Commonwealth Scientific and Industrial Research Organization, Division of Plant Industry, Horticulture Unit, PO Box 350, Glen Osmond, SA 5064, Australia
* Author for correspondence (e-mail: anna.koltunow{at}csiro.au)
Accepted 8 April 2004
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SUMMARY |
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Key words: C2H2 zinc finger, KNUCKLES, Parthenocarpy, Floral development, Fruit development
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Introduction |
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Arabidopsis thaliana floral meristems each give rise to four concentric whorls of determinate lateral organs: four sepals, four petals, six stamens, and two fused carpels (the gynoecium). Third- and fourth-whorl floral organs contain the reproductive male (micro-) and female (mega-) gametophytes, respectively. Each megagametophyte or embryo sac is enclosed by a specialized sporophytic structure, the ovule, which may give rise to a seed if fertilized.
After anthesis (pollen shedding) and fertilization, the gynoecium lengthens
considerably and develops into a seed-containing silique, the
Arabidopsis fruit (Fig.
1A). Elongation of the silique is accomplished primarily by
longitudinal expansion of cells of the exocarp, schlerenchyma, and endocarp
layers, whereas cell division accounts for most growth within the mesocarp
(Vivian-Smith and Koltunow,
1999). A dehiscence zone allows for separation of the valves from
the replum and seed dispersal (Ferrandiz
et al., 1999
). In the absence of fertilization, the gynoecium may
undergo restricted post-anthesis elongation, but it remains a determinate
organ that eventually abscises. Parthenocarpic Arabidopsis mutants,
in which the gynoecium undergoes post-anthesis fruit development without
fertilization, have been isolated (Ito and
Meyerowitz, 2000
; Vivian-Smith
et al., 2001
). The application of exogenous plant growth
regulators to emasculated flowers induces fertilization-independent fruit
development in Arabidopsis
(Vivian-Smith and Koltunow,
1999
), and transgenic Solanaceae producing elevated levels of
auxin within placental tissue and ovules are also parthenocarpic
(Rotino et al., 1997
;
Ficcadenti et al., 1999
). It
is likely that parthenocarpic mutants either produce abnormal levels of growth
regulators or respond inappropriately to these compounds.
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A tenet of the ABC model is that A- and C-class regulators are
antagonistic, and thus have cadastral as well as organ identity functions
(Gustafson-Brown et al., 1994;
Drews et al., 1991
). Other,
purely cadastral genes do not themselves confer primordium identity but
mutations in them lead to homeotic transformations of floral whorl organs
because the expression of floral organ identity genes extends beyond wild-type
whorl boundaries. Recessive mutations at the SUPERMAN (SUP)
locus, for example, lead to an extension of B-class gene expression and result
in supernumerary stamen production at the expense of fourth whorl carpel
development (Bowman et al.,
1992
; Sakai et al.,
1995
). It is theorized that SUP exerts its control over the
boundary between whorls three and four by regulating the balance of cellular
proliferation between meristematic regions fated to give rise to stamens or
carpels (Sakai et al., 1995
;
Sakai et al., 2000
). SUPERMAN
is a C2H2 zinc finger protein and possesses additional putative motifs typical
of a transcriptional regulator.
The floral meristems of plants triply mutant for A-, B- and C-class genes
are indeterminate and produce whorls of leaf-like structures neither
recognizable nor functioning as floral organs
(Bowman et al., 1991). This
homeotic phenotype substantiates a long-held belief that floral organs,
including the two fused carpels that comprise the whorl four gynoecium, are
modified leaves. Like leaves, the carpels may be described in terms of three
developmental axes along which pattern elements or specific tissue types
differentiate: adaxial-abaxial, medial-lateral, and basal-apical
(Fig. 1B, drawing). Because of
congenital fusion, the adaxial face of each carpel is inside the gynoecium,
whereas the abaxial surfaces make up its exterior. Four narrow bands of
placental tissue form along the interior of the gynoecium where the adaxial
faces of the two carpels meet and fuse. The gynoecium is bisected into two
locules by a false septum of adaxial origin that fuses post-genitally. Two
placentae develop in each carpel and thus two rows of ovules form on opposite
sides of each locule, interdigitating as they grow toward one another
(Fig. 1B). Abaxial-adaxial
polarity in carpel development is regulated redundantly by CRABS CLAW (CRC),
KANADI (KAN), and GYMNOS (GYM) (Bowman and
Smyth, 1999
; Eshed et al.,
1999
).
