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First published online 25 July 2007
doi: 10.1242/dev.004788
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Department of Biology, University of Washington, Seattle WA 98195 USA.
Author for correspondence (e-mail:
ktorii{at}u.washington.edu)
Accepted 20 June 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Arabidopsis, Integument growth, Ovule, Receptor-like kinase, Haploinsufficient, Cell proliferation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Potter and Xu,
2001). The Arabidopsis ovule has a relatively simple
structure and a specific, well-defined differentiation pattern, making it a
useful model for understanding the regulation of growth and organogenesis. The
ovule originates from the placenta as an elongate protrusion with a defined
proximal/distal axis that can be separated into three regions. The distal
portion, called the nucellus, is the site of megasporogenesis and embryo sac
development. The proximal region differentiates into the funiculus or stalk,
which attaches the ovule to the carpel wall. Two integuments initiate at the
central chalazal region and eventually envelop the nucellus and form the seed
coat. Similar to the process of leaf laminar expansion, initiation and
expansion of the integument requires the juxtaposition of abaxial and adaxial
factors such as those encoded by PHABULOSA (PHB),
KANADI1 (KAN1), KAN2 and KAN3, ABERRANT TESTA
SHAPE (also known as KAN4 - TAIR), and the YABBY-family member
INNER NO OUTER (Eshed et al.,
2001; Eshed et al.,
2004; McAbee et al.,
2006; Sieber et al.,
2004; Villanueva et al.,
1999). In addition, transcription factors such as AINTEGUMENTA and
NOZZLE (also known as SPOROCYTELESS), and the mitochondria ribosomal protein
HUELLENLOS, have been identified as necessary for integument initiation
(Elliott et al., 1996;
Schneitz et al., 1998;
Skinner et al., 2004;
Villanueva et al., 1999).
A second group of loci affect the progression of integument growth after
initiation. Several genes, including PRETTY FEW SEEDS 2
(PFS2), SHORT INTEGUMENTS 1 and 2 (SIN1
and 2) and TSO1, affect cell proliferation or expansion of
the integuments, and their loss-of-function mutations result in reduction or
loss of fertility (Hauser et al.,
2000; Park et al.,
2004; Park et al.,
2005; Schneitz et al.,
1997). Embryo sac failure is a secondary consequence of the
absence of integuments; the gametophyte fails to develop in cases where the
nucellus is not enclosed (Gasser et al.,
1998). The cause of gametophyte abortion is not clear, but is
likely to lie in the requirement for tight coordination of cell division and
expansion within the ovule and communication between gametophytic and
sporophytic tissue (Gasser et al.,
1998). Relatively little is known about the genes involved in the
coordination of growth or cell-cell communication within the ovule. This might
be due to redundancy among the genes involved in this process, or to
pleiotropic effects resulting from the loss of these genes. For instance,
TSO1, TOUSLED (TSL) and SIN1 have multiple,
sometimes detrimental, effects on vegetative and floral development in
addition to ovule defects (Ehsan et al.,
2004; Lang et al.,
1994).
ERECTA (ER) and its two paralogs, ERECTA-LIKE 1
(ERL1) and ERL2, regulate organ shape and inflorescence
architecture and are members of the leucine-rich repeat receptor-like kinase
(LRR-RLK) gene family in Arabidopsis
(Shiu and Bleecker, 2001;
Torii, 2004;
Torii et al., 1996). Based on
phylogeny, it was suggested that ERL1 and ERL2 evolved by
recent gene duplication and are functionally related to ER, while
maintaining overlapping but unique transcript expression patterns
(Shpak et al., 2004). We
report here that in the absence of functional ER and ERL1,
Arabidopsis plants heterozygous at the ERL2 locus exhibit
specific defects in integument development. Based on data from genetic and
gene expression analysis, the ER family plays a key role in ovule development
and fertility by regulating cell proliferation in the integuments. Our study
highlights the unequal redundancy and dosage compensation among
ER-family genes, and further reveals a potential molecular
consequence of their reduced dosage for cell division control of integument
development and fertility. Finally, genetic interactions of the ER
family with PFS2, a WUSCHEL (WUS)-type homeodomain
gene, revealed an unexpected intersection of two pathways required for proper
integument growth and embryo sac development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The erl1-1 allele (YJ133) was identified as an
enhancer-trapped line by Dr John Bowman (University of California, Davis, CA)
and kindly provided as a gift. The line was originally in the Landsberg
erecta (Ler) background and outcrossed into Col three times.
