haswell slides - washington university department of genetics

Plants: The Other Eukaryotic Organism

Elizabeth Haswell

Bio 5491

January 29, 2009

OUTLINE

I. IntroductionII. Forward geneticsIII. Reverse geneticsIV. Genomic resources and

strategies

OUTLINE

I. IntroductionA. Why study plant genetics?B. Model organismsC. Arabidopsis thaliana

1. Life cycle2. Genome3. Tools

A. WHY STUDY PLANTS?

According to the Food and Agriculture Organization of the United Nations, more than 25,000 people died of starvation every day in 2003 and about 800 million people were chronically undernourished.

1. Practical Value of Plant Studies: Plants are the Foundation of Our Diet

Genetic engineering of plants:Pest ResistanceEnhanced Nutrition

1. Practical Value of Plant Studies: Plants can be Green Machines

• Plants are a source of biofuel.

Derived from recently living biomass:– Wood– Biodiesel (rapeseed)– Bioethanol (corn)

New plants with high biomass yield: – Switchgrass (prairie grass)– Miscanthus– Algae

switchgrass

2. Value of Plant Genetic Studies for Basic Biology

1. For comparison

2. As additional examples

3. Because they are part of the natural world

4. Aesthetics

a. Plants share a common eukaryotic ancestor with animals.

Examples:

Chromatin

Cytoskeleton

Golgi, ER, usual organelles

Gene expression components

Ga = giga-annum = billion yearsFrom Meyerowitz, 2002

Ga = giga-annum = billion years

b. Plants evolved multicellularity independently from animals.

Implications for:pattern formation cell-cell communication.

Example: flower development

From Meyerowitz, 2002

c. Plants underwent two endosymbiotic events.

Horizontal transfer of bacterial genes that integrate into eukaryotic system

Example: chloroplast division proteins

Ga = giga-annum = billion yearsFrom Meyerowitz, 2002

HOW DO WE STUDY PLANT BIOLOGY?

B. Plant Genetic Model Systems

• Crop plants– Rice

– Alfalfa

– Tomato

– Grapevine

– Sugarcane

– Tobacco

– Maize

Considerations•Genome size•Polyploidy•Translation to crop plants•Synteny

• Model systems–Mosses –Algae –Poplar–Arabidopsis

C. Arabidopsis thaliana: A model system for flowering plants

Advantages:

1. Life cycle • 6 weeks• Small plant, easy to grow• High fecundity (10,000

seed/individual)• Self and cross-fertilization

2. Genome• Diploid• 125 Mb, smallest known in plant

kingdom• Little repetitive DNA

3. Tools• Agrobacterium transformation• RFLP map between ecotypes• Tiling arrays

Arabidopsis is a member of the mustard (Brassicaceae) family, which includes cultivated species such as cabbage and radish.

Meyerowitz. Ann. Rev. Genet. 21 : 93-111(1987)

1. Arabidopsis Life Cycle

Life cycle of higher plants.(A) The dominant diploid generation(B) flowers(C) male and the female reproductive structures (anthers and siliques) (D) Gametes produced by meiosis: pollen and ovule (E) Fusion of pollen and ovule to form new diploid generation (embryo).

• Haploid generation is multicellular• No dedicated germline

1. Two Features of Plant Development Relevant to Genetic Analyses

• Gametes in plants are formed by a separate multicellular haploid generation called the gametophyte. – Multiple rounds of division in the haploid phase.– Implication for essential genes.

• Plants have no dedicated germline. Instead, cells

giving rise to the germline develop de novo from the somatic tissues. – Implications for environmental inputs into the production of the

germline.

(Nature, 408:796-815; 2000)

2. The Arabidopsis genome

•Sequenced by an international consortium:•European Union•Riken•US (CSH/WU/ABI did parts of chromosomes 4 and 5)

•Strategies: BAC-end sequencing, physical map-based approaches

•Error rate is < 1 error per 20 Kb

• 125 Mb

• Current status:The TAIR8 release (April 08) contains 27,235 protein coding genes(~same as humans)– 1 gene / 4.4 Kb.– ~30% unknown.– 11,000 families.