The replum defines the medial axis of the bilaterally symmetrical ovary.
The replum remains attached to the plant after ripening and dehiscence of the
lateral valves that enable seed release
(Fig. 1B). SPATULA (SPT) and
other factors are required for complete development of medial tissues
(Alvarez and Smyth, 1999;
Heisler et al., 2001
;
Alvarez and Smyth, 2002
).
Carpel pattern elements occur as follows along the basal-apical axis:
gynophore, ovary, style, and stigma (see drawing,
Fig. 1). Mutations at the
ETTIN locus have been shown to alter the development of these
elements along the proximo-distal or basal-apical axis, such that apical
elements extend basally at the expense of ovary development
(Sessions and Zambryski, 1995
;
Nemhauser et al., 2000
).
We isolated a recessive, conditionally male-sterile Arabidopsis mutant, knuckles (knu). A fraction of knu flowers produce ectopic stamens and carpels in a reiterating pattern from placental tissue near the base of a primary fourth-whorl carpel, and this indeterminacy appears to be necessary for parthenocarpic silique development. We report the identification of the KNUCKLES locus that, like SUPERMAN, encodes a small protein containing a single C2H2 zinc finger and probably functions as a transcriptional repressor. KNU likewise appears to have a cadastral regulatory function in the developing flower. Our observations indicate that KNU expression occurs early in the development of the gynoecium and persists near its base until after ovule primordia appear. KNU suppresses overgrowth of basal gynoecial structures such as the nectaries and gynophore to allow for full development of the ovaries, and prevents the placentae therein from acquiring floral meristem identity.
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Materials and methods |
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Histology
Buds, flowers and siliques were fixed, embedded and sectioned essentially
as described in Koltunow et al. (Koltunow
et al., 1998). Sections were usually stained in 0.1% toluidine
blue in 0.02% sodium carbonate and photographed under bright field on a Zeiss
Axioplan microscope using a Spot digital camera (SciTech Pty). Lactophenol
clearing of whole-mount tissues was allowed to proceed for 3-5 hours at room
temperature prior to microscopy.
Mapping of the knuckles mutation
Ws plants homozygous for the recessive knu mutation were crossed
with Ler. F2 progeny of this cross were scored for the
presence of knuckled siliques. Mutant F2 progeny comprised only 8%
rather than the expected 25% of this mapping population, probably as a result
of reduced male and female fertility, and decreased viability of homozygous
knu seeds. Genomic DNA was prepared from leaf tissue using the
protocol of Edwards et al. (Edwards et
al., 1991). Preliminary mapping using published SSLP markers
(Bell and Ecker, 1994
)
indicated that the knu mutation was linked to NGA151 on chromosome 5.
The Cereon database of DNA polymorphisms between the Columbia and Ler
ecotypes (maintained by The Arapidopsis Information Resource,
http://www.arabidopsis.org)
was used to design a series of insertion/deletion (INDEL) PCR markers flanking
NGA151. In all, 24 primer pairs were tested; half were polymorphic between WS
and Ler. Markers were named according to the AGI genomic clone they
overlapped. The nearest left marker at which heterozygosity was detected was
MXE10a, amplified by the primer set MXE10a-F (5'-GCG CTT AAC AAC GGT TTG
TTG-3') and MXE10a-R (5'-CAT TTG GGT GCC TGC ACA TTG-3') and
based on CER457604. The nearest heterozygous marker flanking the mutation on
the right was F18O22b, which also overlaps the P1 clone MUA22. This marker
corresponds to CER478399 and is amplified by the primer set F18O22b-F
(5'-CTT GAA ACT TGA AAG CAA ACC AG-3') and F18O22b-R (5'-GGG
CCT AAA AAT TGT AAC TGT AG-3'). These markers defined an interval of 123
kb spanned by three AGI P1 clones: MXE10 (AB011484), MAC12 (AB005230) and
MUA22 (AB007650).
Complementation of the knuckles mutant
Twenty-five overlapping genomic subclones derived from three AGI P1 clones
spanning the 123 kb knu interval were produced in the pGEM derivative
pSHUTTLE (Wang et al., 1998).
Insert ends were sequenced to verify the identity of the inserts before
further subcloning of the genomic fragments into the binary T-DNA vector
pWBVec8 (Wang et al., 1998
).
knu seedlings grown at 16°C were transformed via the floral dip
method (Clough and Bent,
1998
). After dipping, plants were covered and kept at room
temperature overnight, then returned uncovered to a 16°C growth chamber
until seeds were harvested. Transgenic seedlings were selected on MS agar
plates supplemented with 20 mg/L hygromycin and 150 mg/L Timentin. Between 5
and 40 transgenic seedlings harboring each construct were transplanted to soil
and grown at non-permissive temperatures for evaluation of phenotype.
