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First published online 10 July 2006
doi: 10.1242/dev.02463
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1 Department of Biology and the Huck Institutes of Life Sciences, the
Pennsylvania State University, University Park, PA 16802, USA.
2 Department of Gene Technology, Tallinn University of Technology, Akadeemiatee
15, Tallinn 19086, Estonia.
3 Institute of Plant Biology, Zollikerstrasse 107, Zurich CH-8008,
Switzerland.
Author for correspondence (e-mail:
hxm16{at}psu.edu)
Accepted 30 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Pollen development, DYT1, SPL/NZZ, EMS1/EXS, Tapetum, bHLH Transcription factor
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Molecular genetic
studies have revealed that the B and C functions of the wellknown ABC model
together determine the stamen identity, whereas the C function alone specifies
the carpel identity (Coen and Meyerowitz,
1991; Ma, 2005).
In Arabidopsis, APETALA3 (AP3) and PISTILLATA
(PI) are two essential B function genes, and AGAMOUS
(AG) is required for C function
(Coen and Meyerowitz, 1991;
Ma, 2005). The
Arabidopsis anther is a bilaterally symmetrical structure with four
lobes. Each lobe comprises four distinct somatic cell layers from outer to
inner: they are the epidermis, endothecium, middle layer and tapetum
(Goldberg et al., 1993;
Sanders et al., 1999). The
tapetum surrounds meiocytes and generates many proteins, lipids,
polysaccharides and other molecules necessary for pollen development
(Goldberg et al., 1993).
Accordingly, the tapetum is characterized by active protein synthesis and
secretion, a high rate of energy metabolism, and expression of many
tapetum-preferential genes (Dickinson and
Bell, 1976; Hernould et al.,
1998; Liu and Dickinson,
1989; Rubinelli et al.,
1998; Scott et al.,
2004; Taylor et al.,
1998; Zheng et al.,
2003).
Several genes have been identified that are required for normal anther
development (Ma, 2005). For
example, the SPOROCYTELESS/NOZZLE (SPL/NZZ) gene is required
for cell type specification in both male and female reproductive organs
(Schiefthaler et al., 1999;
Yang et al., 1999). The
spl/nzz mutant anthers lack the endothecium, middle layer, tapetum
and meiocytes. Recently, Ito et al. (Ito
et al., 2004) found that SPL/NZZ is a direct target of
AG, and that ectopic expression of SPL/NZZ, independent of AG,
induces the formation of anther locule with pollen grains
(Ito et al., 2004).
SPL/NZZ encodes a putative transcription factor
(Schiefthaler et al., 1999;
Yang et al., 1999), suggesting
that SPL/NZZ regulates anther cell differentiation by activating
downstream genes.
Furthermore, EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS CELLS
(EMS1/EXS) and TAPETUM DETERMINANT1 (TPD1) are
early (pre-meiosis) genes that encode a receptor-like protein kinase and a
small protein, respectively, and act in the same genetic pathway to control
the tapetal cell identity (Canales et al.,
2002; Sorensen et al.,
2003; Yang et al.,
2003; Yang et al.,
2005; Zhao et al.,
2002). Recently, the SERK1 and SERK2 genes
encoding closely related receptor-like protein kinases were shown to have
redundant functions in controlling tapetum formation
(Albrecht et al., 2005;
Colcombet et al., 2005).
MALE STERILITY1 (MS1) and ABORTED MICROSPORES
(AMS) act after meiosis and encode a PHD domain-containing protein
and a bHLH transcription factor, respectively; they are essential for late
stage functions of the tapetum (Ito and
Shinozaki, 2002; Sorensen et
al., 2003; Wilson et al.,
2001). Remarkably, the `early' gene EMS1/EXS is expressed
in both stamen and gynoecium, whereas the `late' genes MS1 and
AMS are anther specific. However, it is not known how the
`male-specific' MS1 and AMS expression is regulated.
