Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype.

The mutants that we discover correspond to mutated genes.

Why is this extremely useful?

• Which of these seedlings is insensitive to the plant hormone ethylene?

1. Seedling 12. Seedling 23. Seedling 3

1 2 3

Noethylene

+ ethylene

• Which seedling is a “constitutive ethylene-response” mutant?

1. Seedling 12. Seedling 23. Seedling 34. Seedling 4

1 2 3 4

Noethylene

+ ethylene

+ ACC

No ACC

Wild type Mutant

Ethylene insensitive mutants (“ein”)

*

+ ACC

No ACC

Wild type Mutant

Constitutive ethylene response mutants (“ctr”)

*

Ethylene responses in Arabidopsis

“TripleResponse”

Inhibition of leaf cell expansion

Senescence

Ethylene-induced gene expression

A dormancy mutant called abi1-1Wild type abi1-1

Dormancy(No germination)

ABA

NO Dormancy (Seeds germinate)

NO ABA

NO Dormancy (Seeds germinate)

NO ABA

ABA

NO Dormancy (Seeds germinate!)

What kind of mutant is this abi1-1 mutant?

Wild type abi1-1

Dormancy(No germination)

ABA

NO Dormancy (Seeds germinate)

NO ABA

NO Dormancy (Seeds germinate)

NO ABA

ABA

NO Dormancy (Seeds germinate!)

1. ABA overproduction mutant 2. ABA constitutive response mutant 3. ABA insensitive mutant

abi1-1 mutants are ABA-insensitive in all their responses

Leung, J., Bouvier-Durand, M., Morris, P., Guerrier, D., Chefdor, F., and Giraudat, J. (1994). Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase. Science 264: 1448-1452; Meyer, K., Leube, M., and Grill, E. (1994). A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana. Science 264: 1452-1455.

Root growth is not inhibited on

ABA

Guard cells are not ABA-

responsiveWildtype abi1

Germination is not inhibited on

ABA

Wild type abi1-1

Dormancy(No germination)

ABA

NO Dormancy (Seeds germinate)

NO ABA

Dormancy (No germination)

NO ABA

ABA

Dormancy (No germination)

What would be the phenotype of an ABA constitutive response mutant?

Plant growth, development, and survival depend on appropriate responses to a diverse array of constantly fluctuating external and internal signals

ctr1 (recessive)

(eto1)

ein2 ein3 ein5 (recessive)ein6 ein7

Constitutive-response mutants

Ethylene-insensitive mutants

etr1 etr2 ein4 (dominant)

Ethylene-Response Mutants in Arabidopsis

air

C2H4

CHECK YOUR PLATES – Can you find any mutants?

Some of the proteins in the ethylene signaling pathway

CTR1

EIN2

Kinase domain

Soluble domainMembrane domain

Regulatory domain

ETR1Signaling domainEthylen

e binding domain

An ethylene receptor

ETR2Signaling domainEthylen

e binding domain

An ethylene receptor

A protein kinase

A protein of unknown function

Yeast two-hybrid assay shows interaction of ETR1 and ERS ethylene receptors with the CTR1 protein kinase

Clark K L et al. PNAS 1998;95:5401-5406

Yeast colonies

Introduction to transcription activation

DBAD

X Y

UAS

Promoter sequence

Coding Sequence

Coding sequence of a gene

Inside the NUCLEUS of the

yeast cell

DNA

transcription

mRNA

translation

ProteinDB

AD = transcription activator

Y

UAS

Promoter sequence

DNA

Transcriptional activators have 2 domainsDB = DNA binding domainAD = Activation domain

DB

AD

Introduction to transcription activation

Coding Sequence

Underlying principle of the Yeast Two-Hybrid Assay

DB

AD Y

HIS3 or lacZUAS

X

Promoter Reporter Gene

Reporter will be expressed X

AD

Y

Interaction of X and Y proteins

DBHIS3 or lacZ

UAS

Promoter Reporter Gene

Underlying principle of the Yeast Two-Hybrid Assay

DB

AD Y

HIS3 or lacZUAS

X

Promoter Reporter Gene

“BAIT”

“PREY”

The “BAIT” is defined as the protein fused to the DB

The “PREY” is defined as any protein fused to the AD

Underlying principle of the Yeast Two-Hybrid Assay

X

DBHIS3 or lacZ

UAS

Promoter Reporter Gene

X

AD

z No transcription

Y

HIS3 or lacZUAS

Promoter Reporter Gene

AD Y

DB

X

Interaction of ETR1 and ERS ethylene receptors with the CTR1 protein kinase in the yeast two-hybrid assay.

Clark K L et al. PNAS 1998;95:5401-5406

How do we get these proteins into yeast cells so that we can test whether they interact?

X

DBHIS3 or lacZ

UAS

Promoter Reporter Gene

X

AD

z No transcription

Y

HIS3 or lacZUAS

Promoter Reporter Gene

AD Y

DB

X

First we have to clone our bait and prey genes into yeast plasmids to express the proteins fused to the DB and AD

*Transform the plasmids into yeast cells

Bait

Prey

Plasmids that are constructed in the lab

Resulting proteins that are produced by the yeast cells

LAB: Yeast 2-hybrid assays with ethylene signaling proteins

CTR1

EIN2

Kinase domain

Soluble domainMembrane domain

Regulatory domain

ETR1Signaling domainEthylen

e binding domain

An ethylene receptor

ETR2Signaling domainEthylen

e binding domain

An ethylene receptor

A protein kinase

A protein of unknown function

4 and 5

8

1

7

2

3 = empty prey plasmid; 6 = empty bait plasmid

RECORD YOUR DATA

Ethylene Responsive Gene Expression

ETR1

Nucleus

-

Cytoplasm

ER

EIN2

N

Lumen

C

ETR2

Ethylene signaling pathway

N

C2H4

N

Cu+

Cu+

1 CTR1

785 42

CTR1

78

Ethylene Responsive Gene Expression

ETR1

Nucleus

-

Cytoplasm

ER

EIN2

N

Lumen

C

ETR2

Ethylene signaling pathway

N

C2H4

N

Cu+

Cu+

15 42 78CTR1

Ethylene Responsive Gene Expression

ETR1

Nucleus

-

ER

Lumen

Ethylene signaling pathway

N

C2H4

N

Cu+

EIN2

N

C14

ETR2Cu+

2

Cytoplasm

78CTR1

5

RESULTS FROM CHANG LAB

++ ++

++++++ ++

+ +

? ?

nonono

nonono

nonono

nonono

no no

nono

Did anyone have this one turn blue?