Signal transduction in higher plants I

; A gaseous hormone, ethylene (C2H4)

Sang-Dong Yoo

Ethylene (C2H4)

Major References

Bleecker AB, Estelle MA, Somerville C, Kende H (1988)Insensitivity to ethylene conferred by a dominant mutation inArabidopsis thaliana. Science 241: 1086-1089Bleecker AB and Kende H (2000) Ethylene: a gaseous signal Molecule in plants. Annu Rev Cell Dev Biol 16: 1-18Hua J, Meyerowitz EM (1998) Ethylene responses are negatively Regulated by a receptor gene family in Arabidopsis. Cell 94: 261-271Schaller GE and Kieber JJ (2002) Ethylene. www.aspb.org/publication/arabidopisisWang Q, Hall AE, O’Malley R, Bleecker AB (2003) Canonical histidineKinase activity of the transmitter domain of the ETR1 ethylene Receptor from Arabidopsis is not required for signal transmission.PNAS 100: 352-357Cho YH, Yoo SD (2007) ETR1 Histidine kinase activity of ArabidopsisPromotes plant growth. Plant Physiol. In press.

Ethylene (C2H4)

Discovery of ethyleneIlluminating gas - senescence and abscission -ethyleneBioassay (triple responses)

Chemical nature of ethyleneGas (gas chromatography)Membrane permeableWater insoluble

Biological significance of ethylene in plantsGerminationSeedling growthOrgan senescence and abscissionFruit ripening (climacteric fruits - biosynthesis of ethylene)

Transcriptional regulation

Yang’s cycle

(e.g. CDPK)

(ETO1)

Triple response of etiolated seedlings with ethylene

• short hypocotyl

• thick radial growth

•apical hook formation

Screening of ethylene response mutants

Constitutive ethylene response in air (CER)

Ethylene resistant/insensitive in ethylene (ETR/EIN)

ETOCTR

etr1-1 with ethylene in dark

Screening of mutagenized seed population with ethylene in dark

etr1-1 in C2H4

C2H4air

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

etr1-1 in C2H4

C2H4air

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

EIN3

EBS 1º responsive genes

?

2º responsive genesGCC

nucleus

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)

etr1-1 in C2H4

C2H4air

Col-0 in C2H4

ctr1-1

ein2-1

ein3-1

Air C2H4

ein2-1

etr1-1 in C2H4

C2H4Air

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

Genetics of ethylene signaling + C2H4

ETR1

CTR1

EIN3

EBS1º responsive genes

EIN2

?

2º responsive genesGCC

(~ 6 more)

(4 more)

(~ 60 more)

(>120 RAPs)

etr1-1 in C2H4

C2H4air

Col-0 in C2H4

ctr1-1

ein2-1

ein3-1

etr1-1 in C2H4

C2H4air

Col-0 in C2H4

ctr1-1

ein2-1

ein3-1

etr1-1;ctr1-1 is bigger than ctr1-1- CTR1 is not a sol player- CTR1 independent ETR1 function

Membrane spanning domainEthylene binding site through copper ion

Histidine kinase domain

Receiver domain

CTR1 interacting domain

GAF domain

Putative signal peptide

ER localization

(tobacco homolog-PM localization)

Dominance of etr1-1 mutants Not clear whether gain/loss of function

– neo function by gain of functionNegative regulator of ethylene signaling ???

Isolation of loss of function mutants for ethylene receptorsLack 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

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/etr1*(wt phenotype-intragenicinsensitive - extragenic_etr1)

F1 etr1-1/etr1* (recessive)

ERS1

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 responseadult plant – normal growth and development with smaller leaves (-suc; severe phenotype)

Single etr2, ein4 and ers2 loss of function mutants do not appear to have ethylene response defectsLight/dark grown seedlings – normal growth and development – wild type like ethylene response – failure to find mutants in original genetic screening

wt

Recessive singleetr1 mutants

wt

recessive single etr 2, ers2 or ein4 mutants

Screening of mutagenized seed population with ethylene in dark

GF etr1-1

LF etr1

LF ers1LF ers2

LF ein4

LF etr2

etr1; ein4 double loss of function mutants have an ethylene response-like phenotype in airLight 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

3x

2x

ctr1-1

etr1, etr2 and ein4 triple mutants display strong ethylene response phenotype in airlight grown plants in air - reduced in size of cotyledon, first few sets of true leaves, epinatic growth --- alleviated symptoms latter stageAVG or Ag2+ can enhance the hypocotyl growth but it is still 1/2 of wt sizedark grown plants in air – very short hypocotyls, roots exaggerated apical hooks – typical ethylene response

ers2, etr2, ein4 ?

