Genetic Analysis of Organ Fusion in Arabidopsis thaliana

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Copyright1998 by the Genetics Society of America

Genetic Analysis of Organ Fusion in Arabidopsis thaliana

Susan J. Lolle, Wendy Hsu and Robert E. Pruitt

Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138 Manuscript received December 31, 1997

Accepted for publication March 3, 1998

ABSTRACT

Postgenital organ fusion occurs most commonly during reproductive development and is important in many angiosperms during genesis of the carpel. Although a number of mutants have been described that manifest ectopic organ fusion, little is known about the genes involved in regulating this process. In this article we describe the characterization of a collection of 29 Arabidopsis mutants showing an organ fusion phenotype. Mapping and complementation analyses revealed that the mutant alleles define nine different loci distributed throughout the Arabidopsis genome. Multiple alleles were isolated for the four complemen-tation groups showing the strongest organ fusion phenotype while the remaining five complemencomplemen-tation groups, all of which show only weak floral organ fusion, have a single representative allele. In addition to fusion events between aerial parts of the shoot, some mutants also show abnormal ovule morphology with adjacent ovules joined together at maturity. Many of the fusion mutants isolated have detectable differences in the rate at which chlorophyll can be extracted; however, in one case no difference could be detected between mutant and wild-type plants. In three mutant lines pollen remained unresponsive to contact with the mutant epidermis, demonstrating that organ fusion and pollen growth responses can be genetically separated from one another.

I

N plants the outermost layer of cells covering the dermis to physical contact with other epidermal cells and occurs most commonly during floral ontogenesis shoot and root surfaces offers the first site of contact

with biotic as well as abiotic factors present in the sur- (Cusick1966). Postgenital fusion represents a special case where the developmental potential of the epider-rounding environment. The barrier presented by this

epidermal layer is dynamic and selective, permitting mis is altered so that it no longer remains unresponsive to contact. In Catharanthus roseus, where this develop-some biological interactions while blocking others.

Dur-ing plant growth, for example, epidermal cell interac- mental response has been studied most extensively, it has been demonstrated that organ fusion involves the tions play an important role in the elaboration of the

exchange of small, water-soluble morphogenetic factors shoot by regulating organ fusion, thus contributing to

(SiegelandVerbeke1989). Although many of the epi-variation of the body plan, especially in flowers. During

dermal cells along the fusion suture in C. roseus respond reproductive development epidermal derivatives

pro-to contact by forming a tight cell wall association, a vide a receptive surface for the hydration and

germina-subset of approximately 400 cells dedifferentiate and tion of pollen, an important first step in the fertilization

redifferentiate into parenchyma cells (Walker 1975 process. In some species the specialized epidermal cells

a,b,c;VerbekeandWalker1985, 1986). Both cell wall that interact with pollen (the stigmatic papillar cells)

adhesion and the cellular redifferentiation response are provide the selection point where self-pollen is blocked

known to require recognition events because noncarpel during early development (Ockendon 1972;de

Net-epidermal derivatives will not undergo any of the tancourt 1977;Bell1995), thereby selecting against

changes associated with this contact-mediated response self-fertilization and promoting outcrossing. Clearly,

(Siegel and Verbeke 1989). The details of how this mediating the responsiveness of the shoot epidermis to

developmental pathway is regulated and information external factors is critical to the viability, proper

elabora-about the signals and signalling cascades involved re-tion and growth of the plant, as well as the fertilizare-tion

main elusive. process itself.

To date six mutations have been described which In many plant species primordial structures are known

cause ectopic expression of an epidermal fusion re-to unite following initiation at the shoot apical

meri-sponse: adherent1 (Kempton1920), adherent2 (Neuffer stem. This process, known as postgenital fusion, is

et al. 1997) and crinkly4 (Becraftet al. 1996) in maize achieved by a change in the responsiveness of the

epi-and fiddlehead (Lolleet al. 1992), wax1 and wax2 (Jenks et al. 1996) in Arabidopsis. In all six cases the mutations were identified by virtue of some impairment of normal Corresponding author: Robert E. Pruitt, Department of Molecular and

expansive growth in mutant plants. In maize plants har-Cellular Biology, Harvard University, 16 Divinity Ave., Cambridge,

MA 02138. E-mail: pruitt@billie.harvard.edu boring the crinkly4 (cr4) mutation, for example,

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mal cells adhere to one another early in development, occur on all mutants showing the organ fusion pheno-type, supporting the notion that organ fusion and pollen culminating in significantly stunted and misshapen

adult plants (Becraftet al. 1996). Closer examination growth promotion define distinct biological processes. of cr4 mutants reveals that the epidermis itself is atypical

and forms graft-like fusions. The CR4 gene has been

MATERIALS AND METHODS

isolated and encodes a putative receptor protein kinase

(Becraftet al. 1996). In the Arabidopsis fdh-1 mutant, _{Plant growth conditions:}_{Plants were maintained under a} 24-hr light regimen and were illuminated with a mix of

fluo-epidermal morphology is normal (with the exception

rescent and incandescent lights (100–175mmol m22sec21at

of the fusion sutures) although plant growth is also

pot level). Plants were grown in CustomBlen Plus (Griffin

severely affected (Lolleet al. 1992). In addition, analysis

Greenhouse Supplies, Tewksbury, MA) soil mix and watered

of the fdh-1 mutant revealed that the entire shoot surface _{as needed with distilled water. The environment in the Percival}

