310
Review
9 Kaya, B. et al. (2000) Use of the Drosophila wing
spot test in the genotoxicity testing of different
herbicides. Environ. Mol. Mutagen. 36, 40–46
10 Amanuma, K. et al. (2000) Transgenic zebrafish
for detecting mutations caused by compounds in
aquatic environments. Nat. Biotechnol. 18, 62–65
11 Winn, R. et al. (2000) Detection of mutations in
transgenic fish carrying a bacteriophage lambda
cII transgene target. Proc. Natl. Acad. Sci. U. S. A.
97, 12655–12660
12 Gomot, A. and Bispo, A. (2000) Methods for
toxicity assessment of contaminated soil by oral or
dermal uptake in land snails. 1. Sublethal effects
on growth. Environ. Sci. Technol. 34, 1865–1870
13 Wagner, E. et al. (1998) Analysis of mutagens with
single cell gel electrophoresis, flow cytometry, and
forward mutation assays in an isolated clone of
Chinese hamster ovary cells. Environ. Mol.
Mutagen. 32, 360–368
14 Feron, V. et al. (1999) Long- and medium-term
carcinogenicity studies in animals and short-term
genotoxicity tests. Int. Agency Res. Cancer Sci.
Publ. 131, 103–129
15 Mahler, J.F. (2000) The use of genetically altered
animals in toxicology. Toxicol. Pathol. 28, 447–449
16 George, S. et al. (2001) Use of a Salmonella
microsuspension bioassay to detect the
mutagenicity of munitions compounds at low
concentrations. Mutat. Res. 490, 45–56
17 Powell, R. (1997) The use of vascular plants as
‘field’ biomonitors. In Plants for Environmental
studies (Wang, W. et al., eds), pp. 335–357, CRC
Lewis Publishers, Boca Raton, New York, USA
18 Xiao, L.Z. and Ichikawa, S. (1998) Mutagenic
interactions between X-rays and two
promutagens, o-phenylenediamine and Nnitrosodimethylamine, in the stamen hairs of
TRENDS in Plant Science Vol.6 No.7 July 2001
19
20
21
22
23
24
25
26
27
Tradescantia clone BNL 4430. Mutat. Res. 413,
177–186
Kovalchuk, O. et al. (1998) The Allium cepa
chromosome aberration test reliably measures
genotoxicity of soils of inhabited areas in the
Ukraine contaminated by the Chernobyl accident.
Mutat. Res. 415, 47–57
Kovalchuk, I. et al. (1998) Transgenic plants are
sensitive bioindicators of nuclear pollution caused
by the Chernobyl accident. Nat. Biotechnol. 16,
1054–1057
Grant, W.F. (1999) Higher plant assays for the
detection of chromosomal aberrations and gene
mutations – a brief historical background on their
use for screening and monitoring environmental
chemicals. Mutat. Res. 426, 107–112
Kong, M. and Ma, T. (1999) Genotoxicity of
contaminated soil and shallow well water
detected by plant bioassays. Mutat. Res. 426,
221–228
Rossman, T.G. et al. (1991) Performance of 133
compounds in the lambda prophage induction
endpoint of the Microscreen assay and a
comparison with S. typhimurium mutagenicity
and rodent carcinogenicity assays. Mutat. Res.
260, 349–367
Friedlender, M. et al. (1996) Cell divisions in
cotyledons after germination: localization, time
course and utilization for a mutagenesis assay.
Planta 199, 307–313
Kovalchuk, I. et al. (2000) Genome-wide variation
of the somatic mutation frequency in transgenic
plants. EMBO J. 19, 4431–4438
Swoboda, P. et al. (1994) Intrachromosomal
homologous recombination in whole plants.
EMBO J. 13, 484–489
Kovalchuk, O. et al. (2000) Plants experiencing
28
29
30
31
32
33
34
35
36
chronic internal exposure to ionizing radiation
exhibit higher frequency of homologous
recombination than acutely irradiated plants.
Mutat. Res. 449, 47–56
Ries, G. et al. (2000) Elevated UV-B radiation
reduces genome stability in plants. Nature 406,
98–101
Kovalchuk, O. et al. A sensitive transgenic plant
system to detect toxic inorganic compounds in the
environment. Nat. Biotechnol. (in press)
Knasmuller, S. et al. (1998) Use of metabolically
competent human hepatoma cells for the
detection of mutagens and antimutagens. Mutat.
Res. 402, 185–202
Gonzalez, C.A. et al. (2000) Biomonitoring study
of people living near or working at a municipal
solid-waste incinerator before and after two years
of operation. Arch. Environ. Health 55, 259–267
Tsongas, T. et al. (2000) Risk analysis of PCB
exposure via the soil–food crop pathway, and
alternatives for remediation at Serpukhov,
Russian Federation. Risk Anal. 20, 73–79
Fomin, A. et al. (1999) Assessment of the
genotoxicity of mine-dump material using the
Tradescantia-stamen hair (Trad-SHM) and the
Tradescantia-micronucleus (Trad-MCN)
bioassays. Mutat. Res. 426, 173–181
Hinton, T. et al. (1996) Foliar absorption of
resuspended Cs-137 relative to other pathways of
plant contamination. J. Environ. Radioact. 30,
15–30
Raskin, I. et al. (1997) Phytoremediation of metals:
using plants to remove pollutants from the
environment. Curr. Opin. Biotechnol. 8, 221–226
Bizily, S. et al. (2000) Phytodetoxification of
hazardous organomercurials by genetically
engineered plants. Nat. Biotechnol. 18, 213–217
Relearning our ABCs: new twists on
an old model
Thomas Jack
Over the past decade, the ABC model of flower development has been widely
promulgated. However, correct flower-organ development requires not only the
ABC genes but also the SEPALLATA genes. When the SEPALLATA genes are
expressed together with the ABC genes, both vegetative and cauline leaves are
converted to floral organs. Most of the ABC genes and all three SEPALLATA
genes encode MADS transcription factors, which bind to DNA as dimers. Here,
amendments to the ABC model are considered that incorporate both the
SEPALLATA genes and the ability of MADS proteins to form higher-order
complexes.
