Testing the ABC floral-organ identity model: expression of B

function genesObjectives:

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 Type

1 2 3 4

B (AP3, PI)

C (AG)

sepal petal stamen carpel

A (AP1, AP2)

Inflorescence SEM Inflorescence Section in situ hybridization

AP3 expression in wild type

Bowman et al., 1992 Development 114: 559

AP3 expression in wild type

AP3 expression= Whorls 1,2,3

AP3 expression= Whorls 2,3

Inflorescence Section in situ hybridization

AP3 (and PI) expression in wild type

Mature Flower

Jack et al., 1992, Cell 68: 683

Inflorescence SEM Inflorescence Section in situ hybridization

PI expression in wild type

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?

1 2 3 4

sepal petal stamen carpel

Stage 3

Stage 4

AP3

PI

B AP3, PI

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?

1 2 3 4

AP3

PI

sepal petal stamen carpel

B AP3, PI

Stage 3

Stage 4

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

PIAP3

Class B Organ-Identity Gene Products are Autoregulatory

AP3P

PIP

AP3 PI

PIAP3

PI

1 2 3 4

Test autoregulatory model using ectopic expression of class B genes.

sepal petal stamen carpelStage 3

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

AP335S AP3

AP3

Structure of wild type and mutant Arabidopsis flowers

Whorl 1 Whorl 2 Whorl 3 Whorl 4

WT SEPAL PETAL STAMEN CARPEL

35S EAP3 O

35S EPI O

35S EAP3 O+35SPI

SEPAL PETAL STAMEN STAMENSEPAL PETAL STAMEN STAMEN

Test autoregulatory model using ectopic expression of class B genes.

1 2 3 4

sepal petal stamen carpelStage 3

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

PI35S PI

PI

AP3

Structure of wild type and mutant Arabidopsis flowers

Whorl 1 Whorl 2 Whorl 3 Whorl 4

WT SEPAL PETAL STAMEN CARPEL

35S EAP3 O

35S EPI O

35S EAP3 O+35SPI

SEPAL PETAL STAMEN STAMENSEPAL PETAL STAMEN STAMEN

PETAL PETAL STAMEN CARPELPETAL PETAL STAMEN CARPEL

Test autoregulatory model using ectopic expression of class B genes.

1 2 3 4

sepal petal stamen carpelStage 3

PI35S PI

AP335S AP3

PI

AP3

Structure of wild type and mutant Arabidopsis flowers

Whorl 1 Whorl 2 Whorl 3 Whorl 4

WT SEPAL PETAL STAMEN CARPEL

35S EAP3 O

35S EPI O

35S EAP3 O+35SPI

SEPAL PETAL STAMEN STAMENSEPAL PETAL STAMEN STAMEN

PETAL PETAL STAMEN CARPELPETAL PETAL STAMEN CARPEL

PETAL PETAL STAMEN STAMENPETAL PETAL STAMEN STAMEN

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 loss-of-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.)