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

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 (and PI) expression in wild type
Mature Flower
Inflorescence Section
in situ hybridization
Jack et al., 1992, Cell 68: 683
AP3 expression in wild type
AP3 expression
= Whorls 1,2,3
AP3 expression
= Whorls 2,3
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 wild type
AP3 expression
= Whorls 1,2,3
AP3 expression
= Whorls 2,3
What is the expected expression in a Pi mutant? Ap3 mutant?
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 wild type
PI expression
= Whorls,2,3,4
PI expression
= Whorls 2,3
What is the expected expression in a Ap3 mutant? Pi mutant?
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
PETAL
STAMEN
STAMEN
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
PETAL
STAMEN
STAMEN
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
PETAL
STAMEN
STAMEN
STAMEN
STAMEN
35S E PETAL
PI
O PETAL
PETAL
PETAL
STAMEN
STAMEN
CARPEL
CARPEL
35S E PETAL
AP3 O PETAL
+
35S
PI
PETAL
PETAL
STAMEN
STAMEN
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.)