The ULTRAPETALA1 gene functions early in Arabidopsis development to restrict shoot apical meristem activity and acts through WUSCHEL to regulate floral meristem determinacy.

Abstract

Shoot and floral meristem activity in higher plants is controlled by complex signaling networks consisting of positive and negative regulators. The Arabidopsis ULTRAPETALA1 (ULT1) gene has been shown to act as a negative regulator of meristem cell accumulation in inflorescence and floral meristems, as loss-of-function ult1 mutations cause inflorescence meristem enlargement, the production of extra flowers and floral organs, and a decrease in floral meristem determinacy. To investigate whether ULT1 functions in known meristem regulatory pathways, we generated double mutants between ult1 alleles and null alleles of the meristem-promoting genes SHOOTMERISTEMLESS (STM) and WUSCHEL (WUS). We found that, although the ult1 alleles have no detectable embryonic or vegetative phenotypes, ult1 mutations restored extensive organ-forming capability to stm null mutants after germination and increased leaf and floral organ production in stm partial loss-of-function mutants. Mutations in ULT1 also partially suppressed the wus shoot and floral meristem phenotypes. However, wus was epistatic to ult1 in the center of the flower, and WUS transcriptional repression was delayed in ult1 floral meristems. Our results show that during the majority of the Arabidopsis life cycle, ULT1 acts oppositely to STM and WUS in maintaining meristem activity and functions in a separate genetic pathway. However, ULT1 negatively regulates WUS to establish floral meristem determinacy, acting through the WUS-AG temporal feedback loop.

Full text

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Copyright  2004 by the Genetics Society of America
DOI: 10.1534/genetics.104.028787
The ULTRAPETALA1 Gene Functions Early in Arabidopsis Development to
Restrict Shoot Apical Meristem Activity and Acts Through WUSCHEL to
Regulate Floral Meristem Determinacy
Cristel C. Carles, Kvin Lertpiriyapong, Keira Reville1and Jennifer C. Fletcher2
Department of Plant and Microbial Biology, University of California, Berkeley, California 94720
Manuscript received March 15, 2004
Accepted for publication April 21, 2004
ABSTRACT
Shoot andfloral meristem activity inhigher plants iscontrolled by complex signalingnetworks consisting
of positive and negative regulators. The Arabidopsis ULTRAPETALA1 (ULT1) gene has been shown to
act as a negative regulator of meristem cell accumulation in inflorescence and floral meristems, as loss-
of-function ult1 mutations cause inflorescence meristem enlargement, the production of extra flowers
and floral organs, and a decrease in floral meristem determinacy. To investigate whether ULT1 functions
in known meristem regulatory pathways, we generated double mutants between ult1 alleles and null alleles
of the meristem-promoting genes SHOOTMERISTEMLESS (STM) and WUSCHEL (WUS). We found that,
although the ult1 alleles have no detectable embryonic or vegetative phenotypes, ult1 mutations restored
extensive organ-forming capability to stm null mutants after germination and increased leaf and floral
organ production in stm partial loss-of-function mutants. Mutations in ULT1 also partially suppressed the
wus shoot and floral meristem phenotypes. However, wus was epistatic to ult1 in the center of the flower,
and WUS transcriptional repression was delayed in ult1 floral meristems. Our results show that during the
majority of the Arabidopsis life cycle, ULT1 acts oppositely to STM and WUS in maintaining meristem
activity and functions in a separate genetic pathway. However, ULT1 negatively regulates WUS to establish
floral meristem determinacy, acting through the WUS-AG temporal feedback loop.
H
at their growing tips, called apical meristems. During
the developmentof Arabidopsisthaliana, theshoot apical
meristem (SAM) provides all of the cells for above-
ground organ formation while simultaneously main-
taining a reservoir of pluripotent stem cells (Steeves
and Sussex 1989). The stem cell population resides at
the very apex of the meristem and replenishes those
cellsthat arelost duringorganogenesis onthe meristem
flanks. The SAM forms during embryonic development,
but generates the vast majority of the lateral organs
after germination. The SAM generates leaves during
the vegetative phase of development, followed by stem
tissue,axillarymeristems,andanindeterminatenumber
of flowers during the reproductive phase. Flowers arise
from floral meristems, which sequentially produce se-
pals, petals, stamens, and carpels in a whorled pattern
from the outside to the inside of the flower. Unlike
SAMs, floral meristems are determinate structures that
IGHER plants continuously produce organs such
as leaves and flowers from small groups of cells
terminate stem cell activity at the time of carpel forma-
tion.
Overlapping networks of meristem-promoting and
meristem-restricting factors regulate shoot apical and
floral meristem activity during Arabidopsis development.
One key meristem-promoting factor is SHOOTMERI-
STEMLESS (STM). Plants carrying strong stm alleles fail
to maintain a functional SAM during embryogenesis
(Barton and Poethig 1993), while plants carrying
weaker stm alleles have reduced shoot and floral meri-
stem function (Clark et al. 1996). STM encodes a Knot-
ted1-like homeobox (KNOX) gene that is expressed only
in shoot and floral meristem cells (Long et al. 1996).
STM activity prevents SAM cells from undergoing differ-
entiationbyrestrictingtheexpressionoftheASYMMET-
RIC LEAVES1 (AS1) and AS2 genes to organ primordia,
thereby preventing the inappropriate development of
leaves across the shoot apex (Byrne et al. 2000, 2002).
Thus, STM provides an environment in which stem cell
derivatives can become amplified to the appropriate
extent prior to their incorporation into organ pri-
mordia.
The homeodomain transcription factor WUSCHEL
(WUS; Mayer et al. 1998) maintains stem cell identity
as part of a spatial negative feedback loop that functions
in both shoot and floral meristems. WUS is expressed in
a small group of cells in the interior of these meristems
(Mayer et al. 1998), called the organizing center, where
1Present address: Department of Medicine and Therapeutics, Conway
Institute of Biomolecular and Biomedical Research, University Col-
lege, Dublin 4, Ireland.
2Corresponding author: U. S. Department of Agriculture, Plant Gene
Expression Center, 800 Buchanan St., Albany, CA 94710.