Of 15 hygromycin-resistant knu seedlings containing the p8MUASAL1-3 construct produced, five were wild-type in appearance. The remaining 10 displayed a spectrum of weak knu phenotypes characterized by reduced knuckling and (in all but two plants) partial restoration of fertility. The genomic insert in this clone is an 8751 bp SalI fragment subcloned from the P1 clone MUA22 (AB007650) from an area of overlap with the P1 MAC12 (AB005230). It contains two annotated genes, MAC12.2 (At5g14010, GI 18417266) and MAC12.3 (At5g14000, GI 18417263). PCR products from the coding region of each gene were amplified from wild-type WS and knu seedlings and sequenced using BIG DYE Version 3.0 dideoxy terminators. Upon identification of the mutation in MAC12.2, further subcloning of the complementing fragment was performed to separate the two genes. The insert in pMUASAL1H-5 is a HindIII fragment of 5092 bp containing the MAC12.2 coding region and 2010 bp of 5' and 2596 bp of 3' sequence. Half of the transgenic knu plants harboring this construct were wild-type in appearance, and half displayed weak knu phenotypes. None of the knu transgenics harboring a HindIII fragment containing the adjacent MAC12.3 gene (pMUASAL1H-2) were complemented. The smallest segment of genomic DNA confirmed to be capable of complementing knu was cloned into pWBVec8 as a HindIII-PstI fragment. In this construct, p8KNU, sequence 3' to the KNU coding region was reduced to 1403 bp.
5' and 3' RACE
The GeneRacer kit for full-length, RNA ligase-mediated rapid amplification
of 5' and 3' cDNA ends
(Maruyama and Sugano, 1994;
Schaefer, 1995
;
Volloch et al., 1994
)
(Superscript II RT version) and the TOPO TA cloning kit (both from Invitrogen
Life Technologies) were used essentially as per the manufacturer's
instructions. Template for cDNA synthesis was total RNA extracted from
wild-type Ws inflorescence apices composed of pre-anthesis flower buds,
prepared with an Rneasy Plant Mini kit (Qiagen). An on-column RNase-free DNase
protocol was performed. For 5' RACE the following KNU-specific primers
were used in combination with those provided by the manufacturer of the
GeneRacer kit: MAC12.2RTR (5'-TCG TCT TCT TCC ATA ACG CC-3') and
MAC12.25R2 (5'-GTA GAA CTT TCG AGG ACA GTA CTG-3'). 3' RACE
primers were: MAC12.2ZF1 (5'-CAG TAC TGT CCT CGA AAG TTC-3') and
12.2GR3PR (5'-CTC AAG CTC TCG GCG GTC ACC AAA A-3').
In situ hybridization
Templates for generation of RNA probes were the plasmids XKNUX-4 and
ICRTR-2. The full-length KNU insert in XKNUX-4 was amplified by PCR
from the p8MUASAL1H-5 plasmid using the primer set XhoIKNUATG (5'-CCC
CTC GAG CCC ATG GCG GAA CCA CCA CCG TC-3') and 3'KNUXbaI
(5'-GGG TCT AGA TAA CTT ATA AAC GGA GAG AAA-3') and cloned into
pGEM-T Easy (Promega). The 204 bp of KNU sequence inserted into
pICRTR-2 was amplified by PCR from pMUASAL-1 using the primer set MAC12.2IC
(5'-CAA CAA CAC GTT TCT TCG TCC-3') and MAC12.2RTR (5'-TCG
TCT TCT TCC ATA ACG CC-3') and cloned into pGEM-T Easy. Plasmids were
sequenced to verify identity and orientation of inserts. The full-length
XKNUX-4 probe template was restricted with XbaI or SalI, and
DIG-labelled probes produced with SP6 (sense) or T7 (antisense) RNA
polymerases, respectively, as per the manufacturer's instructions. The
pICRTR-2 template was restricted with NcoI or NdeI, and the
RNA polymerases SP6 (sense) and T7 (antisense) used respectively to generate
probes. Samples of the probes were electrophoresed in TAE-agarose gels and
capillary blotted on nylon membrane to check for integrity or extent of
carbonate hydrolysis and success of labelling before being used in
hybridization experiments. The in situ hybridizations were performed as
described previously (Tucker et al.,
2003) except that once probes were added to the formamide-based
hybridization solution and cover slips applied, the slides were heated at
80°C for 2 minutes prior to hybridization overnight at 42°C.