(I-37LLVL and I-60LLVL) growth chambers (Percival

Manu-was competent to promote pollen hydration,

germina-facturing Company, Boone, IA) was maintained at 258

tion and tube growth (LolleandCheung1993). The

(618) and 50% relative humidity.

pollen response seen on the fdh-1 shoot surface

reiter-Mutagenesis and plant manipulations:All of the new

muta-ates a pollen-stigma type interaction in that the pollen _{tions isolated in our laboratory as a part of this study were} growth response involves recognition events that limit _{recovered from mutagenesis with ethyl methanesulfonate}

(EMS). Seeds were treated with EMS essentially as described

the spectrum of pollen species that can grow on the

in Robinson-Beerset al. (1992). M₁ plants were grown on fdh-1 surface to other members of the mustard family

soil and a single silique was harvested from each plant. All of

(LolleandCheung1993). Furthermore, like the

wild-the seeds from a single M1plant were grown as a family and

type stigma, fdh-1 epidermis is unable to bypass the hy- _{screened for the segregation of organ fusion mutants. In some} dration block imposed by some of the cer mutations _{cases mutations could be recovered directly from selfing of}

homozygous mutant plants, while in others they were

recov-(Preusset al. 1993;Hu¨ lskampet al. 1995; Lolleet al.

ered through heterozygous siblings present in the family.

Ap-1997). The wax1 and wax2 Arabidopsis mutants, on the

proximately 15,000 M1 families were screened and 21 new

other hand, show altered epicuticular wax as well as an

organ fusion mutations were recovered (see Table 1).

organ fusion phenotype (Jenkset al. 1996). Whether

Lines polymorphic for molecular markers were generated

or not these wax mutants interact with pollen has not _{by manually crossing homozygous or heterozygous mutant}

plants (in one of three ecotype backgrounds: Landsberg,

Co-been determined.

lumbia or Wassilewskija) to an appropriate wild-type line. F1

Two distinct mechanisms for promoting organ fusion

plants were allowed to self pollinate and set seed. F2plants

are suggested by the studies done on the cr4 and fdh-1

homozygous for the mutation of interest were then collected

mutants. It seems plausible in light of the sequence _{and tissue stored at}₂₈₀₈_{until processed for DNA-based} map-homology shown by the CR4 gene that fusions in these _{ping analyses.}

Complementation analyses were done by crossing

homozy-maize mutants result from disruption of the signalling

gous or heterozygous mutant plants to one another and the

pathway directing epidermal differentiation such that

resulting F1plants scored for the presence or absence of the

cells either do not achieve or do not maintain a terminal

mutant phenotype. In some instances, lines polymorphic for

and unresponsive developmental state typical of epider- _{molecular markers were used to construct trans-heterozygous} mal derivatives. In fdh-1 mutants, on the other hand, a _F₁_{plants in order to facilitate identification of both F}₁_{and F}₂

progeny using the molecular markers.

change in the permeability barrier of the outer

epider-Determination of genetic map positions:DNA was prepared

mal cell wall and cuticle is correlated with and may be

from individual F2plant samples according toEdwardset al.

responsible for organ fusion (Lolle et al. 1997). It is

(1991). Briefly, tissue was ground in microcentrifuge tubes

postulated that the factors promoting fusion become _{using disposable plastic pestles. Following grinding, 400}_m_{l of} accessible and are exchanged across fdh-1 cell walls be- _{extraction buffer (200 m}mTris-HCl pH 7.5, 250 mmNaCl, 25 mm EDTA, 0.5% SDS) were added and samples vortexed.

tween contacting cells, initiating the cascade of

develop-Samples were centrifuged for 1 min in a microcentrifuge, 300

mental events leading to cell wall adhesion and organ

ml of the supernatant transferred to a fresh tube and an equal

fusion. In the former case a putative receptor in the

volume of isopropanol added. Samples were vortexed and left

signal transduction pathway has been identified while _{for 2 min at room temperature. Following a 5-min} centrifuga-in the latter case early events centrifuga-in signallcentrifuga-ing have become _{tion, supernatants were discarded and pellets air-dried for}

5–10 min at room temperature. DNA preparations were then

constitutive.

resuspended in 100 ml TE (10 mm Tris-HCl pH 8.0, 1 mm

In this article we describe the isolation and

character-EDTA) and stored either at 48or at2208.

ization of 29 independently derived mutations causing

Amplification of simple sequence length polymorphisms

organ fusion in Arabidopsis. The mutations fall into _(SSLPs;BellandEcker1994) or codominant amplified

poly-nine complementation groups putatively identifying _{morphic sequences (CAPS;} Konieczny andAusubel1993) was performed using polymerase chain reaction (PCR).

Oligo-nine genes. In the majority of mutants recovered, a

nucleotide primers were obtained from Research Genetics

change in the permeability barrier of the epidermal cell

Inc. (Huntsville, AL) and Taq DNA polymerase and reaction

wall cosegregates with the organ fusion phenotype but

buffer from Perkin-Elmer (Norwalk, CT). Deoxynucleotide

in one case it does not, suggesting that organ fusion _{triphosphates (dNTPs) were purchased from GIBCO-BRL} can be achieved by at least two distinct mechanisms _{(Gaithersburg, MD). Each 20-}_m_{l reaction mixture contained}

1ml of DNA, buffer, 200mmol dNTPs, 0.5 units of AmpliTaq

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DNA polymerase (Perkin-Elmer, Norwalk, CT) and 5 pmol _{screen, which would allow the recovery of any fully male} each of the forward and reverse primers. _{and female sterile mutant through heterozygous sibling}

Conditions for amplification of SSLPs were as follows: 1

plants. As shown in Table 1, 21 mutants were isolated in

cycle of 2 min at 948, 15 sec at 558, 30 sec at 728, followed by

this screen, which displayed an organ fusion phenotype.