Here, we report the isolation of a new Arabidopsis mutant, which is male sterile and defective in tapetum differentiation and function. Because the mutant has an abnormal tapetum, we named the gene DYSFUNCTIONAL TAPETUM1 (DYT1). DYT1 encodes a putative bHLH transcription factor and is preferentially expressed in tapetal cells as early as anther stage 5, spatially similar to, but temporally earlier than, MS1 and AMS. Furthermore, our results suggest that DYT1 probably acts downstream of SPL/NZZ and EMS1/EXS, and is required for normal expression of AMS, MS1 and other tapetumpreferential genes. We propose that DYT1 is a component of a genetic network for tapetum differentiation and function.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
ems1 (Zhao et al.,
2002) and tpd1 (Yang
et al., 2003) mutants were kindly provided by Drs W. Yang, D. Zhao
and D. Ye, respectively. The dyt1 mutant was isolated from the
progeny of a Ds insertional line (ET4262). The ems1 dyt1
double mutant was obtained by pollinating the dyt1 pistil with pollen
from an ems1/+ plant. Plants were grown under long-day conditions (16
hours light/8 hours dark) in a 
22°C growth room.
Characterization of mutant phenotypes
Plants were photographed with a Sony digital camera, DSC-F707 (Sony Corp.,
Tokyo, Japan). Flower pictures were taken using a Nikon dissecting microscope
(Nikon Corp., Tokyo, Japan) with an Optronics digital camera (Optronics,
Goleta, CA, USA). To determine pollen viability, mature anthers were stained
with the Alexander solution (Alexander,
1969) and photographed under an Olympus BX-51microscope (Olympus,
Tokyo, Japan) with a SPOT II RT camera (Diagnostic Instruments, Sterling
Heights, MI, USA). Chromosome spreading and DAPI staining were performed as
described (Ross et al., 1996).
Wild-type and mutant inflorescences were collected and fixed as described
(Zhao et al., 2002). Floral
buds were embedded in Spurr's resin; semi-thin (0.5 µm) sections were made
by using an Ultracut E ultramicrotome (Leica Microsystems, Nussloch, Germany),
stained with 0.05% of Toluidine Blue O for 40 to 60 seconds, and photographed
under the Olympus BX-51 microscope with the SPOT II RT camera.
Mapping and functional complementation
The dyt1 mutation was found to be distinct from the Ds
insertional locus (not shown). The dyt1 mutant (in the Ler
background) was crossed with Columbia to obtain F1 and F2 seeds. About 500 F2
plants with mutant phenotypes were genotyped by using the SSLP and dCAPS
markers (Li et al., 2001).
Several predicted genes were found in the mapped region flanked by
recombination events; these genes were amplified from wild-type and
dyt1 plants and sequenced. TAIL PCR
(Liu and Whittier, 1995) was
used to determine the sequences of a retrotransposon insertion in the
dyt1 mutant. To verify the DYT1-coding region, we used the
primers oMC1872/oMC1873 (see Table
1 for all primer sequences), designed according to the annotated
At4g21330 locus, to amplify wild-type Ler cDNA.
