Arabidopsis Experiments

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

2. Arabidopsis • Arabidopsis 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. • Fecund. One Arabidopsis plant yields thousands of seeds. • Genome size. 125 megabases. Maize has 5000 mb, tobacco1500 mb. • Diploid. Relatively simple genome. • 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.

3. ArabidopsisHistory Linnaeus

5. Forward vs. Reverse Genetics • Treat thousands of organisms with a mutagen, - random mutagenesis, • Identify an individual with a phenotype of interest, • Identify the gene. Forward • Treat thousands of organisms with a mutagen (usually), • random mutagenesis, • Identify an individual with a genotype of interest, • Identify the phenotype. Reverse

7. Ethylene“the gaseous hormone” • Egyptians gassed figs in order to stimulate ripening, • 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 • Dimitry Neljubow (1901) showed that the active component was ethylene. • R. Gane (1934) reported that plants synthesize ethylene. H2C = CH2

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

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

10. ACC: 1-aminocyclopropane-1-carboxylate

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

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

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, With ethylene present, or the receptor “absent”, or the CTR1 or the gene mutated, • the triple response is activated.

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

15. Friday’s Work Sterilizing/Planting Germinating Breaking Dormancy H2O/Imbibition,  O2/Aeration,   Cold/Prechilling "stratification”  Inducing Germination Light • 70% ETOH/0.1% Triton X • 95% ETOH • Murishige and Skoog Media (MS), • plant minimal medium • 0.5x strength

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

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

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

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

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

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

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

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

24. Mutate DNA Sequence Genetically Link Reverse GeneticsFunctional Genomics Function Gene DNA Sequence Phenotype Analysis Gene Disruption Development Physiology Cell Biology

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

26. Agrobacterium tumefaciens Ti Plasmid (Tumor inducing) neoplastic transformation opaline, nopaline virulence genes hormone genes Mother Nature wt plant chromosome hormone genes (i.e. auxins) opaline virulence genes Ti Plasmid (from agro) Agro food nopaline

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

28. To Do Germination • Breaking Dormancy • H2O/Imbibition,  • O2/Aeration, • Cold/Prechilling "scarification”   • Inducing Germination • Light Surface Sterilize Seeds Plant on Nutrient Media Germinate 1. EMS Treated Seeds on MS/ACC media. 2. aha3-1 on MS media.

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

30. Knockology Plants/Pools DNA/Pools

31. Set-UpDNA Pooling Maintain lines as pools of seed. Seeds (9) Germinate and grow seeds in liquid culture. Seedlings (225) Extract DNA, DNA (225) Super Pool DNA, Super Pools (2025) 1 2 3 4 5 6 …30 PCR Screen

32. 5’--GCATGCATTAT 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’ 3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’ 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ CTGATCGTGAC--5’ CTGATCGTGAC--5’ 5’--GCATGCATTAT Denature Step ~30 seconds 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ Annealing Step ~30 seconds 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ CTGATCGTGAC--5’ 5’--GCATGCATTAT 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 94o Synthesis ~1 minute/kb 72o PCR ~65o

33. PCR Strategy • 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. Reaction: 5’ 3’ Your gene Product: Your gene amplified

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

35. PCR Screens for Mutants

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

37. Find the Plant You are ~here

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

39. L t T homozygote wt L t T heterozygote 5’ 5’ 5’ 3’ 3’ 3’ L t T 5’ 5’ 5’ 3’ 3’ 3’ homozygote mutant PCR Genotyping

40. TT TT TT Tt Tt Tt T T t t T t Tt Tt Tt tt tt tt T T T t t t 1 : 2 : 1 1 wt : 2 het 1 wt : 1 het Not Lethal Lethal Gametophyte Lethal Genetic AnalysisF2 Segregation