E-mail: fletcher@nature.berkeley.edu
Genetics 167: 1893–1903 (August 2004)

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1894C. C. Carles et al.
it is required to specify the overlying cells as stem cells
(Schoof et al. 2000). In floral meristems, WUS is also
required to induce the expression of its own repressor,
AGAMOUS (AG; Lenhard et al. 2001; Lohmann et al.
2001). Repression of WUS by AG is necessary to termi-
nate stem cell activity at the appropriate time during
flower development to permit the cells in the center of
the flower to differentiate into carpels.
Thestem-cell-promotingactivityofWUSismodulated
by the CLAVATA (CLV) signaling pathway. The func-
tion of the CLV pathway is to restrict excess stem cell
accumulation by limiting the size of the WUS expression
domain (Brand et al. 2000; Schoof et al. 2000). The
CLV3 gene is expressed in the stem cell population of
shoot and floral meristems and encodes a small, se-
creted signaling molecule (Fletcher et al. 1999; Rojo
et al. 2002). CLV3 protein spreads through the extracel-
lular space to the interior regions of the meristems,
where it is proposed to interact with a receptor complex
(Trotochaud et al. 1999) consisting of the leucine-
rich-repeat (LRR) receptor kinase CLV1 (Clark et al.
1997) and the LRR receptor-like protein CLV2 (Jeong
et al. 1999). CLV3 represses WUS transcription in the
stem cells and their lateral neighbors; however, the
movement of CLV3 protein into the meristem interior
is limited by CLV1, allowing WUS to be transcribed in
the organizing center (Lenhard and Laux 2003).
An additional negative regulator of meristem cell ac-
cumulation is the ULTRAPETALA1 (ULT1) gene. ULT1
encodes a novel cysteine-rich protein with a B box-like
domain and is expressed throughout shoot and floral
meristems and in developing stamens and carpels (C.
Carles, D. Choffnes-Inada, K. Reville, K. Lertpiriy-
apong and J. Fletcher, unpublished results). The loss-
of-function ult1-1 and ult1-2 mutations cause the repro-
ductive (inflorescence) meristems to produce more
floral meristems than normal and the floral meristems
to produce extra organs (Fletcher 2001). These phe-
notypes correlate with an increase in the size of ult1
inflorescence and floral meristems and an enlargement
of the CLV1 expression domain in the interior of the
ult1 inflorescence meristem. ult1 mutations also lead to
the partial loss of floral meristem determinacy, such
that supernumerary whorls of carpels, stamens, and/or
undifferentiated tissue are observed in the center of
ult1 gynoecia.
The relationship between ULT1 and the CLV loci, all
of which negatively regulate shoot and floral meristem
cell accumulation, was investigated using genetic analy-
sis (Fletcher 2001). Double mutants generated be-
tween ult1 alleles and strong clv1 or clv3 alleles showed
synergistic effects on inflorescence meristem size, indi-
cating that these genes have overlapping functions in
controllingSAMgrowthbutactinseparategeneticpath-
ways.Inaddition, thecentraltissueofsome ult1clv1and
ult1 clv3 double-mutant flowers developed into entirely
new inflorescence meristems, revealing that ULT1 and
the CLV genes also interact to regulate floral meristem
determinacy. One point at which the ULT1 pathway
and the CLV pathway might intersect is at the level of
WUS regulation, as limiting WUS activity is essential
for both proper SAM maintenance and floral meristem
determinacy.
ULT1 acts to restrict shoot and floral meristem cell
accumulation, while STM and WUS both function to
promote meristem activity. To further investigate the
role of ULT1 in meristem growth control, we have ana-
lyzed the interactions between ULT1 and the STM and
WUS genes at the genetic and molecular levels. We find
that ULT1 functions in a genetic pathway separate from
STM and WUS in restricting shoot and floral meristem
size, but that ULT1 and WUS act antagonistically in the
same pathway to control floral meristem determinacy.
MATERIALS AND METHODS
Plant materials and growth conditions: Plants were grown
in a 1:1:1 mixture of perlite:vermiculite:topsoil under 140
mmol/m2sec of continuous constant illumination at 22? and
watered daily with a dilute (1:1500) solution of Miracle-Gro
20-20-20 fertilizer. Putative double-mutant plants were identi-
fied in the F2generation and confirmed through segregation
analysis in the F3generation. All plants were in the Landsberg
erecta (Ler) ecotype. The stm-11 allele was provided by Jeff
Long and Kathy Barton, and the wus-1 allele by Thomas Laux.
Microscopy: Confocal laser scanning microscopy was per-
formed as described previously (Running et al. 1995) using a
Zeiss 510 confocal microscope. Scanning electron microscopy
was performed as described previously (Bowman et al. 1989)
using a Hitachi 4700 electron microscope with digital imaging
capability.
Histology: Tissues were fixed for histological analysis by
formaldehyde acetic acid vacuum infiltration, dehydrated,
processed through paraffin wax, sectioned at 8 ?m thickness,
stained with toluidine blue, and visualized using a Zeiss Axio-
phot microscope. Images were acquired with a 12-bit Micro-
Max cooled CCD camera operated by IPLab software.
In situ hybridization: A WUS antisense probe for in situ hy-
bridization was generated as described previously (Mayer et
al. 1998) using a digoxigenin labeling mix (Roche). Tissue
fixation and in situ hybridization were performed as described
previously (Jackson 1991). Slides were prehybridized at 55?
for 5 hr, and 200 ?g of probe per slide was then added for
an overnight incubation at 55?. After two washes in 0.2? SSC
0.1% SDS of 10 min each at 55?, the slides were treated with
10 ?g/ml RNAse A for 30 min at 37?. Two more washes (0.2?
SSC 0.1% SDS, 10 min each at 55?) were then performed.
For signal detection the NBT/BCPIP reagents (Roche) were
applied for 42 hr.
