1. 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?

2. Which of these seedlings is insensitive to the plant hormone ethylene? 1.Seedling 1 2.Seedling 2 3.Seedling 3 1 2 3 No ethylene + ethylene

3. Which seedling is a “constitutive ethylene- response” mutant? 1.Seedling 1 2.Seedling 2 3.Seedling 3 4.Seedling 4 1 2 3 4 No ethylene + ethylene

4. + ACC No ACC Wild typeMutant Ethylene insensitive mutants (“ein”) *

5. + ACC No ACC Wild typeMutant Constitutive ethylene response mutants (“ctr”) *

6. Ethylene responses in Arabidopsis “Triple Response” Inhibition of leaf cell expansion Senescence Ethylene-induced gene expression

7. A dormancy mutant called abi1-1 Wild typeabi1-1 Dormancy (No germination) ABA NO Dormancy (Seeds germinate) NO ABA NO Dormancy (Seeds germinate) NO ABA ABA NO Dormancy (Seeds germinate!)

8. What kind of mutant is this abi1-1 mutant? Wild typeabi1-1 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

9. 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. 1448-14521452-1455 Root growth is not inhibited on ABA Guard cells are not ABA- responsive Wild type abi1 Germination is not inhibited on ABA

10. Wild typeabi1-1 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?

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

12. 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 C2H4C2H4

13. CHECK YOUR PLATES – Can you find any mutants?

14. Some of the proteins in the ethylene signaling pathway CTR1 EIN2 Kinase domain Soluble domainMembrane domain Regulatory domain ETR1 Signaling domain Ethylene binding domain An ethylene receptor ETR2 Signaling domain Ethylene binding domain An ethylene receptor A protein kinase A protein of unknown function

15. 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

16. Introduction to transcription activation DB AD X Y UAS Promoter sequence Coding Sequence Coding sequence of a gene Inside the NUCLEUS of the yeast cell DNA transcription mRNA translation Protein DB AD = transcription activator

17. Y UAS Promoter sequence DNA Transcriptional activators have 2 domains DB = DNA binding domain AD = Activation domain DB AD Introduction to transcription activation Coding Sequence

18. Underlying principle of the Yeast Two-Hybrid Assay DB AD Y HIS3 or lacZ UAS X PromoterReporter Gene Reporter will be expressed X AD Y Interaction of X and Y proteins DB HIS3 or lacZ UAS PromoterReporter Gene

19. Underlying principle of the Yeast Two-Hybrid Assay DB AD Y HIS3 or lacZ UAS X PromoterReporter 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

20. Underlying principle of the Yeast Two-Hybrid Assay X DB HIS3 or lacZ UAS PromoterReporter Gene X AD z No transcription Y HIS3 or lacZ UAS PromoterReporter Gene AD Y DB X

21. 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

22. How do we get these proteins into yeast cells so that we can test whether they interact? X DB HIS3 or lacZ UAS PromoterReporter Gene X AD z No transcription Y HIS3 or lacZ UAS PromoterReporter Gene AD Y DB X

23. 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

25. LAB: Yeast 2-hybrid assays with ethylene signaling proteins CTR1 EIN2 Kinase domain Soluble domainMembrane domain Regulatory domain ETR1 Signaling domain Ethylene binding domain An ethylene receptor ETR2 Signaling domain Ethylene 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

26. RECORD YOUR DATA

27. Ethylene Responsive Gene Expression ETR1 Nucleus - Cytoplasm ER EIN2 N Lumen C ETR2 Ethylene signaling pathway N C2H4C2H4 N Cu + 1 CTR1 7 8 5 4 2 7 8

28. Ethylene Responsive Gene Expression ETR1 Nucleus - Cytoplasm ER EIN2 N Lumen C ETR2 Ethylene signaling pathway N C2H4C2H4 N Cu + 1 5 4 2 7 8 CTR1

29. Ethylene Responsive Gene Expression ETR1 Nucleus - ER Lumen Ethylene signaling pathway N C2H4C2H4 N Cu + EIN2 N C 1 4 ETR2 Cu + 2 Cytoplasm 7 8 CTR1 5

30. RESULTS FROM CHANG LAB ++ + + ? ? no Did anyone have this one turn blue?