• Recent large-scale genome duplication events.– Recent tetraploid ancestor, now reducing.

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2. The Arabidopsis genome

Duplications: red (recent) and blue (old) sister regions

2. The Arabidopsis genome

Blanc, et al. Genome Res. 13 : 137-44 (2003).

Agrobacterium tumefaciens & crown gall tumor

Agrobacterium is a genus of soil bacteria that infects wounded plants and leads to gall formation

Galls = benign tumor that feeds the extracellular agrobacteria

3. Arabidopsis Molecular Genetic ToolsA. Agrobacterium-mediated plant transformation

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Agrobacteria induce tumors by genetically engineering plant cells at the wounded site

Ti plasmid

~200 Kb

LB RB

Virulence genes LB RB

T-DNA introduced into plant chromosomal DNA

LB RB

T-DNA plasmid for transformation

Selectable marker

T-DNA acts as an insertional mutagen

Agrobacterium-mediated plant transformation

“Floral Dip”

SelectionT1

Solution of Agrobacterium harboring a T-DNA plasmid

• Complementation experiments• Method of mutagenesis• GFP fusions, reporter genes, etc.

T-DNA plasmid

Gene A Gene B Gene C

Examples of possible insertions:

LB Selection RBYFG

T-DNA insertion at random locations in the genome

*

• A complete database of polymorphisms between Columbia and Landsberg ecotypes (and others!)

– CAPS and SSLPs– Point mutations– Insertions or deletions– All occur randomly in one ecotype and not in the other.

Jander, 2002; Lukowitz, 2000

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3. Arabidopsis Molecular Genetic Tools B. Polymorphism database

Positional Cloning in Arabidopsis: Why it feels good to have a genome initiative working for you

Positional Cloning in Arabidopsis: Why it feels good to have a genome initiative working for you

OUTLINE

I. IntroductionII. Forward geneticsIII. Reverse geneticsIV. Genomic resources and

strategies

II. Forward genetics

B. Generate a

mutant population

C. Identify

interesting mutants

A. Select a biological process

pi-1

D.Map and

clone mutation

Infer mechanism &Infer mechanism &Generate hypothesesGenerate hypotheses

Mutant phenotype Responsible gene

A. Floral Organ Development in Arabidopsis

4 sepals

4 petals

6 stamenMale gametespollen

1 carpelFemale gametesovules

A. Arabidopsis Floral Organs are Arranged in Whorls

• Four concentric whorls of organs• Stereotyped pattern of number and position.

cast pe

se

II. Forward genetics

B. Generate a

mutant population

C. Identify

interesting mutants

A. Select a biological process

(Flower development)

pi-1

D.Map and

clone mutation

Mutagens commonly used in Arabidopsis

• EMS• Insertional mutagenesis

– T-DNA– Transposon

• Irradiation• Fast neutron

• Natural variation

B. Generate a mutant population

B1. Chemical Mutagenesis with EMS

EMS = ethylmethanesulfonateGenerates G/C to A/T mutations

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Advantages:• wide range of mutants possible• mutagenesis easy to perform• high mutation rate• can combine many lesions in the same line

Disadvantages:• background mutations• cloning the gene can be time-consuming• not all mutations are transmitted to the second generation

B2. T-DNA insertional Mutagenesis

ATG STOP

A random gene

Advantages:• tagged, therefore easier to isolate gene involved

Disadvantages:• low mutation rate• unlinked mutations• preference for promoter regions• will limit type of alleles; often severe loss of function alleles

II. Forward genetics

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B. Generate a

mutant population

C. Identify

interesting mutants

A. Select a biological process

Flower development

pi-1

D.Map and

clone mutation

EMS

C. Identify Interesting Mutants

DESIGN IS CRITICAL• Selection vs. Screen• False positives• False negatives• Etc.

Our example: visual screen for floral development mutants of Arabidopsis.

•Deviations in floral organ number• Deviations in floral organ location

Arabidopsis Homeotic Mutants

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Homeotic mutations cause conversion from one organ to another.

