abc model of flower development

In the name

of allah

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Molecular Biology of Floral

Organogenesis

Dr.Hassanpour

By: Z.esmaillou

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Introduction

Our understanding of the molecular nature of floral organ development has

increased tremendously over the last 15 years.

In the early 1990s, genetic studies on Arabidopsis thaliana and Antirrhinum

majus led to the isolation and characterization of floral organ-identity genes

(also called floral homeotic genes) and the establishment of the seminal ABC

model for flower development.

This model proposed that different organ-identity genes act alone and in various

combinations to specify each of the four types of floral organs.

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Floral Organ-identity Genes and the ABC Model

During reproductive development, the shoot apical meristem (SAM) initiates

floral meristems (FMs) along its flanks.

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The identities of these different organs are specified by the actions of floral

organ-identity genes in different regions of a developing flower.

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The ABC model for flower development proposed that three classes of these

organidentity genes function in overlapping domains to specify

whorl one=sepals

whorl two=petals

whorl three= stamens

whorl four= carpels

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Basic ABC Model of Floral Organ Development

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The A class genes= APETALA1 (AP1) and APETALA2 (AP2)

Act to specify sepal and petal development

the B class genes= APETALA3 (AP3) and PISTILLATA (PI)

Act to specify petal andstamen development and

the C class gene= AGAMOUS (AG)

Acts to specifystamen and carpel development

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Mutations in the class A gene AP2 result in C activity spreading into whorls one

and two and consequently homeotic transformations in organ identity with carpels

replacing sepals in whorl one and stamens replacing petals in whorl two

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Mutations in the class C gene AG result in class A activity in all four whorls.

The ag mutants produced indeterminate flowers repeating the pattern of organs (Se

Pe Pe)n

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Mutations in the class B genes result in flowers with sepals in the outer two

whorls and carpels in the inner two whorls.

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ABC triple mutants produce indeterminate flowers consisting only of leaf-like

organs.

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The organ-identity genes are expressed at the RNA level in spatially restricted

regions of a FM consistent with the activities of these genes as proposed in the

ABC model.

gene AG is expressed in cells that will give rise to the third and fourth whorls of a

flower and subsequently in developing stamen and carpel primordia.

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B genes are expressed in largely overlapping domains that correspond to second

and third whorl cells and later in petal and stamen primordia.

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gene AP1 is expressed throughout very young FMs

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Ectopic Expression Studies Provide Support for the ABC Model

Misexpression of the class C gene AG, under the control of the cauliflower

mosaic virus 35S promoter, demonstrated that this gene is sufficient to turn

off class A function when it is expressed in the outer two floral whorls

35S::AG plants closely resemble ap2 mutants.

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35S::PI 35S::AP3 plants produce flowers with petals in whorls one and two and

stamens in whorls three and four.

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SPT and CRC Promote Carpel Identity Independently of AG

Carpelloid features are present on floral organs of ap2-2 ag-1 double mutants and

ap2-2 pi-1 ag-1 triple mutants, suggesting that other genes besides AG can

specify certain aspects of carpel development.

These features are lost when the triple mutant is combined with mutations in

SPATULA (SPT) and CRABS CLAW (CRC)

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Individual mutations in either SPT or CRC show defects in carpel fusion

in the upper part of the organ and small effects on carpel morphology.

The crc spt double mutants show a much more severe phenotype with further

reductions in carpel fusion and decreased amounts of many carpel tissue types

including stigma, style, septum, transmitting tract and ovules.

CRC is a member of the YABBY gene family that specifies abaxial identity

while SPT encodes a bHLH transcription factor.

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SEPALLATA Genes Work with ABC Genes to Specify Organ Identity

A significant revision of the ABC model was the addition of the class E or

SEPALLATA (SEP) genes, which were first identified in tomato and petunia.

Arabidopsis has three SEP genes (SEP1, SEP2 and SEP3) that are

functionally redundant.

SEP1 and SEP2 are expressed in all four

whorls of the flower while SEP3 mRNA is

detected in whorls two, three and four

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Mutations in any one or two of these genes do not significantly alter floral organ

identity but sep1 sep2 sep3 triple mutants produce indeterminate flowers

consisting only of sepals.