The erl1-4 allele was isolated from ethyl
methanesulfonate-mutagenized Arabidopsis er-105 erl2 seeds and
outcrossed to Col. The pfs2-1 allele was a gift from Dr Bernard
Hauser (University of Florida, Gainesville, FL) and was in the Ler
background and outcrossed to Col. The pfs2-1 mutant was crossed with
the er-105 erl1-2 (+/-) erl2-1 mutant for further analysis.
All plants were grown under long-day conditions as described previously
(Shpak et al., 2003).
Genotyping
PCR-based genotyping for er-105, erl1-2 and erl2-1
mutations was described previously (Shpak
et al., 2004). Detection of the erl1-1 T-DNA insertion
was performed with PCR primers: ERLK-185.rc,
5'-CGTAGGTCTCCAATACGTGGA-3' and GUS R,
5'-CAGTTGCAACCACCTGTTGAT-3'. The erl1-4 mutation was
detected by derived Cleaved Amplified Polymorphic sequences (dCAPs) using
primers: erl1-4dcaps1151, 5'-GACGATGTTCACAACAGTGACTTGTGTTCTAG-3'
and erl1-4dcaps1447.rc, 5'-CATCGAAATCAACAGAGAAAGAAAGGG-3', and
subsequent digestion with XbaI for 2 hours at 37°C, which cuts
the mutant erl1-4 allele sequence.
In situ hybridization
Tissue preparation and in situ hybridization were performed as described
previously (McAbee et al.,
2006). Probe template construction was performed as follows. For
the ERL1 probe template, the ERL1 sense and antisense probes
were produced by linearizing plasmid pLJP562 (contains ERL1 kinase
domain) with BamHI or XhoI and transcribing in vitro with T3
or T7 RNA polymerase, respectively. For the ER probe template, the
ER sense and antisense probes were synthesized by linearizing pLJP131
(contains the ER kinase domain and partial 3'UTR) with
XhoI or BamHI and transcribing in vitro with T3 or T7 RNA
polymerase, respectively. The WUS sense and antisense probes were
produced by linearizing pSMB106 (nt 627-816 of the WUS cDNA) with
EcoRI and XbaI and transcribing in vitro with T3 or T7 RNA
polymerase, respectively. PFS2 probe template construction and
hybridization conditions were according to Park et al.
(Park et al., 2005). Sense and
antisense probes for PHB were produced by linearizing plasmid pPHB (a
gift from Dr Kiyotaka Okada, Kyoto University, Kyoto, Japan) with
SacI or KpnI and transcribing in vitro with T7 or T3 RNA
polymerase, respectively.
GUS histochemical analysis
proERL1::GUS and proERL2::GUS constructs were described
previously (Shpak et al.,
2004). Histochemical staining for ß-glucuronidase (GUS)
activity was performed as described previously
(Sessions et al., 1999).
RNA extraction and quantitative real-time RT-PCR
Total RNA was isolated from Arabidopsis stage 12 gynoecia
(Schneitz et al., 1995) or
from ovules isolated from stage 12 gynoecia using the RNeasy Plant Mini Kit
(Qiagen, Valencia, CA) and treated with DNase I (Amp Grade, Invitrogen).
First-strand cDNAs were generated by ThermoScript Reverse Transcriptase
(Invitrogen) and random hexamers using 1.0 µg of RNA according to
manufacturer's instructions. Real-time PCR was performed with a Chromo4
thermocycler (BioRad, Hercules, CA) and analyzed using Opticon Monitor 3
software (BioRad). Triplicate reactions were performed in 25 µl total
volume containing iTaq SYBR Green mix (BioRad) and 1.0 µl of the
first-strand reaction. ACTIN 2 was used as an internal control in
parallel reactions for each run. The relative level of expression of each
target gene was calculated with respect to ACTIN 2 expression for
each genotype. Real-time reactions were repeated in two biological replicates
and the average fold-difference relative to wild type was calculated.
Oligomers and DNA sequences used for PCR are shown in
Table 1.