RESULTS
ult1 embryonic and vegetative development: Prior
analysis revealed that ult1 mutants accumulate excess
SAM cells during the reproductive phase of develop-
ment—both inflorescence and floral meristems are
larger in ult1-1 and ult1-2 mutant plants than in wild-
type plants, and the flowers produce more whorls and
floral organs than normal (Fletcher 2001). The ult1-1

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1895ULTRAPETALA1 Gene Functions
Figure 1.—Embryonic shoot apical meri-
stemformationinwild-type,ult1,stm,andwus
mutant plants. (A) Wild-type Ler embryonic
SAM appears as a dome-shaped structure at
the base of the two cotyledons. (B) ult1-2
embryonic SAM resembles that of the wild
type. (C) stm-11 embryos lack a dome-shaped
SAM structure at the base of the cotyledons
(arrow). (D) ult1-2 stm-11 embryos resemble
stm-11 single-mutant embryos. (E) wus-1 em-
bryos have only a few densely staining cells
at the base of the cotyledons (arrow) and
lacka dome-shapedSAM structure.(F) ult1-2
wus-1 embryos resemble wus-1 single-mutant
embryos.
mutation has more severe effects on meristem size and
on sepal and petal number than the ult1-2 mutation.
The phenotypes of plants carrying the ult1-3 mutation,
which is caused by a T-DNA insertion that abolishes
ULT1 transcription (C. Carles, D. Choffnes-Inada, K.
Reville, K. Lertpiriyapong and J. Fletcher, unpub-
lished results), are statistically indistinguishable from
those of ult1-2 plants. Thus, ult1-2 is a phenotypic null
allele for the ULT1 locus, while the ult1-1 allele has
slightly more severe effects on meristem size and sepal/
petal number than the ult1-3 knockout allele and is
weakly semidominant (C. Carles, D. Choffnes-Inada,
K. Reville, K. Lertpiriyapong and J. Fletcher, un-
published results).
To determine at what stage plant development is first
affected by mutations in ULT1, we examined wild-type
Ler, ult1-1, and ult1-2 embryos and seedlings. Shoot api-
cal meristem cell layering, organogenesis, and organ
morphology appear normal in ult1-1 and ult1-2 embryos
andseedlings(Figure1Banddatanotshown).Confocal
laser scanning microscopy reveals that the ult1-2 mature
embryonic meristem size and cell number is not signifi-
cantlydifferentfromthatofwild-typemeristems(Figure
1, A and B). ult1-1 mutant embryonic meristems on
average are 33.0 ? 1.2 ?m wide and 8.5 ? 0.5 ?m tall
(n ? 7), while Ler embryonic meristems on average are
31.2 ? 1.2 ?m wide and 9.3 ? 0.4 ?m tall (n ? 12).
Seven-day-old ult1-1 seedlings (52.4 ? 2.0 ?m wide,
22.4 ? 1.1 ?m tall, n ? 18) likewise have approximately
the same size shoot apical meristem as Ler seedlings
(48.9 ? 2.3 ?m wide, 23.9 ? 1.8 ?m tall, n ? 8). ult1-3
null mutant plants are also indistinguishable from wild-
type plants during the embryonic and vegetative peri-
ods. Thus ult1 mutant phenotypes are not detectable
until the reproductive phase of development.
ult1 interactions with stm: Since ult1 mutants accumu-
late excess meristem cells during reproductive develop-
ment,wehypothesizedthattheult1allelesmightrestore
shoot and/or floral meristem activity to stm mutants,
which fail to maintain meristem cells in an undifferenti-
atedstate.Thishasbeenshowntobethecasefortheclv1
and clv3 mutants, which partially suppress stm mutant
phenotypes in a dose-dependent manner (Clark et al.
1996). To test this hypothesis, we generated double
mutantsbetweentheult1allelesandstrongandweakstm
allelesandfollowedtheirembryonicandpostembryonic
development.
Plants homozygous for the stm-11 null allele (Long
and Barton 1998) form a pair of normal embryonic
leaves (cotyledons) during embryogenesis, but fail to
develop a densely staining dome of meristematic cells
at the base between them (Figure 1C). Among the prog-
eny of ult1-2 F2plants that segregated stm-11, we found
that ?25% of embryos lacked a dome-shaped SAM be-
tween the cotyledons (Figure 1D). In addition, progeny
of ult1-1 F2plants that segregated stm-11 also yielded
?25%embryos lackinga discernibleSAM. Thusneither
the ult1-1 mutation nor the ult1-2 mutation rescues the
stm-11 embryo shoot-meristemless phenotype.
Following germination, stm-11 mutant seedlings do
not produce postembryonic organs between the cotyle-
dons (Figure 2, A and C) and completely lack a dome-
shaped shoot apical meristem (Figure 2E). Double
mutants generated between stm-11 and either ult1-1 or
ult1-2 initially resembled stm-11 single-mutant plants.
After 7 days of growth, the ult1-1 stm-11 and ult1-2 stm-11
double-mutant seedlings showed no evidence of organ
formation between the cotyledons (Figure 2G). How-
ever, after 10 days ?15% of the ult1-1 stm-11 and ult1-2
stm-11 double-mutant plants began to develop leaves
(Figure 2, B, D, and G). Scanning electron microscopy
and sectioning revealed that the leaves produced by the
double-mutant plants originated at the seedling shoot
apex from the flanks of a dome of meristematic cells

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1896C. C. Carles et al.
Figure 2.—Shoot apical
meristem activity in ult1 stm-
11 seedlings. (A) stm-11
seedling14daysaftergermi-
nation. No organs have
formed between the cotyle-
dons. (B) ult1-2 stm-11 seed-
ling 14 days after germina-
tion. Leaves are developing
from between the cotyle-
dons. (C) SEM of an stm-11
seedling10daysaftergermi-
nation.(D)SEMofanult1-1
stm-11 seedling 10 days after
germination.
forming between the cotyle-
dons at the position where
they are normally produced
by the shoot apical meri-
stem in wild-type plants. A
pair of developing leaves
arch over the SAM. (E) Sec-
tionthroughanstm-11 seed-
ling 10 days after germina-
tion. There is no evidence
of a dome-shaped SAM. (F)
Section through an ult1-2
stm-11 seedling 10 days after
Leavesare
germination. A correctly positioned SAM is producing leaves from its flanks, and the organization of the meristem cell layers is
intact. (G) Percentage of ult1-1 stm-11 (red, n ? 46) and ult1-2 stm-11 (yellow, n ? 29) mutant seedlings that formed a shoot
apical meristem and produce true leaves following germination and growth under constant light. No stm-11 plants (blue, n ?