?

?

3x

4x

0 3x 4x ctr1-1

etr1, etr2, ein4, ers2 quadruple loss of function mutants have constitutive ethylene responseslight 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

constitutive response phenotype of the ers1;etr1 double loss of function mutnatslight 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

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

Putative signal peptide

Canonical histidine kinase

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

.Genetic redundancy between members of the ethylene receptor gene family

.Ethylene responses are negatively regulated by a receptor gene familyLoss 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

ctr1-1 - activated MAPK

ETR1

CTR1

MKK

MPK

EIN3 (EIL1)

EBS 1º responsive genes

EIN2 ?

?

2º responsive genesGCC

+ C2H4

MKKK x60

MKK x10

MPK x22

?

Key Question:MAPK Cascade inEthylene Signaling?

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.

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.

Does ETR1 function more than ethylene signaling?

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)

Let’s look at ETR1 again!

• 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.

Membrane spanning domain

ETR1

659353

H G1 G2 DGAF

HK Receiver

• 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 AHP1-3 --- 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).

• 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).

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

ETR1 HK function not in ethylene signaling

ACC10 M

ACC1M

Control

0

5

10

15

20

25

30Control

1M ACC

10M ACC

Hy

po

co

tyl

len

gth

(m

m)

*

*

*

**

*****

***

***

W T

etr

1-

7 cE

TR

1 etr

1-1 etr

1-

2

cH

35

3Q

gE

TR

1

1 2 3 4 5 6 7

W T

etr

1-

7 cE

TR

1 etr

1-1 etr

1-

2

cH

35

3Q

gE

TR

1

ETR1

RBC

WT

cH35

3Q

cET

R1

gE

TR

1

Transgenic etr1-7

etr1

-7

• Transgenics with or without functional HK make no difference in ACC (ethylene )-triple responses!

ETR1 HK function in ethylene independent

growth

• 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.

W T

etr

1-

7 cE

TR

1 etr

1-1 etr

1-

2

cH

35

3Q

gE

TR

1

1 2 3 4 5 6 7

Control

Ag+

100M

Hyp

ocoty

l le

ng

th (

mm

)

0

5

10

15

20

25

30

1 2 3 4 5 6 7

Control100M Ag+

W T

etr

1-

7 cE

TR

1 etr

1-1 etr

1-

2

cH

35

3Q

gE

TR

1

**** ***

***

***

*

*

ETR1 HK function in ethylene independent

growth

• Transgenics with or without functional HK make growth rate difference under grwth promoting high light condition (bottom of A).

A

WT etr1-7 cETR1 gETR1 cH353Q

Transgenic etr1-7

Transgenic etr1-7

0

1

2

3

4

5

Col etr1-7 cETR gETR H353Q

Ro

se

tte

Siz

e

(cm

)

75

250

B

Col etr1-7 cETR gETR H353Q etr1-1 WT etr1-7 cETR1 gETR1 cH353Q

ETR1 HK in cellular organization

- 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 ?

etr1-7

WT etr1-1 etr1-7

cETR1 gETR1 H353Q

A type ARR is activated- two component system is on?

• Transgenics with or without functional HK make difference in ARR5 transcription-indicating activation of two component systems in the transgenics with intact ETR1 HK.

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5

0

1

2

3

4

5

6

7

8

1 2 3 4 5

Rel

ativ

e g

ene

exp

ress

ion

ERF1 ARR5

cH35

3Q

cET

R1

etr1

-1

cH35

3Q

cET

R1

etr1

-7

etr1

-1WT

WT

A

etr1

-7

ETR1 HK function in the two component system

• ETR1 HK can activates two component system through the conserved H and D residues

0

30

60

90

120

150

180

cont ETR1WT ETR1HQ ETR1DA ETR1DE

ETR1

ARR6LUC/NOSGUS

Rel

ativ

e p

rom

ote

r ac

tivi

ty

Cont ETR1WT H353Q D659A D659E

ARR10/etr1-7

B

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.

THANKS for your ATTENTION