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First published online October 10, 2008
doi: 10.1242/10.1242/dev.026708
Institute of Molecular Plant Sciences, School of Biology, University of Edinburgh, Rutherford Building, Mayfield Road, Edinburgh EH9 3JH, UK.
Authors for correspondence (e-mails:
justin.goodrich{at}ed.ac.uk;
gwyneth.ingram{at}ed.ac.uk)
Accepted 4 September 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Endosperm, Embryo, Epidermis, Arabidopsis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In many
dicotyledonous plants, such as Arabidopsis, the endosperm is largely
transient and is consumed during seed development. In Arabidopsis, a
single specialized cell layer, which has been compared with the aleurone layer
of monocotyledonous plants, is all that remains of the endosperm by seed
maturity. Although the importance of the endosperm in embryo/seedling
nutrition is uncontested, it may also provide developmental signals to the
young embryo (Berger, 2003;
Berger et al., 2006). In both
monocotyledonous and dicotyledonous species, the region of the endosperm that
directly surrounds the developing embryo (the embryo surrounding region or
ESR) expresses a unique complement of genes, supporting the idea that it has a
specialized function. Although several of these ESR-specific genes encode
proteins with obvious metabolic roles, others encode small secreted peptides,
some of which are related to the precursor of CLAVATA3 (CLV3), the likely
ligand of the CLAVATA1 receptor-like kinase, supporting the supposition that
the ESR may be involved in signalling
(Opsahl-Ferstad et al., 1997;
Sharma et al., 2003). However,
no precise developmental role for any of these peptides has yet been
demonstrated. Recent studies have also shown that the developing embryo can
send developmental signals to the endosperm, as fertilization of the egg cell
alone is sufficient to trigger proliferation of the central cell
(Nowack et al., 2006).
One potentially important role that the endosperm could play in
embryogenesis is in defining the continuous cuticular layer at the boundary
between the developing embryo and the endosperm. This structure is necessary
to prevent organ fusion after germination, and it may also prevent fusion of
the embryo surface with surrounding endosperm tissues during seed development,
thus allowing normal embryo growth. In addition, the presence of a watertight
cuticle surrounding the mature embryo is crucial in protecting it from
desiccation upon germination (reviewed by
Jeffree, 2006). Although the
production of cuticle is generally considered to be a strictly epidermal
property, a cuticularized cell wall has been reported to surround the
unicellular zygote in Citrus, well before a defined protodermal cell
layer is formed (Bruck and Walker,
1985). Interestingly, the expression of epidermal markers, such as
the transcription factors ATML1 and PDF2, which are required
for epidermal specification in Arabidopsis, is also detected well
before protoderm formation (Abe et al.,
2003; Lu et al.,
1996). Thus, the outer layer of embryonic cells display epidermal
characteristics, including the presence of cuticularized tissue, from an
extremely early stage in embryogenesis.
Several recent publications have supported a role for the endosperm in
embryonic cuticle production and the specification of embryonic epidermal
identity in Arabidopsis. A secreted subtilisin-like serine protease
ABNORMAL LEAF SHAPE1 (ALE1), which is expressed predominantly in the ESR
appears to be required for normal cuticle production in Arabidopsis
embryos (Tanaka et al., 2001).
Plants that lack ALE1 produce embryos that adhere to the endosperm
during development. Moreover, germinating seedlings are extremely sensitive to
desiccation owing to abnormal cuticle production on their cotyledons. However,
if ale1 seedlings are grown in vitro in high humidity, and then
transferred to soil, they survive and their adult leaves have a normal cuticle
structure, consistent with their cuticle defects being derived from
abnormalities arising during embryogenesis. Mutations in either of two
embryonically expressed genes encoding RLKs, ACR4 or ALE2,
leads to a significant enhancement of the relatively mild ale1 phenotype. In
double mutants, epidermal specification (as judged by the expression of the
epidermal markers such as ATML1) is also partially lost
(Tanaka et al., 2007;
Watanabe et al., 2004).
