APETALA1 and SEPALLATA3 interact to promote flower development

APETALA1 and SEPALLATA3 interact to promote ¯owerdevelopment

Soraya Pelaz1, Cindy Gustafson-Brown1, Susanne E. Kohalmi2, William L. Crosby3 and Martin F. Yanofsky1,*

1Section of Cell and Developmental Biology, University of California at San Diego, La Jolla, CA 92093-0116, USA,2Department of Plant Sciences, University of Western Ontario, 151 Richmond Street N., London, ON N6A 5B7, Canada,

and3Plant Genomics NRCC Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada

Received 4 December 2000; revised 5 March 2001; accepted 5 March 2001.*For correspondence (fax +1 858 822 1772; e-mail [email protected]).

Summary

In Arabidopsis, the closely related APETALA1 (AP1) and CAULIFLOWER (CAL) MADS-box genes share

overlapping roles in promoting ¯ower meristem identity. Later in ¯ower development, the AP1 gene is

required for normal development of sepals and petals. Studies of MADS-domain proteins in diverse

species have shown that they often function as heterodimers or in larger ternary complexes, suggesting

that additional proteins may interact with AP1 and CAL during ¯ower development. To identify proteins

that may interact with AP1 and CAL, we used the yeast two-hybrid assay. Among the ®ve MADS-box

genes identi®ed in this screen, the SEPALLATA3 (SEP3) gene was chosen for further study. Mutations in

the SEP3 gene, as well as SEP3 antisense plants that have a reduction in SEP3 RNA, display phenotypes

that closely resemble intermediate alleles of AP1. Furthermore, the early ¯owering phenotype of plants

constitutively expressing AP1 is signi®cantly enhanced by constitutive SEP3 expression. Taken together,

these studies suggest that SEP3 interacts with AP1 to promote normal ¯ower development.

Keywords: Arabidopsis, ¯owers, development, MADS-box.

Introduction

Following the transition from vegetative to reproductive

development in Arabidopsis, ¯ower meristems arise on

the ¯anks of the shoot apical (in¯orescence) meristem and

subsequently develop into ¯owers with four organ types

(sepals, petals, stamens and carpels). Flower meristem

identity is speci®ed in part by the APETALA1 (AP1),

CAULIFLOWER (CAL) and LEAFY (LFY) genes. In ap1

mutants, the sepals are transformed to leaf-like organs

and the petals fail to develop. In the axils of these leaf-like

organs, secondary ¯owers arise which repeat the same

pattern as the primary ones. Although cal single mutants

appear as wild type, ap1 cal double mutants display a

massive proliferation of in¯orescence-like meristems in

positions that would normally be occupied by solitary

¯owers. The functional redundancy shared by AP1 and

CAL can be explained in part by the fact that these two

genes encode related members of the MADS-box family of

regulatory proteins (Bowman et al., 1993; Gustafson-

Brown et al., 1994; Kempin et al., 1995; Mandel et al., 1992).

Genetic studies led to the proposal of the landmark ABC

model that explains how the individual and combined

activities of the ABC genes specify the four organ types of

the typical eudicot ¯ower. A alone speci®es sepals; A and

B specify petals; B and C specify stamens; and C alone

speci®es carpels. In Arabidopsis, the A-function genes are

AP1 and APETALA2 (AP2); B-function genes are APETALA3

(AP3), PISTILLATA (PI); and the C-function gene is

AGAMOUS (AG). In addition, recent studies have shown

that a trio of closely related genes, SEPALLATA1/2/3 (SEP1/

2/3), are required for petal, stamen and carpel identity, and

are thus necessary for the activities of the B- and C-

function genes (Pelaz et al., 2000). Remarkably, with the

exception of the AP2 gene, all the other organ-identity

genes belong to the extended family of MADS-box genes,

a family that is known to include more than 44 distinct

sequences in Arabidopsis (Alvarez-Buylla et al., 2000a;

Davies and Schwarz-Sommer, 1994; Purugganan et al.,

1995; Rounsley et al., 1995).

