976) A potent inhibitor of ethylene action in plants. Plant Physiol. 58:

2. 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

3. When germinating in the dark, impeded seedlings produce ethylene which confers a characteristic “triple response”
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

4. Ethylene is derived from methionine via ACC
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: with permission from Elsevier.

5. Ethylene synthesis
Shang Fa Yang
1932 – 2007
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: with permission from Elsevier.; Image sources: University of California; Crenim

6. The two key enzymes, ACS and ACO, are rare and unstable
ACS is ACC synthase
ACO is ACC oxidase
Both are unstable proteins. Normally ACS is continually synthesized and continually degraded, maintaining a very low level of ethylene
Proteasome
Stress-induced protein phosphorylation stabilizes ACS and increases ethylene accumulation
ACS P
WOUNDING, STRESS, PATHOGEN ATTACK
Reprinted from Chae, H.S., and Kieber, J.J. (2005). Eto Brute? Role of ACS turnover in regulating ethylene biosynthesis. Trends Plant Sci.10: with permission from Elsevier.

7. 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

8. Ethylene response – receptors and downstream signaling
Normal triple response
No ethylene response – ethylene insensitive
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: reprinted with permission from AAAS; photo by Kurt Stepnitz, Michigan State University.

9. 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
ETR1
histidine kinase
receiver
GAF
ethylene
binding
Arabidopsis has five ethylene receptors, with differing signaling strengths
Ethylene-binding, membrane-spanning domain

10. The etr1-1 mutation is dominantWT
etr1-1
ETR1
Introduction of the mutant etr1-1 allele into a wild-type plant causes an ethylene insensitive phenotype
(The level of sensitivity varies as shown in two independent lines).
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.

11. How can a mutant receptor have a dominant phenotype???
Responses ON ) ( H
Ethylene
OFF
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
We still don’t know what the signaling mechanism is, but we have a general idea about how the receptors function. The receptors are negative regulators of ethylene responses. That means, in the absence of ethylene binding, the receptors are signaling by an unknown mechanism to keep responses off. When ethylene is bound, the receptors turn off, presumably thru a conformational change
This model comes from analysis of null mutants and gain-of-function mutations.

12. A receptor that always shuts off signaling is dominant
Responses ON
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

13. Genetic epistasis studies determined the order of action of the genes+ =
etr1
ctr1
etr1 ctr1
ETR1
CTR1
responses
ethylene
The double mutant has the same phenotype as ctr1, indicating that it acts downstream from ETR1

14. CTR1 is a negative regulator of ethylene signaling
Air
Ethylene
Wild type
ctr1
The 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
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: with permission from Elsevier.

15. CTR1 acts through EIN2, a positive regulator of ET signaling
+ Ethylene ER
nucleus
C-END
In ethylene, the C-terminus of EIN2 is cleaved and moves to the nucleus
EIN2 has 12 membrane spanning domains and is ER localized
EIN2
Responses ON )
cytoplasm
CTR1 (inactive)
Genetic studies show that EIN2 acts downstream of CTR1
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: with permission from Elsevier.

16. 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 ethylene-responsive genes
EIN2
C-END ?
GCC
EBS
ERF1
EIN3/EIL1
Nucleus
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: with permission from Elsevier.

17. Transcription factors mediate many of ethylene’s functions
Fruit ripening genes
FLS2, encoding a receptor that recognizes pathogens
C2H4 Responsive Gene
GCC
EBS
ERF1
EIN3/EIL1
Defense response genes
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: with permission from Elsevier.

18. Ethylene’s roles in whole-plant processes
Shoot and root elongation
Reproductive development
Sex determination
Petal and leaf senescence
Fruit ripening
Flooding responses –
Aerenchyma formation, leaf epinasty
Deepwater rice
Pathogen responses
Interactions with other hormones

19. Ethylene restricts elongation of the shoot and root in the dark
C2H4

20. Ethylene synthesis increases dramatically during fruit ripening
Ethylene accumulation
On the x-axis of the graph, dpa is days post anthesis
Giovannoni, J.J. (2004). Genetic regulation of fruit development and ripening. Plant Cell 16: S

21. Ethylene synthesis increases upon hypoxia caused by flooding
C2H4 O2
Ethylene induces cell death or cell separation and formation of aerenchyma – air channels through which oxygen can move into roots
When flooded, roots cannot take up oxygen, and become hypoxic – oxygen deprived.
Normally, soil has air pockets from which plant roots can take up oxygen.
Hypoxia induces ACC synthase and ethylene production
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.

22. In deepwater rice, ethylene induces internode elongation
Preserved deepwater rice specimen
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: , copyright Photo credit Moto Ashikari, Nagoya University.
Deepwater
Dramatic movies showing rice extension growth after flooding are available as supplemental material for Hattori et al. (2009) at
(Pra Nakorn Sri Ayutthaya Rice Research Center, Thailand).

23. Ongoing research - 1 ACS ACO
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?

24. Ongoing research - 2
CTR1
What are the roles of MAP kinases in synthesis and signaling?
How is EIN2 cleaved and de-phosphorylated?
EIN2
EIN3/EIL1
ACS
What role if any is played by the histidine kinase domain in the receptors? What do the different receptor isoforms do? S
S S S P
ETR1
Many other ethylene-response mutants are being characterized and integrated into the pathway – what do they 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: , by permission of Oxford University Press.