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). To
examine ovules using differential interference contrast (DIC) optics, carpels
were dissected from flowers and incubated in 9:1 (v:v) ethanol:acetic acid
overnight, rinsed with water and placed in choral hydrate (1:8:1 v:v
glycerol:chloral hydrate:water). Cleared ovules and in situ hybridization
sections were viewed under DIC optics using an Olympus BX51 microscope
equipped with a DP70 digital camera.
Cell size and number measurements
Cell size was measured from images of plastic-embedded sections of
wild-type and er erl1-2 erl2-1/+ carpels. The length and width of the
first ten cells (nucellar to chalazal end) of the outer layer of the outer
integument were measured using pixel distance in Photoshop Pro (Adobe Systems,
San Jose, CA). Images used for these measurements corresponded to similar
cross-sections through the nucellus of mature wild-type and er erl1-2
erl2-1/+ ovules. Cell numbers were counted from cleared ovules at stages
1-II, 2-IV, 3-II and 3-VI (Schneitz et
al., 1995). At stages 2-IV, 3-II and 3-VI, counting started at the
tip of the outer integument (nucellar end) and stopped where cells of the
funiculus appeared obvious. At stage 1-II, all cells in the L1 layer of the
ovule primordia were counted.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
(Fig. 1A-D). To unravel the
specific functions of ERL1 and ERL2 during inflorescence and
floral organ growth, we investigated the phenotypes conferred by reduced
dosages of ERL1 and ERL2 in the absence of ER. For
this purpose, 100 self-fertilized progenies of er-105 erl1-2/+
erl2-1/+, pre-selected for the presence of the erl1-2 T-DNA
insertion, were grown to maturity. This population segregated seven extremely
dwarf and sterile plants, consistent with the expected 8.3% ratio for the
er-105 erl1-2 erl2-1 triple mutant (dwarf plants/total=7/100;
2=0.221, P=0.637). Seventeen additional plants from
this population were completely sterile and produced no seeds. However, unlike
the triple er-family mutants, they did not exhibit severe dwarfism,
disrupted phyllotaxis or floral organ patterning defects. A reciprocal
cross-pollination with wild type revealed that these plants were female
sterile (data not shown).
To determine the genetic basis for this novel phenotype, we next analyzed
the genotypes of all 100 plants in this population. All seventeen
female-sterile, non-dwarf plants were homozygous for er-105 and
erl1-2 and heterozygous for erl2-1 (i.e. er-105 erl1-2
erl2-1/+) (Fig. 1E). This
was highly consistent with the expected ratio of 16.7% for this population
(2=0.006, P=0.936). Unlike er-105 erl1-2
erl2-1/+, the er-105 erl1-2/+ erl2-1 plants were fully fertile
(Fig. 1F). Reintroduction of
functional copies of ER, ERL1 and ERL2 into respective
loss-of-function mutants confirmed these data (see Table S1 in the
supplementary material). The results indicate that, in the absence of
ER and ERL1, ERL2 is haploinsufficient for female fertility,
whereas ERL1 is haplosufficient in the absence of ER and
ERL2. By contrast, the lack of defects in floral morphology (see Fig.
S1 in the supplementary material) and phyllotaxis indicate that a single copy
of ERL2 is sufficient for inflorescence elongation and floral
patterning.
Slight increase in ERL1 activity is sufficient to rescue ERL2 haploinsufficiency
To further investigate the importance of ERL1 function in female
fertility, we generated mutant combinations with two additional erl1
alleles. The erl1-1 allele carries a T-DNA insertion 349 bases
upstream of the initiation codon and represents a weak allele
(Fig. 1I,J). The
erl1-4 allele has a G-to-A substitution at nucleotide position +750.
This creates a stop codon at amino acid 61, leading to a premature truncation
of the protein prior to the LRR domain
(Fig. 1I). Therefore,
erl1-4 is a null allele.
The er-105 erl1-1 erl2-1, er-105 erl1-2 erl2-1 and er-105 erl1-4 erl2-1 triple-mutant plants were almost indistinguishable from each other (data not shown). Similar to er-105 erl1-2 er2-1/+ plants (Fig. 1E), er-105 erl1-4 erl2-1/+ plants were completely female sterile (Fig. 1G). By contrast, er-105 erl1-1 er2-1/+ plants were fertile and produced viable seed (Fig. 1H). These results demonstrate that a slight increase in ERL1 activity is sufficient to rescue the haploinsufficiency of ERL2. However, because erl1-1 failed to promote organ growth, proper floral patterning or fertility in the absence of ER and ERL2, the results highlight a strict ER-family signaling threshold required to promote female fertility.