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RT-PCR and real-time PCR
A set of genes were selected for expression analysis by RT-PCR because they
are known to be important for male reproduction: SPL/NZZ
(Schiefthaler et al., 1999;
Yang et al., 1999),
EMS1/EXS (Canales et al.,
2002; Zhao et al.,
2002), TPD1 (Yang et
al., 2003; Yang et al.,
2005), AMS (Sorensen
et al., 2003), MS1
(Ito and Shinozaki, 2002;
Wilson et al., 2001), MALE
STERILITY2 (Aarts et al.,
1997), MALE STERILITY5
(Glover et al., 1998),
AtMYB32 (Preston et al.,
2004), AtMYB103
(Higginson et al., 2003), A6
and A9 (Sorensen et al.,
2003), AtMYB33 and AtMYB65
(Millar and Gubler, 2005),
SOLO DANCERS (SDS) (Azumi
et al., 2002), and ROCK-N-ROLLERS/AtMER3
(RCK/AtMER3) (Chen et al.,
2005; Mercier et al.,
2005). A second group of 16 genes was identified using microarray
data of wild-type and ems1 mutant anthers we had obtained in our
laboratory (W.Z., Y.S., A. Wijeratne and H.M., unpublished). The genes that
were expressed in the ems1 anther at levels that were one half or
less of those in the wildtype anthers were regarded as candidate
tapetum-preferential genes because the ems1 anthers lack tapetum. In
addition, UBQ1 and AtMYB4
(Vannini et al., 2004) were
used as constitutive expression controls. The primers for RTPCR and relevant
microarray data are provided in the Tables
1 and
2, respectively. The primers
for real-time PCR are listed in Tables
1 and
3. The PCR and data treatment
were carried out as described previously
(Ni et al., 2004). Plant
tissue was collected and quickly frozen in liquid nitrogen. The anthers at
approximately anther stages 4 to 7 were collected under a dissection
microscope. Total RNA was extracted using the RNeasy Plant Kit (Qiagen,
Valencia, CA) from young inflorescences (approximately floral stage 1-10). 1-2
µg total RNA was used for reverse transcription according to the
manufacturer's instruction to synthesize cDNA, which was used directly as PCR
templates (Invitrogen, Carlsbad, CA).
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). A 624 bp
DYT1 cDNA fragment was amplified using DYT1-specific primers
with XbaI and BglII sites at the 5' end: oMC2238 and
oMC2239, respectively. The PCR product was confirmed by sequencing and cloned
into the pGEM-T vector (Promega, Madison, WI) to yield plasmid pMC2949.
Plasmid DNA was completely digested by XbaI or BglII and
used as template for transcription with SP6 or T7 RNA polymerases,
respectively (Roche, Mannheim, Germany). Images were obtained using the
Olympus BX-51 microscope with the SPOT II RT camera and edited using PHOTOSHOP
5.0 (Adobe, San Jose, CA).
Overexpression of DYT1
A DYT1 cDNA fragment was amplified using gene-specific primers
oMC1872 and oMC1873 (Table 1),
then cloned into pGEM-T vector to yield plasmid pMC2941. After verification by
sequencing, the DYT1 cDNA fragment was subcloned into pCAMBIA1300
downstream of the CaMV 35S promoter to produce the plasmid pMC2942. An
Agrobacterium strain C3581 carrying pMC2942 was used to transform
wild-type, ems1/+ and spl/+ plants. The transgenic plants
were selected by hygromycin resistance and verified using PCR with oMC1872 and
oMC1873. EMS1 gene-specific primers oMC499 and oMC500,
SPL/NZZ gene-specific primers oMC2044 and oMC2045, plus Ds5-specific
primer oMC490 were used to identify ems1 and spl
heterozygous and homozygous plants, respectively. Paraffin sections were
prepared as described for in situ experiments (above) and photographed as
described for semi-thin sections.
Phylogenetic analysis of the DYT1 subfamily
The protein sequences of the nine genes from group 9 of the
Arabidopsis bHLH family, including DYT1, were used to search
for the closest homologs in both the rice genome
(http://tigrblast.tigr.org/euk-blast/index.cgi?project=osa1)
and Populus genome
(http://www.floralgenome.org/cgi-bin/tribedb/tribe.cgi)
using both BLAST and TBLASTN programs with a cutoff of 1E-15
(Heim et al., 2003;
Toledo-Ortiz et al., 2003).