cDNA synthesis and RT-PCR
Total RNA was prepared from freshly harvested floral tissues using Trizol
reagent (Invitrogen), and treated with RQ1 RNase-free DNase (Promega). Some of
each preparation (2 µg) was used as template for first-strand cDNA
synthesis with an oligo dT primer and Thermoscript (Invitrogen) in 20 µL
reactions. KNUCKLES and ß-tubulin cDNAs were amplified
in separate, otherwise identical 50 µL PCR reactions containing 2.5 µL
first-strand reaction, 10 mM Tris pH 8.3, 50 mM KCl, 2 mM MgCl2,
0.1 mM dNTPs, 0.5 µM forward and reverse primers, and 2.5 units AmpliTaq
(Applied Biosystems). After an initial denaturation step of 5 minutes at
94°C, reactions were subjected to amplification cycles consisting of 30
seconds at 94°C, 30 seconds at 56°C, and 1 minute at 72°C, with a
final incubation of 10 minutes at 72°C. Thirty and 40 cycles of
amplification were performed on ß-tubulin and KNUCKLES
reactions, respectively. ß-tubulin (At5g23860) primers were
essentially as described in Tucker et al.
(Tucker et al., 2003), and
provided a necessary control for the efficacy of the DNase treatment because
KNUCKLES does not contain introns. KNUCKLES was amplified
with MAC12.2ZF1 and MAC12.2RTR. Sequenced plasmid templates were used as
positive amplification controls. Samples were electrophoresed on a 3% TAE
agarose gel loaded to normalize the ß-tubulin band across
lanes.
Construction of the KNUCKLES:GUS fusion plasmids and GUS staining of transgenic floral tissues
In p12.2GUS2-1, a pBI101.2 derivative, the 2010 bp of upstream sequence
present in the complementation construct pMUASAL1H-5 and all but 19 bp of the
annotated coding sequence of KNU was fused in-frame to the E.
coli uidA (GUS) gene. In p8KNUGUS the full-length KNU coding
sequence is fused to GUS, whereas in p8KPGUS, GUS alone is placed under
transcriptional control of KNU 5' and 3' sequence.
p12.2GUS2-1 was constructed as follows: pMUASAL-1, a pSHUTTLE derivative
and the source of the complementing insert in p8MUASAL1-3, was prepared from a
dam-strain of E. coli and restricted with the enzymes
HindIII and XbaI. The desired fragment was then subcloned
into pBI101.2 (Jefferson et al.,
1987) cut with the same restriction enzymes.
We subsequently produced two additional binary T-DNA constructs containing
the GUS reporter: p8KNUGUS and p8KPGUS. The HindIII-PstI
restriction fragment present in p8KNU was cloned into pALTER-1 (Promega), and
the phosphorylated oligonucleotides MutKNUATG (5'-GGT GGT TCC GCC ATG
GTT GAG AGG TTG TTA AGC-3') and MutKNUXbaI (5'-CAA AAC AGA GAA GAA
AGT CTA GAT AAC TTA TAA ACG-3') were used to introduce an NcoI
site overlapping the KNU start and a methylation-insensitive
XbaI site four nucleotides downstream of the KNU stop codon
with the Altered Sites II kit (Promega). The doubly mutant insert was then
cloned into a version of the pBluescript II KS+ (Stratagene) in which the
XbaI site had been destroyed by ligation to the adjacent
SpeI site, to create pBSSXKNU-5. The GUS reporter gene from
pCAMBIA1381xa was used as a template to amplify a modified version of the gene
with NcoI and XbaI sites, and this was sequenced and cloned
into pBS
SXKNU-5 using the same sites to create pBSKPGUS-1. A version of
the KNU coding sequence lacking the stop codon was amplified by PCR
to incorporate NcoI sites overlapping its start and immediately
3' of its final codon. The amplified fragment was cloned, sequenced, and
then subcloned in front of GUS in pBSKPGUS-1 to create pBSKNUGUS. Finally, the
KNU cassette containing GUS and the full-length KNU:GUS
fusion were cloned into pWBVec8 as HindIII-NotI fragments to
create the binary T-DNA constructs p8KPGUS and p8KNUGUS, respectively. These
constructs were transformed into knu and wild-type Ws plants.