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) (Fig.
2). Similar to wild type, er-105 erl1-2/+ erl2-1
exhibited typical amphitropic ovule morphology
(Fig. 2A,B,E). Ovules of
er-105 erl1-1 erl2-1/+ were fertile, but had slightly shorter
integuments than wild type, resulting in the micropyle resting slightly away
from the funiculus (Fig. 2C).
By contrast, the mature ovules of er-105 erl1-2 erl2-1/+ displayed
aberrant morphology. Growth of both inner and outer integuments as well as the
funiculus was dramatically compromised, leading to small, stunted ovules that
often had an exposed nucellus (Fig.
2D,F). The embryo sac of mature er-105 erl1-2 erl2-1/+
ovules was absent and replaced by a disorganized mass of small cells
(Fig. 2F).
Initiation and polarity of integuments was not disrupted in er-105
erl1-2 erl2-1/+ (Fig. 2G).
To determine whether integument initiation was defined normally, we used
WUS and PHB as a nucellar regional marker and adaxial
marker, respectively (Fig.
2H-K). WUS is expressed exclusively in the nucellus of
wild-type ovules and promotes integument initiation in the chalazal region
proximal to its expression domain
(Groß-Hardt et al.,
2002), whereas PHB expression marks the inner integument
(Sieber et al., 2004). No
alteration in WUS or PHB expression was detected between
wild type and er-105 erl1-2 erl2-1/+. These data suggest that loss of
ER-family signaling does not disrupt regional domain specification or radial
patterning.
Integument outgrowth is compromised in er-105 erl1-2 erl2-1/+ mutants
To determine the onset of developmental defects, wild-type and er-105
erl1-2 erl2-1/+ ovules were examined at sequential developmental stages
(Fig. 3)
(Schneitz et al., 1995). Early
ovule initiation and patterning were normal in er-105 erl1-2
erl2-1/+, consistent with the normal PHB and WUS
expression patterns (Fig.
2I,K). Similar to wild type, ovule primordia initiated and
elongated from the placental wall (stage 1-II;
Fig. 3A,E,I). At stage 2-II to
2-V, outer and inner integument primordia arose from the chalaza and grew
toward the nucellus (Fig.
3B,F,J). However, starting at stage 3-I, the cells of er-105
erl1-2 erl2-1/+ outer integuments had a more disorganized appearance than
wild type (Fig. 3C,G,K).
Enlargement of the megaspore mother cell (MMC) and tetrad formation proceeded
as in wild type within the nucellus of er-105 erl1-2 erl2-1/+ ovules
(Fig. 3I,M). However, mutant
ovules arrested at a two-nucleate embryo sac
(Fig. 3N). We never observed
er-105 erl1-2 erl2-1/+ embryo sacs beyond this two-nucleate stage.
Based on our observations, we conclude that progression of integument growth
in the absence of ER is dependent on ERL1 and 2 in
a dosage-dependent manner. At a minimum, ERL1 in a heterozygous state
or ERL2 in a homozygous state is required.
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We next assessed the possibility of endoreduplication in mutant integument
cells by observing the nuclear size using 4',6-diamidino-2-phenylindole
(DAPI). Endoreduplication is the repetitive duplication of chromosomal DNA
without mitosis or cytokinesis, resulting in an increase in cell ploidy level
(Kondorosi et al., 2000). In
plants, a positive correlation between ploidy level and cell size has been
observed (Kondorosi et al.,
2000; Melaragno et al.,
1993). No qualitative difference in the size (area) of nuclei
between wild-type and er-105 erl1-2 erl2-1/+ outer integument cells
was observed (see Fig. S2 in the supplementary material), consistent with the
absence of a cell size difference between wild-type and mutant
integuments.
To investigate whether er-105 erl1-2 erl2-1/+ ovule integument defects were due to a decrease in cell division, we quantified the number of cells contributing to the epidermis at stage 1-II and to the outer integument at three subsequent stages (see Materials and methods). At stages 1-II and 2-II, no difference in cell number was observed between er-family combination mutants and wild type (Fig. 4D). However, er-105 erl1-2 erl2-1/+ outer integuments had significantly fewer cells per outer integument than wild type from stage 3-III (31% reduction) to maturity (57% reduction). A similar trend was observed for er-105 erl1-2/+ erl2-1 (15% reduction) and er erl1-1 erl2-1/+ (30% reduction) ovules, which had an intermediate number of integument cells between that of wild type and er-105 erl1-2 erl2-1/+ at maturity (Fig. 4D). Together, these data provide evidence that a reduction in cell division is responsible for the abnormal integument morphology of er-105 erl1-2 erl2-1/+ ovules, and that the number of cell divisions is sensitive to the dosage of ERL1.