39 cycles of 15 sec at 948, 15 sec at 558, 30 sec at 728. Conditions

for amplification of CAPS were as follows: 1 cycle of 2 min at During the course of our genetic screen we also

ob-948, 15 sec at 558, 2 min at 728, followed by 39 cycles of 30 _{tained an additional eight mutants from the sources} sec at 948, 15 sec at 558, 2 min at 728. Restriction digests of _{indicated in Table 1. All of the mutant phenotypes} de-CAPS products were carried out using the restriction enzymes

scribed segregated in a manner consistent with

mono-specified inKoniecznyandAusubel(1993). PCR products

genic, recessive mutations. Each mutant line was

out-were size-separated by Tris-Borate-buffered agarose gel

elec-trophoresis. Agarose (4%) gels were used to resolve SSLP crossed to an appropriate wild-type line to generate an

products and 1.5–2% agarose gels to resolve CAPS products. _F₁ _{line bearing polymorphisms for molecular markers} Linkage to specific SSLP or CAPS markers was determined _{suitable for use in DNA-based mapping procedures.} by scoring a sufficient number of F2individuals to give an

Analysis of F2plants for each of these outcrossed mutant

LOD score larger than 3.0 using Mapmaker/EXP version 3.0

lines revealed linkage to nine different SSLP or CAPS

(Landeret al. 1987). Mutations which mapped to similar map

positions were tested for allelism by complementation analysis. DNA markers distributed throughout the genome.

Phenotypic analyses of organ fusion, fertility, porosity and _{Complementation analysis between mutants with} simi-pollen hydration:Plants were visually inspected for evidence of _{lar map positions confirmed that our collection of} fu-organ fusion during seedling, juvenile, adult and reproductive

sion mutants defines nine distinct genes. The genetic

developmental stages. Fusion was scored as positive if two

map positions of these complementation groups are

organs adhered to one another and could not easily be

sepa-rated by gentle physical manipulation. Ovules were analyzed illustrated in Figure 1 and their morphological

pheno-by dissecting open the ovary walls of the gynoecium and view- _{types are described individually as follows:}

ing ovules under a dissecting microscope (Leica/Wild M3C, _{airhead (ahd) complementation group:}_{Only a single} Heerbrugg, Switzerland). Samples analyzed by scanning

elec-allele was isolated in this complementation group. Of all

tron microscopy were fixed in FAA (50% ethanol, 3.7%

formal-the fusion mutants we have characterized, this mutant

dehyde, 10% acetic acid) and dehydrated through a graded

ethanol series to 100% ethanol. Samples were critical point showed one of the weakest organ fusion phenotypes,

dried using a Samdri-PVT-3B unit and sputter coated with _{which is manifested only in fusion between floral organs} gold. Samples were viewed in an AMR 1000 scanning electron _{(Table 2). Fusions are most frequently observed} be-microscope at 10 kV.

tween sepals and petals, resulting in poor emergence

Mutant plants were tested for male fertility by outcrossing

of the latter. Although interorgan fusions within the

to a male sterile/female fertile line (TH154; R. E. Pruitt,

unpublished results) and scoring for silique elongation and flowers are common, no morphological aberrations

seed set. Female fertility was tested by pollinating mutant _{have been observed in the ovules. Fertility is normal} plants with wild-type pollen and scoring for silique elongation _{(see Table 2).}

and seed set. In cases where floral organ fusion was severe

bulkhead (bud) complementation group:Only one

al-mutant flowers had to be dissected open to reveal the

gynoe-lele belonging to this complementation group was

iden-cium.

The rate at which chlorophyll could be extracted as a mea- tified. Like airhead, this allele causes only weak fusion

sure of porosity was determined as described inLolle_{et al.} _{where fusion events are essentially restricted to floral} (1997). Tissue samples were collected following elongation of _{organs (Figure 2). A striking feature of this mutant,} the inflorescence bolt (approximately 4 wk after planting).

however, is seen in the severity of the ovule phenotype.

In all cases where fusion did not prevent physical separation,

As shown in Figure 3, C and D, ovule morphology is

the rosettes were removed from the inflorescence bolt and

the two tissue samples immersed separately in 80% ethanol. highly abnormal. Although the funiculus can be

identi-Samples were agitated gently on a rotating platform and 100- _{fied for each individual ovule the remaining structure}

ml aliquots of the ethanol solution removed at 10 min, 20 _{is severely compromised and shows little morphological} min, 40 min, 60 min, 80 min and 24 hr following immersion.

integrity. Not surprisingly, this mutant is a tight female

Total chlorophyll content in each sample was determined

sterile (Table 2).

using absorption readings taken at 647 and 664 nm using a

UV-1201 spectrophotometer (Shimadzu Corp., Tokyo). Pollen conehead (cod) complementation group:Plants

homo-hydration assays were carried out as described previously _{zygous for the conehead-1 mutation display either a weak} (Lolleet al. 1997).