41) grown at the same time under the same conditions produced any leaves or any sign of a shoot apical meristem. (H) Percentage
of stm-2 (blue, n ? 26), ult1-1 stm-2 (red, n ? 35), and ult1-2 stm-2 (yellow, n ? 27) mutant seedlings that formed a shoot apical
meristem and produced true leaves following germination and growth under constant light. Nearly all of the single and double-
mutant plants produced organs from the shoot apex, but the ult1-1 and ult1-2 mutations increased the rate at which stm-2 mutants
developed postembryonic leaves. Bars: 100 ?m, C and D; 30 ?m, E and F.
between the cotyledons (Figure 2, D and F). In contrast,
leaves that are occasionally produced by single-mutant
plants carrying the weaker stm-1 allele arise from the
hypocotyl region (Clark et al. 1996). After 21 days
nearly 90% of ult1-1 stm-11 and ult1-2 stm-11 plants dis-
played this “restored” phenotype, while none of the stm-
11 single mutants showed signs of postembryonic organ
formation (Figure 2G). Thus, a shoot apical meristem
structure and organogenic capability is eventually re-
stored to stm-11 mutant plants when ULT1 activity is
absent. This experiment also reveals that ULT1 is active
during the vegetative phaseof development, despite the
fact that the ult1 mutants lack a detectable vegetative
phenotype.
Ultimately, 100% of the restored ult1-1 stm-11 plants
and 86% of the restored ult1-2 stm-11 plants produced
oneor moreabnormalinflorescence meristemsbearing
one to a few flowers (Figure 3). Compared to wild-
type (Figure 3A) and ult1-1 (Figure 3B) inflorescences,
which produced flowers in a stereotypical spiral phyllo-
taxy, ult1-1 stm-11 inflorescences consisted of disorga-
nized aerial structures with abnormal phyllotaxy, con-
sistingofleavesandreducednumbersofflowers(Figure
3C). The flowers produced by the double-mutant inflo-
rescences were also abnormal. Wild-type flowers nor-
mally form four sepals, four petals, five or six stamens,
and two carpels that fuse to form the central gynoecium
(Smyth et al. 1990). In contrast, ult1-1 stm-11 and ult1-2
stm-11 flowers often contained fused and/or mosaic or-
gans, such as fused stamens and sepal/petal, petal/sta-
men, and stamen/carpel mosaics (see supplemental
table at http:/ /www.genetics.org/supplemental/). More-
over, while sepal number was similar to that observed
in the wild type, the petals, stamens, and carpels were
either reduced in number or absent (Figure 4A). Al-
though the center of the flower was the most severely
affected,rareult1-1stm-11flowerswithcentralstructures
bearing ovules and/or stigmatic tissue were observed
(Figure 3D), indicating that stm-11 plants are capable
of forming all organ types when ULT1 activity is lost.
ult1-1 stm-11 and ult1-2 stm-11 plants displaying these
inflorescence and floral phenotypes resembled plants
homozygousfortheweakstm-2allele(Clarketal.1996).
Thus, significant inflorescence and floral meristem ac-
tivity is restored to stm null mutant plants in the absence
of ULT1 function.
stm-2 mutant plants retain some meristematic activity,
as evidenced by their ability to form abnormal rosettes

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1897ULTRAPETALA1 Gene Functions
Figure
meristem and flower pheno-
types of ult1 stm and ult1 wus
double mutants. (A) A wild-
type Ler inflorescence meri-
stem. (B) An ult1-1 mutant in-
florescence meristem, which
generates flowers containing
additional organs of all types,
predominantly sepals and pet-
als.(C) ult1-1stm-11 plantspro-
duce inflorescence meristems
thatgeneratealimitednumber
of abnormal flowers. (D) Flow-
ers produced by an ult1-1 stm-
11inflorescencemeristemcon-
tain fewer petals, stamens, and
carpels than wild-type flowers
doandresembleflowersgener-
ated by plants carrying weak
stm alleles. Infrequently, flow-
ers form carpeloid structures
in the center of the flower
(arrow). (E) Plants carrying
the weak stm-2 allele produce
inflorescence meristems that
generate a limited number of
abnormal flowers. (F) Flowers
produced by an stm-2 inflor-
3.—Inflorescence
escence meristem lack the full complement of internal organs and fail to generate carpels. (G) ult1-1 stm-2 plants produce
inflorescence meristems that generate more flowers than stm-2 inflorescence meristems do. (H) Flowers produced by an ult1-1
stm-2 inflorescence meristem contain internal organs and can form unfused carpels or a normal, fused gynoecium (arrows). (I)
A wus-1 inflorescence meristem, which generates a small number of abnormal flowers in a disorganized phyllotactic pattern. (J)
ult1-1 wus-1 inflorescence meristems form many more flowers than do wus-1 single-mutant meristems in a normal spiral phyllotaxy.
(K) Flowers produced by wus-1 plants lack the full complement of organs and generally terminate in a solitary stamen (arrow).
(L) Flowers produced by ult1-1 wus-1 plants can form more sepals and petals than either wild-type or wus-1 flowers, but fail to
form carpels and generally terminate in a solitary stamen (arrow).
of leaves followed by inflorescence stems that produce
reduced numbers of flowers (Clark et al. 1996). To
determine whether the ult1 mutations affected the pro-
duction of these postembryonic structures, we gener-
ated ult1 stm-2 double mutants and compared their
growth with that of stm-2 single-mutant plants. Because
nearly all stm-2 plants eventually form leaves, we com-
pared the rate of postembryonic leaf production be-
tween the different genotypes. After 6 days of growth,
43% of ult1-1 stm-2 and 37% of ult1-2 stm-2 seedlings
had produced one or more leaves, compared to 17%
of stm-2 seedlings (Figure 2H). After 10 days of growth,
100% of ult1-1 stm-2 and ult1-2 stm-2 seedlings had pro-
duced leaves, compared to 82% of stm-2 seedlings (Fig-
ure 2H). Therefore, stm-2 seedlings generate leaves at
a slightly accelerated rate when ULT1 activity is reduced
or absent.