Specific misregulation of cell-cycle regulators is associated with the arrested ovule development in er-105 erl1-2 erl2-1/+ mutants
Because cell divisions were reduced in er-105 erl1-2 erl2-1/+
ovules, we examined the expression of cell-cycle-regulatory genes to determine
if their transcript accumulation is altered in er-105 erl1-2 erl2-1/+
ovules. The cell cycle is regulated at multiple points by cyclin-dependent
kinases (CDKs) that form complexes with appropriate cyclins
(De Veylder et al., 2003;
Dewitte and Murray, 2003).
Plants have a large number of core cell-cycle genes including plant specific
B-type CDKs that are regulated at the transcriptional level
(De Veylder et al., 2003).
Plant cyclin genes can be divided into four groups (A, B, D and H). The
evolution of three A-type subclasses (A1, A2 and A3), compared with only one
in animals, has led to the idea that A-type cyclins might fulfil
plant-specific roles (Caubet-Gigot,
2000; Dewitte and Murray,
2003).
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|
), this
stage of carpel corresponds to the point in ovule development when we observed
noticeable changes in integument morphology between er-105 erl1-2
erl2-1/+ and wild type. Most of the B-type CDKs and cyclin A, B and D
genes did not show a dramatic change in transcript abundance in ER-family
loss-of-function mutants. However, CYCA2;2 showed a pronounced
decrease in expression in er-105 erl1-2 erl2-1/+ (see Fig. S3 in the
supplementary material; data not shown). We next quantified the relative
decrease in CYCA2;2 transcripts using real-time PCR. We found that
CYCA2;2 consistently shows a significant decrease in expression after
loss of ER-family function in both carpels and individual ovules
(Fig. 4E). Conversely,
CYCA2;3 had the opposite trend, showing increased transcript
accumulation after ER-family loss-of-function, perhaps owing to
compensation (Fig. 4E). The
results suggest that reduced levels of ER-family gene function may
impact cell division through disruption of specific cell-cycle-regulatory gene
expression. Alternatively, altered levels of these cyclin genes could be the
consequence of reduced cell division produced by a yet unknown mechanism.
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).
ERL1 and ERL2 promoter activity was first detected in the
placental wall prior to ovule primordia initiation (data not shown). They
became broadly active in ovules with a slight increase in activity in the
central chalazal region (Fig.
5A-D). In mature ovules, faint GUS expression was restricted to
the funiculus (data not shown). Interestingly, proERL1::GUS was
notably stronger in the er-105 background than in wild type
(Fig. 5E,F). Because integument growth was sensitive to the dosage of ERL1 and because the promoter activity of ERL1 and ERL2 are similar, we focused our analysis of expression overlap on ER and ERL1. We analyzed the mRNA expression patterns of ER and ERL1 by in situ hybridization (Fig. 6). In reproductive structures, ERL1 transcripts were detected in floral meristems and primordia (see Fig. S4 in the supplementary material). During ovule development, ERL1 transcripts were detected throughout ovule primordia from initiation to stage 3-II, with increased hybridization intensity in the chalazal region and developing integuments (Fig. 6A-C). At maturity, ERL1 hybridization signal was still detectable in the integuments and funiculus (Fig. 6D).
Expression of ER was observed in floral meristems as well as the carpel wall and developing petals (see Fig. S4 in the supplementary material). Similar to ERL1, ER transcripts were detected broadly in the developing ovule, with more intense signal in the chalazal region and developing integuments (Fig. 6E-G). ER expression lessened later in development, but was still detectable in the carpel and ovule integuments at maturity (Fig. 6H). As the probes for ER and ERL1 did not cross-hybridize with each other (see Fig. S4 in the supplementary material), our results suggest that overlapping expression patterns of ER and ERL1 account for their redundant roles in ovule development.