Neither acr4 nor ale2 mutants show a marked seedling
phenotype, although both have cotyledon cuticle abnormalities, and the
synergistic interactions of these genes may indicate that ACR4 and ALE2 are
required to perceive a signal processed by ALE1. Such a function for ALE1
would also be consistent with the known role of several other subtilisin
proteases in processing ligand signals
(Berger and Altmann, 2000;
Cui et al., 1998;
Julius et al., 1984;
Rose et al., 1996). Two other
embryonically expressed receptor-like kinase encoding genes, GASSHO1
and GASSHO2, also act redundantly to allow normal embryonic cuticle
formation (Tsuwamoto et al.,
2008), although how these genes interact genetically with
ALE1, ACR4 and ALE2 has yet to be determined.
In this paper, we show that the transcription factor encoded by the ZHOUPI (ZOU) (Chinese for `shrivelled') gene of Arabidopsis is a key player in controlling ESR-expressed genes involved in the development of the embryo surface, including ALE1. The ZOU gene is widely conserved and occurs in plant groups that lack endosperm (gymnosperms) or seeds (Lycophyta). We propose that ZOU plays a pivotal role in the crosstalk between the embryo and surrounding nutritive tissues. In angiosperms, this is necessary to promote both cuticle formation and the separation of embryo from the surrounding tissues, whereas in more primitive plants it may permit invasive growth of embryos into nutritive tissue of the female gametophyte.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The mutant was backcrossed several times to Ws ecotype
to transfer the zou-1D mutation into a CLF+ background and
all experiments were performed using this line.
The recessive, loss of function zou2-4 alleles were obtained from
the Nottingham Arabidopsis Stock Center. The zou2 allele (Ws
background) arose in the FLAG collection of T-DNA insertions
(Samson et al., 2002) and
corresponded to FLAG 400A08. The zou-3 allele (Ws background) was
obtained from the University of Wisconsin collection of T DNA insertion lines
(Sussman et al., 2000) and
corresponded to WiscDsLox465F5. The zou-4 (Col-0 background) allele
was obtained from the Gabi-Kat collection of T-DNA inserts
(Rosso et al., 2003) and
corresponded to GABI_584D09. The position of the T-DNA inserts was confirmed
by PCR amplification and sequencing of genomic DNA flanking the inserts. The
ale2-1 and ale1-1 mutations were provided by Hirokazu Tanaka
(Tanaka et al., 2001;
Tanaka et al., 2007). The
acr4-2 mutation has been described previously
(Gifford et al., 2003). The
ARF12 and ARF21 reporter lines were provided by Dolf
Weijers. The reporter line N9185 was obtained from the Haseloff collection of
enhancer trap lines
(http://www.plantsci.cam.ac.uk/Haseloff/construction/GAL4Frame.html).
The marker lines pATML1::GFP-ATML1 and pACR4::H2B-YFP have
been described previously (Gifford et al.,
2003).
To propagate zou mutants, seeds were sterilized and germinated on sterile tissue culture medium comprising 0.5 MS salts (Duchefa), 0.3% sucrose, 1% agar. Seedlings were transferred to soil about 10 days after germination, when first leaves were easily visible.
Protein sequence alignments
Protein sequences similar to ZOU were retrieved using the BLASTP program
(http://www.ncbi.nlm.nih.gov/blast/Blast.cgi)
to search plant genome databases
(http://www.plantgdb.org/).
The Selaginella moellendorffi sequence was retrieved using the
TBLASTN program to query the Selaginella DNA sequence database
(http://selaginella.genomics.purdue.edu/cgi-bin/blast_tmpl_s.cgi).
We used the software NetGene2
(http://www.cbs.dtu.dk/services/NetGene2/)
(Brunak et al., 1991;
Hebsgaard et al., 1996) and
comparisons with the ZOU sequence to predict intron/exon boundaries in the
Selaginella sequence. The protein sequence alignments were generated
using the programs T-Coffee and ClusalW2 available at
http://www.ebi.ac.uk/t-coffee/.