Thomas Jack
Dept Biological Sciences,
Dartmouth College,
Hanover, NH 03755, USA.
e-mail: thomas.jack@
dartmouth.edu
In the early 1990s, Elliott Meyerowitz, Enrico Coen
and colleagues proposed the ABC model of floral
organ identity1. This model, based on genetic
experiments in Antirrhinum and Arabidopsis, was
striking in its simplicity and is applicable in a wide
range of angiosperm species. The Arabidopsis flower,
like most angiosperm flowers, consists of four organ
types that are arranged in a series of concentric rings
or whorls. From outside to inside, the flower consists
of sepals in whorl one, petals in whorl two, stamens in
whorl three and carpels in whorl four. The ABC model
postulates the existence of three activities in the
flower, referred to as A, B and C, that function in
adjacent whorls – A activity in whorls one and two, B
activity in whorls two and three, and C activity in
whorls three and four (Fig. 1).
Two genes known to produce A activity in
Arabidopsis are APETALA1 (AP1) and APETALA2
(AP2) (Box 1; Table 1). In ap1 and ap2 mutants,
organs in floral whorls one and two fail to develop
with the correct identity. In addition to organ identity
defects, ap1 mutants also exhibit defects in floral
http://plants.trends.com 1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)01987-2
Review
Fig. 1. The ABC and SEP
genes specify floral organ
identity. The three SEP
genes function
redundantly and are
necessary for petal,
stamen and carpel
development. A revised
version of the ABC model
postulates that, in whorl 1,
A-class activity specifies
sepals; in whorl 2,
A + B + SEP activities
specify petals; in whorl 3,
B + C + SEP activities
specify stamens; and, in
whorl 4, C + SEP activities
specify carpels.
TRENDS in Plant Science Vol.6 No.7 July 2001
Box 1. ABC and SEP genes
B
AP3
PI
A
C
AP1
AP2
AG
SEP1
SEP2
SEP3
Sepals
1
Stamens
3
Petals
2
311
Carpels
4
AG
AP1
AP3
DEF
GLO
PI
PLE
SEP1
SEP2
SEP3
SQUA
AGAMOUS (Arabidopsis C class)
APETALA1 (Arabidopsis A class)
APETALA3 (Arabidopsis B class)
DEFICIENS (Antirrhinum AP3 orthologue)
GLOBOSA (Antirrhinum PI orthologue)
PISTILLATA (Arabidopsis B class)
PLENA (Antirrhinum AG orthologue)
SEPALLATA1
SEPALLATA2
SEPALLATA3
SQUAMOSA (Antirrhinum AP1 orthologue)
Whorl
TRENDS in Plant Science
Necessity and sufficiency
meristem identity (partial conversion of flowers to
shoots). Mutants of the two B-class genes,
APETALA3 (AP3) and PISTILLATA (PI ), exhibit an
identical phenotype, namely the conversion of petals
in the second whorl to sepals and of stamens in the
third whorl to carpels. Mutants for the C-class gene
AGAMOUS (AG) exhibit organ identity defects in
whorls three and four as well as a loss of floral
determinacy. In ag mutants, the third whorl develops
as petals and the fourth whorl as sepals (or,
alternatively, as the first whorl of an inner flower);
this pattern can repeat itself many times, resulting in
large, indeterminate flowers that can consist of >100
floral organs. The accepted ABC model postulates
that sepals are specified by A activity alone, petals by
a combination of A and B activities, stamens by a
combination of B and C activities, and carpels by C
activity alone (Fig. 1).
By the mid-1990s, the central tenets of the ABC
model were supported by molecular experiments. All
ABC genes encode proteins resembling transcription
factors. The general rule is that the ABC genes are
persistently expressed in the region of the flower that
exhibits defects in mutants. For example, AP3 and PI
RNA, and AP3 and PI protein accumulate in the
precursor cells of the petals and stamens and
throughout these organs as they mature2–4. However,
there are exceptions to this rule, for example, AP2 is
ubiquitously expressed5. A second line of
experimentation showed that ABC gene expression
correlates with organ identity. For example, in an ag
mutant, the A-class gene AP1 is persistently
expressed in whorls three and four, and these whorls
develop as petals and sepals, respectively6.
Table 1. Floral organ identity genes in Arabidopsis and Antirrhinum
Arabidopsis thaliana
Antirrhinum majus
A class
AP1, AP2
SQUAMOSA (SQUA)
B class
AP3, PI
DEFICIENS (DEF ), GLOBOSA (GLO)
C class
AG
PLENA
SEPALLATA
SEP1, SEP2, SEP3
DEFH84, DEFH200, DEFH72
http://plants.trends.com
The failure of floral organs to develop with the correct
identity in ABC mutants shows that the ABC genes
are necessary to specify floral organ identity. As a test
of sufficiency, the ABC genes were ectopically
expressed under the control of the broadly expressed
cauliflower mosaic virus 35S promoter. The most
straightforward of these ectopic expression
experiments involves the B-class genes.
Overexpression of both B-class genes together (i.e.
