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First published online 13 March 2008
doi: 10.1242/dev.012807
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1 Department of Botany, University of British Columbia, Vancouver, British
Columbia, V6T 1Z4, Canada.
2 Department of Molecular Biosciences, University of Oslo, PO Box 1041 Blindern,
N-0316 Oslo, Norway.
3 Division of Biological Sciences, 303 Life Sciences Center, University of
Missouri, Columbia, MO 65211, USA.
Author for correspondence (e-mail:
haughn{at}interchange.ubc.ca)
Accepted 6 February 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Abscission, BLADE-ON-PETIOLE, Flower development, INFLORESCENCE DEFICIENT IN ABSCISSION
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Patterson,
2001; Roberts et al.,
2002). Separation of organs from the plant body occurs at
anatomically distinct cell files called abscission zones (AZs). Differentiated
AZ cells are small, isodiametric and cytoplasmically dense compared with
surrounding cells, and are responsive to signals promoting abscission. These
signals induce a directed, enzymatic dissolution of the middle lamellae
between AZ cell walls, resulting in a loss of adhesion between the organ and
plant body (Addicott, 1982;
Sexton and Roberts, 1982).
Many enzymes have been suggested to be involved in the separation process,
including cellulases, polygalacturonidases (PGs) and expansins
(Roberts et al., 2002). After
the organ is shed, the cells exposed at the AZ on the plant body differentiate
to form a protective surface (Patterson,
2001).
Arabidopsis petals, stamens and sepals develop an abscission zone
four to six cell layers thick, where the bases of the organs meet the
receptacle (Bleecker and Patterson,
1997). Following fertilization, these floral organs senesce and
abscise. Various genes are specifically expressed at floral organ AZs in
Arabidopsis. For example, the promoter from an Arabidopsis
abscission-related PG (PGAZAT) gene was able to drive floral organ
AZ-specific expression of β-glucuronidase (GUS) during abscission
(Gonzalez-Carranza et al.,
2002). Furthermore, GUS constructs driven by the soybean
(Glycine max) chitinase (CHIT) promoter or the
bean (Phaseolus vulgaris) abscission cellulase
(BAC) promoter are also upregulated in abscission zones during floral
organ abscission (Bleecker and Patterson,
1997; Butenko et al.,
2006; Chen and Bleecker,
1995; Patterson and Bleecker,
2004).
In several plant species, ethylene promotes abscission, whereas auxin
inhibits it (Roberts et al.,
2002). Although considerably delayed, floral organ abscission
occurs in ethylene-insensitive mutants of Arabidopsis, suggesting
that ethylene signalling is important for the timing of abscission but is not
essential for it to occur (Patterson and
Bleecker, 2004). Numerous loci are proposed to modulate floral
organ abscission in Arabidopsis, including HAESA
(Jinn et al., 2000),
AGAMOUS-LIKE 15 (Fernandez et
al., 2000), the DELAYED ABSCISSION loci
(Patterson and Bleecker,
2004), genes for the actin-related proteins ARP4 and ARP7
(Kandasamy et al., 2005b;
Kandasamy et al., 2005a),
several AUXIN RESPONSE FACTOR genes
(Ellis et al., 2005;
Okushima et al., 2005),
INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)
(Butenko et al., 2003), and
HAWAIIAN SKIRT (Gonzalez-Carranza
et al., 2007). Disruption of the expression of these genes results
in delayed floral organ abscission, with the exception of ida plants,
which retain floral organs indefinitely
(Butenko et al., 2003). Floral
organs of ida develop AZs; however, the middle lamellae only
partially dissolves. IDA encodes a putative secreted peptide ligand
suggested to act late in abscission to promote final middle lamellae
dissolution (Butenko et al.,
2003). Interestingly, ectopic and overexpression of IDA
in 35S::IDA transgenic plants lead to precocious floral organ
abscission and ectopic abscission at the vestigial AZs of pedicels and cauline
leaves (Stenvik et al., 2006).
Restricting IDA expression to the flower is likely to be crucial to
prevent abscission at vestigial AZs.