The multiple sequence alignment of full-length protein sequences was performed
using ClustalX (Plate-Forme de Bio-Informatique, Illkirch Cedex, France) with
a combination of GOP=4.0 and GEP=0.1. The bHLH domain region and additional
conserved regions were aligned and used to perform neighbor joining (NJ)
analyses with the `pairwise deletion' option, `P-distance' model and 1000
bootstrap replicates test using MEGA version 3.0
(Kumar et al., 2004)
(http://www.megasoftware.net/index.html).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The dyt1 mutant is defective in tapetum development
Detailed analyses were performed to understand the mutant developmental
defects. Chromosome spread experiments were performed and revealed that normal
meiotic features could be observed in the mutant meiocytes, demonstrating that
meiotic nuclear divisions can proceed normally
(Fig. 2). We also generated
semi-thin anther sections to investigate the mutant anther development
(Fig. 3). From anther stage 1
to 3, the dyt1 anthers appeared normal (data not shown). At stage 4,
the mutant anther was similar to the wild-type anther in cell layer and cell
number, but had a slightly different shape from the wild-type anther and was
vacuolated in more cells than normal. In addition, the mutant sporogenous
cells appeared more deeply stained than wild-type cells
(Fig. 3A,B). At early stage 5,
the dyt1 anther lobe had four cell types interior to the epidermis,
similar to the wild type. The wild-type anther at late stage 5
(Fig. 3E) was also vacuolated
in more cells than earlier and had deeply stained meiocytes. Therefore, mutant
anthers at stage 4 and early stage 5 exhibited these morphological features
precociously (Fig. 3A-D).
In the wild-type anther at late stage 5 (Fig. 3E), the tapetum had significantly larger cells than earlier. In the mutant (Fig. 3F), additional vacuoles were observed in tapetal cells, with a reduction of the cytoplasm. The vacuoles in the wild-type tapetum at this time are fewer and smaller than those in the mutant. In addition, the mutant middle layer maintained its thickness with vacuolation, unlike the reduced thickness of the wild-type middle layer (Fig. 3E,F). At stage 6, a thick callose wall forms around the meiocytes (Fig. 3G). By contrast, the mutant meiocytes had very thin callose cell walls (Fig. 3G,H). At this stage, most of the mutant meiocytes were undergoing meiosis (Fig. 2), but some of them had collapsed. At stage 7 and stage 8, the completion of wild-type meiosis results in the formation of tetrads and then microspores, but mutant tapetal and middle layer cells swelled with expanded vacuoles and filled the center of the locules where the meiocytes had collapsed and degraded (Fig. 3I-L).
DYT1 encodes a putative bHLH transcription factor
To gain further insights into its function, we cloned the DYT1
gene. The dyt1 mutant was from a Ds insertional line, but
the dyt1 mutation was genetically separable from the Ds
element (data not shown). To clone the gene, we mapped the dyt1 locus
by analyzing 500 mutant F2 progenies from a cross between the
dyt1 mutant (Ler) and Columbia wild-type plant. The mapping
results indicated that the DYT1 gene was on chromosome 4, between
At4g21220 and At4g21360 (data not shown). We sequenced candidate genes in this
region from both wild-type and the dyt1 mutant, and found that only
one locus, At4g21330, had an insertion mutation 109 bp upstream of the
predicted translation initiation codon
(Fig. 4A). We performed TAIL
PCR to obtain the DNA at the 5' and 3' ends of the insertion and
found that they each partially matched the same region of a putative
retro-transposon At5g33382 in the Columbia genomic sequence. Further PCR and
sequence analysis indicated that the insertion at the DYT1 locus
matched exactly and completely to a seemingly intact retro-transposon in the
Ler genome (W.Z., Y.S. and H.M., unpublished). To verify that
At4g21330 is DYT1, we cloned an At4g21330 genomic fragment into a
modified pCAMBIA1300 plasmid and used it to transform dyt1/+ plants.
Fifty independent transgenic plants were analyzed; 12 lines were found to be
homozygous for the dyt1 insertion. All 12 lines were fertile,
including nine lines with normal fertility
(Fig. 1E,H), confirming that
At4g21330 is the DYT1 gene.
|
).