35S::AP3 35S::PI) results in a flower that consists of
two outer whorls of petals and two inner whorls of
stamens7 (Table 2). Based on this, it was concluded
that AP3 and PI are sufficient, within the flower, to
direct petal and stamen identity. However, AP3 and
PI together are not sufficient to convert rosette leaves
to petals, but cauline leaves do exhibit a slight
conversion to petals.
Additional evidence that AP3 and PI are active
within but not outside the flower comes from analysis
of the expression of a B-class target gene promoter
fused to a reporter gene encoding β-glucuronidase
(AP3::GUS). In 35S::AP3 35S::PI flowers, AP3::GUS
is activated throughout the flower but it is not
activated in cauline leaves, rosette leaves or roots.
This experiment shows that components other than
AP3 and PI are required for B-class function outside
the flower7. However, based on this experiment alone,
it is not clear whether there is a negatively acting
factor (or factors) present outside the flower that
prevents AP3 and PI from functioning, or if there is a
flower-specific positive factor (or factors) that is
necessary, in combination with AP3 and PI, to direct
petal and stamen identity.
Three SEP genes are necessary for floral organ identity
Most ABC genes in Arabidopsis are members of the
MADS family of transcription factors, including the
A-class gene AP1, the B-class genes AP3 and PI, and
the C-class gene AG. With the completion of the
sequence of the Arabidopsis genome8, it is now known
that there are >80 MADS genes in Arabidopsis9,10.
The MADS genes that have been studied to date are
involved in diverse aspects of plant development11
including flowering time control [e.g. FLC, AGL20
312
Review
TRENDS in Plant Science Vol.6 No.7 July 2001
Table 2. Ectopic expression of ABC and SEP genes
Overexpressed
Whorl 1
Whorl 2
Whorl 3
Whorl 4
Cauline leaves
AP3, PI
Petals
Petals
Stamens
Stamens
Slightly petaloid
Normal
AP3, PI, SEP1
Petals
Petals
Stamens
Stamens
Petals
Slightly petaloid
AP3, PI, SEP2
Petals
Petals
Stamens
Stamens
Petals
Slightly petaloid
AP3, PI, SEP3
Petals
Petals
Stamens
Stamens
Petals
Slightly petaloid
AP3, PI, AP1
Petals
Petals
Stamens
Stamens
Petals
Slightly petaloid
AP3, PI, SEP3, AP1
Petals
Petals
Stamens
Stamens
Petals
Petaloid
AP3, PI, SEP2, SEP3, AP1
Petals
Petals
Stamens
Stamens
Petals
Petals
AP3, PI, SEP3, AG
Stamens
Stamens
Stamens
Stamens
Staminoid
Normal
AP3, PI, AG
Petaloid staminoid
Stamens
Stamens
Stamens
Slightly petaloid
Normal
(also known as SOC1) and SVP], meristem identity
(CAL), fruit dehiscence (SHP1 and SHP2) and root
development (ANR1) (reviewed in Ref. 11). In spite of
efforts to determine the function of all MADS genes in
Arabidopsis, fewer than a quarter have been
correlated with a loss-of-function phenotype.
For the past decade, Martin Yanofsky and coworkers have been systematically characterizing the
Arabidopsis MADS genes12–14. Initially, MADS genes
were identified based on homology to AG. Thus, the
MADS family members were referred to by
AGAMOUS-LIKE (AGL) designations. Three MADS
genes, SEP1 (previously referred to as AGL2), SEP2
(previously AGL4) and SEP3 (previously AGL9), are
similar in sequence and exhibit similar temporal
expression patterns. During early and intermediate
stages of flower development, SEP1 and SEP2 are
expressed in all four floral whorls15,16, whereas SEP3 is
expressed only in whorls two, three and four17. Loss-offunction mutations have been obtained in all three SEP
genes18. Neither sep single mutants nor combinations of
sep double mutants exhibit a dramatic developmental
phenotype. By contrast, the sep1 sep2 sep3 triple
mutant exhibits a phenotype that is similar to a BC
double mutant such as pi ag or ap3 ag. Specifically, the
sep1 sep2 sep3 triple mutant consists entirely of sepallike organs and the flowers are indeterminate.
There are two important conclusions from this
result. First, all three SEP genes together are
necessary for proper development of petals, stamens
and carpels; in the absence of all three SEP genes, the
inner three whorls of the flower develop as sepals.
Second, the SEP genes function redundantly;
mutation of one or two SEP genes does not affect
floral organ identity but simultaneous removal of all
three SEP genes results in dramatic organ identity
defects (Fig. 1). For two reasons, however, it seems
unlikely that the SEP genes function completely
redundantly. First, if they were completely redundant
then there would be no selective advantage in
maintaining functional copies of all three genes, yet
evolutionary analysis suggests that the SEP genes
have been maintained for millions of years. Second,
SEP3 has a different expression pattern compared
with SEP1 and SEP2, suggesting that these genes
might have independent functions.
http://plants.trends.com
Rosette leaves
Converting leaves into floral organs
As a test of sufficiency, the SEP genes were ectopically
expressed in combination with the ABC genes19,20. By
themselves, 35S::SEP1, 35S::SEP2 or 35S::SEP3 do
not alter the organ identity of the cauline and rosette
leaves. However, ectopic expression of the SEP genes
together with the ABC genes converts cauline and
rosette leaves to petals or stamens (Table 2). For
example, in 35S::SEP3 35S::AP3 35S::PI plants,
cauline leaves are completely converted to petals, and
rosette leaves are partially transformed to petals
(Fig. 2b). Cauline leaves are also converted to petaloid
organs when SEP1, SEP2 or AP1 is ectopically
expressed in combination with AP3 and PI.