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apetala1 (ap1)apetala2 (ap2)

cast

stca

ca

se

ca

se pese

pe

pistillata (pi)apetala 3 (ap3) agamous (ag)

The ABC Model of Floral Development

Coen and Meyerowitz, Nature 1991

Three classes of homeotic genes• A function (AP1, AP2)-->sepals, petals• B function (AP3, PI)-->petals, stamen• C function (AG)--stamen, carpels

ap3 or pi

A C

B

ca

se

ca

se

B-function genes are required for the production of petals and stamens

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ap2

A C

B

cast

stca

A function genes are required for the production of sepals and petals

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ag

A C

B

pese

pe

C-function genes are required for the production of stamen and carpels

ABC Model of Floral Organ Development

• Provides framework for future work.• Comparison to other species.

II. Forward genetics

B. Generate a

mutant population

C. Identify

interesting mutants

A. Select a biological process

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pi-1

D.Map and

clone mutation

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Flower developmentEMS

ag-1

pi

1. Uses linkage analysis: test for genetic linkage between previously identified genetic markers and your gene.

2. Based on the principle that the frequency of recombination between genes decreases along

with the distance between them.

How to identify mutant alleles? Genetic Mapping

C1. Map and Clone Mutation--EMS

Map-based cloning:– Mutant plants are crossed to another ecotype– The mutant gene is identified by virtue of its

association with nearby genetic markers that differ between ecotypes (polymorphisms).

Ecotype B

Ecotype Amutant

Wild-type

Wild-type

mutantX

mutant

mutant

Look for co-segregation of the mutant phenotype and the ecotype A marker

Michelmore, 1991

First, map to a chromosome arm:

Then, narrow down further and further.

C1. Map and Clone Mutation--EMS

A B het

C2. Map and Clone Mutation--T-DNA

ATG STOPYour favorite mutant

LB Selectable Marker RB

•Degenerate PCR•Sequence PCR product•Test for altered mRNA or protein production•Complement with transgene

The floral homeotic genes encode MADS box transcription factors that activate organ-specific gene expression in a combinatorial manner.

Homeotic mutants in Drosophila

• Homeotic mutants– Antennapedia

• Genes direct anterior-posterior positioning of the embryo.

• Hox genes.

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Wild type Antennapedia

Thus homeotic genes arose once in animals and once in plants, accomplishing the same function using different types of transcription factors.

II. Forward Genetics

Uses:– Unbiased search for genes involved in a biological

process.

Pitfalls :– You get what you screen for – Will miss redundant or essential genes

phenotype genotype

III. Reverse genetics

Uses:– can get additional alleles to corroborate data on previous EMS

alleles– can study genes that are interesting because of evolutionary,

biochemical, or expression data.– Can do a step-by-step analysis of the redundant functions of

members of a gene family

Pitfalls:– Requires guessing.– Common outcome: no phenotype because gene is redundant

or conditionally required.

genotype phenotype

Reverse genetics

B. Generate

mutant plants

C. Evaluate mutant

phenotype

A. Identify gene or genes of interest

pi-1

D. Identify the function of the genes

Infer mechanism &Infer mechanism &Generate hypothesesGenerate hypotheses

Mutant phenotype Responsible gene

1. Sequence-indexed T-DNA insertion/transposon linesSALK, GABI-Kat, CHSL, RIKEN, SAIL, Wisconsin, etc.

2. TILLING.

3. Engineered Post-Transcriptional Gene Silencing.

4. Overexpression of wild-type or dominant-negative alleles.

5. Gene replacement.

III. Reverse genetics: the Arabidopsis toolbox

Available SALK insertion lines for AP3

AP3

SIGnAL= Salk Institute Genomic Analysis Laboratoryhttp://signal.salk.edu/

III. Reverse genetics: the Arabidopsis toolbox

Available SALK insertion lines for PI

PI

III. Reverse genetics: the Arabidopsis toolboxSIGnAL= Salk Institute Genomic Analysis Laboratoryhttp://signal.salk.edu/

A high-throughput strategy for generating and isolating point mutations in your favorite gene

Exploits a nuclease that recognizes and digests heteroduplexes.