Isolation of ZOU gene and transgene construction
Southern analysis showed that zou-1D plants carried a single
pSKI074 T DNA insertion (data not shown). The T DNA contained a selectable
marker (kanamycin resistance) that co-segregated with zou-1D (data
not shown), suggesting that the insertion was the cause of the mutation. The
sequences flanking the pSKI074 T-DNA insertion at zou-1D were
isolated using the plasmid rescue technique described previously
(Weigel et al., 2000). We
confirmed the predicted intron-exon structure of ZOU by isolating a
cDNA from silique tissue and determining its sequence, which matched that in
databases (Swissprot Accession Number, NM 103864). Total RNA was extracted
using Trizol Reagent (Invitrogen) and RNeasy columns (Qiagen) according to
manufacturer's instructions. First-strand cDNA was prepared from 0.5 µg
total RNA primed with oligo-dT primer using ImProm-II reverse transcription
system (Promega) according to the manufacturer's instructions. The
ZOU cDNA was amplified by PCR using primers ZOU-F
5'-GGCTCTAGATGACTAATGCTCAAG and ZOU-R 5'-CAGGTCGACAACTCAAACCGAAGC.
The resulting product was cloned in the plasmid vector pGEM-T (Promega) to
generate clone pSY3.
To generate the 35S::ZOU construct, the ZOU cDNA was
excised from pSY3 by digestion with XbaI and SalI, and
subcloned into the binary vector pFP101 (generous gift from Francois Parcy)
under control of CaMV 35S promoter to generate clone pSY5. To generate the
ZOU::ZOU-GFP reporter, the Gateway modified primers ZOUGWF
(5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCCTGACCATAACAACCTATATCTC) and ZOUWGR
(5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTAGAGATGAAAAATATAACACCAGTTC) were used
to amplify the ZOU genomic region from plant DNA (Col-0 ecotype) by
PCR with the hi-fidelity Phu DNA polymerase (NEB). The PCR product
was cloned into the Gateway entry vector pDONR207 by recombination with BP
enzyme mix (Invitrogen) to create clone pSY13. The clone pSY13 was then
recombined with the Gateway compatible binary vector pMDC111
(Curtis and Grossniklaus,
2003) to create clone pSY14 encoding an in-frame fusion of GFP to
the C terminus of the ZOU protein. To create the ZOU::H2B-YFP
reporter, a 1.6 kb region upstream of the ZOU ATG codon was amplified
by PCR (as above) using primers ZOUSALIF (5'-GTCGACTGTGGTGGCATAATACGA)
and ZOUSALIR (5'-GTCGACTGCTCATTTTACCCTTTT). The resulting product was
subcloned into the vector pSC-B (Stratagene), excised by digestion with
SalI and cloned into the binary vector pMD4
(Gifford et al., 2003)
containing the H2B-YFP fusion.
Analysis of gene expression
RT-PCR analysis of gene expression was performed as described previously
(Chanvivattana et al., 2004)
using the following primers: ZOU ZOUD3F,
5'-GCTGACTATCTGTGGGAATG; ZOUD3R, 5'-AACTCGGATTTACCTGTGCT;
EiF4A EiF4AF, 5'-TTCGCTCTTCTCTTTGCTCTC; and EiF4A-R,
GAACTCATCTTGTCCCTCAAGTA. In situ hybridization analysis was as described
previously (Chanvivattana et al.,
2004), with the exception that plant tissue was wax embedded using
an automated processor (Leica Tissue Processor TP1050) and embedding centre
(Leica EG 1160). The ZOU probes were made from plasmid pSY3. To make
the ALE1 probe, a region of ALE1 was amplified from silique cDNA
using primers ALE5-2 (5'-TGAAACTAATGACAACATACACTCCC) and ALE3-2
(5'-ACATATCACGATACTTCCAAAAACTGC). The resulting product was subcloned
into pGEM-T vector (Promega). Plasmids were linearized by digestion with
appropriate restriction enzyme, and RNA probes made using T7 or SP6 RNA
polymerase as described previously
(Chanvivattana et al.,
2004).
For real-time PCR measurements, RNA was extracted from seedlings and siliques using Trizol Reagent (Invitrogen) and RNeasy columns (Qiagen) according to manufacturers instruction's. First-strand cDNA was prepared from 0.5 µg total RNA primed with oligo-dT primer using ImProm-II reverse transcription system (Promega) according to manufacturer's instructions. PCR reactions were performed in triplicate and the products quantified using a Rotor gene RG-3000 real-time PCR machine and associated software (Corbett Research) to assay SYBR green fluorescence. PCR reactions were made using SYBR green Jump-Start mix (Sigma). ZOU was amplified using primers ZOUD3F and ZOUD3R (above). ALE1 was amplified using primers ALE1F (5'-CTTCTCAGGCCAAGAAACTC) and ALE1R (5'-TTTGCCAGACTTGTTGAGGA). AtSUC5 was amplified using primers AtSUC5-F (5'-ATCGAAGAAACTTTACGACCAAGG) and AtSUC5-R (5'-TTAACGCTAAGACTCCACTAACC). Results were normalized using the EiF4A gene primers described above.