These flowers resemble those produced in ap3 ag and pi ag double mutants,

indicating that the B and C functions are not active.

Wild-type

sep1 sep2 sep3 triple mutant

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Three SEP genes function in combination with the ABC genes to specify petal,

stamen and carpel identity.

Cepal= A

Petal= A+B+E

Stamen=B+C+E

Carpel=C+E

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MADS-domain Protein Complexes

All of the class A, B, C and E genes except for AP2 code for members of the MADS-

domain family of dimeric transcription factors.

MADS is an acronym for the first four identified members of the

family:

MCM1=yeast

AG = Arabidopsis

DEFICIENS (DEF) = Antirrhinum

SRF = humans

These proteins share a 56 amino acid DNA-binding domain called the MADS

domain and bind to CArG box [CC(A/T)6GG] sequences

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In plants, there are two classes of MADS-domain proteins based on sequence

similarity outside of the MADS domain.

MADS - DNA binding

I - intervening region: contributes to dimerization specificity

K - protein-protein interactions: form a coiled-coil structure and is also

involved in dimerization of these proteins

C - carboxy-terminal domain: acts as a transcriptionalactivation domain

in some family members

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Each whorl of the flower contains a unique combination of MADS-domain organ-

identity proteins that act as transcription factors regulating the expression of different

sets of target genes.

Early studies showed that although all of the class A, B and C MADS-domain

proteins could interact with each other; only certain combinations (AP1

homodimers, AP3/PI heterodimers and AG homodimers in Arabidopsis) could bind

DNA in vitro.

The quartet model proposes that distinct complexes of four MADS domain

proteins function in each whorl of the flower.

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second whorl =AP3-PI-AP1-SEP

third whorl=AP3-PI-AG-SEP

fourth whorl=AG-AG-SEP-SEP

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Regulation of Floral Organ-identity Gene Expression

A key advance in the field of flower development has been the uncovering of a direct

role for the FM-identity gene LEAFY (LFY) in activation of the floral organ-identity

genes.

LFY encodes a novel plant-specific DNA-binding protein that can bind

in vitro to the promoters of several floral organ-identity genes

LFY alone is sufficient to activate expression of the class A gene AP1

throughout the flower.

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Expression of each of the floral organ-identity genes continues throughoutmost of

flower development

Since LFY is not expressed after stage 5, other factors presumably act to maintain

their expression.

The plant hormone gibberellin (GA) appears to be an important regulator of

homeotic gene expression in later stages of flower development.

The Gadeficient mutant ga1 produces flowers that have organs with the correct

identity but which are growth arrested and immature.

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Signalling through the GA pathway involves inactivation of a family of DELLA

proteins that act as negative regulators of GAresponses.

Mutations inthese DELLA genes can repress the floral defects of ga1–3, suggesting

that GA responses are essential for completion of floral organ development

Expression of AP3, PI and AG is increased in ga1 flowers after GA treatment and

decreased in dexamethasone-treated plants expressing a steroid-inducible DELLA

protein. Thus GA promotes expression of the class B and C genes and the continued

development of floral organs by negatively regulating DELLA proteins.

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Regulation of AP1 expression

AP1 mRNA is initially detected throughout a young FM and later becomes restricted

to sepal primordia and adjacent second whorl cells.

Expression of AP1 is delayed in lfy mutants increased in flowers expressing a

constitutively active LFY (LFY::LFY-VP16) and activated ectopically in the young

leaves of plants misexpressing LFY.

By stage 3 of flower development, AP1 mRNA is only detected in the first and

second whorls of the flower. The restriction of AP1 expression requires AG

activity as AP1 mRNA is detected in all four whorls of ag mutants

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Initiation of B class gene expression by LFY, UFO and AP1

Both LFY and AP1 act in the initiation of AP3 expression in the second and third whorl

cells of a stage 3 flower.

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Class B gene expression is also reduced in the unusual floral organ (ufo) mutant

suggesting that UFO is another positive regulator of B class gene expression.

