1. Arabidopsis Experiments Forward Genetic Screen (Ethylene Insensitive Mutants) Reverse Genetic Screen / PCR Genotyping (H + - ATPase Mutants)

2. Arabidopsis A rabidopsis thaliana is the predominant model organism used by plant biologists today. Considered a “weed" in nature, this small mustard serves as an experimental subject for everything from root growth to flower development in the laboratory. Arabidopsis has gained prominence as a model organism for several reasons: A rabidopsis thaliana is the predominant model organism used by plant biologists today. Considered a “weed" in nature, this small mustard serves as an experimental subject for everything from root growth to flower development in the laboratory. Arabidopsis has gained prominence as a model organism for several reasons: Generation time. Seed to seed in about 42 days. Generation time. Seed to seed in about 42 days. Fecund. One Arabidopsis plant yields thousands of seeds. Fecund. One Arabidopsis plant yields thousands of seeds. Genome size. 125 megabases. Maize has 5000 mb, tobacco1500 mb. Genome size. 125 megabases. Maize has 5000 mb, tobacco1500 mb. Diploid. Relatively simple genome. Diploid. Relatively simple genome. Tractable. Easily worked and amenable to genetic, molecular genetic, physiological and biochemical studies. Tractable. Easily worked and amenable to genetic, molecular genetic, physiological and biochemical studies. Real plant. Roots, leaves, flowers, seeds, and a full component of physiological and biochemical processes. Real plant. Roots, leaves, flowers, seeds, and a full component of physiological and biochemical processes.

3. Arabidopsis History Linnaeus *Yours Truly

5. Forward vs. Reverse Genetics …we’ll be using both approaches. Treat thousands of organisms with a mutagen, Treat thousands of organisms with a mutagen, - random mutagenesis, Identify an individual with a phenotype of interest, Identify an individual with a phenotype of interest, Identify the gene. Identify the gene. Treat thousands of organisms with a mutagen (usually),Treat thousands of organisms with a mutagen (usually), –random mutagenesis, Identify an individual with a genotype of interest,Identify an individual with a genotype of interest, Identify the phenotype.Identify the phenotype. Forward Reverse

6. First, Forward Genetics Experiment #1 Conditional Screen for Ethylene Insensitivity

8. Ethylene “the gaseous hormone” Egyptians gassed figs in order to stimulate ripening, Egyptians gassed figs in order to stimulate ripening, The ancient Chinese burned incense in closed rooms to enhance the ripening of pears. The ancient Chinese burned incense in closed rooms to enhance the ripening of pears. In 1864, gas leaks from street lights were observed to stunt plant growth, twist plants, and abnormally thicken stems In 1864, gas leaks from street lights were observed to stunt plant growth, twist plants, and abnormally thicken stems Dimitry Neljubow (1901) showed that the active component was ethylene. Dimitry Neljubow (1901) showed that the active component was ethylene. R. Gane (1934) reported that plants synthesize ethylene. R. Gane (1934) reported that plants synthesize ethylene. H 2 C = CH 2

9. Receptor enzyme-linked receptor …found first in bacteria, then in plants, now in most eukaryotes, including mammals. Two-component regulators.

10. Ethylene …promotes fruit ripening, Ethylene signals the transition from unripe to ripe fruits, Ethylene signals the transition from unripe to ripe fruits, cell wall components are broken down, cell wall components are broken down, starches and acids are broken down resulting in “sweetening” and aromatic compounds, starches and acids are broken down resulting in “sweetening” and aromatic compounds, pigmentation may also be induced. pigmentation may also be induced.

11. Ethylene …promotes the “triple response”, …in etiolated seedlings, reduced stem elongation, reduced stem elongation, thicker stem, thicker stem, horizontal growth, horizontal growth, May provide the plant with “behavior” that will provide escape from soil impediments. May provide the plant with “behavior” that will provide escape from soil impediments.