Two recently identified Arabidopsis genes encoding redundant
regulators of leaf and flower patterning, BLADE-ON-PETIOLE 1
(BOP1) and BOP2 (Ha et
al., 2003; Ha et al.,
2004; Ha et al.,
2007; Hepworth et al.,
2005; Norberg et al.,
2005), were also shown to be required for floral organ abscission
(Hepworth et al., 2005;
Norberg et al., 2005).
Loss-of-function bop1 bop2 mutants develop leafy projections from
petioles and form floral bracts. In addition, flowers of bop1 bop2
develop two petalloid sepals instead of a single abaxial sepal. Furthermore,
as in ida mutants, bop1 bop2 mutant floral organs fail to
abscise. However, the molecular and anatomical basis of this defect has not
been studied and it is unclear how the BOP1 and BOP2 genes
function in the abscission process. BOP1 and BOP2 are part of the NPR1
(NON-EXPRESSOR OF PR1) protein family, which is characterised by a series of
conserved cysteines and two protein-protein interaction domains. NPR1 is a
positive regulator of systemic acquired resistance (SAR), a plant immune
response induced following a local infection. During SAR, accumulation of
salicylic acid causes a reductive shift in the cellular redox balance,
prompting NPR1 to preferentially localise to the nucleus where it interacts
with the TGACG sequence-specific binding transcription factors (TGAs) to
activate the transcription of PATHOGENESIS RELATED (PR) genes
(Dong, 2004). Several aspects
of the BOP1/BOP2 signalling mechanism are conserved with NPR1, including
localization to both the cytoplasm and nucleus, and interaction with the TGA
PERIANTHIA, which acts in the same genetic pathway to control perianth
patterning in flowers (Hepworth et al.,
2005). It is unknown whether BOP1/BOP2 activity is controlled by
redox-control of nuclear localization and TGA interaction, as is the case for
NPR1.
This study examines the contribution of BOP1 and BOP2 to abscission zone development. We show that BOP1 and BOP2 are required for abscission in both wild-type and 35S::IDA plants, as well as for all aspects of AZ-related anatomy in both functional and vestigial AZs. These data suggest that BOP1 and BOP2 promote formation of the specialized AZ anatomy necessary for abscission. Interestingly, the expression of abscission-related genes was relatively unperturbed in bop1 bop2 mutants, indicating that activation of AZ-specific gene expression is independent of AZ anatomy. In addition, our analysis determined that BOP1 and BOP2 promote nectary gland formation, which occurs at the receptacle adjacent to developing AZs. Thus, BOP1 and BOP2 regulate the differentiation of multiple cell types in proximal regions of inflorescence lateral organs.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)
and plants were grown in continuous light at 20°C as described
(Dean et al., 2007). Wild type
was the Columbia-0 (Col-0) ecotype, unless otherwise noted. Plants for
35S::IDA experiments were grown at 22°C for 8 hours dark/16 hours
light and wild type was the C24 ecotype. T-DNA mutants bop1-3 (Col-0)
and bop2-1 (Col-0) alleles have been described previously
(Hepworth et al., 2005). T-DNA
ida mutant (C24) and IDA::GUS (C24) lines have been
described previously (Butenko et al.,
2003). The 35S::IDA construct described by Stenvik et al.
(Stenvik et al., 2006) was
transformed into bop1 bop2 plants by floral dip
(Clough and Bent, 1998).
Analysis was conducted on the T2 generation. bop1 bop2 ida
(Col-0xC24) triple mutants were generated by crossing.
BOP1::GUS plants (Col-0) contain transcriptional fusions generated by
fusing 
4 kb upstream of the putative BOP1 start codon to the
β-glucuronidase (GUS) reporter gene in the binary vector pBl101
(Jefferson et al., 1987).
CHIT::GUS (Chen and Bleecker,
1995) and BAC::GUS
(Koehler et al., 1996) marker
lines have been introgressed into Col-0 and were provided by Dr Sara Patterson
(University of Wisconsin, Madison, WA). CRABS CLAW(CRC)::GUS
transgenic plants (Baum et al.,
2001) and crc-1 mutants
(Bowman and Smyth, 1999) were
provided by Dr John Bowman (Monash University, Melbourne, Australia) and are
both in the Landsberg erecta (Ler) background. Floral
developmental stages were determined according to Smyth et al.