The extent of inflorescence and floral meristem de-
velopmentwasalsogreaterinult1stm-2plantscompared
to stm-2 mutants. Approximately one-third of stm-2 plants
formed a solitary, terminal flower. The other two-thirds
produced more than one flower, frequently four or five.
Rarely, an stm-2 plant formed ?10 flowers before termi-
nating (Figure 3E). In contrast, 100% of ult1-1 stm-2 and
ult1-2 stm-2 plants generated inflorescence meristems
bearing more than one flower, and 30–40% of the dou-
ble-mutant plants produced ?10 flowers in a normal
spiral phyllotaxy (Figure 3G). The number of floral
organsgeneratedperwhorl,andperflower,waslikewise
increased in ult1 stm-2 plants compared to stm-2 plants
(Figure4A).Flowersproducedbystm-2plantscontained
reduced numbers of sepals, petals, and stamens and
rarely formed carpels (Figures 3F and 4A). We observed
partial restoration of organogenesis in each whorl of
ult1 stm-2 flowers, including the formation of fused or
unfused carpel structures in the center of the flower
(Figures 3H and 4A). However, we did not detect dose
dependence between ult1 alleles and stm alleles in any
combination, indicating that while these two genes ap-
pear to have opposite activities they do not function in
a directly competitive manner.
We used scanning electron microscopy to determine
the earliest stage at which ult1-1 stm-2 flower develop-
ment deviated from stm-2 flower development. At stage
2, when floral meristems first become distinguishable
as bulges on the flanks of the shoot apical meristem

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1898C. C. Carles et al.
Figure 4.—Organ number in ult1, stm, and wus single- and double-mutant flowers. (A) ult1 mutations restore organogenesis
to stm floral meristems. The mean number of organs in the first 10 flowers of stm single-mutant and ult1 stm double-mutant
plants is shown. If ?10 flowers were produced by an individual plant, then all flowers on the primary inflorescence meristem
were counted. At least 15 flowers were counted for each mean, and the standard error is indicated. (B) ult1 mutations restore
organogenesis to wus-1 floral meristems, except in the carpel whorl in the center of the flower. The mean number of organs in
the first 10 flowers of 10 wus-1 single-mutant and ult1 wus-1 double-mutant plants is shown. If ?10 flowers were produced by an
individual plant, then all flowers on the primary inflorescence meristem were counted. At least 38 flowers were counted for each
mean, and the standard error is indicated.
(stages according to Smyth et al. 1990), we observed
no difference between stm-2 and ult1-1 stm-2 floral meri-
stems (see supplemental figure at http:/ /www.genetics.
org/supplemental/). However, a distinction was clearly
detected when stage 3 floral meristems were compared.
At this stage, wild-type floral meristems assume a dome-
shaped structure, surrounded by four sepal primordia
at the periphery (Smyth et al. 1990). The stage 3 floral
meristems of stm-2 plants formed a very reduced apex
between the developing sepal primordia (see supple-
mental figure at http:/ /www.genetics.org/supplemen
tal/). In contrast, the stage 3 floral meristems of ult1-1
stm-2 plants formed a dome between the developing
sepal primordia and more closely resembled the wild
type (see supplemental figure at http:/ /www.genetics.
org/supplemental/).Thus,theeffectoftheult1-1muta-
tion on stm-2 flower development could be detected at
the time of sepal initiation, after the floral meristems
had formed, consistent with the idea that ULT1 acts
competitively with STM during meristem maintenance
but not meristem initiation.
ult1interactionswithwus:TheWUSCHEL(WUS)gene
is required forproper meristem function, as wus mutant
plantsaredefectiveinshootandfloralmeristemmainte-
nance (Laux et al. 1996). Plants homozygous for the
wus-1nullalleleformanormalpairofcotyledonsduring
embryogenesis, but produce only a few disorganized
meristematic cells at their base (Figure 1E). Because
wus-1 plants are sterile, we crossed ult1-1 and ult1-2
plants to wus-1/? heterozygous plants and identified
homozygous ult1 plants in the F2. Among the progeny
of ult1-1 and ult1-2 F2plants that segregated wus-1, we
found that ?25% of embryos lacked a dome-shaped
SAM between the cotyledons (Figure 1F). Thus neither
the ult1-1 nor the ult1-2 mutation rescues the wus-1
embryoshootapicalmeristemdefect.Aftergermination
the wus-1 plants pause in their development and then
produce multiple abbreviated rosettes of leaves from
the axils of the cotyledons and across the flat shoot
apex (Laux et al. 1996). We compared postembryonic
development between wus-1 plants and ult1 wus-1 plants
and found that the rate of leaf production in the double
mutants was indistinguishable from that of the single
mutants.Thustheult1mutationsdonotacceleratevege-
tative organ formation in wus-1 mutant plants, as they
do in stm mutant plants.
After the transition to flowering, wus mutant plants
produce abnormal inflorescences and flowers due to
reduced meristem activity (Laux et al. 1996; Schoof et
al. 2000). wus-1 inflorescence meristems generate far
fewer flowers than wild-type meristems do, and the
flowers that do form arise in aerial rosettes with a disor-
ganizedphyllotaxicpattern(Figure3I).Underourgrowth
conditions, 45% of wus-1 plants terminated develop-
ment without flowering, and an additional 37% gener-
atedasolitaryflower(Table1).Only18%ofwus-1plants
generated multiple flowers from their adventitious in-
florescence meristems (Table 1). In contrast, 100% of
ult1-1 wus-1 plants and 82% of ult1-2 wus-1 plants pro-
duced one or more flowers in a normal spiral phyllotaxy
(Figure 3J, Table 1), indicating that mutations in ULT1
restore somefunction to wus-1inflorescence meristems.

