Testing the ABC floral-organ identity model: Double Mutants

Objectives:

To test the validity of the ABC model for floral organ identity we will:

1. Use the model to make predictions concerning the phenotype of double or triple loss-of-function mutants and compare with the actual double mutant phenotypes.

2. Clone and sequence the ABC genes. Look for similarities with sequenced genes already in the database.

3. Determine the time and place of expression for each ABC gene and consider whether the expression correlates with the functional domain defined by the loss-of-function phenotype.

4. Test regulatory interactions between ABC genes by examining how the loss-of-function of one gene affects the expression domain of another.

5. Create gain-of-function mutants by generating transgenic plants carrying an ABC gene cDNA under the control of the CaMV35S promoter.

A Model For Control of Organ TypeMutations in Classes B and C function genes

1 2 3 4

B (AP3, PI)

C (AG)A (AP2)

sepal petal stamen carpelWild type

B C Double MutantExpected:

A (AP2)

sepal sepal sepal sepal

Construction of ap3, ag double mutant

ap3/ap3, AG/AG x AP3/AP3,AG/ag

Wild type F1 AP3/ap3, AG/ag self fertilize

F2 99 wild type; 37 Ag; 31 Ap3; 9 AgAp3expected: 99 33 33 11

or AP3/ap3, AG/AG

ap3, ag double mutant

A Model For Control of Organ TypeMutations in Classes B and C function genes

1 2 3 4

sepal petal stamen carpelWild type

B C Double MutantExpected:

Observed:

A (AP2)

sepal sepal sepal sepalsepal sepal sepal sepal

A Model For Control of Organ TypeMutations in Classes A and B function genes

1 2 3 4

B (AP3, PI)

C (AG)A (AP2)

sepal petal stamen carpelWild type

A B Double MutantExpected: carpel carpel carpel carpel

C (AG)

ap3, ap2 double mutant

A Model For Control of Organ TypeMutations in Classes A and B function genes

1 2 3 4

sepal petal stamen carpelWild type

A B Double MutantExpected:

Observed:

carpel carpel carpel carpel

C (AG)

carpel carpel carpel carpel

A Model For Control of Organ TypeMutations in Classes A and C function genes

1 2 3 4

B (AP3, PI)

C (AG)A (AP2)

sepal petal stamen carpelWild type

A C Double MutantExpected: ? ? ? ?

ap2, ag double mutant

A Model For Control of Organ TypeMutations in Classes A and C function genes

1 2 3 4

B (AP3, PI)

sepal petal stamen carpelWild type

A C Double MutantExpected:

Observed: Leaf/sepal/carpel

Petal\Stamen

Leaf/sepal/carpel

Petal\Stamen

? ? ? ?

A Model For Control of Organ TypeMutations in Classes A, B and C function genes

1 2 3 4

B (AP3, PI)

C (AG)A (AP2)

sepal petal stamen carpelWild type

A B C Triple MutantExpected: ? ? ? ?

ap2 ap3, ag triple mutant

A Model For Control of Organ TypeMutations in Classes A, B and C function genes

1 2 3 4

sepal petal stamen carpelWild type

A B C Triple MutantExpected:

Observed:

? ? ? ?leaf leaf leaf leaf

Apetala1 Mutant

APETALA1 Gene

• In the typical Ap1 mutant phenotype the floral perianth organs are replaced by one or two leaves and branches.

• However, some mutant alleles of AP1 result in a phenotype like Ap2 (perianth replaced by stamens and carpels.

• Thus AP1 is considered to regulate two processes: shoot identity and Class A floral organ type.

Structure of wild type and mutant Arabidopsis flowers

Whorl 1 Whorl 2 Whorl 3 Whorl 4WT SEPAL PETAL STAMEN CARPEL

Ap2 CARPEL STAMEN STAMEN CARPEL

Ag SEPAL PETAL PETAL SEPAL

Pi SEPAL SEPAL CARPEL CARPEL

Ap3 SEPAL SEPAL CARPEL CARPEL

Wild type Apetala2

Apetala3 (and Pistillata) Mutant Phenotype

Wild type Apetala3

ap3, ag double mutant

ap3, ap2 double mutant