(Smyth et al., 1990).
Petal breakstrength
Petal breakstrength is the force in gram equivalents required to remove a
petal from the receptacle. Petal breakstrength was measured by the apparatus
described by Lease et al. (Lease et al.,
2006).
Microscopy and histology
For scanning electron microscopy of floral organ and vestigial AZs, sepals,
petals and cauline leaves were removed prior to fixation. Following
critical-point drying, tissues were mounted onto steel stubs, coated with gold
palladium and observed using a Hitachi VP-4600 scanning electron microscope
(Tokyo, Japan). For detection of GUS activity, tissue was fixed in 90%
acetone, treated with heptane and then rinsed with GUS buffer [100 mM
NaH2PO4 (pH 7.0), 10 mM EDTA, 0.1% Triton X-100, 0.5 mM
potassium ferricyanide, 0.5 mM potassium ferricyanide]. Samples were incubated
with GUS buffer supplemented with 0.05% X-gluc (5-bromo-4-chloro-3-indolyl
β-D-glucuronide cyclohexylamine) salt (Rose Scientific, Alberta, Canada)
at 37°C for 2 hours for CRC::GUS and IDA::GUS plants, or
overnight for BOP1::GUS, CHIT::GUS, GLUC::GUS plants. Tissues were
cleared overnight in 70% ethanol, then cleared and mounted in chloral
hydrate:water:glycerol (8:1:2). Tissues for sectioning were embedded in
standard Spurr's resin. BOP1::GUS and IDA::GUS samples were
stained for GUS activity before embedding. For nectary and AZ analyses,
sections were stained in Toluidine Blue.
Reverse transcriptase-mediated expression analyses
Total RNA from green rosette leaves, mature flowers and floral buds was
isolated as described by Stenvik et al.
(Stenvik et al., 2006).
First-strand cDNA synthesis with SuperScript III reverse transcriptase
(Invitrogen, Carlsbad, CA) was performed in a total volume of 20 µl, using
1350 ng of total RNA as template, and incubated at 50°C for 60 minutes.
Reverse transcription was omitted in negative controls. The open reading frame
sequence of ACTIN2 (At3g18780) was used as a positive internal
control. Primers used for ACTIN2 and IDA have been described
by Stenvik et al. (Stenvik et al.,
2006).
Real-time quantitative RT-PCR (qPCR)
Total RNA for qPCR was isolated from position 5-7 floral organ AZs. The
reaction was performed on a LightCycler LC480 instrument (Roche) according to
the manufacturer's protocol. To ensure that primer combinations did not
produce undesired PCR fragments or primer dimers, a SYBER-GREEN qPCR with
melting-point analysis was performed using the LightCycler 480 SYBR Green I
Master Kit (Roche). Probe-based qPCR with these primers was performed using
Universal Probe Library (UPL) hydrolysis probes (Roche), UPL probes 68
(IDA), 82 (HAESA) and 102 (ACTIN2), and the
LightCycler 480 Probes Master Kit (Roche). All samples and reference controls
were performed in two biological replicates and with two technical replicates
each. Primers for amplification were: IDA 68 right,
5'-TCAATGAGGAAGAGAGTTAACAAAAG-3'; IDA 68 left,
5'-CTAAAGGCGTTCCCATTCCT-3'; HAESA 82 right,
5'-GAGAGAGGGAATGGAGAGAAGG-3'; HAESA 82 left,
5'-CATGCTCGTCGGACCTTT-3'; ACT2 102 right,
5'-CCGGTACCATTGTCACACAC-3'; and ACT2 102 left,
5'-CGCTCTTTCTTTCCAAGCTC-3'.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Flowers further down the inflorescence are numbered consecutively. Wild-type
(Col-0) flowers shed sepals, petals and stamens at position 6.55±1.09
(Fig. 1B,
Table 1), whereas bop1
bop2 plants retained all floral organs indefinitely
(Fig. 1C).