12. Ethylene …mutant analysis, wild type ein ein (ethylene present), …ethylene insensitive. wild type ctr (ethylene absent), …constitutive triple response. ctr

13. Ethylene Signal Transduction …negative regulation. Tricky Concept(s) In the absence of ethylene, the enzyme receptor activates CTR1, active CTR1 inhibits the triple response, active CTR1 inhibits the triple response, With ethylene present, or the receptor “absent”, or the CTR1 gene mutated, the triple response is activated. the triple response is activated.

14. ein, etr*, etc, …blocks pathway. ethylene, …or ctr mutant, no ethylene …no triple response. active inactive induces transcription, erf : ethylene response factor. ?

15. Wedsnesday’s To Do, #1 ems-treated seeds Sterilizing/Planting 70% ETOH/0.1% Triton X 95% ETOH Murishige and Skoog Media (MS), plant minimal medium 0.5x strength +/- ACC. Germinating Breaking Dormancy Breaking Dormancy H 2 O/Imbibition, H 2 O/Imbibition, O 2 /Aeration, O 2 /Aeration, Cold/Prechilling "stratification” Cold/Prechilling "stratification” Inducing Germination Inducing Germination Light Light

16. ACC: 1-aminocyclopropane-1-carboxylate

17. Conditional Screen Grow on ACC, Grow on ACC, …in the dark (etiolated). Score for mutants, Score for mutants, Transfer to 0.5X MS (Murisige and Skoog) media (-ACC), Transfer to 0.5X MS (Murisige and Skoog) media (-ACC), Grow in light. Grow in light. NotthisClass.

18. What Next? Backcross to wild-type, Backcross to wild-type, what might the F1 and F2 tell us? what might the F1 and F2 tell us? Complementation tests? Complementation tests? dominant recessive Thought Experiments…

19. Second, Reverse Genetics Experiment #2 PCR genotyping of a t-DNA mutant

20. Wednesday’s To DO, #2 t-DNA insertion mutants Sterilizing/Planting 70% ETOH/0.1% Triton X 95% ETOH Murishige and Skoog Media (MS), plant minimal medium 0.5x strength With ACC. Germinating Breaking Dormancy Breaking Dormancy H 2 O/Imbibition, H 2 O/Imbibition, O 2 /Aeration, O 2 /Aeration, Cold/Prechilling "stratification” Cold/Prechilling "stratification” Inducing Germination Inducing Germination Light Light

21. Proton Pumps in planta Stems transport; sucrose hormones Leaves stomata (gas exchange) sucrose transport Anthers cell elongation Pollen tip growth Embryo/Seeds loading Roots root hair growth mineral uptake Arabidopsis

22. Adapted from Biochemistry and Molecular Biology of Plants, pp. 115 H + (protons) ATP synthase ATP hydrolase (ATPase) Transporters - carriers, - channels.

23. Arabidopsis Genome ~125 Mb (Megabases, million base pairs), Rice: 420 Mb, Human: 3 Gb, Rice: 420 Mb, Human: 3 Gb, 25,498 genes from 11,000 gene families, Rice: 32,000 - 50,000, Human: 25,000 - 66,000. Rice: 32,000 - 50,000, Human: 25,000 - 66,000.

24. PCR Genotyping I.T-DNA Insertion Mutants II.Hs Mt_DAN Hypervariable Region

25. Proton Pumps in planta Stems transport; sucrose hormones Leaves stomata (gas exchange) sucrose transport Anthers cell elongation Pollen tip growth Embryo/Seeds loading Roots root hair growth mineral uptake Arabidopsis

26. Arabidopsis H + -ATPase Gene Family Phylogenetic Family Tree (ClustalW --> Phylip: protdist, fitch) Baxter et al., Plant Physiol, 123, (2003)

27. Reverse Genetics Functional Genomics Gene DNA Sequence Gene Disruption Phenotype Analysis Function Mutate DNA Sequence Development Physiology Cell Biology Genetically Link

28. Agrobacterium Plant Cells Nature Ti-Plasmid T-DNA Hormones Opines Lab Selectable Markers Reporter Genes Genes Out : Ti genes, opine genes, In : DNA of choice. T-DNA

29. wtplantchromosome Ti Plasmid (from agro ) hormone genes (i.e. auxins) opaline nopaline virulencegenes virulencegenes hormone genes opaline, nopaline neoplastic transformation Agrobacterium tumefaciens Ti Plasmid (Tumor inducing) Mother Nature Agrofood

30. Construct T-DNA selection genes virulencegenes infect plant, select(2) for plants with T-DNA T-DNA (Transfer DNA) Laboratory transform, select(1) for agro with T-DNA Agrobacterium …if the T-DNA lands in a gene, the gene is disrupted. …can put other genes.