The phenotype of the sep1 sep2 sep3 triple mutant
strongly suggests that the SEP genes are necessary for
petal identity, but the fact that ectopic expression of
AP1 can substitute for ectopic expression of the SEP
genes suggests that the SEP genes are not absolutely
required for petal identity. One possible reconciliation
is that the SEP genes are required when AP1 is
expressed at normal levels but not when AP1 is
overexpressed (e.g. 35S::AP1). The ability of AP1 to
substitute for the SEP genes is not surprising
considering that AP1 and the SEP genes are grouped
in a common superclade that is clearly distinct from
clades containing AG-, AP3- and PI-like genes. A
second reconciliation is that it is possible that the
endogenous SEP1, SEP2 and/or SEP3 genes are
activated in the leaves of 35S::AP3 35S::PI 35S::AP1.
At present, there is no evidence that the SEP genes
are regulated by the ABC genes, but the RNA
expression patterns of the SEP genes in 35S::AP3
35S::PI 35S::AP1 have not been determined.
Together, the results of the ectopic expression
experiments suggest that it is the absence of a positive
factor such as SEP3 or AP1, rather than the presence of
a negative factor, in non-floral tissues that prevents the
full conversion of leaves to petals in 35S::AP3 35S::PI.
This hypothesis is also supported by experiments that
use the B-class target gene AP3::GUS. 35S::AP3
35S::PI, 35S::SEP3 and 35S::AP1 do not activate
AP3::GUS in non-floral tissues. However, when
35S::SEP3 or 35S::AP1 is present together with
35S::AP3 35S::PI, AP3::GUS is activated throughout
the plant19. Thus, combinations of AP3 + PI + SEP3 or
Review
(a)
(b)
Ct
TRENDS in Plant Science Vol.6 No.7 July 2001
(c)
(d)
RL
RL
Ct
Fig. 2. Conversion of
leaves to petals and
stamens. (a) Wild-type
seedling. Cotyledons and
rosette leaves are
indicated. (b) 35S::AP3
35S::PI 35S::SEP3
seedling. Rosette leaves
are converted to petal-like
organs, but cotyledons
are normal. (c) 35S::AP3
35S::PI 35S::SEP3
35S::AP1 seedling.
Compared with (b),
rosette leaves are more
completely converted to
petals. (d) 35S::AP3
35S::PI 35S::SEP3 35S::AG
inflorescence. Cauline
leaves are converted to
stamen-like organs.
Flowers also consist
primarily of stamen-like
organs. Abbreviations:
CL, cauline leaves; Ct,
cotyledons; F, flowers; RL,
rosette leaves. (b,d)
Reproduced, with
permission, from Ref. 19;
(c) reproduced, with
permission, from Ref. 20.
RL
F
CL
AP3 + PI + AP1 are sufficient to activate downstream
target genes required for petal development.
A more dramatic conversion of rosette leaves to
petals occurs when both SEP3 and AP1 are ectopically
expressed in combination with 35S::AP3 35S::PI; in this
case, rosette leaves are converted almost completely to
petals (Fig. 2c; Table 2). The conversion to petals is
slightly more complete when SEP2 is also misexpressed
to create a quintuple ectopic expression line20. SEP2
enhances the leaf phenotype, suggesting that SEP2 and
SEP3 are not completely redundant and that SEP2
might possess a function that is independent of SEP3.
An alternative explanation is that the organ identity
transformations are sensitive to levels of SEP2 and
SEP3 expression, which might not be equivalent owing
to transgene copy number and/or position effects.
In a second set of experiments, the C-class gene AG
was ectopically expressed in combination with AP3,
PI and SEP3. In 35S::SEP3 35S::AG 35S::AP3
35S::PI, cauline leaves are converted to stamens19
(Fig. 2d; Table 2). In this line, however, rosette leaves
are not converted to stamens, suggesting that at least
one additional factor is required to convert a rosette
leaf into a stamen.
Molecular interaction between SEP and ABC MADS
genes
The ABC genes and the SEP genes encode MADS
transcription factors, and both groups of genes are
required for proper development of petals, stamens and
carpels in the flower. However, the SEP genes and the
ABC genes, exhibit different temporal expression
profiles, suggesting that there might be a regulatory
relationship between these two classes. For example,
transcription of the ABC genes might be dependent on
the SEP genes, or vice versa. However, there is no
transcriptional relationship between the SEP genes
and the ABC MADS genes. RNA from the SEP genes
accumulates normally in single mutants for A-, B- and
C-class genes15,16. Similarly, B- and C-class gene
expression is activated normally in the sep1 sep2 sep3
triple mutant18. Based on these results, the organ
identity defects in ABC mutants and the sep1 sep2 sep3
mutant cannot be explained by a failure of either the
ABC genes or the SEP genes to be transcribed. Thus,
the requirement for these two groups of genes must be
mediated by a different mechanism.
A second possibility is that there is a direct
interaction between the SEP proteins and the ABC
proteins. Plant MADS proteins bind to DNA in vitro as
dimers, either homodimers or heterodimers. However,
the in vivo significance of different dimer combinations
is not well understood. MADS proteins bind to a
10 bp consensus binding site called the CArG box
http://plants.trends.com
313
(5′-CC[AT]6GG-3′). In Arabidopsis, AG binds to a CArG
box sequence as either a homodimer or a heterodimer
with SEP1 (Ref. 21). By contrast, AP3 and PI do not
form DNA-binding homodimers but instead bind to
DNA only as a heterodimer22,23. AP1 and the SEP
proteins do not form DNA-binding heterodimers with
AP3 or PI, which rules out heterodimerization as an
explanation of why petal and stamen development
requires both the SEP genes and the B-class genes.