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BOP1 and BOP2 are necessary for the formation of floral AZ and vestigial AZ anatomy
The abscission defect of bop1 bop2 was further characterized using
a petal breakstrength meter (Lease et al.,
2006), which measures the amount of force required to remove a
petal from the receptacle (Craker and
Abeles, 1969). Owing to the progressive degradation of the middle
lamellae in the AZ, breakstrength force decreased as the organs approach
abscission (Fig. 1D).
Dramatically, bop1 bop2 showed no decrease in breakstrength at any
position, suggesting that cell-wall adhesion at the organ-receptacle boundary
does not weaken in bop1 bop2 (Fig.
1D).
We used scanning electron microscopy (SEM) to examine the organ-receptacle
boundary of bop1 bop2. Petals were removed at the receptacle if not
yet abscised from wild-type and bop1 bop2 flowers at positions 2, 4,
6 and 12. The petal fracture surface of wild-type and bop1 bop2
position 2 flowers was composed of cells that ruptured upon petal removal
(Fig. 2A,B, arrows). Owing to
weakening of the middle lamellae, petal removal at position 4
(Fig. 2A) did not cause cell
rupture but revealed a smooth fracture cavity that is typical of wild-type AZ
cells (Bleecker and Patterson,
1997). At position 6, petals readily abscised to reveal a mature
AZ characterised by spherical elongated cells (arrow). By position 12, the
exposed plant body had differentiated into a protective surface. By contrast,
the fracture surface of bop1 bop2 showed no evidence of AZ
activation; cells ruptured during petal removal from all positions
(Fig. 2B).
To determine more precisely the differences in cellular morphology between bop1 bop2 and wild type, longitudinal sections of flowers at anthesis were examined. Cytoplasmically dense AZ cells were visible in the stamen filament-receptacle, petal-receptacle and sepal-receptacle boundaries of wild type (Fig. 2C-E, arrows), but such a layer never was observed in bop1 bop2 (Fig. 2G-I, arrows). Interestingly, the abaxial petalloid sepals of bop1 bop2 did not form an obvious junction with the floral receptacle - the cell layers were fused (Fig. 2J, arrow). In summary, analyses via petal breakstrength, SEM and light microscopy strongly suggest that cytologically distinct and active floral AZs do not form in bop1 bop2.
Vestigial AZs develop in Arabidopsis at branching points and at
the base of pedicels and cauline leaves
(Stenvik et al., 2006). Given
the absence of floral AZs in bop1 bop2, we investigated whether
bop1 bop2 also lacks vestigial AZs. To this end, SEM was performed on
the vestigial AZs exposed after removal of cauline leaves. Fully expanded
cauline leaves were removed from the stem at three maturation phases based on
leaf colour: green, yellowing (50%) and yellow. The exposed fracture plane on
wild type and bop1 bop2 stems following removal of green cauline
leaves showed breakage of cells along the surface
(Fig. 3A,D). Removal of
yellowing cauline leaves from wild-type stems revealed enlarged rounded cells
at the edges of the attachment point, indicative of an AZ
(Fig. 3B), and when fully
yellow, revealed rounded cells, although the vasculature cells remain broken
(Fig. 3C). Corresponding
surfaces of bop1 bop2 lacked any signs of these enlarged AZ cells
(Fig. 3E,F). The bases of
attached green cauline leaves were also examined. Abaxial surfaces of
wild-type leaves at the stem showed a furrow of narrowed cells
(Fig. 3G,H, arrows) flanked by
stipules (Fig. 3H, arrowhead).
This boundary furrow corresponded to the lower cleavage site formed when
wild-type leaves are removed. Abaxial surfaces of bop1 bop2 cauline
leaves lacked an obvious boundary furrow
(Fig. 3J,K). Sections through
expanded green wild-type cauline leaves revealed the narrowed cell layers of
the leaf-stem boundary furrow (Fig.