The Plant Journal (2001) 26(4), 385±394

ã 2001 Blackwell Science Ltd 385

MADS-domain proteins, well characterized in yeast

(MCM1, Ammererer, 1990) and mammals (SRF, Norman

et al., 1988), form dimers that bind to DNA and form

ternary complexes with many unrelated proteins (Lamb

and McKnight, 1991; Shore and Sharrocks, 1995). A

number of studies have shown that heterodimers and

ternary complexes of plant MADS-domain proteins can

occur and, given the overlapping expression pattern of

numerous MADS-box genes, such interactions greatly

increase the regulatory complexity of MADS-box genes

(Davies et al., 1996; Egea-Cortines et al., 1999; Fan et al.,

1997). The regulatory speci®city of these genes is achieved

through protein±protein interactions and not through

different intrinsic DNA-binding speci®cities (Krizek and

Meyerowitz, 1996; Shore and Sharrocks, 1995). MADS-box

proteins are composed of four different domains, desig-

nated M, I, K and C. The MADS (M) domain is highly

conserved among these proteins, and is responsible for

binding to DNA in addition to its participation in homo-

dimer formation of some proteins. The I region also

participates in homodimer formation (Krizek and

Meyerowitz, 1996; Riechmann et al., 1996). Adjacent to

the I region is the K domain, so named due to its similarity

to the coiled-coil domain of keratin. It is absent in the non-

plant proteins, and has been implicated in protein±protein

interaction (Fan et al., 1997; Krizek and Meyerowitz, 1996;

Mizukami et al., 1996; Moon et al., 1999; Riechmann et al.,

1996). The C-terminal region has been proposed to be

involved in transcriptional activation (Huang et al., 1995),

and also to play a role in the formation of ternary

complexes (Egea-Cortines et al., 1999).

In order to identify candidate proteins that interact with

AP1 and CAL, we used a modi®ed version of the yeast two-

hybrid system (Bartel et al., 1993; Chien et al., 1991; Fields

and Song, 1989; Fields and Sternglanz, 1994; Kohalmi

et al., 1998). These screens resulted in the isolation of ®ve

MADS-box genes as well as two additional genes. Here we

present the results of these screens, as well as the

molecular and genetic analyses of one of these interacting

genes, SEP3. Our results suggest that the observed

interactions in yeast re¯ect functional interactions that

occur in plants.

Results

Proteins that interact with CAL

Yeast two-hybrid screens were performed to identify

candidate genes whose products interact with AP1 and

CAL. Using a full-length CAL cDNA as bait, 23 interacting

clones were identi®ed, rescued from yeast and trans-

formed into Escherichia coli. Sequence analyses showed

that they fell into four classes, all previously identi®ed as

AGAMOUS-like (AGL) genes (Figure 1a).

The ®rst class, SEP3, included four clones all of which

began within the I region. Because the cDNA library was

poly (T) primed, the clones all comprised varying lengths

of the 3¢ end of the gene. SEP3 is ®rst expressed in the

central dome of stage two ¯oral primordia, and is main-

tained in the inner three whorls of the ¯ower (Mandel and

Yanofsky, 1998). SEP3 acts redundantly with SEP1 and

SEP2 and is necessary for the development of petals,

stamens and carpels (Pelaz et al., 2000).

The second class identi®ed was the SUPPRESSOR OF

CO OVEREXPRESSION 1 (SOC1) gene, and included seven

clones. The starting point of these clones varied. One clone

began with the ATG start codon, another started near the

end of the MADS box, and the remaining clones started at

5¢ ends of the I region. SOC1 is expressed in the

in¯orescence meristem, as well as in the two inner whorls

of the ¯ower beginning in late stage two, and is involved in

promoting ¯owering (Samach et al., 2000).

The third class was the SHORT VEGETATIVE PHASE

(SVP) gene, and included four clones. Of the clones from

this screen, one started in the MADS box and three began

in the I region. SVP was identi®ed as an Arabidopsis-

expressed sequence tag with homology to the MADS-box

family (Alvarez-Buylla et al., 2000a), and was also cloned

by Hartman et al. (2000) through transposon tagging. SVP

is a repressor of ¯owering and is expressed in young

leaves and throughout the shoot apical meristem during

vegetative development. After the transition to ¯owering,

it is expressed in young ¯ower primordia until stage 3

(Hartman et al., 2000).

The last eight clones were identi®ed as AGL24. One of

these clones began within the MADS box and three within

the I region. In addition, the 5¢ ends of four clones lie in the

®rst third of the K box, representing the shortest clones

isolated in the screen. AGL24 was ®rst identi®ed in a

previous yeast two-hybrid screen as a clone that interacts

with AG (S.E.K. and W.L.C., unpublished results; Alvarez-

Buylla et al., 2000a). AGL24 is expressed in in¯orescences

and young ¯oral primordia.

