1. Signal transduction in higher plants I
; A gaseous hormone, ethylene (C2H4)
Sang-Dong Yoo

2. Ethylene (C2H4) Major References
Bleecker AB, Estelle MA, Somerville C, Kende H (1988)
Insensitivity to ethylene conferred by a dominant mutation in
Arabidopsis thaliana. Science 241:
Bleecker AB and Kende H (2000) Ethylene: a gaseous signal
Molecule in plants. Annu Rev Cell Dev Biol 16: 1-18
Hua J, Meyerowitz EM (1998) Ethylene responses are negatively
Regulated by a receptor gene family in Arabidopsis. Cell 94:
Schaller GE and Kieber JJ (2002) Ethylene.
/arabidopisis
Wang Q, Hall AE, O’Malley R, Bleecker AB (2003) Canonical histidine
Kinase activity of the transmitter domain of the ETR1 ethylene
Receptor from Arabidopsis is not required for signal transmission.
PNAS 100:
Cho YH, Yoo SD (2007) ETR1 Histidine kinase activity of Arabidopsis
Promotes plant growth. Plant Physiol. In press.

3. Ethylene (C2H4) Discovery of ethylene
Illuminating gas - senescence and abscission -ethylene
Bioassay (triple responses)
Chemical nature of ethylene
Gas (gas chromatography)
Membrane permeable
Water insoluble
Biological significance of ethylene in plants
Germination
Seedling growth
Organ senescence and abscission
Fruit ripening (climacteric fruits - biosynthesis of ethylene)

4. Yang’s cycle
Transcriptional
regulation

5. (ETO1)
(e.g. CDPK)

6. Triple response of etiolated seedlings with ethylene
short hypocotyl
thick radial growth
apical hook formation

7. Screening of ethylene response mutants
Constitutive ethylene response in air (CER)
ETO
CTR
Ethylene resistant/insensitive in ethylene (ETR/EIN)

8. etr1-1 with ethylene in dark
Screening of mutagenized seed population with ethylene in dark

9. Epistatic analysis between CER (ctr1-1) and ETR/EIN mutants
etr1-1 in C2H4
C2H4
air
Col-0 in C2H4
ctr1-1
ein2-1
ein3-1
Epistatic analysis between CER (ctr1-1) and ETR/EIN mutants
– linear signaling pathway
Biochemistry of ethylene signal transduction
gene cloning – protein identity
protein ID based functional positioning in signaling pathway
manipulation of gene action in planta

10. etr1-1 in C2H4
C2H4
air
Col-0 in C2H4
ctr1-1
ein2-1
Air C2H4
ein3-1
EIN3
- a mutigen family (5-6?) of transcription factors, targeting EBS in ERF1 (ethylene primary responsive-AP2/EREBP-transcription factors)
- acts as a homodimer (in vitro) to activate ERF1 transcription - ERF1 binds GCC box in ethylene 2nd responsive genes (e.g. PDF 1.2)
- pre-existing TF, but not induced by ethylene; protein modification or accumulation is important

11. EIN3
EBS
1º responsive genes ?
nucleus
2º responsive genes
GCC

12. - Nramp family of 12 membrane spanning motif metal ion-transporter
etr1-1 in C2H4
C2H4
air
Col-0 in C2H4
ctr1-1
ein2-1
ein3-1
Air C2H4
EIN2
- Nramp family of 12 membrane spanning motif metal ion-transporter
- functional positioning ??? – metal ion as a secondary messenger???
- C-terminal end (cytosolic portion) can complement the mutant (not all, but subset of ethylene responses)

13. - Raf-like ser/thr protein kinase (MKKK); initiate a MAPkinase cascade
etr1-1 in C2H4
C2H4
Air
Col-0 in C2H4
ctr1-1
ein2-1
ein3-1
CTR1
- Raf-like ser/thr protein kinase (MKKK); initiate a MAPkinase cascade
- CTR1 directly interacts with ethylene receptor class proteins, ETR1 and ERS1
- Recessive nature of ctr1, showing constitutive ethylene response indicates its negative regulation on ethylene response

14. etr1-1 in C2H4
C2H4
air
Col-0 in C2H4
ctr1-1
ein2-1
ein3-1

15. Col-0 in C2H4 etr1-1 in C2H4 air ctr1-1 ein2-1 ein3-1 C2H4
etr1-1;ctr1-1 is bigger than ctr1-1
- CTR1 is not a sol player
- CTR1 independent ETR1 function
Col-0 in C2H4
etr1-1 in C2H4
air
C2H4
ctr1-1
ein2-1
ein3-1

16. GAF domain
Histidine kinase domain
CTR1 interacting domain
Receiver domain
Membrane spanning domain
Ethylene binding site through copper ion

17. Putative signal peptide
ER localization
(tobacco homolog-PM localization)
Putative signal peptide

18. Dominance of etr1-1 mutants
Not clear whether gain/loss of function
– neo function by gain of function
Negative regulator of ethylene signaling ???
Isolation of loss of function mutants for ethylene receptors
Lack of recessive allele
1) Lethal mutation (e.g. embryo mutants)
2) Gene functional redundancy (many cases in plants)
3) Not involved in signaling (neo function by mutation)
2),3) – intragenic suppressor will show wild type response to ethylene