Interestingly, a preliminary search of public databases using the DYT1 protein
sequence with the BLAST program showed that the DYT1 protein has the highest
similarity to the Arabidopsis AMS protein
(Sorensen et al., 2003).
To gain additional insights into the phylogenetic relationship between
DYT1, AMS and other close homologs, we performed phylogenetic
analysis of bHLH genes, including DYT1, AMS and a recently reported
rice gene, UNDEVELOPED TAPETUM (OsUDT1), which is required
for normal tapetum development (Jung et
al., 2005). The phylogenetic analyses with the bHLH domain region
alone, with both bHLH domain and conserved regions
(Fig. 4C), or with the
full-length sequences yielded trees with very similar topologies (others not
shown). Our result indicates that DYT1, AMS, OsUDT1, Os02g02820 and
PtDYT1-Like (Populus trichocarpa) form a separate clade
within a group of related members of the bHLH gene family. Among them,
AMS and Os02g02820 are supported as an orthologous pair, as are
DYT1 and PtDYT1-like. In Arabidopsis, DYT1 and
AMS are most closely related to each other, in agreement with the
preliminary BLAST results. The rice OsUDT1 gene could be the ortholog
of the DYT1 and PtDYT1-like genes.
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17% of the wild-type level
(Fig. 5B). These results
suggest that DYT1 might be downstream of SPL/NZZ and
EMS1/EXS. We performed RNA in situ experiments to verify this
possibility. In the spl anther, little DYT1 expression could
be detected (Fig. 5J). A weak
signal could be detected in the meiocytes in the ems1 anther at late
stage 5 (Fig. 5K) and early
stage 6, but, unlike the wild-type anther, the strong tapetal signal was not
found in the ems1 anther (compare
Fig. 5H with 5K). Therefore,
the strong DYT1 expression in the tapetum requires EMS1/EXS.
Both the real-time PCR and the RNA in situ hybridization results indicate that
SPL/NZZ is essential for DYT1 expression and that
EMS1/EXS promotes the high-level DYT1 expression specific to
the tapetum.
The expression of anther genes in the dyt1 mutant is altered
The finding that DYT1 encodes a bHLH-type putative transcription
factor suggests that DYT1 controls gene expression required for
normal anther development. To test this hypothesis, we performed RT-PCR using
primers for anther genes. We obtained results for a total of 32 genes
(Fig. 6); among these,
SPL/NZZ, EMS1/EXS and TPD1 are known early anther
development genes (Canales et al.,
2002; Schiefthaler et al.,
1999; Yang et al.,
1999; Yang et al.,
2003; Yang et al.,
2005; Zhao et al.,
2002). The other genes were chosen for their tapetum-preferential
expression according to either previous reports or to gene expression data
obtained in our laboratory (Table
2). We found that for 21 genes out of 32, the expression was
significantly reduced in the dyt1 mutant compared with the wild type
(Fig. 6), indicating that
indeed the normal expression of a large number of genes depends on the
DYT1 gene function. In particular, two regulatory genes, MS1
and AMS, which are important for tapetum development
(Ito and Shinozaki, 2002;
Sorensen et al., 2003;
Wilson et al., 2001),
exhibited greatly reduced levels of expression, suggesting that MS1
and AMS act downstream of DYT1. By contrast, some tapetum
preferential genes, such as A6 and A9, were still expressed in the
dyt1 mutant at slightly reduced levels. In addition, the expression
of SPL, TPD1, EMS1/EXS, AtMYB33 and AtMYB65 was not
dramatically different in the dyt1 mutant, indicating that their
expression does not require DYT1. To verify the RT-PCR results,
selected genes were further analyzed using real-time PCR and the results
(Fig. 6D) support the
conclusion that AMS and MS1 expression requires
DYT1 function.