To con®rm the speci®city of the observed interactions,

the longest and shortest clone of each class was trans-

formed back into a yeast strain that contained either the

CAL bait; the bait vector; or an inert control bait, cruciferin.

The strains containing the CAL bait tested positive for both

b-Gal activity and HIS prototrophy. The strains containing

the bait vector or the cruciferin bait were negative in both

assays, as they were not able to grow on plates lacking

histidine and the yeast colonies were completely white in

the b-Gal assay (not shown).

AP1 forms dimers in yeast with CAL interactors

The structural and functional similarities between CAL and

AP1 suggested that they may interact with an overlapping

386 Soraya Pelaz et al.

ã Blackwell Science Ltd, The Plant Journal, (2001), 26, 385±394

set of proteins. In order to explore this possibility, we

constructed an AP1 bait by inserting the intact AP1-coding

region into the pBI-880 vector. As in the Finley and Brent

system, the full-length AP1 bait activated transcription

independently. To overcome this problem, a deletion

construct was made encoding residues 1±196 of AP1

(AP1D1), thus eliminating the putative trans-activating

Figure 1. Yeast two-hybrid assays using AP1 and CAL proteins fused tothe GAL-4 DNA binding domain (BD).(a) Strength of interactions with CAL and AP1D1.(b) The K box alone is suf®cient for interactions of SEP3, SOC1, SVP andAGL24 with CAL and AP1 baits, although these interactions are strongerwhen approximately half the C-terminal domain (C/2) is added to the Kbox. Shown here are the SOC1 prey interactions with CAL and AP1 baits,although similar results were also found with SEP3, SVP and AGL24preys (data not shown).

Figure 4. 35S::SEP3 antisense phenotypes.(a) Wild-type ¯ower.(b) 35S::SEP3 antisense ¯ower showing green petals (pe).(c) Another ¯ower of a 35S::SEP3 antisense plant bearing an axillary¯ower (af) fused to a sepal (se).

Figure 5. Genetic interaction of SEP3 and AP1.(a,b) 35S::SEP3 and 35S::AP1 plants ¯ower early after producing onlyfour or ®ve curly rosette leaves (rl). Two very curly cauline leaves (cl),each subtending a solitary ¯ower, are also present. co, cotyledons.(c,d) 35S::SEP3 35S::AP1 double hemizygous plants show an extremelyearly ¯owering phenotype after producing only two rosette leaves.(e) The early ¯owering phenotypes of t¯1-1 single mutants and of35S::SEP3 plants is dramatically enhanced in t¯1-1 mutants that alsohave the 35S::SEP3 transgene.(f) Close-up view of a t¯1-1 35S::SEP3 plant reveals a phenotype that issimilar to 35S::SEP3/+35S::AP1/+plants (compare f with c,d).Scale bar: (a,b,e) 1 cm; (c,d,f) 2 mm.

AP1 and SEP3 interact to promote ¯owering 387


C-terminus. In contrast to the full-length AP1 clone, the

deletion derivative did not activate the reporter on its own.

The longest clone of each class was transformed into yeast

in combination with the AP1 deletion bait. In every case

both reporters were strongly activated, suggesting that all

four CAL-interacting proteins also interact with AP1 (Figure

1a).

Domain for protein±protein interactions

Previous studies have shown that the MADS domain and I

regions may be important for homodimer formation by AG

and AP1 (Krizek and Meyerowitz, 1996; Mizukami et al.,

1996; Riechmann et al., 1996), and that the I region and K

domain are needed for the formation of AP3/PI hetero-

dimers (Krizek and Meyerowitz, 1996; Riechmann et al.,

1996). In addition, the K domain of AG is suf®cient to

promote interactions with SEP1, SEP2, SEP3 and AGL6 in

yeast (Fan et al., 1997). Since many of the CAL- and AP1-

interacting clones isolated as part of our study lacked the

MADS domain and I regions, we tested if the K domain

itself was suf®cient to promote the observed interactions.