Problem of functional specificity
For some time, the nature of the functional specificity
of the plant MADS proteins has been unclear.
Evidence suggests that the functional specificity of
the MADS proteins does not lie in DNA-binding-site
selection or affinity as determined by the ABC
proteins alone24. The ABC MADS proteins that
specify organ identity (i.e. AG, AP1 and AP3–PI)
exhibit in vitro DNA-binding specificities that are
largely overlapping22,23, yet these proteins function
differently in directing floral organ identity.
In a series of illuminating experiments24, the DNAbinding portion of the MADS domains of AP3–PI were
replaced with the diverged MADS domain of the
mammalian MADS protein Mef2A. The Mef2A MADS
domain has a slightly different consensus DNAbinding sequence compared with AP3–PI as
determined by in vitro DNA-binding assays. As
expected, the chimeric Mef2a–AP3 and Mef2A–PI
heterodimers bound in vitro to the Mef2A consensus
binding site, but not to the AP3–PI consensus.
However, when expressed under the control of the 35S
promoter in transgenic plants, the chimeric proteins
behaved identically to 35S::AP3 and 35S::PI. These
experiments definitively show that DNA-binding-site
affinity, as determined by the AP3 and PI proteins
alone, does not determine functional specificity.
One possibility is that MADS dimers themselves,
either homodimers or heterodimers, interact
specifically with ternary or accessory factors that are
essential for function. According to this model,
different MADS dimer combinations could interact
with different accessory factors and the accessory
factors, in turn, might determine the functional
specificity of the complex by altering its DNA-binding
specificity, activating transcription or some other
molecular mechanism. Ternary factors are essential
for the function of the yeast MADS protein MCM1
and the human MADS protein serum response factor
(SRF). For example, the DNA-binding proteins α1
and α2 are essential for the ability of MCM1 to
determine cell-type specificity in yeast25. Similarly,
ETS-domain DNA-binding proteins such as Elk-1 are
important for the specificity of the interaction of SRF
with DNA26.
Higher order complexes of MADS proteins
Recent evidence suggests that MADS proteins in plants
might associate in complexes larger than dimers27. One
line of evidence comes from yeast two-hybrid
314
Review
TRENDS in Plant Science Vol.6 No.7 July 2001
(a)
CArG box
AP1 SEP3
C
C
C
C
AP3
PI
CArG box
(b)
SEP3
C
C
C
AP3
PI
CArG box
(c)
C
C
AP3
PI
CArG box
C
C
SEP3 API
CArG box
TRENDS in Plant Science
Fig. 3. Three molecular models explain how the ABC proteins and the SEP
proteins interact with each other and with DNA to direct the transcription
of genes that determine floral organ identity. Different target genes might
be controlled by different molecular mechanisms. For simplicity, this
figure summarizes the molecular interactions that might occur in petals.
(a) The first model, referred to as the ‘quartet’ model34,35, postulates that
MADS tetramers bind to two CArG box sequences and direct
transcription of target genes. In petals, the AP3–PI heterodimer is
postulated to bind to one CArG box and an AP1–SEP3 heterodimer to a
second CArG box. Present evidence suggests that the C-terminal
domains (C) of the MADS proteins mediate protein–protein interactions
between dimers (dots). However, it is possible that some ternary
interactions might be mediated by the K domain. (b) The second model
postulates that a multimeric complex of MADS proteins binds to a single
CArG box sequence. In petals, the AP3–PI heterodimer binds to the CArG
box sequence. A ternary MADS factor, such as SEP3 or AP1, interacts via
the C-terminal domain with the AP3–PI heterodimer, possibly altering
DNA-binding affinity or specificity, or providing a transcriptional
activation domain to the complex. (c) A third model, which at present
cannot be ruled out by the data, is that dimers of MADS proteins bind to
adjacent CArG box sequences in a cooperative manner that does not
involve a protein–protein interaction between MADS dimers.
experiments in Antirrhinum and Arabidopsis. A twohybrid screen using AG as bait identified SEP1, SEP2
and SEP3 as interacting proteins28. Similar screens in
Antirrhinum using the AG homologue PLENA
(Table 1) as bait led to the isolation of several SEPlike genes29. Two-hybrid screens using either AP3 or
PI alone (or the Antirrhinum homologues DEF and
GLO; Table 1) resulted in the isolation of only the
partner protein (i.e. when DEF was used as bait, only
GLO clones were isolated, and vice versa29).
A more sophisticated screen was carried out by
looking for proteins that could interact with the
AP3–PI heterodimer but not with AP3 and PI proteins
alone19. In this experiment, a single plasmid expressed
both PI and an AP3–lexA fusion. Interacting proteins
were isolated from a library of floral cDNAs fused to
http://plants.trends.com
the GAL4 activation domain. In this screen, SEP3 and
AP1 were isolated (as well as PI, which is, in this case,
a positive control). Expression of either AP1 or SEP3,
in combination with AP3 and PI together, but not with
either AP3 or PI alone, led to the activation of the
reporter. Similar interactions were observed in yeast
with the Antirrhinum proteins DEF, GLO and SQUA
(Ref. 30). Interactions between AP3, PI and AP1, and
between AP3, PI and SEP3 were confirmed by coimmunoprecipitation experiments19, lending support
to the hypothesis that MADS proteins form complexes
that consist of more than two monomers.
