testing the abc floral-organ identity model: expression of ... outlines/biol 433 lecture 26... ·...
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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.)