Unequal dosage compensation among ER-family genes
Given the overlapping expression patterns of ER-family genes in
the ovule and their ability to functionally substitute for one another
(Shpak et al., 2004), we
tested whether loss of specific ER-family gene function would lead to
changes in expression of the other family members. To assess changes in
transcript abundance, quantitative real-time RT-PCR analysis was performed on
RNA from stage 12 carpels (Fig.
6Q). An increase in both ERL1 and ERL2
transcripts was observed in the er-105 background as compared with
wild type and, reciprocally, the accumulation of ER transcripts was
higher in the erl1 erl2 mutant background. In situ hybridization
determined that the spatial expression patterns of ER and
ERL1 were not disrupted in the er-105 and erl1 erl2
mutant backgrounds (Fig. 6I-P),
respectively.
|
) that ER-family members are able to substitute for each other
if their temporal and spatial expression patterns overlap. Fertility in the
null er single mutant or erl1 erl2 double mutant might
require dosage compensation by the remaining family members to maintain
signaling above a required threshold. This threshold is not met in the
er-105 erl1 erl2/+ mutant, resulting in the arrest of integument
growth.
Interaction of pfs2 with er erl1-2 erl2-1/+
PFS2, a WUS-related homeobox gene also known as
WOX6, affects ovule patterning and regulates integument cell
proliferation and gametophyte differentiation
(Park et al., 2004;
Park et al., 2005). It has
been well-documented that the competing actions of the transcription factor
WUS, and the CLAVATA (CLV) LRR-RLK signaling pathway, maintain meristem size
in Arabidopsis (Brand et al.,
2000). Numerous WOX genes display region-specific expression, but
relatively little is known about their regulation
(Haecker et al., 2004). To
investigate whether a mechanism including PFS2 and ER
signaling directs integument size during ovule development, we produced the
combination mutant er-105 erl1-2 erl2-1/+ pfs2-1 and characterized
the phenotype.
The pfs2-1-null mutation causes the production of ovules with
similar characteristics to er-105 erl1-2 erl2-1/+. pfs2-1 ovules have
aberrant integument growth after initiation and embryo sac arrest
(Park et al., 2004)
(Fig. 7A). However, growth
defects are variable in pfs2-1 mutants: some ovules complete various
stages of embryo sac development and a low percentage of viable seed are
produced (Park et al., 2005).
Similar to pfs2-1 alone, we observed a variable degree of integument
development in the er-105 erl1-2 erl2-1/+ pfs2-1 mutant
(Fig. 7C,D). The phenotype
ranged from an outer integument that covered the inner integument to a near
absence of the outer integument. In addition, subtle changes in integument
development were observed including lobing of the outer integument during
development (Fig. 7C,D). Such
variability in integument growth was not observed in er-105 erl1-2
erl2-1/+, in which the phenotype showed little variation
(Fig. 7B). Surprisingly, some
ovules of er-105 erl1-2 erl2-1/+ pfs2-1 developed beyond the
two-celled nuclei stage typical of er-105 erl1-2 erl2-1/+ ovules,
although no viable seed could be collected from these plants
(Fig. 7E-G). Therefore, the
loss of PFS2 function partially rescues the er-105 erl1-2
erl2-1/+ ovule defects.
Because pfs2-1 ovules show aberrant integument growth, we performed in situ hybridization using ER and ERL1 probes in the pfs2-1 ovules to determine whether loss of ER-family signaling contributed to the abnormal integument phenotype of pfs2-1 ovules (Fig. 7H-K). Loss or change in expression domain of ER or ERL1 was not detected in the pfs2-1 mutant, suggesting that the required threshold of ER-signaling is met in the pfs2-1 mutant.
The phenotypic rescue of er-105 erl1-2 erl2-1/+ ovules by additional pfs2 mutation implies that it is advantageous not to have PFS2 under the sub-threshold level of ER-family signaling. To test whether the er-105 erl1-2 erl2-1/+ phenotype was due in part to misexpression of PFS2, we performed in situ hybridization using a PFS2 probe (Fig. 7L-O). PFS2 expression was detected broadly in developing carpels and initiating ovule primordia (data not shown) with strong expression in the inner integument during initiation and growth (Fig. 7L-O). The expression domain of PFS2 was not altered in the er-105 erl1-2 erl2-1/+ ovules (Fig. 7P,Q), indicating that the gametophytic defects in er-105 erl1-2 erl2-1/+ are not due to PFS2 misexpression. However, quantitative real-time RT-PCR detected an increase in PFS2 transcript abundance in er-105 erl1-2 erl2-1/+ stage 3-II ovules compared with wild type, indicating a potential inhibitory role for the ER family in PFS2 expression. Phenotypic rescue of er-105 erl1-2 erl2-1/+ by the addition of the pfs2 mutation suggests that ovule development requires a proper balance of both ER family and PFS2 function.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Vision et al., 2000).