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1899ULTRAPETALA1 Gene Functions
TABLE 1
extra whorls of floral organs within the normal fourth
whorl of carpels (Fletcher 2001; Figure 5B: compare
with wild type in Figure 5A). Since no fourth whorl
carpels or internal fifth whorl organs are formed by
ult1-1 wus-1 double mutants, the partial loss of determi-
nacyobservedinult1-1flowersdependsuponWUSactiv-
ity. To confirm this result, we used in situ hybridization
to visualize the WUS expression pattern in developing
wild-type and ult1-1 mutant flowers.
In wild-type floral meristems, WUS transcripts are first
detected in stage 2 primordia budding from the flanks
of the inflorescence meristem. In wild-type stage 2 floral
meristems, WUS expression is restricted to the interior
cells in the central zone (Figure 5E). This area of the
meristem has been proposed to act as an organizing
center, specifying the overlying neighbor cells to main-
tain their pluripotent state (Mayer et al. 1998). WUS
expression in ult1-1 floral meristems at this stage of
development (Figure 5F) is indistinguishable from that
in the wild type. WUS transcription is repressed in wild-
type floral meristems after stage 6 (Mayer et al. 1998),
when two carpel primordia are initiated in the center
of the flower and meristematic activity ceases (Figure
5G).Theformationofadditional whorlsoffloralorgans
in ult1-1 and ult1-2 flowers is correlated with the pres-
ence of a dome of cells that separate the two developing
carpel primordia detectable in stage 6 and stage 7 flow-
ers (Figure 5D). In contrast, the carpel primordia pro-
duced by wild-type flowers abut each other (Figure 5C).
In ult1-1 stage 7 flowers displaying such a dome of tissue
between the carpel primordia, WUS transcription is still
detectable in cells underlying this dome (Figure 5H).
This WUS expression domain corresponds to cells for
which floral organ identity specification has been de-
layed, on the basis of our observation that AG induction
in the center of the floral bud occurs later in ult1-1 than
in wild-type plants (Fletcher 2001). These data show
that ULT1 negatively regulates WUS to establish floral
meristem determinacy and that the partial loss of deter-
minacy observed in ult1 flowers depends on WUS ac-
tivity.
Terminal structures produced by wus-1 plants and
ult1 wus-1 double-mutant plants
Structurewus-1 (%) ult1-1 wus-1 (%) ult1-2 wus-1 (%)
Basal rosette
Aerial rosette
Solitary flower
Multiple flowers
15
30
37
18
0
0
0
18
18
64
28
72
Numbers represent the percentage of plants with the indi-
cated structure as a terminal phenotype. Plants terminating
in either a basal or an aerial rosette did not produce flowers.
wus-1, n ? 33; ult1-1 wus-1, n ? 36; and ult1-2 wus-1, n ? 27
plants scored.
wus-1 floral meristems generate reduced numbers of
floralorgans,producingonaveragethreetofoursepals,
three to four petals, and zero to one stamen per flower
(Figures 3K and 4B). wus-1 flowers do not contain more
than four sepals and petals and never form carpels (Fig-
ure 4B). ult1-1 wus-1 and ult1-2 wus-1 flowers have slightly
more sepals and petals on average than wus-1 flowers
(Figure 4B). However, unlike wus-1 flowers, ult1-1 wus-1
flowers and ult1-2 wus-1 flowers can contain up to six
or seven sepals and petals (Figure 3L). The sepal and
petal number increase observed in ult1 flowers is there-
fore at least partially independent of WUS.
In contrast to the other wus-1 reproductive meristem
phenotypes, the premature floral meristem termination
phenotype is not rescued by the ult1-1 or ult1-2 muta-
tions. wus-1 flowers contain an average of less than one
stamen per flower and completely lack carpels (Figures
3K and 4B). Similarly, ult1-1 wus-1 flowers contain an
average of less than one stamen per flower and lack
carpels (Figures 3L and 4B; of 88 counted, a single
carpel-likestructurewasformedin1ult1-1wus-1flower).
Examination of floral meristem development using
scanning electron microscopy revealed that the floral
primordia of single- anddouble-mutant plants are indis-
tinguishable atboth stage 2and stage 3(see supplemen-
tal figure at http:/ /www.genetics.org/supplemental/).
Like wus1 stage 3 floral meristems, ult1-1 wus-1 stage 3
floral meristems lack a detectable dome of meristematic
cells interior to the developing sepal primordia (see
supplementalfigureathttp:/ /www.genetics.org/supple-
mental/). Thus, wus is epistatic to ult1 in the center of
the floral meristem, revealing that the WUS and ULT1
genes play antagonistic roles in the same genetic path-
way that controls floral meristem determinacy.
wus and ult1 mutant plants have opposite phenotypes
in the center of the flower. wus-1 floral meristems are
smaller than those of the wild type, generate reduced
numbers of stamens, and terminate prematurely prior
to carpel formation (Laux et al. 1996; Schoof et al.
2000). ult1-1 and ult1-2 floral meristems, in contrast,
are larger than those of the wild type and can generate
DISCUSSION
Shoot and floral meristem maintenance in Arabi-
dopsis depends upon the activity of networks of meri-
stem-restricting and meristem-promoting factors. Our
previous experiments have shown that ULT is an impor-
tantmeristem-restrictingfactorthatlimitstheaccumula-
tion of cells in both inflorescence and floral meristems.
STM and WUS represent two meristem-promoting fac-
tors that act in separate genetic pathways, with the STM
pathwaymaintainingmeristemcellsinanuncommitted,
proliferative state and the WUS/CLV pathway main-
taining stem cell fate at the meristem apex. To deter-
mine the genetic interaction between the meristem-
restrictingULT1factorandtheSTMandWUSpathways,

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1900C. C. Carles et al.
Figure 5.—WUS expression in wild-type and ult1-1 floral meristems. (A) A wild-type silique dissected lengthwise to reveal the
ovules within one valve. (B) An ult1-1 silique dissected lengthwise to reveal the presence of supernumerary whorls of stamens
and carpels (arrowheads). (C) Wild-type stage 7 flower with two carpel primordia (c) developing in the center of the flower.