First, we subcloned the K-box regions of SEP3, SOC1, SVP

and AGL24 into the prey vector, and tested their ability to

Figure 2. Comparison of the abaxial surfaceof petals in sep3 mutants, 35S::SEP3antisense and ap1 mutants.The sep3-1 and sep3-2 petals show a partialtransformation toward sepals, as indicatedby the presence of stomata (arrows) and therectangular and elongated shape of thecells, in contrast to the rounded cells of thewild type. The abaxial side of 35S::SEP3antisense petals and of intermediate allelesof ap1 (ap1-2, ap1-4 and ap1-6) show asimilar transformation of petals towardsepals. Petals, pe; sepals, se.



activate the reporter using either the empty bait or the

cruciferin gene cloned into the bait plasmid. As expected,

these K-box regions did not activate the reporter. In

contrast, when these K-box prey constructs were intro-

duced into yeast strains that contained each of the CAL or

AP1 bait plasmids, reporter activity signi®cantly above

background levels was consistently observed (Figure 1b;

data not shown). Furthermore, the addition of approxi-

mately half the C-terminal domain of the SOC1 protein was

suf®cient to greatly strengthen the interaction, similarly to

what has previously been shown to occur for AG and its

interactors (Figure 1b; Fan et al., 1997). Taken together,

these studies suggest that the ability of CAL and AP1 to

interact with SEP3, SOC1, SVP and AGL24 is largely

mediated by the K domain. However, other protein

domains appear to enhance these interactions as the

level of reporter gene activation is higher when larger

constructs are used.

Proteins that interact with AP1

In order to ®nd additional proteins that could interact with

AP1, the library was screened with the truncated AP1 bait

(1±196), and 13 clones that tested positive for b-Gal activity

were characterized. As expected, we found three clones of

SOC1, ®ve clones of SVP, and one clone of AGL24.

In addition we found one clone of a new MADS-box

gene designated AGL27 (Alvarez-Buylla et al., 2000a;

Alvarez-Buylla et al., 2000b); two different clones encoding

a putative RNA-binding protein (GI 10178188); and one

clone encoding a novel protein (GI 3157943) (Figure 1a).

We determined that these three newly isolated genes have

overlapping expression patterns with that of AP1, consist-

ent with the idea that they may interact with AP1 in planta

(not shown). To con®rm the speci®city of these inter-

actions, the longest clone of each class was transformed

back into yeast with the AP1 bait; the bait vector; and an

inert control bait, cruciferin. The strains containing the AP1

bait tested positive for both b-Gal activity and HIS

prototrophy. The strains containing the bait vector or the

cruciferin bait were negative in both assays (not shown).

We then tested if the three new AP1-interacting clones

could also interact with CAL, as they had not been isolated

in the CAL library screen. However, AGL27, the RNA-

binding protein, and the novel protein were unable to

interact with CAL in yeast (Figure 1a).

sep3 mutants resemble intermediate alleles of AP1

As a start in determining if the observed interactions in

yeast re¯ect functional interactions in vivo, we character-

ized loss-of-function and gain-of-function alleles of SEP3. If

some of the activities of AP1 require an interaction with

SEP3, then mutations in SEP3 might be expected to

resemble mutant alleles of AP1. We recently identi®ed

two independently derived En-1 transposon insertion

alleles of SEP3, and have described the phenotype of

sep1 sep2 sep3 triple mutants in which the three inner

whorls of organs become sepaloid (Pelaz et al., 2000).

The ¯owers of sep3-1 and sep3-2 single mutant plants

have petals that are partially transformed into sepals, and

axillary ¯owers infrequently develop at the base of the

®rst-whorl sepals (not shown). When examined by SEM,

the abaxial cells of these transformed petals resemble cells

that are a mixture of abaxial wild-type sepal and abaxial

wild-type petal cells (Figure 2a±d). The abaxial side of the

wild-type sepals has rectangular cells of varying size, some

of which are very long, reaching 300 mm in length (Figure

2a). These long cells can be more than 10 times the length

of the smallest sepal cells. Numerous stomata are visible

throughout wild-type sepals, but are never found on wild-

type petals (Figure 2a,b). Cells on the abaxial side of wild-

type petals all have a uniformly small, rounded appear-

ance, and are typically about half of the size of the smallest

sepal cells (Figure 2b). Unlike wild-type petals that have

rounded cells, the abaxial side of the sep3 petals consists

of rectangular cells resembling those found on sepals.

Although these mutant petal cells are larger than their

wild-type counterparts, they are still smaller than the wild-

type sepal cells. Several stomata are interspersed on the

surface of these petals (Figure 2c,d), further suggesting a

partial transformation of these petals into sepals.