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Genetic analysis
For double mutant construction, zou-4 mutants were crossed as
females to acr4-2, ale1-1 or ale2-1 homozygotes (all alleles
are in Colombia background, ale2-1 mutants are female sterile). In
all cases, zou-4 homozygotes were pre-selected in the resulting F2
generation by only germinating the shrivelled (zou) seeds. To confirm that
these plants were zou-4 homozygotes, we also tested that their F3
progeny was 100% resistant to sulphadiazine antibiotic (the T-DNA allele at
zou-4 confers resistance). The zou4 acr4-1 mutant was
identified from F2 plants whose immature F3 seeds were round (phenotype
maternally determined by acr4-1 mothers). The ale1-1 zou-4
double mutant was identified by a PCR-based genotyping assay using primers
ALE1GenoF (5'-CGTGCTAGAATAGACGAAGG), ALE1GenoR
(5'-CGTGGTGGAGATGGCAG) and ALE1Ds (5'-CCGTTTTGTATATCCCGTTTCCGT).
The ALE1+ allele produced a 388 bp fragment, whereas ale1-1
harbours a Ds transposon insertion and produces a 300 bp fragment
(Tanaka et al., 2001). Because
ale2 mutants are sterile, we first identified zou-4/zou-4
ale2-1/+ F2 individuals using primers ALE2F
(5'-AGGAACGCTTGATTGGGATG) and ALE2R (5'-GAAGTCAGCAGAGTCTGGTA) (the
ale2-1 mutation introduces a novel XhoI site within the
amplified ALE2 fragment). These plants gave rise to F3 seeds that were
germinated in sterile tissue culture and segregated about one-quarter of
seedlings with severely defective cotyledons. We confirmed that these were
zou-4 ale2-1 double mutants by PCR-based genotyping.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (see Materials and methods; see Fig. S1 in the
supplementary material). The zou-1D mutation harboured a T-DNA
insertion in the promoter of At1g49770, 119 nucleotides upstream of
the ATG start codon (Fig. 1A).
The insertion caused ectopic expression of At1g49770 in leaves, but
did not affect the expression of At1g49760, the other gene
neighbouring the insertion (Fig.
1B). The mis-expression of At1g49770 caused leaf curling
(see Fig. S1 in the supplementary material). However, the biological relevance
of this phenotype is unclear as ZOU is not normally expressed in
leaves and the leaf curling is not obviously related to the normal function of
ZOU. In this study we therefore concentrate on the analysis of
loss-of-function mutations.
The ZOU gene is predicted to encode a transcription factor of the
basic helix-loop-helix (bHLH) class, designated bHLH95 in previous analyses of
the Arabidopsis genome (Heim et
al., 2003; Toledo-Ortiz et
al., 2003). Genome database searches revealed that the ZOU protein
is widely conserved in plants (see Fig. S2 in the supplementary material):
homologues were found both in other dicotyledonous species, including grape
(Vitis vinifera) and barrel medic (Medicago truncatula) and
also in more distantly related angiosperms (e.g. rice, a monocot);
furthermore, ZOU homologues were also found outside flowering plants in the
gymnosperms [Sitka spruce (Picea sitchensis)] and club mosses
(Selaginella moellendorffii). Protein sequence alignments showed that
two regions of the ZOU protein were well conserved: the bHLH domain (residues
66-124) and a region of unknown function near the C terminus of the protein
(residues 215-301, see alignment in Fig. S2 in the supplementary material). It
is likely that these proteins are ZOU orthologues as each is more similar to
ZOU than to any other Arabidopsis protein. Using these criteria, no
clear ZOU orthologues were identified in the bryophyte Physcomitrella
patens or in the green alga Chlamydomonas reinhardtii. The
ZOU gene is therefore ancient and is likely to have arisen early in
vascular plant evolution. In addition, the Arabidopsis ZOU protein is
more similar to homologues in other plant species than to any of the other
Arabidopsis bHLH proteins [estimated to be between 132 and 146 in
number (Heim et al., 2003;
Toledo-Ortiz et al., 2003)],
suggesting that it is a unique gene in Arabidopsis.