(D) ult1-1 stage 7 flower with a dome of tissue (arrow) between the two developing carpel primordia (c). (E–H) Wild-type and
ult1-1 floral meristems hybridized with an antisense WUS riboprobe. (E) A wild-type stage 2 floral meristem. WUS expression is
detected in a small group of cells beneath the outermost two cell layers (arrow). (F) An ult1-1 stage 2 floral meristem. The
pattern and level of WUS expression (arrow) are similar to those observed in wild-type stage 2 floral meristems. (G) A wild-type
stage 7 flower. After the carpel primordia have emerged, WUS transcription is not detected in the center of the flower. The
arrowhead points to the signal detected in the inflorescence meristem, testifying to the integrity of the tissue section. (H) An
ult1-1 stage 7 flower. WUS expression is observed in the cells within the dome of tissue between the two carpel primordia (arrow).
The inset shows the region depicted by the arrow at higher magnification. Bars, 30 ?m.
we generated double mutants between strong and weak
ult1 alleles and stm and wus alleles. We determined that
while the ult1 alleles cause no detectable phenotypes
during embryonic or vegetative development, they can
partially suppress the vegetative and inflorescence meri-
stem defects that result from reduced meristem activity
in both stm and wus mutant plants. ult1 mutations also
restored organogenic potential to stm floral meristems,
leading to increased organ production in all whorls.
However, we found that wus mutations are epistatic to
ult1 mutations in the center of the flower and that WUS
transcripts persist longer than normal in developing
ult1 flowers. Thus, ULT1 controls floral determinacy
by negatively regulating WUS expression during floral
meristem development.
ULT1 function early in Arabidopsis development:
Plants carrying loss-of-function ult1 alleles are indistin-
guishable from wild-type plants during embryonic and
vegetative development. This may be because either
ULT1doesnotfunctionpriortotheinflorescencephase
or its activity earlier in development is masked by the
activity of another gene or genes. We find that loss of
ULT1activityrestorespostembryonicSAMstructureand
organogenesis function to stm mutant plants and also
partially suppresses the wus vegetative terminal meri-
stem phenotype. Thus our analysis of ult1 stm and ult1
wus double mutants reveals that ULT1 is functional dur-
ing vegetative development, prior to the stage at which
anult1single-mutantphenotypeisdetectable.However,
lack of ULT1 activity does not restore embryonic SAM
structure in stm-11 and wus-1 mutants.
ULT1 regulation of shoot and floral meristem activity:
Genetic and molecular studieshave defined the homeo-
box genes STM and WUS as essential regulators of shoot
and floral meristem formation and maintenance. WUS
and STM are induced independently of one another in
embryonic SAMs (Long and Barton 1998; Mayer et
al. 1998), and the evidence to date indicates that these
two genes promote meristem activity in independent
butcomplementarywaysandfunctionindistinctregula-
tory pathways (Lenhard et al. 2002). The STM pathway
suppresses cell differentiation throughout the meri-
stem, while the WUS pathway specifies a subset of cells
at the meristem apex as stem cells. These pathways ulti-
mately converge to maintain meristem cells in an undif-
ferentiated state (Gallois et al. 2002; Lenhard et al.
2002).
The CLV loci act to restrict meristem activity, having
the opposite effect to STM and WUS on shoot and floral
meristems. Genetic analysis showed that wus mutations
were epistatic to clv mutations in both shoot and floral
meristems, placing WUS and the CLV genes in the same
genetic pathway (Schoof et al. 2000). In contrast, clv
mutations partially suppressed the stm mutant pheno-

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1901ULTRAPETALA1 Gene Functions
types and vice versa, and the suppression occurred in
adominant fashion(Clark etal. 1996).The occurrence
of dominant interactions between clv and stm mutations
was interpreted to mean that these genes play opposing
and possibly competitive roles in shoot and floral meri-
stem regulation.
Like the CLV loci, ULT1 acts opposite to STM and
WUS in that it functions to restrict the excess accumula-
tion of cells in shoot and floral meristems. We have
shown that ult1 mutations restore organized vegetative
shoot apical meristems to stm mutant plants, allowing
postembryonic organ formation to proceed, albeit in
an abbreviated manner. In addition, we observe restora-
tion of floral meristem and floral organ formation in
both ult1 stm-11 and ult1 stm-2 plants, including the
development of carpel tissue in the latter genotype. In
sum, the ult1 alleles reverse many of the effects of weak
and strong stm alleles, but do not suppress them com-
pletely. Similarly, the stm mutations partially suppress
the ult1 phenotypes, showing that a wild-type level of
STM activity is necessary for excess meristem cell accu-
mulation in ult1 plants. The ult1 stm double-mutant
phenotypes can therefore be considered additive, from
which we conclude that STM and ULT1 act oppositely
through separate genetic pathways to regulate shoot
and floral meristem activity. However, the lack of dose
dependency between stm and ult1 alleles suggests that
the two genes do not function competitively to regulate
the same process.
Theinteraction betweenULT1 andWUS ismore com-
plex.Similartostm-2plants,wus-1plantsproducedsome
lateral organs from disorganized meristems that initiate
randomly across the entire differentiated shoot apex
(Laux et al. 1996). Yet unlike stm-2 plants, the rate at
which wus-1 plants produced leaves was not accelerated
in the absence of ULT1. However, ult1 wus-1 double
mutants bolted at a higher frequency and formed many
more floral meristems than did wus-1 single mutants.
These results indicate that ult1 mutations partially sup-
presswus,restoringagreateramountofshootandfloral
meristem activity. However, ult1 wus-1 double-mutant
inflorescences still terminated prematurely and pro-
ducedflowerswithfewerorgansthanult1singlemutants
did, indicating that the wus-1 mutation also partially
suppresses the ult1 mutations. We also observe signifi-
cantsepalandpetalrestorationinult1-1wus-1andult1-2
wus-1 flowers, and, in fact, supernumerary sepals and
petals could be produced by ult1 floral meristems even
in the absence of WUS. Thus ULT1 acts in a separate
pathway from WUS to control shoot apical meristem
activity, and the sepal and petal number increase in ult1
flowers is largely WUS independent. However, the wus-1
mutation is epistatic to the ult1 mutations in the inner
two whorls of the floral meristem, indicating that WUS is
absolutely requiredfor the formationof supernumerary
whorls of organs by ult1 floral meristems.