3I, asterisk). Wild-type sections also showed darkened vestigial
AZ cells at the adaxial leaf-stem boundary
(Fig. 3I, arrow) and between
the primary and axillary stem branching point
(Fig. 3I, arrowhead). Wild-type
vestigial AZs were readily apparent in serial sections through the sides of
the leaf-stem boundary, but were less obvious through the vasculature (see
Fig. S1 in supplementary material). However, neither cauline leaf nor
branching point vestigial AZs were present in any position for expanded green
bop1 bop2 cauline leaves; cells at the junction remained large and
vacuolated (Fig. 3L). In
summary, these data indicate that BOP1 and BOP2 are
essential for vestigial AZ formation. Interestingly, stipules that flank the
wild-type leaf fracture plane (Fig.
3A-C, arrow) and intact wild-type cauline leaves
(Fig. 3H, arrowhead) were never
observed flanking bop1 bop2 fracture planes
(Fig. 3D-F) or intact leaves
(Fig. 3K).
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;
Hepworth et al., 2005;
Norberg et al., 2005). We
examined the expression pattern in more detail using a GUS reporter
gene under the control of the BOP1 promoter. BOP1::GUS was
expressed at the base of developing floral organs in stage 9/10
(Fig. 4A), stage 12
(Fig. 4B) and stage 14
(Fig. 4C) flowers, agreeing
with previous results (Norberg et al.,
2005). In addition, bases of recently shed organs had GUS
activity (Fig. 4D).
BOP1::GUS receptacle expression was detected for a prolonged period
following abscission (Fig. 4E).
The adaxial regions of young pedicel-stem junctions showed GUS activity
(Fig. 4F) that spreads around
the entire boundary in older pedicels (Fig.
4G). BOP1::GUS expression exhibited similar patterns in
bop1 bop2 as in wild type (data not shown), showing that
BOP1 expression does not depend on functional BOP proteins.
Interestingly, BOP1 was ectopically expressed in the bop1
bop2 pedicel where the ectopic bract develops
(Fig. 4H, compare with 4G). In
addition, expression of BOP1::GUS was detected at the bases of
cauline leaves (Fig. 4I), as
previously reported (Norberg et al.,
2005).
To further characterise BOP1 expression, BOP1::GUS
stained tissue was sectioned. Corresponding to in situ hybridisation results
of BOP2 (Hepworth et al.,
2005), BOP1::GUS was expressed in early floral organ primordia
(Fig. 4J). As the stamens
become stalked, GUS activity was basally restricted
(Fig. 4K,L), while the petal
primordia, which develop later, displayed diffuse staining
(Fig. 4L). The petal expression
also became restricted to the petal base as petals grew out (data not shown).
Mature flowers showed strong staining through the AZ
(Fig. 4M) agreeing with
BOP2 in situ hybridisation
(Hepworth et al., 2005;
Norberg et al., 2005). In
summary, patterns of BOP1::GUS expression correlate with a putative
role in abscission zone development.
Suppression of ida and 35S::IDA phenotypes by bop1 bop2
The ida mutant shows a complete lack of abscission; however,
unlike bop1 bop2, ida flowers differentiate floral organ AZs and have
reduced petal breakstrength (Butenko et
al., 2003). BOP1 and BOP2 are essential for anatomical
differentiation of the AZ, suggesting that these roles are fulfilled prior to
IDA function. To test this hypothesis, triple mutants were constructed and
examined. The triple mutant bop1 bop2 ida, did not differentiate AZs
or show decreases in petal breakstrength (see Fig. S2 in the supplementary
material), demonstrating that bop1 bop2 is epistatic to
ida.
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). These plants also exhibited activation of vestigial cauline
AZs (Fig. 5F) and ectopic
abscission at pedicels and branching points
(Stenvik et al., 2006).
Similar to bop1 bop2 plants (Fig.
5C), 35S::IDA bop1 bop2 plants showed no floral organ
abscission (Fig. 5D),
indicating that bop1 bop2 is completely epistatic to
35S::IDA. Furthermore, ectopic abscission at vestigial AZs in
pedicels, branching points (data not shown) and cauline leaves
(Fig. 5F) was lost in
35S::IDA bop1 bop2 plants, which resembled bop1 bop2
(Fig. 5E). Both C24 and Col-0
backgrounds demonstrate these phenotypes when transformed with
35S::IDA, suggesting that the lack of 35S::IDA phenotype in
35S::IDA bop1 bop2 is not due to the Col-0 ecotype. In addition, the
35S::IDA construct was not transcriptionally silenced in bop1
bop2 (Fig. 5H). Thus,
earlier and ectopic expression of IDA is not sufficient to promote
abscission in bop1 bop2 because of an absence of proper AZ
architecture.
