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

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.

Arabidopsis History Linnaeus

Forward vs. Reverse Genetics 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

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

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

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.

ACC: 1-aminocyclopropane-1-carboxylate

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.

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

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 or the gene mutated, the triple response is activated. the triple response is activated.

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

Friday’s Work Sterilizing/Planting 70% ETOH/0.1% Triton X 95% ETOH Murishige and Skoog Media (MS), plant minimal medium 0.5x strength 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

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.

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…

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

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

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, Human: 25, ,000. Rice: 32, ,000, Human: 25, ,000.

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

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

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

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

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

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

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

To Do Germination  Breaking Dormancy  Breaking Dormancy  H 2 O/Imbibition,  H 2 O/Imbibition,  O 2 /Aeration,  Cold/Prechilling "scarification”  Cold/Prechilling "scarification”  Inducing Germination  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.

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

Knockology Plants/PoolsDNA/Pools

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

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

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

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

PCR Screens for Mutants

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

Find the Plant You are ~here

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

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

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

Midterm Review on Wednesday, Review on Wednesday, 1 hour midterm, 1 hour midterm, Look for Ethylene-Insensitive Mutants. Look for Ethylene-Insensitive Mutants.