fusion phenotype similar to bulkhead-1 mutants or a se-vere fusion phenotype like that seen in fdh mutants with approximately equal frequency (Figure 2). We were

RESULTS

unable to separate the weak and strong fusion pheno-types even after 3 backcrosses; when the two phenopheno-types

Mutant isolation and complementation analysis: The

isolation of the original fdh-1 mutant demonstrated the were mapped independently they mapped to the same location. These two distinct phenotypes may represent possibility of identifying genes whose products are

re-quired to suppress interorgan fusion through the isola- modification of the cod mutant phenotype by a second segregating locus, but we have not yet attempted to tion of mutations that promote such fusions. In order

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TABLE 1

Summary of organ fusion mutations

Gene Allele Genetic background Mutagen Source

airhead 1 Columbia fast neutrons Alan Sessions(University of California, Berkeley, CA)

bulkhead 1 Landsberg erecta EMS This study

conehead 1 Landsberg erecta EMS This study

deadhead 1 Landsberg erecta EMS This study

2 Landsberg erecta EMS This study

Chris Somerville(Carnegie Institute of Washington,

3 Columbia ? Stanford, CA)

4 Columbia ? Lawrence Hobbie(Adelphi Univeristy, Garden City, NY)

eceriferum10 1 Landsberg erecta X ray Koornneffet al 1989

fiddlehead 1 Landsberg erecta EMS Lolleet al. 1992

7 Landsberg erecta EMS David Smyth

hothead 1 Landsberg erecta EMS This study

9 Wassilewskija T-DNA Stock center (Arabidopsis Biological Resource Center)

10 Landsberg erecta EMS David Smyth(Monosh University, Clayton, Australia)

pothead 1 Landsberg erecta EMS This study

thunderhead 1 Landsberg erecta EMS This study

deadhead (ded) complementation group:Four alleles ed a wild-type phenotype while others showed only a waxless phenotype or both a waxless and organ fusion were identified which fell into this complementation

group. All of the mutants in this group show a marked phenotype. Because of the wax phenotype observed in ded mutants, all of these mutations were also comple-surface luster consistent with a waxless or eceriferum

phenotype. As summarized in Table 2, for the ded-1 and mentation tested against cer5-1, the only cer mutant known to map in the same vicinity. All of these crosses ded-2 alleles, fusion can occur upon emergence of the

first true leaves. Only a small fraction of these mutant produced only wild-type F1progeny. Mapping data

con-firmed that cer5-1 mapped to a nearby but distinct map plants grows to produce an inflorescence as fusion often

limits normal vegetative growth. Early fusion events are position relative to the DED gene.

eceriferum10 (cer10) complementation group: The also typical for the ded-4 allele but not the ded-3 allele

where fusion events are commonly limited to the inflo- cer10 locus has been described previously byKoornneef et al. (1989). These mutants have a pleiotropic pheno-rescence and flowers. Ovules show a strongly aberrant

phenotype in ded-1 whereas ovule defects seen in ded-2 type including reduced plant height (Koornneefet al. 1989). However, in these descriptions no mention was mutant plants are less severe. No ovule phenotype was

observed in ded-4 mutant plants (ded-3 ovaries were not made of the organ fusion phenotype. As indicated in Table 2, cer10 mutants manifest a fusion phenotype simi-surveyed). ded-1 and ded-2 are female semi-sterile (Table

2), whereas ded-3 and ded-4 mutants show normal fer- lar to the bulkhead-1 mutant. Although fusion is re-stricted to floral organs, the ovules are not affected tility.

Complementation analyses revealed unusual allelic (Table 2). These mutants are male semisterile, as pre-viously noted byKoornneefet al. (1989) but are female interactions at this locus. As a consequence all possible

pair-wise allele combinations were constructed and F1 fertile (Table 2).

fiddlehead (fdh) complementation group:Five new al-plants scored for their wax and fusion phenotype. As

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Figure 1.—Schematic representation of genetic map positions for the nine genetic loci. The distance of each locus from a linked molecular marker is incated, together with the di-rection from that marker, if known. Three of the loci map to chromosome 1 and two each to chromosomes 2, 3 and 5, respectively.

one additional allele was obtained from a colleague Both fdh-5 and fdh-6 manifest a defective ovule pheno-type (Table 2) and all alleles tested (fdh-1, fdh-3, fdh-5 (see Table 1). Like the original fdh-1 mutant, described

previously byLolleet al. (1992), all of these new alleles and fdh-6) are female semisterile (Table 2).

hothead (hth) complementation group:Eleven alleles show a strong organ fusion phenotype (Figure 2).

Al-though organ fusion is variable from one individual define the HTH locus. The fusion phenotype of these mutants is intermediate in strength with organ fusion plant to the next, for every allele it is clear that all organs

are competent to fuse, including the first true leaves. generally being limited to flowers (Table 2). The floral

TABLE 2

Summary of mutant phenotypesa

Juvenile Adult Wild-type

rosette leaf rosette leaf Floral Ovule Spontaneously Female Male pollen cer pollen