Following a gene duplication event, decreased selection pressure on one of the
gene copies can produce proteins with novel function or expression resulting
in partial redundancy among gene family members
(Hardtke et al., 2004). If the
shared function of duplicated genes becomes unequally distributed between gene
copies, unequal redundancy among the gene family members may result, as
described for the ER family, where both erl1 and
erl2 confer no growth phenotype but enhance the er growth
defects in a unique manner (Kempin et al.,
1995; Shpak et al.,
2004). In many cases, unequal redundancy is produced by
differences in the expression level or expression patterns between gene
copies, where the duplicated gene(s) may have redundant function with the
ancestral gene, but the expression might be lower or restricted. A good
example is the three Arabidopsis brassinosteroid receptor genes. The
major receptor, BRI1, is broadly expressed at high levels, whereas
expression of BRL2 and BRL3 is restricted to vasculature
(Cano-Delgado et al., 2004).
Successive loss of family members enhances the phenotype of the ancestral gene
and a true null phenotype is observed only in the absence of the entire family
as has been shown for ER, ERL1 and ERL2
(Shpak et al., 2004). Our study uncovered unequal contributions and dosage compensation among gene family members with overlapping expression patterns. The most significant difference between er-105 erl1-2/+ erl2-1 and er-105 erl1-2 erl2-1/+ plants is the loss of female fertility owing to reduced cell division and outgrowth of the integuments in er-105 erl1-2 erl2-1/+. The expression pattern for all three family members overlaps in the distal, central and proximal regions, consistent with all three regions being reduced when successive family members are removed. An erl1-2 erl2-1 double or er single mutant shows no obvious defects in integument development, indicating that the upregulation of ER or ERL1 and 2 in these backgrounds can maintain expression above the threshold necessary to direct integument development (Fig. 6Q). However, as successive family members are lost, low levels of ERL2 are insufficient to maintain signaling, and integument growth is compromised. Consistent with this dosage-dependence model, the ovules of er-105 erl1-2 erl2-1/+ are significantly larger and more developed than those of the ER-family triple mutant (see Fig. S5 in the supplementary material).
ERL2 haploinsufficiency specifically affects cell division of the ovule integuments
The ER-family LRR-RLKs probably maintain cell-cell communication, a
reduction of which results in the irregular and reduced cell division during
integument outgrowth in er-105 erl1-2 erl2-1/+ ovules. They do not
regulate ovule primordia patterning or integument initiation, as these aspects
are not affected even by the complete loss of ER-family function
(Fig. 2 and see Fig. S5 in the
supplementary material).
Our results suggest that the ER-family RLKs promote cell division after
integument initiation through potential regulation of core cell-cycle genes,
specifically CYCA2;2 (Fig.
4). Among over 30 cyclins present in Arabidopsis, ten
belong to the A-type (Dewitte and Murray,
2003; Vandepoele et al.,
2002). Recent studies suggest that Arabidopsis cyclin A2
families act as negative regulators of endoreduplication
(Burssens et al., 2000;
Dewitte and Murray, 2003;
Imai et al., 2006;
Yu et al., 2003). For example,
loss-of-function mutations in CYCA2;3 led to elevated ploidy levels
in mature organs (Imai et al.,
2006). Consistently, the dominant, activation-tagged mutation in
INCREASED LEVEL OF POLYPLOIDY1, a conserved repressor of
CYCA2 transcription, caused an increase in endoreduplication
(Yoshizumi et al., 2006).
However, cyclin A2 families may also function to regulate mitosis. In
Medicago, CYCA2;2 (MedsaCYCA2;2) is required for meristem
formation or activity but dispensable for endoreduplication
(Roudier et al., 2000).
