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Which of these seedlings is insensitive to the plant hormone ethylene? Seedling 1 Seedling 2

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?.

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Which of these seedlings is insensitive to the plant hormone ethylene? Seedling 1 Seedling 2

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  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? • Seedling 1 • Seedling 2 • Seedling 3 No ethylene + ethylene 1 2 3

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

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

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

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

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

  8. What kind of mutant is this abi1-1mutant? Wild type abi1-1 ABA ABA NO Dormancy (Seeds germinate!) Dormancy (No germination) NO ABA NO ABA NO Dormancy (Seeds germinate) NO Dormancy (Seeds germinate) ABA overproduction mutant ABA constitutive response mutant ABA insensitive mutant

  9. abi1-1 mutants are ABA-insensitive in all their responses Germination is not inhibited on ABA Root growth is not inhibited on ABA Guard cells are not ABA-responsive Wild type abi1 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.

  10. What would be the phenotype of an ABA constitutive response mutant? Wild type abi1-1 ABA ABA Dormancy (No germination) Dormancy (No germination) NO ABA NO ABA NO Dormancy (Seeds germinate) Dormancy (No germination)

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

  12. air Ethylene-Response Mutants in Arabidopsis Ethylene-insensitive mutants etr1 etr2 ein4 (dominant) ein2 ein3 ein5 (recessive) ein6 ein7 C2H4 Constitutive-response mutants ctr1 (recessive) (eto1)

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

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

  15. Yeast two-hybrid assay shows interaction of ETR1 and ERS ethylene receptors with the CTR1 protein kinase Yeast colonies Clark K L et al. PNAS 1998;95:5401-5406

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

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

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

  19. Underlying principle of the Yeast Two-Hybrid Assay “PREY” AD Y “BAIT” X DB HIS3 or lacZ UAS Promoter Reporter Gene 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 Y AD DB HIS3 or lacZ Y UAS Promoter Reporter Gene AD z X X No transcription DB HIS3 or lacZ UAS Promoter Reporter Gene

  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 Y AD DB HIS3 or lacZ Y UAS Promoter Reporter Gene AD z X X No transcription DB HIS3 or lacZ UAS Promoter Reporter Gene

  23. First we have to clone our bait and prey genes into yeast plasmids to express the proteins fused to the DB and AD Bait *Transform the plasmids into yeast cells Prey Resulting proteins that are produced by the yeast cells Plasmids that are constructed in the lab

  24. LAB: Yeast 2-hybrid assays with ethylene signaling proteins 4 and 5 ETR1 An ethylene receptor Ethylene binding domain Signaling domain 2 ETR2 An ethylene receptor Ethylene binding domain Signaling domain 8 7 CTR1 A protein kinase Regulatory domain Kinase domain 1 EIN2 A protein of unknown function Membrane domain Soluble domain 3 = empty prey plasmid; 6 = empty bait plasmid

  25. RECORD YOUR DATA

  26. Ethylene signaling pathway C2H4 Lumen ETR2 ETR1 EIN2 N N ER Cu+ Cu+ N CTR1 CTR1 C 5 4 2 8 8 1 7 7 - Nucleus Cytoplasm Ethylene Responsive Gene Expression

  27. Ethylene signaling pathway C2H4 Lumen ETR2 ETR1 EIN2 N N ER Cu+ Cu+ N C 5 2 7 1 4 8 - CTR1 Nucleus Cytoplasm Ethylene Responsive Gene Expression

  28. Ethylene signaling pathway C2H4 Lumen ETR2 ETR1 EIN2 N N ER Cu+ Cu+ N C 7 2 5 1 4 8 - CTR1 Nucleus Cytoplasm Ethylene Responsive Gene Expression

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

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