Use:

• Mutations in genes that are not found in the insertional database.

• Partial loss of function.

• Conditional alleles.

2. TILLING Targeted Induced Local Lesions IN Genomes

2. TILLING Targeted Induced Local Lesions IN Genomes

EMS-mutagenized plant population

**

CEL1

**

**

**

xx

x

Heat, anneal wild type and mutant versions

Digest with CEL1 endonucleasedigests heteroduplexes ONLY

**** ** **

Pooled DNA from individual plants in 96 well plates

PCR amplify your gene with fluorescently tagged primers from DNA in each pool

Pool#104

Nature Biotechnology 18: 455-457 (2000).

Run on gelScreen individual samples, sequence

TILLING Targeted Induced Local Lesions IN Genomes

pools

• Arabidopsis• Other plants• Animals

Reverse genetics gave additional insight into floral development

• We know MADS-box genes are required for floral development:

• But they are not sufficient.• What are the missing factors?

The SEPELLATAS: A fourth class of floral organ identity genes

From Current Opinion in Genetics and Development 2001 11 : 449

•SEP1 identified as an AP3-interacting protein in the Y2H.

•SEP1,2,3,4 are all highly similar MADS-box proteins.

SEP4

The SEPALLATA genes are required for floral organ formation

Wild type sep1sep2sep3sep4

Ditta, et al. Current Biology 14 :1935 (2004).

Turning Leaves into Petals

Wild type

Expression of AP1, AP3, PI, and SEP is sufficient to convert seedling leaves into petals.

Pelaz, et al. Current Biology 11 : 182-184 (2001)

The ABCE Model of Floral Development

OUTLINE

I. IntroductionII. Forward geneticsIII. Reverse geneticsIV. Genomic resources and

strategies

IV. Genomic Resources and Strategies

1. genome gene genotype phenotype

2. Whole genome vs. one-by-one strategies

-OMIC APPROACHES

• Genomics • Transcriptomics• Proteomics

• Phenomics = the total phenotypic outcome of disrupting the genome

• Epigenomics = the total epigenetic modifications made in a genome

• Metabolomics = totality of metabolites in an organism

Changes in response to environmental or genetic conditions

Occurrence of Related Genes in Clusters of Co-expressed Genes during Floral Development

• Five clusters of genes with similar expression profiles during flower development.

• Black bars indicate the percentage of closely related sequences in each cluster.

• White and gray bars are randomly chosen genes.

• Suggests genetic buffering by functional redundancy.

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Wellmer et al. PLOS Genetics 2(7) 1012-1024 (2006).

MetabolomicsStudy of the

complement of metabolites:

ionsosmolytessugarsbiosynthetic intermediatesetc.

Ionomics

High-throughput, quantitative mass spectroscopyThe altered ionome of a mutant plant can reflect defects in transport or metabolism

Nature Biotechnology 21, 1215 - 1221 (2003)

frd-3 WT msl9-1; msl10-1

msl9-1; msl10-1msl4-1; msl5-2; msl6-1

msl4-1; msl5-2; msl6-1

Fe transporter

Other ions assayed:Na, Mg, K, Ca, Mn, Co, Ni, Cu, Zn, Mo, B, P

Epigenomics• Characterizing the “methylome”:

– Wild type and methylase/demethylase mutants– Natural variation in methylation sites.

– Genome-wide bisulfite sequencing and subsequent mapping to Arabidopsis genome (Lister et al., 2008).

– Chromatin IP with antibodies against methyl-C, then hybridize to a microarray covering entire genome (Zilberman et al., 2006).

– Digestion with methylation-sensitive enzymes, then hybridize to microarray covering the entire genome Zhang et al., 2008).

TAKE-HOME MESSAGES1. Plants are interesting and important.

practical valueopportunity to gain insight into basic life processes

2. Arabidopsis is an excellent model system for molecular genetics

but--inefficient homologous recombination --redundancy

3. Forward and reverse genetic approaches help reveal the genes involved in a particular biological process

4. State of the art genomics-level resources are available to understand the interaction between plant genomes and their environment.