GUS staining of tissues from plants harbouring the constructs were
performed in tissue culture dishes containing the staining buffer described in
Vielle-Calzada et al. (Vielle-Calzada et
al., 2000). Samples were routinely incubated at 37°C for 16-20
hours. Subsequent clearing of tissues consisted of treatment with a 1:1
mixture of acetic acid and ethanol for 1-2 hours followed by mounting in an
ovule-clearing solution of (v/v) 20% lactic acid and 20% glycerol, or direct
mounting in the latter solution, depending upon extent of dissection and type
of tissue to be examined. Observations described herein are based on an
examination of 24 p12.2GUS2-1 T1 transgenics and five
representative T2 seedling sets derived from these. The p8KNUGUS
and p8KPGUS results are based on 16 and 17 T1 plants,
respectively.
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Results |
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In vitro culture (not shown), dissections and sectioning of these knuckle
structures from flower buds and earlier stages of silique development revealed
that they represent an indeterminate repetition of ectopic stamens and carpels
(Fig. 1D-G). Ectopic carpel
structures may remain green for the life of the knu mutant plant,
even after ripening and abscission of primary valve tissues, and stay firmly
attached to the replum by a fused vascular network that might extend to the
pedicel (Fig. 1E,F). The
cross-section of a knuckled silique shown in
Fig. 1G reveals at least three
reiterations of carpelloid tissue. We have elected to describe these ectopic,
partly carpelloid growths as `knuckles' to differentiate them from the
superficially similar `carpel-like structures' (CLSs) that sometimes develop
in bel1 siliques (Modrusan et
al., 1994).
The ectopic floral organs of the knu mutant are of placental origin. We used bright field microscopy and Nomarski optics to examine thin sections and lactophenol-cleared gynoecia of wild-type and mutant plants as shown in Fig. 2A-G. Ectopic organs arose from placental tissues in the basal third or half of the developing gynoecium. Unlike bel1 mutants they were not derived from ovule primordia. Abnormalities in carpel development were first observed around stage 8 in the basal portion of knu carpels, with the proliferation of placental tissue adjoining the zone of ovule primordia formation (Fig. 2C). The planes and patterns of cell division in ectopic primordia differed from those found in later developing ovules (Fig. 2C,D). Within a primary carpel, we observed that ectopic organ development was often more advanced than that of surrounding ovules. The ectopic structure in Fig. 2E appears to be composed of three primordia and is significantly larger than surrounding ovule primordia. We believe that the ectopic structure in the gynoecium from a stage 9 bud shown in Fig. 2F is an ectopic floral meristem flanked by two developing stamens. Within the primary knu gynoecium pictured in Fig. 2G well-developed ectopic stamens are intermingled with developing ovules from which inner and outer integuments have recently been initiated. The dome of an ectopic gynoecium is also evident. Although ectopic organs tended to originate in the basal third to half of the knu silique, the presence of ovules basal to the ectopic floral organs indicated that meristematic activity was not merely a consequence of direct whorl 4 indeterminacy.
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We investigated the effects of reduced growing temperature on the knuckling phenotype and fertility of our mutant. When 169 mature siliques from knu plants grown at 16°C were examined, 30% were found to contain knuckles. We also observed that male fertility was partially restored and that anthers dehisced in knu plants grown at 16°C (Fig. 3A). Sections of anthers from stage 10 buds dissected from knu mutants grown at reproductively non-permissive temperatures showed that microspores often formed (Fig. 3B) as in wild-type anthers (not shown); however, at flower maturity, indehiscent anthers did not contain recognizable pollen grains but a degenerated mass on the inside of the anther wall (Fig. 3C). Numbers of seeds formed in siliques of self-pollinated knu mutants grown at 16°C ranged from 0 to 51, with a mean of 23 (±13) seeds/silique (n=100 siliques). By contrast, 13 to 65 seeds were found in siliques of self-fertilized Ws plants grown simultaneously at 16°C, with a mean of 47 (±10) seeds/silique (n=100 siliques).