Loss-of-function mutations show that ZOU normally functions during seed development
A previous study of Arabidopsis bHLH genes suggested that
ZOU (there termed bHLH95) expression was specific to flowers and/or
siliques (Heim et al., 2003).
Quantitative RT-PCR (Fig. 1C)
confirmed and extended this: ZOU was not expressed in whole
seedlings, rosette leaves or unopened flower buds, but was detected at a low
level in opened flowers and more strongly in siliques, suggesting that it
normally functions in siliques or seed development. To determine the
loss-of-function phenotype, we obtained three independent alleles (zou-2,
zou-3 and zou-4) from collections of T-DNA insertion lines
(Fig. 1A). All three alleles
conferred very similar phenotypes, with zou-2 being most severe,
consistent with the location of the T-DNA insertion in the first exon. Plants
heterozygous for zou were normal; however, when self-pollinated about
one quarter of their seeds (see Table
1) were mis-shapen and shrivelled at maturity
(Fig. 2A,B). In zou-2
homozygotes, all seeds were shrivelled, but when they were crossed either as
male or female parent to wild type, the resulting seeds were normal
(Table 1). Together, these
observations indicated that zou-2-4 are recessive loss-of-function
mutations with normal transmission through male and female gametophytes and
that ZOU acts zygotically (rather than maternally) to control seed
development. The seed shrivelling phenotype co-segregated with antibiotic
resistance conferred by the T DNA insertion at zou-2 (data not shown)
and could be complemented by transformation with a genomic ZOU clone
(Fig. 2C;
Table 1), confirming that it
was caused by loss of ZOU function. We renamed the
At1g4977/bHLH95 gene ZHOUPI (Chinese for `shrivelled') in
reference to the seed phenotype.
|
) have
been attributed to impaired cuticle formation during embryogenesis. To examine
surface development in zou embryos, seeds were embedded in resin,
stained with osmium tetroxide and ultra-thin sections examined using
transmission electron microscopy (TEM). Wild-type torpedo stage embryos were
covered by a cell wall with a continuous electron dense cuticularized layer
(Jeffree, 2006) (CL). An
overlying translucent cuticle proper
(Jeffree, 2006) (CP) was also
present, although visualization of this at high resolution in seed tissue is
challenging. We did not observe any obvious discontinuities in the CL of
zou mutant embryos at the same developmental stage, but no CP was
apparent, and the cuticularized cell wall was extensively fused to endosperm
cell wall (Fig. 2K,L).
The separation of embryo from endosperm during angiosperm embryogenesis
correlates with breakdown of the endosperm cells surrounding the developing
embryo (Briggs, 1993). As this
separation does not occur efficiently in zou mutants, we examined
whether the endosperm is more persistent in zou seeds than in wild
type. After imbibition of mature wild-type seeds, the embryo could easily be
separated from the single layer of residual endosperm cells by extrusion. By
contrast, in zou mutant seeds, the embryo and endosperm adhered
strongly. Furthermore, the chalazal pole of the endosperm, which was not
invaded by the embryo, formed a sac-like structure. When the embryo was cut
away, a considerable quantity of abnormal paste-like tissue was extruded from
the endosperm cavity (see Fig. S3 in the supplementary material).
ZOU is needed for normal epidermal development in seedlings
The shrivelled zou seeds were viable and germinated to produce
seedlings that survived when grown in conditions of high humidity, but
desiccated and died when grown in low humidity. This phenotype is commonly
found in mutants with defects in cuticle or epidermis that impair water
retention. We therefore stained whole seedlings with Toluidine Blue, as this
has been shown to provide a rapid test for cuticle and/or epidermal defects
(Tanaka et al., 2004). Whereas
wild-type plants were unstained, in zou mutants the hypocotyl and the
cotyledons, but not the leaves, stained strongly
(Fig. 3A,B). This suggested
that the defects in zou mutants were restricted to organs that
initiate during embryogenesis (i.e. hypocotyls and cotyledons, but not
leaves), consistent with the seed-specific expression of ZOU
(Fig. 1C;
Fig. 4). Furthermore, when
zou mutants were grown in tissue culture and transferred to soil
after the first leaves had initiated, the mutants recovered and gave rise to
normal plants, again suggesting that ZOU did not affect
post-embryonic organ development.