Because the sep3 petal phenotype resembles that

observed for intermediate alleles of ap1 (Bowman et al.,

1993), we compared second-whorl organs of sep3 mutants

to those of intermediate alleles of ap1, including ap1-2,

Figure 3. SEP3 expression is reduced in 35S::SEP3 antisense lines.RNA was isolated from Col wild-type in¯orescences and from two35S::SEP3 antisense lines, SP70.1 and SP70.2. SEP3 RNA levels werereduced in the 35S::SEP3 antisense lines. In contrast, AP1 RNA levelsappear unchanged.



ap1-4 and ap1-6. The abaxial cells of these ap1 mutant

petals are very similar to those of the sep3 mutants, and

consist of a blend between petal and sepal cells. These ap1

mutant cells are larger and more elongated than the wild-

type petal cells, but they do not reach the length of the

longer wild-type sepal cells. As was observed for sep3

mutants, petals of these intermediate alleles of ap1

develop several stomata, further indicating the sepal-like

identity (Figure 2f±h). The similarities of sep and ap1

mutants are consistent with the idea that some of the

activities of AP1 are compromised in sep mutants, con-

sistent with the possible loss of AP1/SEP interactions.

If the interaction between SEP and AP1 is necessary for

AP1 activity, then a reduction in SEP expression would be

predicted to produce some or all of the ap1-mutant

phenotypes. To test this idea we generated transgenic

antisense lines in which the 5¢ end of the SEP3 gene was

expressed in the antisense orientation from the double 35S

promoter (see Experimental procedures). Two independ-

ent transgenic lines (SP70.1 and SP70.2) were tested for

reduction in the amount of SEP3 mRNA accumulation. As

expected, the amount of SEP3 mRNA in these antisense

lines was reduced in comparison to the wild type (Figure

3). The resulting lines underexpressing SEP3 showed

green petals whose cells appeared partially transformed

into sepal cells (Figures 2e and 4). These plants also

occasionally had axillary ¯owers arising from the base of

the ®rst-whorl sepals (Figure 4c). These phenotypes are

consistent with a reduction in AP1 activity, as intermediate

alleles of ap1 produce similar phenotypes. This activity

reduction does not mean less AP1 transcription; the levels

of mRNA in these antisense lines are comparable to those

of wild-type ¯owers (Figure 3). Interestingly, the green-

petal phenotype of these SEP3 antisense lines is more

extreme than that observed for sep3 single mutants, based

on the color change, suggesting that the SEP3 transgene

may also have downregulated other closely related genes

such as SEP1 and SEP2.

Constitutive expression of SEP3

Previous studies have demonstrated that constitutive

expression of AP1 (35S::AP1) results in plants that ¯ower

considerably earlier than wild-type plants (Mandel and

Yanofsky, 1995). If some of the activities of AP1 require an

interaction with SEP3, as the loss-of-function studies

above indicate, then it might be expected that constitutive

SEP3 expression would further enhance the 35S::AP1 early

¯owering phenotype. To test this hypothesis, and to

provide further evidence that SEP3 interacts with AP1 in

planta, we generated 35S::SEP3 sense lines that express

SEP3 constitutively throughout the plant (data not shown).

35S::SEP3 transgenic plants are early ¯owering and bolt

after producing only four or ®ve rosette leaves, in contrast

to wild-type plants which bolt after producing approxi-

mately 10 leaves under the same growth conditions. In

addition to the early ¯owering phenotype, 35S::SEP3

plants have curled rosette leaves as well as two or three

very curled cauline leaves, each of which typically sub-

tends a solitary ¯ower. The primary in¯orescence usually

produces only a few ¯owers before terminating (Figure

5a). Some of the phenotypes caused by ectopic SEP3

expression are similar to those conferred by ectopic

expression of several other MADS-box genes. However

ectopic expression of these other genes often produces

additional phenotypes, including alterations in ¯ower

organ identity and fruit development that are not seen in

the 35S::SEP3 plants.

Genetic interactions between 35S::SEP3 and 35S::AP1

transgenes.

To provide genetic evidence that SEP3 and AP1 interact,

we crossed the 35S::SEP3 transgene into 35S::AP1 plants.

Whereas 35S::AP1 plants ¯ower early after producing four

to ®ve rosette leaves (Figure 5b), 35S::AP1 35S::SEP3

doubly transgenic plants ¯ower after producing only two

rosette leaves, often developing a terminal ¯ower directly

from the rosette. Occasionally, these plants produce a very

short in¯orescence with two cauline leaves that subtend

solitary ¯owers, a terminal ¯ower at the apex, and very

little internode elongation (Figure 5c,d). The strong

enhancement of the early ¯owering phenotypes conferred

by each single transgene is consistent with the suggestion

that AP1 and SEP3 interact in planta.