Locus fusionb _fusionb _fusionb _abnormalityc _fertiled _fertilee _fertilee _hydrationf _hydrationf

wildtype 2 2 2 2 11 1 1 2 2

airhead 2 2 1/2 2 11 1 1 1 2

bulkhead 2 2 1 1 2 2 1 2 2

conehead 2 1 1 2 1/2 1 1 1 2

deadhead 1 1 1 1/2 1/2 1/2 1/2 1 2

eceriferum10 2 2 1 2 2 1 2 1 2

fiddlehead 1 1 1 1 2 2 1 1 2

hothead 2 2 1 1/2 1 1 1 1 2

pothead 2 2 1/2 2 11 1 1 2 2

thunderhead 2 1 1 1 2 1/2 1 1/2 2

a_{The representative alleles scored for each locus were: ahd-1, bud-1, cod-1, ded-1, ded-2, ded-4, cer10-1, fdh-3, fdh-5, fdh-6, hth-4,}

hth-8, hth-10, phd-1, thd-1 and thd-2.

b_{For each of the fusion phenotypes:}₁_{, fusion events strong enough to seriously distort the shape of the organ;}₁_/₂_{, fusion}

events which do not distort the shape of the organ;2, no fusion.

c_{Each representative allele was scored for the presence or absence of morphologically abnormal ovules (}₁_or₂_).₁_/₂_{, some}

alleles showed an ovule defect while others did not.

d_{Spontaneously fertile refers to the ability of the flowers to set seed without human intervention.}₁₁_{, wild-type levels of seed}

set;1, seed set less than 50% of wild-type levels;2, no seed set (less than 1 seed/flower);1/2, variation between alleles tested.

e_{Male and female fertility were scored relative to wild-type in reciprocal crosses.}1_{, at least 50% of wild-type fertility;}2_{, less}

than 50% of wild-type fertility;1/2, variation between the alleles tested.

f_{Samples were scored as positive for hydration if 50% or more of the applied pollen grains became spherical in shape within}

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Figure2.—Light micrographs showing the fusion phenotypes associated with various mutant alleles. As shown in A, wild-type Landsberg er inflorescences show a typical spiral phyllotaxy where contact between neighboring buds decreases as development progresses. As individual flowers mature petals elongate and emerge unimpeded by the surrounding sepals. In bulkhead (B), hothead (G) and pothead (H) mutant petals are restricted in their growth but do emerge from some flowers (arrow in G). In mutants showing stronger fusion phenotypes, like conehead (C), deadhead (D), thunderhead (E), and fiddlehead (F) mutants, organ fusion alters the configuration normally seen in the inflorescence and blocks petal and anther emergence. The pistil, however, usually protrudes out from the individual floral buds. In thunderhead-1 mutants the first 3–4 rosette leaves (as shown in E) do not adhere and have a normal phyllotactic relationship. Upon emergence of subsequent leaves, however, fusion commences resulting in a central trumpet-like clustering of leaves (arrow in E). The inflorescence is completely sequestered within these leaves but often will emerge as the plant continues to grow. Magnification of panel A, B, D, F and H is the same. Magnification of panels C and G is the same. Bars, 1 cm.

organ fusion phenotype is stronger than that seen in plants was confirmed by demonstrating that they were homozygous for at least three SSLP markers that were either bud-1 or ahd-1 but not strong enough to

com-pletely block petal emergence (see Figure 2) or self- homozygous in the F1 plants and represented both

Landsberg and Columbia alleles. All of these plants were fertilization. Although the majority of gynoecia show no

ovule defects, approximately 10–20% of the gynoecia also progeny tested and shown to segregate 3:1 for the fusion phenotype after selfing. As shown in Table 3 only surveyed in the hth-8 and hth-10 mutants showed

evi-dence of ovule abnormalities while no defects were seen F2progeny derived from hth-8/hth-10 transheterozygotes

segregated wild-type plants. No wild-type progeny were in any of the ovaries sampled from hth-4 mutants.

Mapping data from the HTH locus showed that a found among F2 plants derived from the hth-4/hth-10

transheterozygotes. subset of hth alleles recombined with a flanking SSLP

marker at a higher frequency than the remaining group pothead (phd) complementation group: Only one al-lele for this locus was isolated. Organ fusion in the phd-1 of hth alleles, although crosses between these two groups

of alleles failed to show complementation in the F1gen- mutant plants is limited to the flowers and is even weaker

than that seen in ahd-1 (see Figure 2). As in ahd-1, eration. To determine the frequency of recombination

which took place between these different classes of hot- fusion is most often seen between sepals and petals and interferes with the proper deployment of the petals. head alleles, hth-4, hth-8 and hth-10 mutants polymorphic

for Columbia and Landsberg molecular markers were Ovules are normal in appearance and mutant plants are self-fertile (Table 2).

used to generate F1plants. The identity of these F1plants

as heterozygotes was confirmed by demonstrating that thunderhead (thd) complementation group:Organ fu-sions in plants harboring mutations at this locus initiate they were heterozygous for SSLP markers which were

homozygous in each parental line but polymorphic be- late in vegetative development (see Table 2). Of the two alleles isolated, plants homozygous for the thd-1 tween the parental lines. F2plants derived from

individ-ual F1plants were then scored for segregation of wild- allele show a sharper transition to fusion competence.

