Testing the ABC floral-organ identity model: expression of B function genes 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-offunction 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 Type sepal

petal

1

2

stamen

carpel 4

3

B (AP3, PI) A (AP1, AP2)

C (AG)

AP3 expression in wild type

Inflorescence SEM

Inflorescence Section in situ hybridization Bowman et al., 1992 Development 114: 559

AP3 expression in wild type

AP3 expression = Whorls 1,2,3

AP3 expression = Whorls 2,3

AP3 (and PI) expression in wild type

Mature Flower

Inflorescence Section in situ hybridization Jack et al., 1992, Cell 68: 683

PI expression in wild type

Inflorescence SEM

Inflorescence Section in situ hybridization

PI expression in wild type

PI expression = Whorls,2,3,4

PI expression = Whorls 2,3

How can we rationalize the differences in expression of AP3 and PI between stages 3 and 4?

sepal

petal

1

2

stamen 3

AP3 Stage 3

Stage 4

PI

B AP3, PI

carpel 4

AP3 expression in an Ap3 or Pi mutant

Expression of AP3 past stage 3 is dependent on both AP3 and PI function

PI expression in an Ap3 or Pi mutant

Expression of PI past stage 3 is dependent on both AP3 and PI function

How can we rationalize the differences in expression of AP3 and PI between stages 3 and 4?

sepal

petal

1

2

stamen 3

carpel 4

AP3 Stage 3

PI

Stage 4

B AP3, PI

Only cells expressing both AP3 and PI continue to express these genes.

Class B Organ-Identity Gene Products are Autoregulatory

P

P

AP3

AP3

PI

PI

AP3 PI

Test autoregulatory model using ectopic expression of class B genes. Stage 3

sepal

petal

1

2

stamen 3

carpel 4

AP3 PI

35S AP3

AP3

Only cells expressing both AP3 and PI continue to express these genes.

Structure of wild type and mutant Arabidopsis flowers Whorl 1

Whorl 2

Whorl 3

Whorl 4

SEPAL

PETAL

STAMEN

CARPEL

35S E SEPAL AP3 O SEPAL

PETAL

STAMEN

STAMEN

PETAL

STAMEN

STAMEN

WT

35S E PI O 35S E AP3 O + 35S PI

Test autoregulatory model using ectopic expression of class B genes. Stage 3

sepal

petal

1

2

stamen 3

carpel 4

AP3 PI

35S PI

PI Only cells expressing both AP3 and PI continue to express these genes.

Structure of wild type and mutant Arabidopsis flowers Whorl 1

Whorl 2

Whorl 3

Whorl 4

SEPAL

PETAL

STAMEN

CARPEL

35S E SEPAL AP3 O SEPAL

PETAL

STAMEN

STAMEN

PETAL

STAMEN

STAMEN

35S E PETAL PI O PETAL

PETAL PETAL

STAMEN STAMEN

CARPEL CARPEL

WT

35S E AP3 O + 35S PI

Test autoregulatory model using ectopic expression of class B genes. Stage 3

sepal

petal

1

2

stamen 3

AP3 PI

35S AP3

AP3

35S PI

PI

carpel 4

Structure of wild type and mutant Arabidopsis flowers Whorl 1

Whorl 2

Whorl 3

Whorl 4

SEPAL

PETAL

STAMEN

CARPEL

35S E SEPAL AP3 O SEPAL

PETAL

STAMEN

STAMEN

PETAL

STAMEN

STAMEN

35S E PETAL PI O PETAL

PETAL PETAL

STAMEN STAMEN

CARPEL CARPEL

35S E PETAL AP3 O PETAL + 35S PI

PETAL

STAMEN

STAMEN

PETAL

STAMEN

STAMEN

WT

Review: Analysis of Class B expression AP3 and PI are first expressed in stage three flowers in non-identical but overlapping domains. Only cells in the second and third whorl presumptive region (where both genes are expressed) continue to express AP3 and PI past stage 4. Neither AP3 nor PI are expressed past stage 4 in either Ap3 or Pi lossof-function mutants. Expression of PI throughout the flower leads to expression and function of AP3 in whorls 1+2+3 and expression of AP3 throughout the flower leads to expression and function of PI in whorls 2+3+4.

Conclusions: Analysis of Class B expression AP3 and PI function together to autoregulate the expression of their genes. Only those cells in which both AP3 and PI are expressed in stage 3 will continue to express the genes in later stages. (AP3 and PI polypeptides have been shown to interact and bind the MADS-box DNA target sequence CC(A/T)6GG.)