ULT1regulation offloralmeristem determinacy: Nor-
malArabidopsis flowerdevelopmentrequires thatfloral
stem cell activity terminate upon formation of the cen-
tral carpel primordia, which consumes the floral meri-
stem.Floral meristemterminationoccursvia atemporal
autoregulatory loop involving WUS and AG (Lenhard
et al. 2001; Lohmann et al. 2001). AG encodes a MADS
domain transcription factor that is required to termi-
nate floral meristem activity and also to specify stamen
and carpel identity (Yanofsky et al. 1990). Early in
flowerdevelopment, WUSandthefloral meristemiden-
tity factor LEAFY (LFY) activate AG transcription by
binding to adjacent sites in the second intron (Loh-
mann et al. 2001). AG expression is restricted to the
interior two whorls of the flower bud, where the stamen
and carpel primordia form (Drews et al. 1991). At stage
6 of flower development, AG switches off the organizing
center activity by repressing WUS expression, resulting
in the differentiation of the remaining stem cells into
carpel tissues. However, genetic evidence indicates that
AG alone is not sufficient to repress WUS in the center
of the flower, because ectopic activation of AG in the
inflorescence meristem does not cause meristem arrest
(Mizukami and Ma 1997). Therefore AG requires an
additional factor or factors to achieve downregulation
of WUS transcription (Lenhard et al. 2001).
ULT1 also plays a role in specifying floral meristem
determinacy.Matureult1flowerscancontainmorethan
four whorls of organs, such as fifth and sixth whorls of
carpels or a fifth whorl of stamens and a sixth whorl of
carpels (Fletcher 2001; Figure 5B). In this way the ult1
flowers are reminiscent of ag flowers, which produce an
indeterminate number of floral whorls as a result of
active maintenance of a stem cell reservoir and organiz-
ing center at the apex of the floral meristem. When the
stamen and carpel specification functions of AG are
separated from the floral meristem determinacy func-
tion via site-directed mutagenesis, the resemblance is
even more striking: a synthetic partial loss-of-function
ag mutation, AG-Met205, causes production of extra
whorls of stamens and carpels in the ag-3 background
(Sieburth et al. 1995), closely resembling the ult1 mu-
tant phenotype.Transgenic plants carryingan antisense
AG construct in which AG expression is reduced to ap-
proximatelyhalfthenormallevelalsodisplaythenested
stamen and carpel phenotype (Mizukami and Ma
1995). However, ult1 mutant flowers, unlike ag null mu-
tant flowers, are never completely converted to an inde-
terminate fate, and, as expected, ag mutations are epi-
static to ult1 mutations with respect to floral meristem
determinacy (data not shown). Since floral stem cell
terminationeventuallyoccursin ult1mutants,itappears
that AG can partially compensate for the absence of
ULT1, but ULT1 cannot compensate for the absence
of AG.
Our results demonstrate that ULT1 is a new compo-
nent of the AG-WUS temporal feedback loop that con-
trols floral meristem termination. We have shown that

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1902C. C. Carles et al.
ult1 floral meristems contain a dome of cells between
the developing carpel primordia, which normally abut
one another. Proliferating cells that separate the two
carpel primordia are also observed in pAG::WUS trans-
genic plants in which WUS activity is prolonged in the
center of the flower (Lenhard et al. 2001). Similarly,
ag mutants maintain a dome of proliferating cells in the
centeroftheflower,evenaftermultiplewhorlsoforgans
have formed, which sustains WUS expression beneath
the twooutermost celllayers (Lenhardet al.2001; Loh-
mann et al. 2001). In addition, carpel number is not
restored in ult1 wus double mutants, revealing that the
ULT1 indeterminacy phenotype is dependent on the
activityof WUS.wus mutationsarein factepistatic to ult1
mutations with respect to floral meristem determinacy,
indicating that ULT1 acts in the same pathway as WUS
to control floral stem cell termination. Finally, we have
previously observed that AG expression is delayed in
the center of ult1 mutant floral meristems (Fletcher
2001), which correlates with delayed floral meristem
termination. This result suggests that ULT1 repression
of stem cell activity may work through AG.
WecaninvoketwopossiblemodelsforULT1function
in floral meristem termination that are consistent with
our data. In the first model, ULT1 is necessary to induce
AG at the correct time during flower development to
ensure that WUS repression occurs at the time of carpel
initiation. Therefore we propose that in wild-type floral
meristems, ULT1 acts (directly or indirectly) with LFY
and WUS to activate AG at the proper time and place
in the most central region of the flower. AG activation
leadstoWUSrepressionandconsequentstemcelltermi-
nation. In ult1 floral meristems, AG activation in the
central region is delayed, causing a delay in WUS repres-
sion and thus permitting amplification of additional
stem cells that become incorporated into extra whorls
of organs. This model is attractive in that it accounts
for the delayed activation of AG in the center of the
flower, although we note that, because AG is properly
induced in the third whorl even when ULT1 is absent,
some other factor(s) is likely to activate AG in this
region. An alternative scenario is that ULT1 may be
required in addition to and independently of AG to
repress WUS in the center of the flower. In this case,
we predict that,in the absence of ULT1,AG is eventually
able to repress WUS on its own, but only after a delay,
during which the level of AG itself or a secondary activa-
tor may rise sufficiently to terminate WUS transcription.
While we favor the first model because of its simplicity
(it invokes only a single regulatory pathway rather than
two separate pathways), its confirmation or rejection
will await further experiments to determine whether AG
is directly or indirectly activated by ULT1.
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Formation of the shoot
Genes
ASYM-
The
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Stem cell homeostasis in the Arabi-
Termina-
The WUSCHEL and
The development of apical
A
We thank Jeff Long, Kathy Barton, and Thomas Laux for providing
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construct. We are grateful to Karen Osmont, Leor Williams, Dan
Choffnes, Robert Blanvillain, and George Chuck for helpful com-
ments on the manuscript. This work was supported by the National
Science Foundation (IBN-0110667).

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