Expression of both of the class B genes is somewhat reduced in lfy mutants and

more severely reduced in lfy ap1 double mutants

LFY and UFO work together in activation of the class B genes

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UFO is expressed in stage 2 FMs in a pattern suggesting that it could be the

region-specific factor that functions with globally expressed LFY

LFY is not sufficient for AP3 and PI activation as 35S::LFY and LFY::LFY-VP16

plants do not activate AP3 and PI in vegetative tissues or throughout flowers

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ectopic expression of UFO activates AP3 at a slightly earlier time in development

and in an expanded domain as compared to wild-type.

35S::UFO plants produce extra petals and stamens at the expense of sepals and

carpels, similar to the phenotype resulting from misexpression of both of the class

B genes

AP1 also participates in activation of class B gene expression in petals. AP1 is not

sufficient for class B gene activation as 35S::AP1 plants do not show transformations

in organ identity

activated AP1 (AP1-VP16) can turn on AP3 and PI expression in the first whorl

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Maintenance of class B gene expression

The maintenance of AP3 and PI expression in flowers requires the activity of

both AP3 and PI indicating that these two proteins function in a positive

autoregulatory loop.

PI expression is not maintained in ap3 or pi mutants and the early fourth whorl

expression of PI in wild-type flowers is also not maintained in older flowers

Because the AP3 promoter but not the PI promoter contains CArG boxes, it has

been speculated that the autoregulation of AP3 is direct while that of PI is

indirect

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Initiation of AG expression by LFY and WUS

AG mRNA is first detected in the central region of a stage 3 FM

In lfy mutants, AG expression is reduced and delayed suggesting that LFY is a

positive regulator of AG expression

AG expression is activated in all four whorls of these LFY::LFY-VP16 flowers

as well as in vegetative tissues of 35S::LFY-VP16 plants.

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The meristem regulatory protein WUSCHEL (WUS) has been identified as

an LFY cofactor.

The wus mutants have defective SAMs that cannot serve as permanent sources of

stem cells for organ initiation.

These mutants undergo a stop and go pattern of development in which shoot

meristems produce a few leaves and then stop with subsequent adventitious

meristems producing a few leaves and then stopping.

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Both LFY and WUS bind AG regulatory sequences in vitro

Two LFY and two WUS binding sites are found in close proximity within the

AG second intron.

Coexpression of LFY and WUS in yeast stimulated the expression of a reporter

gene under the control of AG regulatory sequence

After being activated by WUS, AG in turn downregulates the expression of WUS in

the centre of the FM

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WUS expression is at its highest levels during stages 2 and 3 of flower development and

is not expressed after stage 6 of flower development when all FM cells have been used

for organ initiation.

In indeterminate ag flowers, WUS expression is maintained in the central region

of the FM

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Transcriptional repression of AG

The lack of AG expression in vegetative tissues and the outer two floral

whorlsis a consequence of both the absence of AG activators and the activity

of AGrepressors.

Repressors of AG expression include

APETALA2 (AP2)

LEUNIG (LUG)

SEUSS (SEU)

STERILE APETALA (SAP)

CURLY LEAF (CLF)

INCURVATA2 (ICU2)

BELLRINGER (BLR)

act to prevent precocious expression of AG in stage 2 FMs.

acts to repress AG expression in inflorescence stems

acts to repress AG in the inflorescence meristem

act to prevent AG expression in leaves

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Mutations in any of these genes result in ectopic AG expression in the first and

second whorl cells leading to partial or complete homeotic transformations of first

whorl sepals into carpels and second whorl petals into stamens or the loss of second

whorl organs

Another factor likely to be involved in AG regulation is AINTEGUMENTA (ANT).

Homeotic changes in organ identity are not usually observed in ant mutants.

Several of the known AG repressors encode proteins that are likely to play roles in

transcriptional repression including DNA-binding proteins, transcriptional

corepressors and chromatin-modifying factors.

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Post-transcriptional regulation of AG

In addition to transcriptional regulation, AG is regulated post-transcriptionally. Two

different genetic screens have identified several factors that act in the processing of

AG pre-mRNA.

The first was an enhancer screen utilizing the partial loss-of-function ag-4 allele.