In addition to the tapetum-preferential genes, we also tested the
expression of two meiosis-specific genes: SDS and RCK/AtMER3
(Azumi et al., 2002;
Chen et al., 2005;
Mercier et al., 2005).
Although the expression level of SDS did not change, the expression
of RCK/AtMER3 was significantly reduced. SDS is known to act
earlier than RCK/AtMER3 in prophase I, suggesting that the
dyt1 mutation might affect the expression of late prophase I genes
more than early prophase I genes.
DYT1 is not sufficient for tapetum development
Both ems1/exs and dyt1 mutations affect tapetum
development. Previous reports and our results suggest that DYT1 acts
downstream of EMS1/EXS. To test this further, we generated the
ems1 dyt1 double mutant and examined its early anther development. We
found that the double mutant resembles the ems1 mutant in that the
double mutant anther also completely lacks the tapetum
(Fig. 7), suggesting that
indeed EMS1/EXS is upstream of DYT1 in the same pathway. In
addition, to test whether DYT1 is sufficient to alter anther
development, we generated transgenic plants carrying a 35S-DYT1
fusion in wild-type, spl/nzz and ems1/exs
backgrounds. Transgenic lines with DYT1 overexpression were
identified by RTPCR (Fig. 6E;
data not shown) and analyzed for their anther morphology. In all cases, the
35S-DYT1 transgenic anthers had morphologies resembling those of the
corresponding genotypes without the transgene (see Fig. S1 in the
supplementary material). Therefore, the overexpression of DYT1 was
not able to suppress the spl/nzz and ems1/exs mutant
phenotypes, indicating that other genes acting downstream of SPL/NZZ
and EMS1/EXS are probably required for normal tapetum development.
Because DYT1 was found to be required for normal expression of a
number of tapetum genes, we tested selected genes by real-time PCR to
determine whether the 35S-DYT1 transgene was able to stimulate the
expression of these genes. Our results indicate that the 35S-DYT1
transgene did not alter the expression of these genes substantially
(Fig. 6E), consistent with the
morphological results.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Canales et al., 2002;
Colcombet et al., 2005;
Yang et al., 2003;
Yang et al., 2005;
Zhao et al., 2002) are
important for the formation of the tapetal layer; therefore, these genes
probably act at earlier stages than DYT1, as supported by the
observed strong tapetal expression of EMS1/EXS and TPD1
(Canales et al., 2002;
Yang et al., 2003;
Zhao et al., 2002) prior to
the strong DYT1 expression in the tapetum. Our expression and double
mutant analyses support the hypothesis that DYT1 acts downstream of
SPL/NZZ and EMS1/EXS.
AMS and MS1 are also important for tapetum function, but
they are required at post-meiotic steps
(Ito and Shinozaki, 2002;
Sorensen et al., 2003;
Wilson et al., 2001). In
ams anthers, meiosis is normal and microspores are formed; however,
the newly formed microspores soon degenerate. In ms1 anthers, pollen
development is abnormal and no normal mature pollen is produced. Compared with
AMS and MS1, DYT1 acts at an earlier stage, before the
completion of meiosis. Therefore, DYT1 is required for a key step in
tapetum development. In other words, tapetum development requires the combined
activities of the EMS1/EXS, SERK1/SERK2, TPD1, DYT1, AMS and
MS1 genes: first EMS1/EXS, SERK1/SERK2 and
TPD1 specify the tapetal cells as distinct from meiocytes at the time
of the cell division that form the tapetal cells, then DYT1 is
required to promote correct tapetal cell fate for proper function, and finally
AMS and MS1 further regulate the tapetal cell function
supporting normal microspore development.