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The basalized phenotype of the knuckles gynoecium suggests that KNUCKLES may establish or maintain a boundary restricting gynophore development
Apart from the ectopic growth of stamens and carpels within the primary
gynoecium, the most striking phenotype of the knu mutant grown at
22-25°C is the formation of `basalized' gynoecia in which the gynophore, a
basal pattern element, may extend apically to replace a portion of the ovary
(Fig. 4). When knu
plants were grown at 16°C, the basal-most portion of the ovary was reduced
rather than replaced: the replum failed to bifurcate and the septum did not
expand, but valves with an exaggerated radial curvature differentiated in this
region and ovules capable of fertilization and development into seeds were
also present. The dried replum of a 16°C-grown dehiscent knu
silique resembled an oar, and the detached valves were spoon-like
(Fig. 5A,B). knu
plants grown at 16°C also produced siliques in which the basal portion of
the ovary was reduced, but in the absence of seed set the relative difference
between expansion of basal and more distal portions of the ovary was less
pronounced. In cross-section the extended gynophore produced by a fraction of
knu siliques that developed at the non-permissive reproductive
temperature possessed a ring of vascular tissue similar to wild-type gynophore
and typical of stem. It may appear that bifurcation of the replum occurs as a
result of internal pressure from ectopic organ growth, but it is important to
note that apically-shifted replum bifurcation occurs even in the absence of
knuckle formation. Although the position at which bifurcation of the replum
and normal expansion of the septum began varied slightly from silique to
silique, the positioning of the origin of the knuckle relative to the
bifurcation was non-random. These morphological observations of the mutant may
be taken to imply that the basalization and knuckling phenotypes are separate
developmental consequences of the loss of KNU-mediated basal domain
maintenance. Alternatively, the knuckling phenotype might have a stochastic
relationship to gynoecial basalization, which seems plausible given that even
consecutive siliques from the same inflorescence stem may be affected to
sharply varying degrees.
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MAC12.2 is predicted to be a small C2H2 zinc finger protein
(Miller et al., 1985) of 161
amino acids. The cysteine residue replaced in the knu mutant is the
second of two required for zinc binding and thus is potentially critical for
function of the zinc finger as a DNA binding or protein-protein interaction
domain (Fig. 6A). In addition
to the single zinc finger, MAC12.2 contains an EAR-like active repression
domain as described by Hiratsu et al.
(Hiratsu et al., 2002
) at its
carboxy terminus (Fig. 6B).
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Transcription of KNUCKLES during floral development
Preliminary characterization of KNU expression by RT-PCR indicated
that the gene was transcribed most strongly in flower buds
(Fig. 7). The patterns of
KNU gene expression examined by in situ hybridization during floral
development were identical in both mutant and wild-type plants. Experiments
utilized two different probes. In all cases KNU transcripts were
detected in a wide range of cell types comprising the floral organs.
Fig. 8 shows data derived from
in situ analysis of wild-type plants. KNU mRNA was detected in the
sepals and pedicels of stage 6 floral buds. KNU transcripts were
absent in the developing petal and stamen primordia at this stage, but
transcripts were localized in a small region towards the base of the
developing carpel primordium (Fig.
8A). Later in development, KNU transcripts were evident
in cells of all floral organs (Fig.
8B-E) including male and female gametophytes. Transcripts were
absent after fertilization in developing seeds (not shown).
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Expression patterns of KNUCKLES:GUS fusions indicate post-transcriptional regulation of KNU
Gene constructions encoding translational fusions of KNU to the
uidA (GUS) gene of E. coli were made in order to examine the
developmental pattern of KNU protein expression within specific floral organs.
p8KNUGUS contained the entire coding sequence of KNU linked to GUS.
p12.2GUS2-1 encoded a fusion that lacked the last 6 amino acids of the
EAR-like domain from the C-terminal portion of the protein. A third construct,
p8KPGUS, completely lacked KNU coding sequences and the GUS gene was
flanked both 5' and 3' by KNU untranslated sequences.
Fig. 9 shows the structure of
these chimeric fusions, the complementation constructs upon which they were
based, and a schematic summary of GUS expression in organs at particular
stages.
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Expression of KNUCKLES:GUS constructs correlate with the knuckles floral phenotype
The patterns of GUS expression in developing stamens and carpels of
wild-type transgenic plants containing p8KNUGUS, p12.2GUS2.1 and p8KPGUS were
conserved, and importantly were restricted to a small number of cell types.
Staining first appeared in the fourth whorl of stage 6 buds as a central spot
(Fig. 10D). This stained area
of the primordium increased in size as the gynoecium differentiated
(Fig. 10E), but as the
gynoecial cylinder lengthened through stages 7 and 8, staining remained strong
only at its base (Fig. 10F).
At stage 9, when ovule primordia arose, staining of the gynoecium was
concentrated at two spots at the base of the carpel that probably corresponded
to the most basal portions of the developing valves
(Fig. 10G,I).