Evidence that these complexes are functional
comes from DNA-binding assays performed in
Antirrhinum. In one study, a probe containing two
CArG box sequences exhibited enhanced DNA binding
in the presence of both SQUA and DEF–GLO
compared with SQUA or DEF–GLO alone30. Based on
this, the authors concluded that DEF–GLO and SQUA
formed a multimeric complex. However, they were
unable to detect a more slowly migrating complex
containing DEF, GLO and SQUA using a DNA
template that contained a single CArG box. Because
there are two binding sites in the probe, it is possible
that the cooperative DNA-binding effects observed are
due to an interaction that does not involve a
protein–protein interaction between dimers. For
example, an alternative model that is consistent with
the data is that binding of DEF–GLO to one binding
site alters DNA structure in a way that enhances the
binding of SQUA to the second CArG box.
Evidence that direct protein–protein interaction of
MADS proteins is functionally important comes from
experiments designed to determine whether MADS
proteins can activate transcription. In yeast, fusions of
AP1 and SEP3 to the GAL4 DNA-binding domain
activate transcription of a reporter controlled by a
GAL4 upstream activating sequence. However, PI, AP3
and AG do not activate transcription in this assay19,31.
Transactivation potential was also tested in transient
co-transfection experiments in onion epidermal cells.
In one experiment, various combinations of MADS
proteins were expressed under the control of the 35S
promoter. The reporter plasmid contained seven copies
of a consensus synthetic CArG box cloned upstream of
a reporter. By themselves, AP1, SEP1 and SEP3
exhibited high levels of activation in this assay,
suggesting that these proteins encode transcriptional
activation domains. SEP2 alone exhibited a moderate
level of transactivation activity, whereas AP3, PI, AG
and AP3–PI did not activate19.
When a version of PI that contained the strong VP16
activation domain was transfected alone, it did not lead
to activation. However, when PI–VP16 was transfected
together with wild-type AP3, strong activation
resulted19. Together, these experiments suggest that the
formation of a higher order complex might provide an
activation domain to MADS dimers that do not encode
an activation domain. For example, in the second whorl,
perhaps the SEP proteins and AP1 provide an activation
Review
TRENDS in Plant Science Vol.6 No.7 July 2001
domain to the AP3–PI heterodimer, which does not have
an activation domain, to allow for the transcriptional
activation of downstream target genes necessary for
petal development. It is curious that the authors did not
test this directly in their co-transfection assay. If this
model were true, one prediction would be that coexpression of one of the SEP genes or AP1 together with
AP3 and PI would increase the levels of transactivation.
The probable reason that the authors failed to do this is
because the SEP proteins and AP1 activate transcription
on their own. Presumably, transcriptional activation by
AP1 (or SEP) alone could be prevented by engineering a
mutation in the MADS domain that would prevent DNA
binding but not the ability of AP1 (or SEP) to function in
a multimeric complex.
Two activities associated with higher order MADS
complexes – transcriptional activation and multimer
association – have been mapped to the C-terminal
domain of MADS proteins. For AP1 and SEP3, the
transcriptional activation domain has been mapped to
the C-terminal domain19. The C-terminal domain is also
necessary for the ternary interaction of AP1 and SEP3
with the AP3–PI heterodimer. Similarly, the C-terminal
domain is important for the ternary interaction of DEF,
GLO and SQUA; removal of the C-terminal domain
from any of these three Antirrhinum MADS proteins
prevents both ternary association in yeast and the
formation of higher order DNA-binding complexes30.
Evidence that the C-terminal domain is necessary for
function in vivo comes from experiments that
ectopically express truncated versions of MADS
proteins; such truncations result either in loss of
activity or a dominant-negative phenotype32,33.
Molecular models
No single model for ABC and SEP gene function
currently explains all the data adequately. However,
three models explain both the necessity of the SEP
genes for specifying floral organ identity and take into
account the evidence that higher order interactions
can occur between MADS proteins. First, the socalled ‘quartet’ model34,35 postulates that tetramers of
MADS proteins determine floral organ identity
(Fig. 3a). Each tetramer consists of two MADS
dimers, each of which binds to a single CArG box. The
tetramers are formed by protein–protein interactions
between MADS dimers, mediated by the C-terminal
domains and resulting in a tetramer of MADS
proteins that is simultaneously bound to two CArG
boxes. There are at least two molecular mechanisms
that explain how these MADS tetramers result in an
active transcription complex. One mechanism is a cooperative DNA-binding effect, in which binding of one
dimer in the tetramer results in increased affinity of
local binding of the second dimer in the tetramer. A
second mechanism is that one or more subunits of the
dimer provide an activation domain to the tetramer to
enable efficient transcriptional activation.
The ‘quartet’ model makes predictions about the
composition of the tetramers in the various whorls.
http://plants.trends.com
315
Specifically, in whorl 1, a tetramer containing at least
one AP1–AP1 homodimer is postulated to specify
sepals. The second dimer of the tetramer could consist
of a second AP1 homodimer but probably does not
include SEP1 or SEP2 because, although these genes
are expressed in whorl 1, they are not necessary for
sepal development18. In whorl 2, a combination of
AP3–PI and SEP3–AP1 is postulated to specify petals;
in whorl 3, AP3–PI and SEP3–AG are postulated to
specify stamens; and, in whorl 4, AG–AG and
SEP3–SEP3 are postulated to specify carpels.
The limitation of the quartet model is the
requirement for two closely linked CArG box
sequences in the promoters of target genes. Certainly,
this is true for some target genes, such as AP3, which
contains three CArG boxes in an 85 bp region of its
promoter36, and GLO, which contains three CArG
boxes within 525 bp of the transcription start37.