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Figure3.—Scanning elec-tron micrographs showing normal wild-type ovules and ovule phenotypes of three representative mutants. In all cases, ovary walls were re-moved to reveal the ovules. The boxed areas in A, C, E and G are shown at higher magnification in the adja-cent and corresponding pan-els (B, D, F, and H). In the wild-type ovary (A and B) each locule contains two parallel rows of ovules (o) on either side of the sep-tum. Pollen grains land on the stigmatic papillar cells (p), germinate and grow tubes which penetrate into the ovary and fertilize the ovules. As shown in B, ov-ules are tightly packed within the ovary but manifest no adhesion. Each ovule is an-chored to the ovary only by the funiculus (f ). In bulk-head-1 mutants (C and D) ovules (o) show little mor-phological integrity. The only identifiable structure is the funiculus (f ). In thunder-head-1 mutants (E and F), although the majority of ovules are physically joined to one another at maturity, some ovules develop rela-tively normally. As shown at higher magnification in F, one ovule has developed without attachments to neighboring structures and not only has a funiculus (f ) but also a micropyle (arrow). In thunderhead-2 mutants (G and H), on the other hand, a larger propor-tion of the ovules remain unattached to their neigh-bors and show normal mor-phology. Bars, magnifica-tion in micrometers.

expansion of the first four to five leaves fuse together, mutant ovaries (Figure 3). Although many ovules are affected adversely in the mutants, some ovules in either as do the inflorescence and flowers (see Figure 2). thd-2

mutants, on the other hand, show a more gradual onset case appear morphologically normal and fully differen-tiated. Mutant plants are female semisterile (Table 2). of late vegetative fusion but display similarly severe floral

organ fusions. In plants homozygous for either mutant Pollen hydration:One goal in characterizing a larger collection of fusion mutants was to determine whether allele, ovules are joined to one another at maturity but

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the organ fusion phenotype. Table 2 summarizes the results of the pollen hydration assays. Some mutants clearly support a rapid hydration response time similar to that seen on fdh-1 plants (cod-1 and thd-2, data not shown). In other instances hydration takes place but is attenuated (ahd, cer10, ded, hth, data not shown). How-ever, three mutants show no hydration response with wild-type pollen: bud-1, phd-1 and thd-1. Two of these, bud-1 and phd-1, manifest a weak fusion phenotype while the thd-1 mutant has a very strong organ fusion pheno-type. Although the thd-1 mutant does not promote hy-dration, pollen grains hydrate within 10 min when ap-plied to the thd-2 mutant surface. No hydration of the

cer1-147 pollen was observed on any of the mutant plants Figure4.—A summary of the complementation phenotypes

tested, indicating that in all cases the pollen hydration _{from all pair-wise crosses of the ded alleles. As shown, one}

allele combination shows full interallelic complementation

observed requires specificity on the pollen side of the

(ded-2 allele crossed to ded-4) while two combinations show

interaction similar to that seen on the Arabidopsis

partial complementation restoring only the fusion phenotype

stigma (Hu¨ lskampet al. 1995;Preusset al. 1993).

to normal (ded-3 crossed to either ded-2 or ded-4). cer5 comple-Permeability to chlorophylls: Representative alleles _{ments all ded alleles.}

from each of the loci identified in this study were assayed for changes in the rate at which chlorophyll could be

extracted. The results are summarized in Figure 5. As mediated organ fusion is the pollen-stigma interaction. In this case a specific recognition reaction also takes is evident from the graphed data, by this criterion there

exists a wide variation in porosity among the mutants place as well as an exchange of factors between the participating cells. Not only do pollen grains adhere to tested. Clearly, the most permeable samples are found

in the fdh and ded complementation groups (Figure 5, the papillar cell (an epidermal derivative) in a manner akin to an organ fusion-like event, but this interaction B and D). Only one mutant showed a relatively intact

chlorophyll permeability barrier (that is, similar to wild triggers the further development of the male gameto-phyte. Interestingly, lipids have recently been high-type): phd-1 (Figure 5G). The remaining loci show a

range of permeabilities, but all show some enhancement lighted as playing regulatory roles in both processes (Hu¨ lskampet al. 1995;Lolleet al. 1997; Preusset al. of permeability to chlorophyll relative to Landsberg. In

cases where the rosette could be assayed independently 1993).

In this article we describe the isolation of 21 new from the inflorescence, the inflorescence usually

show-ed a greater permeability (see Figure 5, A–D, F and mutations and the further characterization of an addi-tional eight mutants which manifest an organ fusion H). thunderhead plant tissues were further subdivided by

separating the juvenile from adult leaves (or adult leaves phenotype. In addition to the original fdh locus de-scribed previously (Lolleet al. 1992), eight additional plus inflorescence in the case of thd-1). As shown in

Figure 5H, the adult leaves of thd-2 show a permeability loci were identified which are involved in mediating organ fusion. Multiple alleles were found for three loci profile similar to the juvenile leaves of the thd-1 allele.

However, both show approximately equivalent perme- (ded, fdh and hth), two alleles for the thd locus but only single alleles at the remaining five loci (ahd, bud, cer10, ability changes in their inflorescence tissues.

cod, phd). With the exception of the conehead mutant, the four genes with only one representative allele are

DISCUSSION

represented by mutants that all manifest weak organ fusion. Due to the subtle nature of the fusions other Examples of postgenital organ fusion can be found

in many angiosperm species; this fusion is thought im- mutations that fall into these complementation groups may have been overlooked in the course of our genetic portant in both increasing developmental flexibility

dur-ing floral ontogeny and facilitatdur-ing the mechanics of screen. It may be possible in the future, however, to manipulate environmental conditions or to use other the pollination process. The most extensive analysis of

the cell biology of postgenital organ fusion has been sensitized genetic backgrounds such that the frequency or severity of organ fusion is enhanced, simplifying iden-done in C. roseus (Walker 1975a,b,c; Verbeke and

Walker1985, 1986). Based on the results of these stud- tification of other alleles at these loci. The relative map positions as determined using DNA-based mapping pro-ies it is known that organ fusion is an epidermis-specific

interaction, involves reciprocal recognition events and cedures reveal that the genes identified in this article are not clustered but map throughout the Arabidopsis is mediated by small diffusible water-soluble molecules

(Siegel and Verbeke1989). Another type of cell-cell genome.