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Genetic interactions of ZOU
We combined zou mutations with other mutations implicated in
epidermal development, including acr4, ale1 and ale2 (see
Materials and methods). The zou-4 mutation was epistatic to
ale1-1 in double mutants, suggesting that ZOU and
ALE1 might act in the same pathway (data not shown). The zou-4
acr4-2 seedlings had more severely defective cotyledons than either
single mutant, suggesting that ZOU and ACR4 may be involved
in a common developmental process (Fig.
3H). The zou-4 ale2-1 seedlings also had more severely
defective cotyledons than either single mutant
(Fig. 3I), consistent with
previous observations that ale1 mutations enhance ale2
(Tanaka et al., 2007).
ZOU is expressed in the ESR of endosperm
To determine where ZOU was expressed, we localized ZOU
mRNA by in situ hybridization to sections of developing seeds. We detected
strong expression in the endosperm in the ESR in seeds containing embryos up
to the torpedo stage, after which expression declined
(Fig. 4A-C). No signal was
detected when sense control probes were hybridized, confirming that the signal
with antisense probes was specific for ZOU
(Fig. 4D). Strikingly, we did
not detect expression either in the embryo itself or in any of the maternal
tissues in the seeds or siliques. The effects of ZOU on the embryonic
epidermis are therefore non cell autonomous. To further characterize
ZOU expression, we made two reporter gene constructs: a gene fusion
in which the entire ZOU genomic region (i.e. upstream promoter and
intragenic sequences) was fused in frame to GFP (ZOU::ZOU-GFP); and a
fusion of the ZOU upstream promoter sequences to a HISTONE 2B-YFP
fusion protein (Boisnard-Lorig et al.,
2001) (ZOU::H2B-YFP). Both constructs showed similar
expression patterns. The ZOU::ZOU-GFP construct complemented
zou-2 mutants (Fig. 2C
and Table 1), confirming that
the ZOU-GFP protein fusion retained ZOU+ activity and that the
construct was expressed correctly. The ZOU-GFP fusion protein was
nuclear-localized, consistent with the predicted function of ZOU as a
transcription factor, and was expressed in the ESR but not in the embryo
(Fig. 4E). The non
cell-autonomous effects of ZOU on the embryonic epidermis are therefore not
due to the ZOU protein moving from endosperm to embryo. The ZOU-GFP fusion
protein expressed from ZOU::ZOU-GFP was below the limits of detection
by confocal microscopy until the early heart stage, possibly owing to
post-transcriptional regulation. For this reason, we characterized the early
expression of ZOU using the ZOU::H2B-YFP construct.
ZOU was not expressed in the ovule prior to fertilization
(Fig. 4F) but was strongly
expressed in the central cell after fertilization
(Fig. 4G). This is consistent
with Q-PCR data which showed that ZOU expression was undetectable in
unopened flower buds, but was detectable in opened flowers shortly after the
point of fertilization (Fig.
1C). It was expressed uniformly throughout the endosperm during
the early stages of its development when it comprised two to eight nuclei
(Fig. 4H,I), but then became
expressed more strongly at the micropylar pole during the 12-16 nuclei stage
(Fig. 4J) and the 24-28 nuclei
stages (Fig. 4K); it was
largely restricted to the ESR by the 44-48 cell stage
[Fig. 4L, stages as defined
previously (Boisnard-Lorig et al.,
2001)].