The ag-4 flowers have a normal third whorl but the fourth whorl carpel is replaced

by a new flower repeating the pattern of organs: sepals, petals and stamens

Two partially redundant genes, HUA1 and HUA2 were identified that when

mutated in combination with ag-4 cause a strong ag-like phenotype

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Mutations in either HUA1 or HUA2 alone do not have any phenotype while

hua1 hua2 double mutants have slight defects in stamen and carpel development

HUA1, HUA2, HEN2 and HEN4 appear to play roles in maturation of AG pre-mRNA

HUA1 encodes a CCCH zinc finger RNA-binding protein while HEN4 encodes a K

homology (KH) domain-containing putative RNA-binding protein

Because HUA1 can bind to AG RNA in vitro, it is likely that HUA1 and HEN4

bind directly to AG pre-mRNA to carry out their function

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Translational repression of AP2

The only floral organ-identity gene that does not encode a MADS-domain protein is

the class A gene AP2.

Although AP2 is expressed at the mRNA level in all four whorls of developing

flowers, its activity is restricted to the outer two whorls of the flower.

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AP2 is regulated post-transcriptionally by the activity of a microRNA (miRNA).

miRNAs are non-coding RNAs of 21–22 nucleotides that are processed from

longer hairpin transcripts and are thought to play important roles in development.

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The miRNAs may regulate target gene expression by binding to regions of

complementarity in the target transcripts and causing RNA cleavage and/or

translational inhibition.

A clue that miRNAs might be involved in regulation of floral homeotic genes came

from the isolation and characterization of an enhancer of the hua1 hua2 double

mutant called HEN1.

The single mutants hen1 have a pleiotropic phenotype with defects in organ

size,leaf shape, number of axillary inflorescences and fertility.

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One of the miRNAs (miRNA172) affected in hen1 has significant sequence

homology to AP2 and three related genes, matching the AP2 sequence in 19

of the 21 positions

The reduced amount of miRNA172 and increased levels of AP2 protein in hen1

suggested that AP2 might be negatively regulated by miRNA172.

35S::miRNA172 plants produce flowers with phenotypes similar to

ap2 mutants

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A possible model for AP2 regulation by miRNA172 can be envisioned in

which expression of miRNA172 in the inner two floral whorls causes

translational inhibition of AP2 mRNA in these whorls

This model is supported by the higher levels of miRNA172 in the inner two

whorls of the flower

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Downstream Targets of Floral Organ-identity Genes

Although we have a good understanding of how floral organ identity is specified

by the ABC class genes and how the ABC class genes are regulated, we know far

less about the events occurring downstream of these factors

Several types of transcriptional profiling techniques such as differential screening,

subtractive hybridization, differential display and expression microarrays have

been performed using transgenic and mutant flowers with altered organ identity

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Genes regulated by the B function

To identify genes directly regulated by AP3 and PI that function in petal

organogenesis, a genetic background of ap3-3 ag-3 35S::PI 35S::AP3-GR was used.

In the absence of steroid these plants produce sepals in all four whorls because they

do not have active class B and C functions.

After steroid treatment, these plants produce petals in all four whorls. Inflorescences

of these plants were treated with steroid and cycloheximide or cycloheximide alone

and RNA populations from both treatments were compared using differential display.

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Targets of AG regulation

A putative direct target of AG in the fourth whorl is SHATTERPROOF2 (SHP2).

SHP1 and SHP2 are closely related and redundant MADS-domain proteins that are

expressed in thin stripes at the boundaries between carpel valves and replums

shp1 shp2 double mutants produce indehiscent fruit that do not shatter to release

seeds

SHP2 has been proposed to be a direct target of AG.

There are several CArG box sites in the SHP2 promoterto which AG binds in vitro and

ectopic expression of AG can activate aSHP2:: b-glucuronidase (GUS) reporter gene in

cauline leaves

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Microarray experiments using 35S::AG-GR ag-1 plants have shown that

SPOROCYTELESS (SPL)/NOZZLE (NZZ) is a direct target of the class C

protein AG

SPL encodes a protein that may function as a transcription factor and ectopic

expression of SPL in an ag-1 background is sufficient to induce microsporogenesis.

This suggests that additional transcriptional regulators turned on by AG mediate

various aspects of AG function in stamen and carpel organogenesis.

abc model of flower development

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