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epidermally-TABLE 3

Summary of hothead intragenic recombination frequencies

Number of individuals Number of wild-type Percent frequency of

Cross with organ fusion individuals recombinationa

hth-103hth-8 1858 30 1.6

hth-103hth-4 1629 0 0

a_{Because the progeny of these crosses are derived from selfing of the F}

1 generation the frequency of

recombination (r) can be estimated from the number of wild-type recombinants: frequency of wild-type recombinants5r/21r/22r2_{/4. Since r is small this latter expression is approximately equal to r.}

notype varies among members in this mutant collection alleles results in both the cer and organ fusion pheno-types only when homozygous or in combination with from a severe phenotype like that of the original

fdh-1-mutant (Lolleet al. 1992), to a very subtle fusion pheno- ded-1. The pattern is further complicated by the fact that plants bearing some combinations of alleles (ded-type such as is the case for the ahd-1 and phd-1 mutants.

Plants with a severe fusion phenotype can suffer serious 3/ded-4; ded-2/ded-3) have an obvious cer phenotype but fail to undergo any organ fusion whatsoever. More growth impairment while plants with a weak phenotype

are relatively normal in appearance. In addition to fu- mutant alleles of this locus are needed to allow a better definition of this complex complementation pattern. sion of organs comprising the external shoot, in some

mutants ovules with abnormal morphology were also Characterization of the mutants presently available as well as molecular analysis of the DED gene may allow found. Although these abnormal structures (in which

adjacent ovules are joined together at maturity) are us to determine the nature of the interallelic comple-mentation seen at this locus.

reminiscent of fused structures seen in other parts of

the plant, determination of the mechanism which leads Of all of the organ fusion mutants we have recovered, only mutations at the DEADHEAD locus show a wax to these structures will require a more detailed

examina-tion of ovule development in these mutants. Surpris- defect. The deadhead eceriferum phenotype suggests a defect in lipid biosynthesis (Aarts et al. 1995; Han-ingly, ovule defects were detected not only in mutants

showing strong fusion between organs but also in mu- noufaet al. 1996; Negruket al. 1996;Xiaet al. 1996), and, as indicated by biochemical analyses of fdh-1 mu-tants like bud-1, which manifest a relatively weak fusion

phenotype. Although the bud-1 mutant is a tight female tants, changes in lipid composition of the outer epider-mal cell wall and cuticle may play some role in promot-sterile, other mutants where ovule abnormalities were

detected (ded-1 and thd-1) are not, indicating that ova- ing organ fusion. Two other Arabidopsis mutants which also show organ fusion and have a wax defect have been ries containing large abnormal structures derived from

many ovules need not block the fertilization process described (Jenkset al. 1996) and may be allelic to our ded mutants, although complementation tests have not in the few remaining normal ovules. The detectable

presence of a funicular structure with relatively normal yet been performed. However, deadhead does not appear to be allelic to cer5 which maps to a similar location in morphology in all mutants that manifest ovule defects

suggests that deviation from normal ovule development the genome. A detailed biochemical analysis of these mutants may reveal the loss or modulation of a lipid occurs after the ovule primordia have elongated and

the integuments have initiated (stage 2-II; Schneitzet component that may be important for maintaining epi-dermal developmental integrity.

al. 1995). Further analysis of ovule development should

help illuminate the ontogeny of these aberrant struc- A second locus which has interesting phenotypic be-havior is CONEHEAD. In this case, plants which are tures and what, if any, cellular consequences these

ab-normalities have for the embryo sac as well as the sporo- homozygous for the mutation fall in equal numbers into two discrete phenotypic classes: those that exhibit strong phytic tissues of the ovule.

The deadhead locus which we have described has an fusion and those that exhibit weak fusion. Both of these phenotypes were present in the original single line fam-interesting pattern of interallelic complementation.

Plants homozygous for any of the individual ded mutant ily in which the mutation was isolated and both pheno-types have persisted through three backcrosses to the alleles have a strong eceriferum phenotype as well as

manifesting relatively severe organ fusion. Heteroallelic parental wild-type line, Landsberg erecta. Although it is possible that this variation in phenotype is due to a combinations have widely varying phenotypes, however,

ranging from completely wild-type plants to plants with genetic modifier which is segregating in this line, it is hard to account for the fact that it has been carried as severe a phenotype as the individual alleles. The

pres-ent complempres-entation analysis fails to define any obvious along through the backcrosses (implying close linkage to the cod mutation) and yet is readily separable from subgroupings of alleles. One allele (ded-1) fails to

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Alterna-tively, it is possible that the mutation in COD is itself of organ fusion is still confined to late adult develop-ment. Plants are known to manifest a variety of morpho-responsible for both phenotypes with variable

expressiv-ity of the gene somehow producing two discrete classes logical and biochemical changes during different phases in their development (Lawson and Poethig of plants. The isolation of additional alleles of this locus

and COD gene isolation may help clarify how these 1995) and it may be that at this locus the juvenile to adult phase transition plays some role in regulating THD mixed phenotypes are achieved.