However, multiple CArG boxes, do not appear to be
present in all targets of MADS proteins; for example,
both NAP1 (Ref. 38), a direct downstream target gene
of AP3–PI, and SHP2, a direct downstream target
gene of AG, contain only a single close match to the
CArG consensus sequence16.
In a second model, the multimers of MADS proteins
associate but only bind to DNA at one binding site
(Fig. 3b). According to this model, a single MADS dimer
contacts DNA and additional MADS protein(s) are
associated with the dimer. If this were true then SEP3
and AP1, which exhibit protein–protein interactions
with the AP3–PI heterodimer, could provide either DNA
binding-site selection or affinity to the complex, or a
transcriptional-activation domain. The limitation of
this model is that these types of complexes have not
been detected in vitro. In the one study that directly
looked for ternary complexes30, they were not detected,
but the failure to detect ternary complexes does not
prove that they fail to form. Specifically, an interaction
of DEF–GLO with SQUA was observed only when two
CArG boxes were present on the DNA fragment used in
the gel shift assay, but no interaction was detected when
a single CArG box was present on the DNA fragment30.
A third model, which is less likely but cannot be ruled
out by present data, is that there are co-operative DNAbinding interactions between MADS dimers bound to
adjacent CArG box binding sites, but these interactions
are not dependent on a protein–protein interaction
between dimers (Fig. 3c). For example, it is possible that
the binding of one MADS dimer to one CArG box alters
DNA structure in a way that increases the affinity of the
second MADS dimer for the second CArG box. This
model does not take into account the two-hybrid and coimmunoprecipitation data, which suggest, for example,
that AP1, SEP2 and SEP3 proteins can interact directly
with the AP3–PI heterodimer.
At present, no molecular model completely
explains the data in hand. Clearly, more needs to be
done to determine both the biochemical composition
of MADS multimers in vivo as well as the function of
these complexes in directing floral organ identity.
316
Review
TRENDS in Plant Science Vol.6 No.7 July 2001
Floral MADS genes in other species
Acknowledgements
I thank members of the
laboratory for discussion
and comments on the
manuscript. Work in the
laboratory is supported
by funds from NSF (MCB0090742).
Genes similar in sequence to the ABC MADS genes
AG, AP1, AP3, PI and AG are present in a wide range of
plant species, including gymnosperms, monocots and
dicots. Similarly, genes that are similar in sequence to
the SEP genes are present in gymnosperms (pine),
monocots (rice and barley) and dicots [e.g. snapdragon
(Antirrhinum majus), pepper (Capsicum annum),
tomato, Petunia, tobacco, sunflower (Helianthus spp.)
and pea]. Although mutants are not available, analyses
of antisense lines suggests that the tomato SEP-like
gene TM5 and the petunia gene FBP2 are involved in
the development of petals, stamens and carpels39,40. In
sunflower (Gerbera hybrida), antisense expression of
the SEP-like gene GRCD1 results in defects in stamen
development41. The broad distribution of SEP-like
genes in dicots suggests that SEP gene function might
be conserved. It is tempting to speculate that the
References
1 Coen, E.S. and Meyerowitz, E.M. (1991) War of
the whorls: genetic interactions controlling flower
development. Nature 353, 31–37
2 Jack, T. et al. (1994) Arabidopsis homeotic gene
APETALA3 ectopic expression: transcriptional
and post-transcriptional regulation determine
floral organ identity. Cell 76, 703–716
3 Kramer, E.M. and Irish, V.F. (1999) Evolution of
genetic mechanisms controlling petal
development. Nature 399, 144–148
4 Samach, A. et al. (1997) Divergence of function
and regulation of class B floral organ identity
genes. Plant Cell 9, 559–570
5 Jofuku, K.D. et al. (1994) Control of Arabidopsis
flower and seed development by the homeotic
gene APETALA2. Plant Cell 6, 1211–1225
6 Gustafson-Brown, C. et al. (1994) Regulation of
Arabidopsis floral homeotic gene APETALA1.
Cell 76, 131–143
7 Krizek, B.A. and Meyerowitz, E.M. (1996) The
Arabidopsis homeotic genes APETALA3 and
PISTILLATA are sufficient to provide the B class
organ identity function. Development 112, 11–22
8 Arabidopsis Genome Initiative (2000) Analysis of
the genome sequence of the flowering plant
Arabidopsis thaliana. Nature 408, 796–815
9 Alvarez-Buylla, E.R. et al. (2000) MADS-box gene
evolution beyond flowers: expression in pollen,
endosperm, guard cells, roots and trichomes.
Plant J. 24, 457–466
10 Riechmann, J.L. et al. (2000) Arabidopsis
transcription factors: genome-wide comparative
analysis among eukaryotes. Science
290, 2105–2110
11 Theissen, G. et al. (2000) A short history of MADSbox genes in plants. Plant Mol. Biol. 42, 115–149
12 Alvarez-Buylla, E.R. et al. (2000) An ancestral
MADS-box gene duplication occurred before the
divergence of plants and animals. Proc. Natl.
Acad. Sci. U. S. A. 97, 5328–5333
13 Ma, H. et al. (1991) AGL1–AGL6, an Arabidopsis
gene family with similarity to floral homeotic
and transcription factor genes. Genes Dev.
5, 484–495
14 Rounsley, S.D. et al. (1995) Diverse roles for
MADS box genes in Arabidopsis development.