|
) and
zou mutants are epistatic to ale1 mutants, suggesting that
they might act in a common pathway. To test whether ZOU regulates
ALE1, consistent with the likely role of ZOU as a transcription
factor, we localized ALE1 expression in young siliques of wild-type
and zou-2 homozygotes. ALE1 was strongly expressed in the
ESR of the endosperm but not in the embryos of wild-type seeds
(Fig. 5A,B). In zou-2
mutants, ALE1 expression was barely detectable at globular and heart
stages (Fig. 5C,D), indicating
that ZOU regulates ALE1 expression. To test whether
ZOU had a general role in regulating ESR identity, or rather was
specific for genes such as ALE1 (which have a role in embryo
development), we analyzed the expression of AtSUC5, a sucrose
transporter that is also expressed specifically in the ESR
(Baud et al., 2005), using
quantitative RT-PCR. Unlike ALE1, which showed an approximately
fivefold decrease in expression, there was no significant change in
AtSUC5 expression in zou mutants (see Fig. S4 in the
supplementary material). Likewise, the expression of reporters for the
ESR-specific AUXIN RESPONSE FACTOR 12 (ARF12) and
ARF21 genes (D. Weijers, personal communication) was unaffected in
zou mutant seeds (data not shown), confirming that ZOU does
not regulate all ESR-specific genes. However, the enhancer trap line N9185,
which is expressed in wild-type ESR
(Ingouff et al., 2005) was not
expressed in zou-4 (Fig.
5E,F), suggesting that the gene responsible for the N9185 pattern
may also be a target of ZOU regulation. The enhancer trap T-DNA in
N9185 is located on chromosome 4 (data not shown), and is therefore not
associated with ALE1 (At1g62340). Because zou mutant seedlings had epidermal defects, we also introduced reporters for ACR4 and ATML1, which show epidermal-specific expression. Both reporters were expressed normally in zou-4 mutant embryos (Fig. 5G-J), consistent with the fact that protoderm specification appears largely normal in cleared and sectioned zou seeds.
ZOU is not sufficient to activate ALE1
The fact that ZOU is needed for ALE1 expression in seeds
raised the possibility that the curled leaves of zou-1D mutants could
be a result of ALE1 being activated by ZOU ectopically in
leaves. To test this, we analysed ALE1 and ZOU expression in
seedlings by quantitative RT-PCR. Consistent with our RT-PCR results
(Fig. 1B), ZOU
expression was not detectable in wild-type seedlings, whereas in
zou-1D/+ seedlings it was readily detectable at about six times the
level in wild-type siliques. No ALE1 expression was detectable in
wild-type or zou-1D seedlings (see Fig. S5 in the supplementary
material). Therefore, ZOU expression is not sufficient to activate
ALE1 outside of the seed and the effects of ZOU
mis-expression may be unrelated to the normal function of ZOU in
seeds.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Massonneau et al.,
2005; van Hengel et al.,
1998; Wiweger et al.,
2003). However, the first concrete evidence that the endosperm
might play a key role in Arabidopsis embryo development, distinct
from its role in embryo nutrition, came only recently, with the
characterization of the ESR gene ALE1
(Tanaka et al., 2001). We show
that ZOU regulates ALE1 expression in endosperm, consistent
with the similar expression patterns of the two genes. However, the zou
phenotype is not simply a consequence of a loss of ALE1 activity
because ale1 mutants are much less severe than zou mutants.
In our growth conditions, ale1 mutant seeds did not shrivel, they
produced normal sized embryos and the epidermal morphology of their seedlings
was only mildly affected. It is likely that ZOU regulates many
targets additional to ALE1, as is supported by the loss of the N9185
reporter gene expression in zou mutants. Previous analyses of the
Arabidopsis bHLH family indicated that all residues crucial for
DNA-binding activity and dimerization are conserved in the ZOU bHLH and that
it is likely to bind the G box sequence CACGTG
(Heim et al., 2003;
Toledo-Ortiz et al., 2003).
Because mis-expression of ZOU in leaves is insufficient to activate
ALE1, ZOU probably requires additional factors for activity. One
possibility is that it acts as a heterodimer with another bHLH member, as is
the case for several plant bHLH proteins
(Heim et al., 2003;
Toledo-Ortiz et al., 2003).
Because ZOU is expressed in rather few cells in seeds (i.e. the ESR of
endosperm), it is technically difficult to demonstrate direct binding in vivo
using techniques such as chromatin immunoprecipitation.