We also used another genetic approach to investigate

the interaction between SEP3 and AP1, avoiding the use of

two different transgenic lines. We took advantage of the

tf1l mutant, in which AP1 is ectopically activated (Bowman

et al., 1993; Gustafson-Brown et al., 1994), producing a

phenotype that closely resembles the 35S::AP1 phenotype

(Figure 5b). As expected, the t¯ mutation in combination

with the 35S::SEP3 transgene produces the same pheno-

types as observed for plants carrying both 35S::AP1 and

35S::SEP3 transgenes. These plants ¯ower after forming

two rosette leaves and produce abbreviated shoots with

very short internodes and a terminal ¯ower (Figure 5e,f).

Discussion

AP1 and CAL share common and distinct interacting

partners.

AP1 and CAL share redundant roles in specifying ¯ower

meristem identity, and later in ¯ower development AP1 is

required for normal development of sepals and petals.

These observations, together with the accumulating data

suggesting that MADS-box gene products typically inter-



act with other proteins (Davies et al., 1996; Egea-Cortines

et al., 1999; Fan et al., 1997; Shore and Sharrocks, 1995),

suggested that AP1 and CAL may share a common set of

protein partners, and that additional proteins may interact

speci®cally with AP1. Using the yeast two-hybrid assay, we

found that AP1 and CAL can each interact with a shared set

of proteins, including SEP3, SOC1, SVP and AGL24. The

¯ower-development roles of SEP3, SOC1 and SVP have all

been recently documented (Hartman et al., 2000; Pelaz

et al., 2000; Samach et al., 2000), whereas the function of

AGL24 has yet to be determined. Given the fact that only

one or a few clones were isolated for some of the genes in

the yeast screens, it is likely that not all of the interacting

factors have been identi®ed. For example, it is possible

that SEP1 and SEP2, which are redundant in function with

SEP3 and very similar in sequence, may also be partners of

AP1 and CAL. AP1 was found to interact with the MADS-

box gene product AGL27 (Alvarez-Buylla et al., 2000a), as

well as with a putative RNA-binding protein and a novel

protein of unknown function. AGL27 is similar in sequence

and expression pattern to FLC (Alvarez-Buylla et al.,

2000b), whereas the RNA-binding protein is related in

sequence to FCA. Both FLC and FCA have previously been

shown to regulate ¯owering time (Macknight et al., 1997;

Michaels and Amasino, 1999; Sheldon et al., 1999). The fact

that all these genes are expressed in patterns that overlap

that of AP1 is consistent with the idea that the observed

interactions re¯ect in vivo functions.

AP1 interacts with SEP3 in planta

Loss-of-function and gain-of-function studies provide sup-

port for the proposed interactions between SEP3 and AP1

in planta. Mutant alleles of sep3 produce petals that

develop some characteristics of sepals, including the

rectangular cells characteristic of sepals, as well as the

formation of interspersed stomata which are never found

on petals. These phenotypes resemble the aberrant petals

that form in intermediate alleles of ap1, suggesting that

the loss of SEP3 function in sep mutants reduces the ability

of AP1 to carry out its petal-identity function. Interestingly,

this partial conversion of petals toward sepals in sep3

mutants is more severe in plants that harbor the 35S::SEP3

antisense transgene. These antisense plants have green

sepaloid petals in their second whorls, indicating that the

SEP3 antisense transgene may be suppressing related

genes as well, thus producing an effect that is stronger

than would be anticipated for speci®c suppression of the

SEP3 activity. In this regard it is important to note that

SEP3 is closely related in sequence and shares an

overlapping expression pattern to the SEP1 and SEP2

genes, and it has recently been shown that sep1 sep2 sep3

triple mutants display a nearly complete conversion of

petals into sepals (Pelaz et al., 2000). Taken together, these

studies suggest that at least one of the three closely related

SEP gene products must interact with AP1 to promote

petal development.