Plant Cell 7, 1259–1269
15 Flanagan, C.A. and Ma, H. (1994) Spatially and
temporally regulated expression of the MADS-box
http://plants.trends.com
16
17
18
19
20
21
22
23
24
25
26
27
28
presence of SEP genes in monocots and gymnosperms
suggests that the SEP genes might play a more general
role in flowering and/or seed development.
Questions for future research
• What is the molecular nature of the higher order
MADS complexes?
• What is the functional significance of higher order
MADS complexes?
• Do higher order MADS complexes alter DNAbinding affinity or specificity?
• Do other members of the MADS family (e.g. the
flowering time MADS proteins SOC/AGL20 and
FLC) form higher order complexes?
• Do the floral MADS genes function similarly in
other flowering plant species?
gene AGL2 in wild-type and mutant Arabidopsis
flowers. Plant Mol. Biol. 26, 581–595
Savidge, B. et al. (1995) Temporal relationship
between the transcription of two Arabidopsis
MADS box genes and the floral organ identity
genes. Plant Cell 7, 721–733
Mandel, M.A. and Yanofsky, M.F. (1998) The
Arabidopsis AGL9 MADS-box gene is expressed
in young flower primordia. Sex. Plant Reprod.
11, 22–28
Pelaz, S. et al. (2000) B and C floral organ identity
functions require SEPALLATA MADS-box genes.
Nature 405, 200–203
Honma, T. and Goto, K. (2001) Complexes of
MADS-box proteins are sufficient to convert
leaves into floral organs. Nature 409, 525–529
Pelaz, S. et al. (2001) Conversion of leaves into
petals in Arabidopsis. Curr. Biol. 11, 182–184
Huang, H. et al. (1996) DNA binding properties of
two Arabidopsis MADS domain proteins: binding
consensus and dimer formation. Plant Cell 8,
81–94
Riechmann, J.L. et al. (1996) Dimerization
specificity of Arabidopsis MADS domain homeotic
proteins APETALA1, APETALA3, PISTILLATA,
and AGAMOUS. Proc. Natl. Acad. Sci. U. S. A.
93, 4793–4798
Riechmann, J.L. et al. (1996) DNA-binding
properties of Arabidopsis MADS domain
homeotic proteins APETALA1, APETALA3,
PISTILLATA and AGAMOUS. Nucleic Acids Res.
24, 3134–3141
Riechmann, J.L. and Meyerowitz, E.M. (1997)
Determination of floral organ identity by
Arabidopsis MADS domain homeotic proteins
AP1, AP3, PI, and AG is independent of their DNAbinding specificity. Mol. Biol. Cell 8, 1243–1259
Dolan, J.W. and Fields, S. (1991) Cell-type-specific
transcription in yeast. Biochim. Biophys. Acta
1088, 155–169
Treisman, R. (1994) Ternary complex factors:
growth factor regulated transcriptional
activators. Curr. Opin. Genet. Dev. 4, 96–101
Egea-Cortines, M. et al. (2000) Beyond the ABCs:
ternary complex formation in the control of floral
organ identity. Trends Plant Sci. 5, 471–476
Fan, H-Y. et al. (1997) Specific interactions
between K domains of AG and AGLs, members of
the MADS domain family of DNA binding
proteins. Plant J. 12, 999–1010
29 Davies, B. et al. (1996) Multiple interactions
amongst floral homeotic MADS box proteins.
EMBO J. 16, 4330–4343
30 Egea-Cortines, M. et al. (1999) Ternary
complex formation between the MADS-box
proteins SQUAMOSA, DEFICIENS, and
GLOBOSA is involved in the control of floral
architecture in Antirrhinum majus. EMBO J.
18, 5370–5379
31 Moon, Y-H. et al. (1999) Identification of a rice
APETALA3 homologue by yeast two-hybrid
screening. Plant Mol. Biol. 40, 167–177
32 Krizek, B.A. and Meyerowitz, E.M. (1996)
Mapping the protein regions responsible for the
functional specificities of the Arabidopsis MADS
domain organ-identity proteins. Proc. Natl. Acad.
Sci. U. S. A. 92, 4063–4070
33 Mizukami, Y. et al. (1996) Functional domains of
the floral regulator AGAMOUS: characterization
of the DNA binding domain and analysis of
dominant-negative mutations. Plant Cell
8, 831–845
34 Theissen, G. (2001) Development of floral organ
identity; stories from the MADS house. Curr.
Opin. Plant Biol. 4, 75–85
35 Theissen, G. and Saedler, H. (2001) Floral
quartets. Nature 409, 469–471
36 Tilly, J. et al. (1998) The CArG boxes in the
promoter of the Arabidopsis floral organ identity
gene APETALA3 mediate diverse regulatory
effects. Development 125, 1647–1657
37 Tröbner, W. et al. (1992) GLOBOSA: a homeotic
gene which interacts with DEFICIENS in the
control of Antirrhinum floral organogenesis.
EMBO J. 11, 4693–4704
38 Sablowski, R.W.M. and Meyerowitz, E.M. (1998)
A homolog of NO APICAL MERISTEM is an
immediate target of the floral homeotic genes
APETALA3/PISTILLATA. Cell 92, 93–103
39 Angenent, G.C. et al. (1992) Cosuppression of the
petunia homeotic gene FBP2 affects the identity
of the generative meristem. Plant J. 5, 33–44
40 Pnueli, L. et al. (1994) The TM5 MADS box
gene mediates organ differentiation in the
three inner whorls of tomato flowers. Plant Cell
6, 175–186
41 Kotilainen, M. et al. (2000) GRCD1, an AGL2-like
MADS box gene, participates in the C function
during stamen development in Gerbera hybrida.
Plant Cell 12, 1893–1902