Shortly after GUS stain was first observed in the gynoecial primordium, it became evident in developing anthers and was prevalent in the stamen predominantly in the developing pollen of anthers at floral stage 9 (Fig. 10F,H). GUS was evident throughout male gametophyte development but there was virtually no detectable staining in third-whorl organs at anthesis. During ovule development, GUS was observed in the archesporial cells. Expression persisted in the megaspore mother cell (Fig. 10J) and was evident during meiosis (Fig. 10K) and the mitotic events of embryo sac formation (Fig. 10L). GUS was evident in mature embryo sacs after fusion of the polar nuclei and absent after fertilization (not shown). Staining of internal portions of the style and of the stigmatic papillae was seen at the apex of the gynoecium as it matured during floral stages 10-13. Little or no staining of gynoecial tissues was observed beyond stage 13 (anthesis). Given the general pattern of KNU transcript accumulation, the conservation of specific cellular staining patterns during stamen and gynoecia development in plants containing the three constructs suggests that mechanisms limiting protein accumulation in these organs might involve 5' sequences flanking the KNU protein coding region as they are common to all three constructs. Early and persistent GUS staining near the base of the carpels late into floral development is consistent with the basalized syndrome of alterations to ovary development seen in the knu mutant. The expression in developing male and female gametophytes is also consistent with the defects in male and female fertility observed in the knu plants. The role of knuckles in female gametophyte development will be investigated elsewhere.
GUS staining patterns of knu transgenics harboring KNU:GUS constructs support hypotheses about the role of KNU in gynoecial development
The p8KPGUS and p8KNUGUS constructs were transformed into homozygous
knu plants, and floral tissues derived from the resulting primary
transgenics were subjected to GUS staining. Neither construct was able to
complement knu. Expression in developing male and female gametophytes
remained unchanged. Interestingly, the GUS staining pattern was expanded
markedly in early developing gynoecia of knu p8KPGUS transgenics
compared with transgenic gynoecia of wild-type plants harboring the same
construct (Fig. 10N). Instead
of the polarized basal expression typically seen in wild-type
(Fig. 10M), staining extended
into a larger proportion of basal gynoecial cells that overlapped the region
where ectopic organ initiation and growth were observed in the mutant.
The p8KNUGUS fusion protein was expressed similarly in developing knu gynoecia. Expression in ectopic floral organs occurred in a developmental pattern comparable to that in stamens and carpels from wild-type plants, confirming that these ectopic floral organs have the same identity and capacity to accumulate KNU as their primary counterparts in the third and fourth whorls (Fig. 10P). The GUS staining patterns observed in the knu background are readily explained if KNU functions to limit proliferation in those cells where it is expressed.
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Discussion |
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The reiterating structure of the knuckle suggests a role for KNU in the regulation of determinacy
The floral phenotype of agamous mutants is the transformation of
third- and fourth-whorl organs to petals and sepals, respectively, as well as
a loss of fourth-whorl determinacy such that a flower-within-flower pattern of
reiterated floral whorls is produced: (sepal, petal, petal)n
(Bowman et al., 1991). Our
observations of the indeterminate floral phenotype of knu can be
interpreted to mean that KNU is involved in the determinacy-promoting activity
of AG, perhaps as a negative regulator of a proliferative or
non-differentiative signal or a stem cell-promoting factor like WUSCHEL (WUS)
(Lenhard et al., 2001
).
Alvarez and Smyth (Alvarez and Smyth,
1999) observed a loss of floral determinacy similar to the ectopic
organ phenotype of knu in plants homozygous for crc-1 and
heterozygous for ag-1. Like knu, crc mutants also produce
many tricarpelloid gynoecia, an observation that has been attributed to a loss
of determinacy (Bowman et al.,
1999
). CRC is a member of the YABBY family, as is INNER NO OUTER
(INO), a regulator of ovule integument development
(Villanueva et al., 1999
).
Interestingly, SUP, which is structurally similar to the KNU protein, has been
shown by Meister et al. (Meister et al.,
2002
) to regulate outer integument growth by negatively regulating
INO transcription. The exaggerated nectaries of knu flowers
(Fig. 4C,D) suggest that KNU
might co-regulate development of this tissue with CRC because crc
mutant flowers lack nectaries altogether.