Abscission-related gene expression in bop1 bop2
We were interested in determining whether BOP1 and BOP2 regulate expression
of genes encoding putative signalling components, such as IDA, and/or
enzymes normally transcribed in AZs. RT-PCR results indicated that IDA,
HAESA and HAWAIIAN SKIRT had the same expression levels in
bop1 bop2 and wild-type flowers (results not shown). Whereas mutation
in HAWAIIAN SKIRT leads to delayed abscission and fusion of sepal
margins that precludes shedding
(Gonzalez-Carranza et al.,
2007), downregulation of IDA or HAESA results in
abscission defects only (Jinn et al.,
2000; Butenko et al.,
2003). The RT-PCR results were confirmed by quantitative PCR for
IDA and HAESA; the expression level in wild-type (Col-0) and
bop1 bop2 floral AZs did not differ as the relative expression was
very close to 1: 0.94 for IDA and 0.95 for HAESA (see Fig.
S3 in supplementary material). To further examine IDA regulation in
bop1 bop2, IDA::GUS plants (C24) were crossed into bop1
bop2. Wild-type IDA::GUS expression was examined in
Col-0xC24 background to compare with bop1 bop2 IDA::GUS plants
generated by crossing. IDA::GUS was expressed shortly following
anthesis in bop1 bop2 flowers and persisted until after the stage
when abscission would normally occur (Fig.
6B) - a temporal profile akin to that observed in wild type
(Fig. 6A). Spatially,
IDA::GUS expression was present at the base of the floral organs in
bop1 bop2, similar to wild type
(Fig. 6C,D).
Two reporter constructs driven by promoters from genes encoding enzymes
that are specifically upregulated in AZs, BAC::GUS and
CHIT::GUS, were also examined. As observed previously in wild type
(Butenko et al., 2006),
BAC::GUS expression appeared early (before anthesis) and throughout
the AZ by position 1 (Fig. 6E).
BAC::GUS was expressed also in the proximal petal and stamen
filaments. Although this temporal pattern was retained in bop1 bop2
(Fig. 6F), BAC::GUS
expression was reduced in intensity, restricted to the bases of the petals and
stamens and absent from single sepal AZ cells, as seen in the wild type
(Fig. 6E, arrow).
|
|
;
Butenko et al., 2006),
CHIT::GUS was expressed in the floral organ AZ cells starting at
position 4 and increased to maximal intensity, coincident with abscission at
position 6, after which expression weakens
(Fig. 6G). Despite the lack of
anatomical AZ structure in bop1 bop2, temporal expression of
CHIT::GUS was maintained, albeit with lower intensity
(Fig. 6H). Often
CHIT::GUS activity was detected earlier in bop1 bop2 but
this was not consistent. CHIT::GUS expression also occurred in the
proximal floral organs in the bop1 bop2 background
(Fig. 6H). These data suggest
that activation signals for BAC::GUS and CHIT::GUS are
operating in bop1 bop2 regardless of a lack of AZ morphology.
Interestingly, ida also shows normal temporal and spatial expression
of both these reporters in AZ cells
(Butenko et al., 2006),
suggesting that signals activating their expression also function
independently of IDA.
BOP1 and BOP2 are necessary for nectary development
Nectaries are secretary organs that develop from receptacle tissue after
maturation of floral organs at stage 9
(Smyth et al., 1990),
appearing as outgrowths at the base stamen filaments and connected to one
another via a ring of nectary tissue that encircles the receptacle
(Davis et al., 1994). Lateral
nectaries surround the base of the lateral stamen filaments and form large
glands subtending the abaxial side. Medial nectaries are smaller and develop
at the abaxial base of medial stamens. As the glands mature, they develop
modified stomata designed for secretion and the cuticle becomes heavily
reticulated (Baum et al.,
2001). Nectaries showed strong BOP1 expression throughout
nectary development (Fig.