We used the gain-of-function approach to further exam-

ine the proposed SEP3/AP1 interaction. Plants harboring

the 35S::SEP3 transgene ¯owered considerably earlier

than wild-type plants, indicating that SEP3 is suf®cient to

promote the transition from vegetative to reproductive

development. Similarly, previous studies have docu-

mented the early ¯owering phenotypes produced by

plants carrying the 35S::AP1 transgene (Mandel and

Yanofsky, 1995). Importantly, these early ¯owering phe-

notypes were dramatically enhanced in plants that con-

tained both the 35S::SEP3 and 35S::AP1 transgenes. These

data indicate that the ability of either transgene to promote

¯owering is considerably stronger when the other trans-

gene is also present, providing additional genetic evidence

in support of the idea that SEP3 and AP1 interact during

¯ower development.

In addition to the interactions between SEP3 and AP1,

recent yeast two-hybrid studies, as well as genetic data,

have shown that SEP3 interacts with the AP3, PI and AG

¯ower-organ identity gene products (Fan et al., 1997;

Homma and Goto, 2001; Pelaz et al., 2000; Pelaz et al.,

2001). Thus the speci®c function of SEP3 during ¯ower

development is determined in part by interactions with

other MADS-box proteins. These observed interactions

appear to be speci®c, as other genes that share an

overlapping expression domain with SEP3 fail to show

an interaction (Cristina FerraÂndiz and M.F.Y., unpublished

results).

The K domain is largely suf®cient for dimer formation

Many of the clones isolated in this screen did not include

the MADS box, and much of the I region was often absent

as well. Four clones of AGL24 also lacked the ®rst N-

terminus portion of the K domain, including part of the ®rst

proposed a-helix. A number of studies have documented

the importance of the MADS and I regions for homodimer

formation (Krizek and Meyerowitz, 1996; Mizukami et al.,

1996; Riechmann et al., 1996), while other studies have

demonstrated that the K domain is important for the

formation of heterodimers (Davies et al., 1996; Fan et al.,

1997; Krizek and Meyerowitz, 1996; Riechmann et al., 1996).

Our studies, together with those previously published,

indicate that the minimal domain required for heterodimer

formation includes the central part of the K domain that

extends through several conserved leucine residues

(Moon et al., 1999). However, the C-terminal domain

appears to stabilize these interactions, as the interaction

is stronger when part or all of this domain is added to the K

domain (Figure 1b; Davies et al., 1996; Egea-Cortines et al.,

1999; Fan et al., 1997; Moon et al., 1999).



Now that the complete genome sequence of Arabidopsis

is available (Arabidopsis Initiative, 2000), it is clear that

functional redundancy is widespread. The three closely

related SEPALLATA genes represent an excellent example

of redundancy, and raise the question as to why these

redundant genes are maintained. Our studies establish the

fact that, while these three genes substantially overlap in

their activities, they may be evolving toward more

specialized functions, as revealed by the subtle pheno-

types conferred by sep3 single mutants.

Experimental procedures

Yeast strain

The two-hybrid library screens were performed in the YPB2 strain[MATa ara3 his3 ade2 lys2 trp1 leu2, 112 canr gal4 gal80LYS2::GAL1-HIS3, URA3::(GAL1UAS17mers)-lacZ] (Kohalmi et al.,1998). Yeast was transformed using a modi®ed version of thelithium acetate method of Schiestl and Gietz (1989).

Plasmid construction.

The two-hybrid cDNA expression library was constructed in thepBI771 (prey) vector using tissue of whole plants at differentstages (Kohalmi et al., 1998; Samach et al., 1997). The baitconstructs were prepared by inserting the intact CAL-codingregion and a truncated form of AP1 into the pBI-880 vector (avariant of pPC62 described by Chevray and Nathans, 1992;Kohalmi et al., 1998) by inserting the corresponding coding regionin-frame at the 3¢ end of the GAL4 (1±147) sequence contained inthe centromere LEU2 plasmid. These baits tested negative for theability to activate transcription of both reporters, alone as well asin combination with each of the prey vector and an inert controlprey, the Arabidopsis cruciferin seed storage protein.