Similar to Medsa;CYCA2;2, all four Arabidopsis cyclin A2
genes show high promoter activity in meristems and young organ primordia
(Burssens et al., 2000;
Imai et al., 2006). Therefore,
they might redundantly control cell division in actively proliferating
tissues, where the ER-family signaling pathway is required. Consistent with
this hypothesis, flow-cytometric analysis of DNA content in er-105
suggested possible cell-cycle arrest
(Shpak et al., 2003).
All members of the ER family are expressed in overlapping domains in
additional areas of the plant including shoot and inflorescence meristems and
floral primordia (Shpak et al.,
2004). The lack of any visual er-105 erl1-2 erl2-1/+
phenotype in these tissues could be due to redundancy by other signaling
factors that are not expressed in the ovule. Alternatively, ovule integuments
might be particularly susceptible to cell-cell signaling disruption. Unlike
meristems and developing leaves and floral organs, integuments are solely
derived from the L1 layer (Schneitz et
al., 1997). Therefore, cell proliferation in integuments is likely
to occur with limited inter cell-layer communication. Such a scenario was
suggested for loss-of-function mutants of the RLK gene ARABIDOPSIS
CRINKLY4, which display localized defects in integument development,
despite its expression in a wider variety of tissues
(Gifford et al., 2003).
Genetic interaction of ER-family genes and PFS2
PFS2 is thought to control ovule primordial patterning by
regulating the timing of cellular differentiation
(Park et al., 2005). In
pfs2-1, premature differentiation of the cells contributing to the
gametophyte or integument primordia could account for decreased integument
length and defects in the embryo sac (Park
et al., 2004). The addition of pfs2-1 to er-105
erl1-2 erl2-1/+ mutations led to increased variability in both integument
and gametophyte development, similar to pfs2-1 alone, indicating that
the pfs2-1 mutation is epistatic to the er-family mutations.
One explanation for these results is that the ER-family RLKs and PFS2 both
contribute to integument outgrowth through distinct but interrelated
mechanisms. The ER family are components of a signaling pathway that sustain
organized cell divisions, potentially through the regulation of core
cell-cycle genes such as CYCA2;2. Without activation of this
signaling pathway, integument cells may differentiate before undergoing
sufficient cell divisions. PFS2 might be responsible for maintaining
integument cells in a state that allows them to respond to factors required
for coordinated cell division. In this scenario, the loss of PFS2 function
would be epistatic to the loss of the ER family.
WOX genes generally have a spatially restricted expression pattern
(Matsumoto and Okada, 2001).
Our in situ hybridization shows that PFS2 expression is spatially
restricted to the inner integument (Fig.
7). Therefore, PFS2 might be required to properly
coordinate the developmental states of the sporophytic integument tissues and
gametophytic embryo sac. If so, the loss of PFS2 uncouples embryo sac
development from abrogated integument growth under the sub-threshold level of
ER-family signaling, therefore partially rescuing the embryo sac defects in
erecta-105 erl1-2 erl2-1/+ ovules.
In the well-established model of CLV/WUS interaction in the shoot
meristem, CLV3 limits the expression of WUS, while
WUS in turn promotes the expression of CLV3, causing a
feedback loop that maintains a stem cell population
(Brand et al., 2000;
Clark, 2001;
Laux et al., 1996). Our
results indicate that the ER family does not spatially restrict PFS2
expression and that factors yet to be identified are likely to be responsible
for the limiting PFS2 expression. However, the ER family might
restrict the expression levels of PFS2, and elevated expression of
PFS2 might account for the abortion of gametophytes in er-105
erl1-2 erl2-1/+ ovules. Consistently, embryo sacs of er-105 erl1-2
erl2-1/+ are filled with a small group of cells, which resembles the
phenotype of PFS2 overexpression described by Park et al.
(Park et al., 2005).
Haploinsufficiency refers to a phenotype associated with the inactivation of a single allele leading to a half-normal amount of gene product, which is insufficient to maintain the wild-type phenotype. In this report, we have shown a novel phenotype for successive ER-family loss-of-function; ERL2 is haploinsufficient in the absence of ER and ERL1 to direct integument growth, but sufficient to drive floral organ growth, elongation and patterning. We further uncovered evidence of dosage compensation within the ER family and their potential regulatory relationships with the WOX-family transcription factor. Our work has identified the specific timing of ERL1 action during integument cell proliferation, which might provide a biological system to further elucidate the signaling components.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/17/3099/DC1
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ACKNOWLEDGMENTS |
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