The ESR of the angiosperm endosperm is distinct structurally, and also in
terms of gene expression, from the rest of the endosperm
(Berger, 2003;
Olsen, 2004). In both cereals
and Arabidopsis, where the first few divisions of the central cell
give rise to a free nuclear syncitium, cellularization is initiated in the
ESR. The ESR endosperm is also densely cytoplasmic and is closely associated
with the developing embryo at early stages. This association is probably key
for provision of nutrients to the young embryo, a supposition that is
supported by the ESR-specific expression of the sucrose transporter AtSUC5,
which is required for normal embryo growth in Arabidopsis
(Baud et al., 2005). ZOU is the
first transcription factor to be identified with an ESR-specific expression
pattern, raising the possibility that it specifies the identity of the ESR
region of the endosperm. We feel that this is unlikely for three reasons:
first, the early morphology and development of zou endosperm,
including the ESR, is indistinguishable from that of wild type; second, the
expression of some ESR genes, such as AtSUC5, ARF12 and
ARF21, is not affected in zou mutants; finally, the
expression of ZOU does not become wholly restricted to the ESR until
the early heart stage. Therefore, rather than specifying the identity of the
ESR region, it seems more likely that ZOU has more specific roles in endosperm
and or embryo development.
|
). We therefore suggest that ZOU regulates
signalling between the endosperm ESR and the embryo that promotes normal
cuticle deposition and is probably mediated by receptor-like kinases such as
GSO1 and GSO2 in the embryo. Such signalling is probably two-way: ZOU
expression becomes gradually restricted to the ESR as endosperm development
progresses. The maintenance of ZOU expression in the endosperm cells
directly in contact with the embryo may therefore be the result of signalling
between the embryonic epidermis and the endosperm. Whether cuticle deposition
is entirely mediated by the embryonic epidermal cells, or involves secreted
compounds originating from the endosperm, is, as yet, unclear.
In Arabidopsis and many other dicotyledonous plants, the
cellularized endosperm progressively degenerates as it is invaded by the
expanding embryo, so that only a single layer - the aleurone - remains at seed
maturity. By contrast, in mature zou seeds, much more endosperm
persists, including non-aleurone tissues. Therefore, a likely second role of
ZOU is to promote the expression of genes involved in degradation of
endosperm cells around the expanding embryo. The ZOU target
ALE1 encodes a subtilisin-like serine protease that has been proposed
to process ligands involved in epidermal specification/function
(Watanabe et al., 2004).
However, it is also possible that ALE1 acts instead in the separation
of the developing endosperm from the embryonic epidermis; for example, by
promoting endosperm autolysis.
ZOU is therefore the first transcription factor to be identified that mediates two poorly understood processes in seed development - separation of the embryo from the endosperm and breakdown of the endosperm. Determining the targets of ZOU; for example, by transcriptional profiling of mutant seeds, should help identify the key factors that mediate these processes.
The ancestral role of ZOU
It is striking that ZOU is so widely conserved in plants. Within
angiosperms, it is likely that the role of ZOU in the ESR is
conserved as the rice ZOU homologue also shows seed-specific
expression (Li et al., 2006).
However, ZOU is also found both in seed plants that lack an endosperm
(e.g. Picea sitchensis, a gymnosperm) and also in more basal vascular
plant groups, such as the club mosses, which lack seeds altogether. A common
feature of all these groups of land plants is that they have a specialized
epidermis with cuticle, and that their embryos enlarge by invading and
digesting surrounding maternal nutritive tissues (see Fig. S6 in the
supplementary material). In gymnosperms, the female gametophyte proliferates
to form a nutrient-rich tissue prior to fertilization, and the growth of the
embryo post-fertilization is invasive (see Fig. S6B in the supplementary
material). Although club mosses such as Selaginella lack a seed, the
embryo is similarly thrust into a nutrient-rich megagametophyte by a suspensor
(see Fig. S6C in the supplementary material). It is therefore possible that
the ancestral role of ZOU is to promote the separation of the embryo
from surrounding gametophytic tissues, and the breakdown of these tissues,
similar to its role in angiosperm endosperm. This would also be consistent
with the current consensus that the endosperm is evolutionarily homologous to
the gymnosperm female gametophyte [see Baroux et al.
(Baroux et al., 2002) for
recent discussion]. To test this further, it will be necessary to determine
whether ZOU is also expressed in the ESR of the female gametophyte in
these groups.
Note added in proof
While this article was under review, Kondou et al.
(Kondou et al., 2008)
published on RETARDED GROWTH OF EMBRYO1. The genes ZHOUPI and
RETARDED GROWTH OF EMBRYO1 both correspond to At1g49770.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/21/3501/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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