31. Probability of Finding an Insert in a Specific Gene thousands of inserts p = 1-(1-f) n p = probability of insertion event f = 1-(Genome/Size of Gene) n = number of T-DNA inserts

32. Knockology Plants/PoolsDNA/Pools

33. Set-Up DNA Pooling Set-Up DNA Pooling Seeds (9) Seedlings (225) DNA (225) 123456…30 Super Pools (2025) Germinate and grow seeds in liquid culture. Extract DNA, Super Pool DNA, Maintain lines as pools of seed. PCR Screen

34. 94 o 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG-- 3’ 5’--GCATGCATTAT CTGATCGTGAC--5’ Denature Step ~30 seconds ~65 o 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG-- 3’ 5’--GCATGCATTAT CTGATCGTGAC--5’ Annealing Step ~30 seconds 72 o 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG-- 3’ 5’--GCATGCATTAT CTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’ Synthesis ~1 minute/kb PCR

35. PCR Strategy 5’ 3’ Polymerase Chain Reaction (PCR), Polymerase Chain Reaction (PCR), with oligonucleotide primers with homology to the 5’ and 3’ ends of your gene, amplify the DNA sequence between the primers. with oligonucleotide primers with homology to the 5’ and 3’ ends of your gene, amplify the DNA sequence between the primers. Your gene Reaction: Product: Your gene amplified

36. Reverse Genetic PCR Strategy T-DNA Reaction: Product: Reaction: Product: none.

37. PCR Screens for Mutants

38. PCR Strategy T-DNA Reaction: Product: T-DNA Reaction: Product:

39. Find the Plant You are ~here

40. T-DNA Mutants Genetic Analysis tagged seed line tagged seed line isolate homozygous mutant isolate homozygous mutant backcross to wildtype backcross to wildtype 2x phenotype analysis phenotype analysis tt x TT (wt) Tt T-DNA Segregation TTTt tt T t T t F2

41. PCR Genotyping LtT 5’5’ 3’3’ 5’5’ 3’3’ heterozygote LtT 5’5’ 3’3’ 5’5’ 3’3’ homozygote wt LtT 5’5’ 3’3’ 5’5’ 3’3’ homozygote mutant

42. Genetic Analysis F2 Segregation Genetic Analysis F2 Segregation 1 : 2 : 1 TTTt tt T t T t Not Lethal 1 wt : 2 het TTTt tt T t T t Lethal 1 wt : 1 het TTTt tt T t T t Gametophyte Lethal

43. Mitochondrial DNA -16, 569 bp, -multiple copies per mt, -100 - 1000 mt per cell, -37 genes; -22 oxidative phosphorylation, -13 tRNA, -2 rRNA, -Mitochondrial Control Region.

44. Mitochondrial Control Region control region, –single promoter on each strand initiates transcription, –ori, D-loop, – replication loop topography, hypervariable region, –mutation rate 10x greater than genome.

45. Mitochondrial Control Region Hair follicle DNA extraction, PCR, Sequencing (at Cold Spring Harbor), Sequence analysis here at WWU. Link Out

46. This Week? “hypervariable” Wednesday (probably) Wednesday (probably) Mt-DNA isolation/PCR (maybe Fri.), Mt-DNA isolation/PCR (maybe Fri.), analyze EMS mutants on ACC screen. analyze EMS mutants on ACC screen. Wasps? Wasps? Friday (maybe, maybe Weds.) Friday (maybe, maybe Weds.) Isolate aha3-1 DNA and PCR genotype? Isolate aha3-1 DNA and PCR genotype? other aha3-1 activity, other aha3-1 activity, Wasps? Wasps?