4B,N).
|
From the onset of nectary development, two distinct nectary cell types
exist: an outer epidermal layer and an inner starch granule-containing
parenchymal tissue (Baum et al.,
2001), as seen in transverse and longitudinal sections
(Fig. 8E,G); a ridge of
connecting nectary tissue also is present
(Fig. 8E, arrow). The
corresponding section of bop1 bop2 lacks distinct epidermal and
parenchymal cells but shows slight bulges where the paired lateral glands
would normally arise (Fig.
8F,H, arrows). Thus, although some residual cell division may
occur, bop1 bop2 lacks differentiation of most nectary tissue
characteristics.
Genetic interaction between CRABS CLAW and BOP1 and BOP2
CRABS CLAW (CRC) is a key gene regulating nectary
development in Arabidopsis and encodes a putative zinc-finger
transcription factor containing a YABBY domain
(Bowman and Smyth, 1999;
Siegfried et al., 1999). While
bop1 bop2 flowers retain residual bulging reminiscent of nectary
glands, crc mutants show a complete loss of nectary development
(Bowman and Smyth, 1999). To
determine whether loss of nectary growth observed in bop1 bop2 was
due to misregulation of CRC expression, CRC::GUS expression
was examined in bop1 bop2. Wild-type CRC::GUS expression was
examined in Col-0xLer background to compare with bop1 bop2
CRC::GUS plants generating by crossing. As described by Baum et al.
(Baum et al., 2001),
CRC::GUS is expressed in stage 7/8 flowers in the nectary gland
anlagen (Fig. 7I); expression
then expands throughout the connecting nectary tissue between the glands and
is maintained after abscission of floral organs
(Fig. 7J,K). CRC::GUS
is also expressed in stage 7/8 bop1 bop2 flowers
(Fig. 7L), suggesting that
BOP1 and BOP2 are not necessary for CRC expression
in the nectary anlagen. As bop1 bop2 flowers mature, receptacle
regions expressing CRC::GUS expand into lateral and medial nectary
regions and in connecting areas although at reduced levels compared with wild
type (Fig. 7M,N).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Several lines of evidence implicate that the specification by BOP1 and BOP2 of abscission zone cells as the earliest known step necessary for abscission. First, BOP1 and BOP2 genes are transcribed in early floral organ primordia, and resolve to a region corresponding to the future AZ prior to other known abscission-related genes. Second, appearance of AZ anatomy, which is absent in bop1 bop2, is the earliest identified event associated with AZ development. Third, the bop1 bop2 phenotype is epistatic to that of both ida and 35S::IDA, suggesting that the BOP1 and BOP2 act upstream of the only other gene known to be absolutely required for abscission.
BOP1 and BOP2 are expressed earlier than IDA,
raising the possibility that BOP1 and BOP2 are positive regulators of
IDA expression. However, we have shown that IDA is expressed
similarly to wild type in bop1 bop2. It is possible that BOP proteins
regulate IDA activity post-transcriptionally but given the requirement of BOP1
and BOP2 for AZ-specific anatomy, we favour a model where BOP1/2 function
early to specify the AZ cell type and IDA acts relatively independently to
finalize the cell separation process. Significantly, as is the case for
IDA (Butenko et al.,
2006), BOP1 and BOP2 are not required for the
correct temporal transcription of known abscission-related genes tested here,
including one encoding a cell wall hydrolytic enzyme.