19. Intragenic suppressor screening
etr1-1/etr1-1
EMS M1 M2
(screening for ethylene response like wt; etr1*) M3
etr1-1/etr1-1 x etr1*/etr1*
ETR1/ETR1 x etr1*/etr1* F1
etr1-1/etr1*
(recessive) F1
ETR1/etr1*
(wt phenotype-intragenic
insensitive - extragenic_etr1)

21. ERS1

22. etr1 loss of function mutants have defects in cell elongation
- wt like phenotype and ethylene dosage-dependent response (saturated by over 10ppm)
- shorter hypocotyl and root than wt–defect in cell elongation (etr1-7, ctr1-1 have a even shorter hypocotyl) ctr1-1 synergistic effect–may not be related to ethylene response
adult plant – normal growth and development with smaller leaves (-suc; severe phenotype)

23. wt
Single etr2, ein4 and ers2 loss of function mutants do not appear to have ethylene response defects
Light/dark grown seedlings – normal growth and development – wild type like ethylene response – failure to find mutants in original genetic screening
Recessive single
etr1 mutants wt
recessive single etr 2, ers2 or ein4 mutants

24. GF etr1-1
LF ers2
LF ers1
LF etr1
LF ein4
LF etr2
Screening of mutagenized seed population with ethylene in dark

25. etr1; ein4 double loss of function mutants have an ethylene response-like phenotype in air
Light grown plants in air – reduced leaf size (resulted from reduced cell size) – shorter roots with more hairs (ethylene response of wt)
Dark grown plants in air – similar to etr1 loss of function mutants
; ein4 synergistic effect only with etr1 in light grown plants

26. 2x
etr1, etr2 and ein4 triple mutants display strong ethylene response phenotype in air
light grown plants in air - reduced in size of cotyledon, first few sets of true leaves, epinatic growth --- alleviated symptoms latter stage
AVG or Ag2+ can enhance the hypocotyl growth but it is still 1/2 of wt size
dark grown plants in air – very short hypocotyls, roots exaggerated apical hooks – typical ethylene response
ctr1-1 3x
ers2, etr2, ein4 ?

27. 3x
0 3x 4x ctr1-1
etr1, etr2, ein4, ers2 quadruple loss of function mutants have constitutive ethylene responses
light grown plants in air – sever phenotype: compact and epinastic small leaves, >1/2 plants wilted and die before bolting, sicker and susceptible to pathogen, delayed flowering timing, shorter inflorescence with smaller flowers 4x ? ?

28. constitutive response phenotype of the ers1;etr1 double loss of function mutnats
light grown plants in air – 15% loss of leaf size compared to wt or ers1, delayed flowering timing, stunt inflorescence with tiny sterile flowers (severity ers1,etr1 = 4x > ctr1 = 3x) – however, not much sever phenotype in dark---- Class 1 receptors caused severe ethylene responsive phenotype

29. ETR1-driven expression of class 1 but not class 2 cDNAs rescues the ers1;etr1 double loss of function mutant phenotype

30. Putative signal peptide
Canonical histidine kinase
Putative signal peptide

31. Kinase-inactive ETR1 genomic clones can also rescue the ers1; etr1 loss of function mutant phenotype
Ethylene saturated phenotype:
ers1;etr1 double mutant w/o C2H4

32. .Genetic redundancy between members of the ethylene receptor gene family
.Ethylene responses are negatively regulated by a receptor gene family
Loss of function mutant causes ethylene responsive phenotype rather than ethylene insensitive phenotype – receptors are actively suppress ethylene response without ethylene.
.Canonical histidine kinase activity is not required for ETR1 function to activate CTR1

33. ctr1-1
- activated MAPK

34. + C2H4 ? MKKK x60 MKK x10 MPK x22 ? Key Question: MAPK Cascade in
ETR1
CTR1
MKK
MPK
EIN3 (EIL1)
EBS
1º responsive genes
EIN2 ? ?
2º responsive genes
GCC
Key Question:
MAPK Cascade in
Ethylene Signaling?

35. ETR1 HK Function in Plant Growth Promotion
Question?
Since ethylene signaling transfers via a CTR1 downstream ser/thr protein kinase (e.g. MAPK cascades) signaling pathway, the functions of well-conserved His kinase of ETR1 has been puzzling.

36. What’s known about ETR1
A lot! Thanks to the late Dr. Bleecker’s excellent contributions in the ethylene signaling field.
ETR1 is an ethylene-binding membrane receptor kinase.
ETR1 is homodimerized.
ETR1 plays a negative regulation on ethylene signaling.
ETR1 has a histidine kinase activity.