A model explaining the positioning of the knuckle along the proximo-distal
axis of the placentae in relation to the ovules could be formulated as
follows: KNU is essential for maintaining aspects of a basal domain or
boundary in the developing gynoecium, such that floral meristematic activity
within the placentae is suppressed and the determinacy of the flower is
maintained. If no, or insufficient, or insufficiently active KNU protein is
made (the latter being most likely for the allele described here, as our
experiments indicate that knu is transcribed), placental or
pre-placental tissue could proliferate to initiate an adventitious floral
meristem before ovule primordia are generated. Ovules develop asynchronously
from the parietal placentae (Gaiser et
al., 1995), and basipetally, such that the most developmentally
advanced ovules occur at the apical end of the gynoecium. KNU expression would
be expected to overlap a region of potential competence to produce floral
meristem in addition to ovule primordia, and ectopic floral meristem
development would not preclude later ovule induction from more basal placental
tissue.
KNUCKLES might be an active repressor of transcription regulating cellular proliferation during floral development relative to a hypothetical fourth whorl/fifth whorl boundary
The recessive knu phenotype, characterised by the production of
ectopic floral organs, genetically defines the KNU protein as a repressor of
non-ovule floral organ development within the context of the placentae of the
pre-stage-9 gynoecium. Similarly, the homozygous sup phenotype is
production of additional whorls of stamens in an inappropriate floral context
(Bowman et al., 1992). Recent
evidence indicates that SUP could be an active transcriptional repressor.
Hiratsu et al. (Hiratsu et al.,
2002
) have identified an EAR-like transcriptional repression motif
near the carboxy terminus of SUPERMAN. Dathan et al.
(Dathan et al., 2002
) found
that basic residues flanking the SUP zinc finger domain on either side were
important to stabilization of its interaction with an oligonucleotide target.
In addition to a zinc finger flanked by small clusters of basic amino acids,
the KNU protein is predicted to encode a consensus-matching carboxy-terminal
EAR-like motif nearly identical to that found at a similar position in SUP.
The presence of a zinc finger and an EAR-like motif provide additional if
indirect evidence that KNU is a transcriptional repressor.
Evidence has accumulated that SUP is a negative regulator of cellular
proliferation, and that the cadastral effects of sup on floral
development result from an overproliferation of the third whorl at the expense
of the fourth (Sakai et al.,
2000). In the developing ovule, SUP negatively regulates adaxial
growth of the outer integument. Hiratsu et al.
(Hiratsu et al., 2002
) found
that Arabidopsis plants overexpressing full-length SUP were severely
dwarfed because of a decrease in cell number rather than cell size. Our
observation that a GUS reporter under the control of KNU flanking
regulatory sequences engenders a larger population of stained cells in
knu vs wild-type transgenics implies that KNU also regulates cellular
proliferation in the basal gynoecial tissues where it is normally
expressed.
Broadly interpreted phenotypic similarities between the sup and
knu mutants as well as the existence of shared protein motifs lead us
to speculate that KNU has a role in determining or maintaining a boundary
between the fourth whorl of the floral meristem and the parietal placentae
which arise from the developing gynoecium. Palaeobotanical studies indicate
that the ovule (an integumented megasporangium) evolved prior to the carpel,
and as Bowman et al. (Bowman et al.,
1999) have pointed out, seeds and therefore ovules are not
inventions of the angiosperms. Unlike the sepals, petals, stamens and carpels
of angiosperm flowers, ovules are not thought to be derived from leaves
(reviewed by Robinson-Beers et al.,
1992
). The placentae which give rise to the ovules in
Arabidopsis are intimately associated with medial adaxial tissue of
the carpels, but in the Solanaceous plant Petunia hybrida the
placentae arise separately, from the central region of the floral meristem.
Angenent and Colombo (Angenent and Colombo,
1996
) have concluded that this central meristematic region
represents an additional whorl. The demonstration that ovule and carpel
development are genetically separable in Petunia, together with the
characterization of MADS box genes (FLORAL BINDING PROTEIN 7 and
11) that might be thought of as providing D function
(Angenent et al., 1995
,
Colombo et al., 1995
), lends
credence to the idea that ovules could be considered fifth-whorl organs. It is
possible that one regulatory function of KNU is analogous to that proposed for
the SUP protein (Sakai et al.,
2000
). KNU might act to maintain a proliferative balance between
the meristematic tissues on either side of a developmentally discontinuous
fourth whorl/placental `whorl' boundary, because in the knu mutant
organs normally present only in the third and fourth floral whorls are
repeated from the parietal placentae that in Arabidopsis would
compose this hypothetical fifth whorl.
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ACKNOWLEDGMENTS |
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