A model of the known essential players in abscission is presented in
Fig. 8. We propose that BOP1
and BOP2 act early to specify AZ-unique anatomy. The characteristics of this
anatomy that make it crucial for abscission remain to be identified but could
include cell shape and/or cell wall structure amenable to middle lamellae
digestion. Upstream factors that regulate initiation of abscission act through
both ethylene-dependent and ethylene-independent pathways that converge to
activate the expression of abscission-related genes, including middle lamellae
degrading enzymes. These enzymes are expressed in the AZ, presumably with
specific spatial and temporal profiles, to progressively weaken the middle
lamellae. This expression is driven independently from BOP1/2-mediated
differentiation of the AZ. As yet, none of these enzymes has individually been
shown as essential for abscission. Finally, IDA is necessary for
abscission and is expressed in the AZ just prior to abscission in response to
an unknown signal. Given that partial enzymatic dissolution of the middle
lamellae occurs in ida mutants, IDA must act downstream from the
initiation of abscission. Expression of IDA alone is insufficient for
abscission as premature abscission of 35S::IDA plants occurs only at
flower positions with differentiated AZ
(Stenvik et al., 2006), and is
dependent on BOP1 and BOP2-mediated differentiation of the AZ. The specific
role of IDA and its putative receptor are unknown but must be involved in the
final essential steps of the cell separation process.
|
).
Several lines of evidence presented here strongly support this hypothesis.
First, cauline leaf AZs have characteristic AZ anatomy and a boundary furrow
that demonstrates progressive degradation of middle lamellae with age similar
to floral AZs. Second, the characteristic anatomy, boundary furrow and
35S::IDA-induced abscission at these putative vestigial AZs are
dependent on BOP activity. Finally, vestigial AZs develop on the adaxial side
of the leaf base, corresponding well to BOP1 and BOP2
expression, as shown here and in other studies
(Ha et al., 2004;
Norberg et al., 2005).Taken
together, these data suggest that vestigial AZs do develop and that their
anatomy is regulated by BOP1 and BOP2.
Nectary formation and the role of BOP1 and BOP2
Nectaries are not entirely absent in bop1 bop2, but rather are
greatly reduced in size and do not differentiate key nectary features such as
parenchymal and secretory tissue, and modified stomata. Our analysis suggests
that bop1 bop2 mutants retain CRC::GUS activity in both the
nectary anlagens and the bulges that later develop, indicating that the lack
of nectary outgrowth is not due to a loss in CRC expression. Similar
to CRC, BOP is expressed very early in nectary development and may be
controlling other downstream elements in conjunction with CRC.
Formation of the third whorl, although not the presence of stamens in this
whorl, is essential for nectary formation
(Baum et al., 2001). Third
whorls in bop1 bop2 often develop an extra medial stamen between the
other two medial stamens and the adjacent petal on the abaxial side
(Hepworth et al., 2005). Given
this additional growth, the nectary phenotype in bop1 bop2 may be a
secondary effect of ectopic growth around the stamen attachment areas.
The role of BOP1 and BOP2 in plant development
Previous research on BOP1 and BOP2 has suggested a role
in defining the identity of the proximal regions of lateral organs
(Ha et al., 2003;
Hepworth et al., 2005;
Norberg et al., 2005). The
receptacle may be thought of as a proximal feature of a flower, just as the
petiole is a proximal area of a leaf. Therefore, the absence of AZs and
reduction in nectaries in bop1 bop2 suggests that BOP1 and
BOP2 regulate multiple differentiation events in the proximal flower.
Prior work has demonstrated that BOP1 and BOP2 may repress class 1 Knox gene
expression in the shoot (Ha et al.,
2003; Ha et al.,
2007). Class 1 Knox genes are important to maintain and establish
shoot meristem identity and are normally downregulated in incipient lateral
organ primordia (Scofield and Murray,
2006). Temporal and/or spatial misregulation of class I Knox genes
in developing AZs may contribute to defects in AZ differentiation.
Although AZs may be thought of as defining the organ-plant body junction,
cauline leaves in bop1 bop2 do not display gross malformations where
they meet the stem. Furthermore, expression of floral organ AZ markers, such
as IDA and HAESA, persists in bop1 bop2, suggesting
the positional information is intact. Thus, bop1 bop2 plants lack
some but not all of the features of the organ-plant body junction, suggesting
that other factors are responsible for overall boundary patterning, such as
CUP-SHAPED COTYLEDONS (Aida and
Tasaka, 2006), while the BOP1 and BOP2 genes are
later effectors of specific aspects of the organ-plant body interface.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/8/1537/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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