37. Does ETR1 function more than ethylene signaling?

38. Loss of function etr1 mutants have defects in cell elongation
Etiolated seedlings show WT- like ethylene response and dosage-dependent response (saturated by over 10ppm) : even shorter hypocotyls and roots than WT---ctr1-1 synergistic effect with ethylene–may be independent of ethylene signaling?
Adult plants–normal growth and development, but smaller leaves (-suc; severe phenotype)

39. Let’s look at ETR1 again!
ETR1
GAF
H G1 G2 D
353
659
Membrane
spanning domain HK
Receiver
ETR1 contains both an N-terminal ligand-binding hydrophobic domain and light signaling-implicated GAF domain (Chang et al., 1993). ETR1 also has a C-terminal histidine kinase (HK) domain fused with a response regulator motif.

40. Functional HK activity of ETR1 (Gamble et al
Functional HK activity of ETR1 (Gamble et al., 1998, 2002; Mousatche and Klee, 2004). the catalytic G1 and ATP-binding G2 domains-dependent autophosphorylation of ETR1 at His353
Protein-protein interaction between ETR1 and AHP ETR1 as an input of two-component phosphor-relay system (Urao et al., 2000).
ETR1-dependent phosphorylation of the B-type ARR2 and ethylene response transcription activation (Hass et al., 2004).

41. BUT! combinations of B-type arr mutants, including arr2 are still sensitive to ethylene (Mason et al., 2005).
ETR1 with inactive HK provides wild-type ethylene responsiveness in single etr1 or double etr1 ers1 null mutants (Gamble et al., 2002; Wang et al., 2003).

42. To Know ETR1 HK function To examine ETR1 HK function in vivo,
transgenic lines of etr1-7
+ a genomic transgene of ETR1 (gETR1)
+ a cDNA of ETR1 (cETR1)
+ a cDNA of ETR1H353Q : the glutamine (Q) replaces the only phosphorylatable histidine (H) 353 (Moussatche and Klee, 2004)
under the control of native 2.2kb ETR1 promoter

43. ETR1 HK function not in ethylene signaling5 10 15 20 25 30
Control
1mM ACC
10mM ACC
Hypocotyl length (mm) * **
***
ACC
10 mM
ACC
1mM 1 2 3 4 5 6 7 WT
etr1-7
cETR1
etr1-1
etr1-2
cH353Q
gETR1
ETR1
RBC WT
cH353Q
cETR1
gETR1
Transgenic etr1-7
etr1-7
Control WT
etr1-7
cETR1
etr1-1
etr1-2
cH353Q
gETR1
Transgenics with or without functional HK make no difference in ACC (ethylene )-triple responses!

44. ETR1 HK function in ethylene independent growth2 3 4 5 6 7 30
Control
Control
100mM Ag+ 25 *
*** 20 * *
***
Hypocotyl length (mm) 15
***
*** 10
Ag+
100mM 5 WT
gETR1
cH353Q
etr1-2 1
etr1-7 2 3 4
cETR1 5
etr1-1 6 7 WT
etr1-7
cETR1
etr1-1
etr1-2
cH353Q
gETR1
Interestingly! Transgenics with or without functional HK make growth rate difference in the endogenous/ambient or in the absence of ethylene.
ETR1 HK promotes etiolated seedling growth.

45. ETR1 HK function in ethylene independent growthA B 75
250 5
Rosette Size (cm) 4 3 2 1
WT etr cETR gETR1 cH353Q
Transgenic etr1-7
WT etr cETR1 gETR1 cH353Q
Col
etr1-7
cETR
gETR
H353Q
etr1-1
Col
etr1-7
cETR
gETR
H353Q
Transgenic etr1-7
Transgenics with or without functional HK make growth rate difference under grwth promoting high light condition (bottom of A).

46. ETR1 HK in cellular organization
WT etr etr1-7
cETR gETR H353Q
etr1-1 show abnormal chloroplasts structure-so as etr1-7; ETR1 function in chloroplasts development have been examined.
Either ETR1 with/without HK makes no difference in this chloroplast phenotype
: A second mutation in etr1-1 ?

47. A type ARR is activated- two component system is on?1 2 3 4 5 6 7 8 9
Relative gene expression
ERF1
ARR5
cH353Q
cETR1
etr1-1
etr1-7 WT A
Transgenics with or without functional HK make difference in ARR5 transcription-indicating activation of two component systems in the transgenics with intact ETR1 HK.

48. ETR1 HK function in the two component system30 60 90
120
150
180
cont
ETR1WT
ETR1HQ
ETR1DA
ETR1DE
ETR1
ARR6LUC/NOSGUS
Relative promoter activity
Cont ETR1WT H353Q D659A D659E
ARR10/etr1-7 B
ETR1 HK can activates two component system through the conserved H and D residues

49. We appreciate excellent contributions of Dr. Tony Bleecker and Dr
We appreciate excellent contributions of Dr. Tony Bleecker and Dr. Hans Kende who revived “plant hormone biology” with their first isolation and identification of plant hormone ethylene receptor. It was simply amazing! You will be remembered forever.

50. THANKS for your ATTENTION