SEP3K, SOC1K, SVPK, AGL24K and SOC1KC/2 were generatedby PCR from the relevant cDNAs using oligos with the appropriaterestriction site for posterior cloning into pBI771. The followingprimers were used:

SEP3-5¢K: 5¢-CCGTCGACCCATGAGCCAGCAGGAGTATCTC-3¢SEP3-3¢Kbox: 5¢-CCGCGGCCGCCTTACTCTGAAGATCGTT-3¢SOC1-5¢K: 5¢-CCGTCGACCCATGAAATATGAAGCAGCAAAC-3¢SOC1-3¢Kbox: 5¢-CCGCGGCCGCCTCCTTTTGCTTGAGCTG-3¢SOC1-C/2: 5¢-CCGCGGCCGCACTTTCTTGATTCTTATT-3¢SVP-5¢K: 5¢-CCGTCGACCCATGAGTGATCACGCCCGAATG-3¢SVP-3¢Kbox: 5¢-CCGCGGCCGCTCCCTTTTTCTGAAGTTC-3¢AGL24-5¢K: 5¢-CCGTCGACCCATGCTTGAGAATTGTAACCTC-3¢AGL24-3¢Kbox: 5¢-CCGCGGCCGCCTCAAGTGAGAAAATTTG-3¢The PCR products were subcloned directly into pCRII

(Invitrogen, Carlsbad, CA, USA), then digested with SalI±NotI forthe next subcloning into pBI-771. All constructs were con®rmedby sequencing.

Two-hybrid screens

CAL screenThe frequency of clones which activated both the HIS3 and lacZreporters from the 30°C plates was 1 / (1.8 3 106) = 5.6 3 10±7.The frequency on the 23°C plates was 22 / (1.8 3 106) = 1.2 3

10±5.

AP1 screen9.2 3 104 total transformants were screened at 23°C, and thefrequency of clones activating both reporter genes was 1.5 3 10±4.

The transformants were selected on supplemented syntheticdextrose medium lacking leucine, tryptophan and histidine butcontaining 5 mM 3-amino-1,2,4-triazole. The colonies growing onthis selective medium were assayed for b-galactosidase activityon nitrocellulose ®lters (Kohalmi et al., 1998). Plasmid DNA frompositive clones was isolated and transformed into E. coli.

35S::SEP3 sense and antisense constructs

SEP3 cDNA was isolated by RT±PCR using the oligos OAM37: 5¢-TAGAAACATCATCTTAAAAAT-3¢ and SEP3-5¢: 5¢-CCGGATCCAA-AATGGGAAGAGGGAGA-3¢.

This cDNA was ®rst cloned into pCRII (Invitrogen), thendigested with BamHI for insertion into the BamHI site ofpCGN18 (which contains 35S promoter) to produce sense lines,and con®rmed by sequencing. The cDNA cloned into pCRII wasdigested with BamHI and BglII, the 363 bp band corresponding tothe 5¢ end of the cDNA was cloned in antisense orientation intothe BamHI site of pBIN-JIT (plasmid carrying two 35S promotersin tandem).

SEM

Flower organs were collected and ®xed overnight in 50% ethanol,5% acetic acid, 3.7% formaldehyde, as described by Gu et al.(1998).

Northern blot

In¯orescences (100 mg) were ground in liquid nitrogen, and themRNA isolated using Dynabeads (Dynal, New Hyde Park, NY,USA) following the manufacturer's protocol. Following standardprocedures, the RNA was separated on a formaldehyde agarosegel and transferred to a nylon membrane (Micron SeparationsInc., Westboro, MA, USA). The blot was hybridized ®rst with a 3¢end of SEP3 cDNA 32P-radiolabelled probe, and afterwards withan AP1 cDNA-labelled probe. The SEP3 probe was obtained bydigesting the pSP47 plasmid with BglII and BamHI, After separ-ation on agarose gel, a band of 430 bp corresponding to the 3¢end of the gene was puri®ed. This part of the SEP3 cDNA is notpresent in the antisense construct. The AP1 probe used has beendescribed by Mandel et al. (1992).

Transgenic plants

35S::SEP3 sense and antisense constructs were introduced intoArabidopsis, ecotype Columbia, by vacuum in®ltration (Bechtoldet al., 1993). Transgenic plants were selected on kanamycin plates.

Plant growth conditions

Plants were grown under continuous light at 23±25°C.

Acknowledgements

We are grateful to Cristina FerraÂndiz for helpful discussions andfor sharing results prior to publication, and we thank Chris Young,Amy Chen and Cheryl Wiley for their excellent technical assist-ance. This work was supported by grants from the National



Science Foundation, the National Institutes of Health, and theUnited States Department of Agriculture (to M.F.Y.), from NSERCand to the NRC Core Biotechnology Program (to W.L.C.), and S.P.was supported by postdoctoral fellowships from the SpanishMinisterio de EducacioÂ n y Ciencia and Human Frontiers ScienceProgram Organization.

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APETALA1 and SEPALLATA3 interact to promote flower development

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