The disproportionately large number of mutations gene expression. Interestingly, the adherent2 locus in maize shows a similar restriction of organ fusion to the recovered in the HOTHEAD complementation group

suggest that this locus may represent an unusually large adult phase of the plant life cycle (Neufferet al. 1997). Although the mutants described in this article were gene (or at least a very large target for the mutagen

EMS). One corroborating piece of evidence comes from all selected on the basis of having an organ fusion phe-notype, epidermal cells in many of them also interact our analysis of recombination frequencies between

dif-ferent hth alleles. Based on estimates derived from these with pollen, as was the case for fdh-1 (LolleandCheung 1993). This fact clearly indicates that these two seem-recombination data the HTH locus may span as much

as 1.6 cM. This is dramatically larger than similar recom- ingly distinct processes are in some way genetically re-lated, since mutations in several different genes bring bination estimates for other Arabidopsis genes (0.07

cM for GA1;Koornneefet al. 1983; 0.01 cM for CSR1; about similar alterations in both processes. On the other hand, three of the new organ fusion mutants which we Mourad et al. 1994). A number of possibilities may

explain this discrepancy. First, it is possible that the have identified, representing three different comple-mentation groups, fail to support pollen hydration on apparent recombinants actually represent reversion of

one of the two mutations (presumably hth-8, since hth- their nonreproductive epidermal surfaces. Taken to-gether, these results indicate that while these two pro-10 was present in both crosses). While we have not

attempted to measure this directly, we consider this an cesses may be related, they are also clearly separable genetically, and it will be very interesting to learn the unlikely explanation since it would indicate a

spontane-ous reversion rate of 1.63 1022_{. Second, it is possible} _{molecular identities of the gene products which are in}

common as well as those which are unique to each of that the HTH locus contains a particularly

recombino-genic region within its borders. In this case the HTH the two pathways.

In the original fdh-1 mutant a striking increase in the locus may be a gene of quite ordinary size despite the

measured recombination frequencies between alleles. permeability of the epidermal cell wall and cuticle was the only alteration detected that distinguished mutant Third, the HTH locus may be split by a large intervening

sequence as is the case in a number of other genes, plants from wild type (Lolleet al. 1997). Although it seemed plausible that this permeability change might such as some of the homeotic genes of Drosophila

(GehringandHiromi1986). Fourth, it remains a for- play a role in the acquisition of fusion competence by the epidermal cells, no corroborating evidence sup-mal possibility that this locus is made up of two distinct

genes that interact in some manner such that they fail ported this hypothesis. The fact that many of the new organ fusion mutations isolated, which fall into a num-to complement one another in a simple cis-trans test.

Although further analysis of recombination frequencies ber of different complementation groups, also show changes in permeability to chlorophyll, however, is con-may reveal how the alleles group on the genetic map,

not until the gene is isolated and characterized will its sistent with this hypothesis. Furthermore, it is clear that thunderhead mutant plants switch to organ fusion compe-structure be revealed.

At the THUNDERHEAD locus, one of the two mutant tence at a discrete time in development and that this switch coincides precisely with a change in permeability. alleles (thd-1) shows a sharp transition from normal

growth to fusion competence, which appears to coincide As such, these data strongly support the notion that changes in the permeability of the epidermal cell wall well with the juvenile to adult phase transition. In these

mutants fusion cannot be detected until emergence of and cuticle to small molecules represent one mecha-nism which can lead to ectopic organ fusion during the fourth or fifth leaf. The mutant plants when mature

show a central trumpet-like clustering of leaves sur- Arabidopsis development.

One of the mutants recovered in this study fails to rounded by a relatively normal rosette. The thd-2 allele

does not show a similarly sharp transition, but the onset show any change in permeability that can be detected

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assistance with various aspects of this project. We thank Edward

with our present assay. This mutant may, of course, have

Seling_{of OEB and MCZ at Harvard for assistance with sample}

prepa-permeability changes below the limit of our detection

ration and use of the scanning electron microscope. We also thank or it may have changes in permeability to the relevant Allen Sessions, David Smyth, Lawrence Hobbie, Chris

Somer-signalling molecules that do not affect the permeabil- ville_{and the Arabidopsis Stock Center for making seeds available}

from mutant lines showing a fusion phenotype. We are grateful to ity of our test molecule, chlorophyll. Alternatively, this

Ueli Grossniklaus_{and members of his laboratory as well as}Graeme

mutant may represent a class of mutations which

Berlyn _{for their continued interest in this project and for many}

allow organ fusion to take place by some other

mecha-helpful discussions. This work was supported in part by a grant from nism, perhaps one related to the cr4 mutant of maize _{the Clark Fund at Harvard University, Harvard College Research} Foun-(Becraftet al. 1996). Interestingly, an Arabidopsis mu- _{dation Fellowships and by National Science Foundation (NSF) Grant} IBN-9405391 awarded to R.E.P. The main body of the work described tant (blasig) having an epidermal phenotype similar to

in this article was supported by NSF Grant IBN-9596044 awarded to cr4 but manifested only in the ovules has previously

S.J.L. and NSF Grant IBN-9723563 awarded to R.E.P. and S.J.L. been characterized in this lab (Schneitz et al. 1997).

A more detailed examination of the epidermal mor-phology of the mutant showing organ fusion without

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