DYT1 is required for normal levels of the expression of tapetum genes
As DYT1 encodes a bHLH putative transcription factor, it is likely
that it regulates the expression of tapetal genes. We found that the
expression of a majority of tapetum-preferential genes tested depends on
DYT1. The greatly reduced expression in the dyt1 mutant of
many tapetum-preferential genes, particularly those encoding transcription
factors, supports the idea that DYT1 is a key component of a
regulatory step in normal tapetum development. The Arabidopsis bHLH
gene family has over 140 members, making this the second largest gene family
of transcription factors (Toledo-Ortiz et
al., 2003). Although DYT1 and AMS clearly have
non-redundant and distinct functions, they are members of the same subfamily.
Phylogenetic analysis performed here including the closest rice homologs of
DYT1 indicates that the rice gene, OsUDT1, is also a member
of this subfamily and a putative DYT1 ortholog. A mutation in the
OsUDT1 gene results in a defective tapetum
(Jung et al., 2005), similar
to the dyt1 tapetum. In addition, the expression pattern of the
OsUDT1 gene (Jung et al.,
2005) is different from that of DYT1. Therefore, this
bHLH subfamily contains phylogenetically and functionally distinct
members.
|
;
Sorensen et al., 2002).
Although the molecular nature of the FAT TAPETUM, GNE1 and
GNE4 genes are unknown at this time, it is likely that additional
loci are involved in defining the tapetal cell fate. In other words,
DYT1 is essential, but not sufficient, for the specification of the
tapetum identity, as supported by our observation that overexpression of
DYT1 did not alter anther phenotypes in wild-type or mutant
backgrounds. Recently, it was reported that the AtMYB33 and
AtMYB65 genes redundantly facilitate tapetum development, with the
double mutant having tapetum defects before the completion of meiosis
(Millar and Gubler, 2005). It
is known that bHLH transcription factors can form homodimers or heterodimers
with other bHLH proteins. In some cases, it has been shown that bHLH proteins
can form complexes with MYB proteins and WD-40 proteins
(Ramsay and Glover, 2005). It
is possible that the AtMYB33 and AtMYB65 proteins may form heterodimers with
DYT1 to regulate tapetum-preferential gene expression. This idea is consistent
with our result that the expression of these two MYB genes is not altered in
the dyt1 mutant.
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DYT1 supports completion of meiosis
It is known that a functional tapetum is required for normal pollen
development following meiosis, as shown by molecular ablation studies and the
characterization of mutants such as ams
(Aarts et al., 1997;
Ito and Shinozaki, 2002;
Sorensen et al., 2003;
Wilson et al., 2001). In the
ems1/exs, serk1 serk2 and tpd1 mutants, the tapetum is
missing and excess meiocytes occupy the position of the tapetum
(Albrecht et al., 2005;
Colcombet et al., 2005;
Yang et al., 2003;
Zhao et al., 2002).
Nevertheless, meiotic nuclear divisions still occur in the ems1
mutant, indicating that the tapetum is not required for meiotic nuclear
events. However, the ems1 meiocytes do not undergo cytokinesis,
suggesting that tapetum might be needed for the completion of meiosis. The
dyt1 mutant phenotypes provide further support for this idea. In
addition to a morphologically abnormal tapetum, the dyt1 mutant
meiocytes were found to have thinner callose cell walls than normal,
suggesting that normal tapetum function is needed for the formation of the
callose wall. Nevertheless, DYT1 expression was detected at a low
level in the meiocytes and expression of some meiotic genes was reduced;
therefore, it is possible that DYT1 might also function in
meiocytes.
A model for DYT1 function and the control of tapetum identity
This and previous studies support a model for the genetic control of
tapetum development and function (Fig.
8), although evidence for biochemical interactions is not yet
available. SPL/NZZ is required for the formation of sporogenous cells
and surrounding somatic cell layers, including the tapetum
(Yang et al., 1999). Recently,
Ito et al. showed that AG is a direct activator of SPL/NZZ
expression (Ito et al., 2004).
EMS1/EXS, TPD1 and SERK1/SERK2 are required for the
formation of tapetum (Albrecht et al.,
2005; Colcombet et al.,
2005; Yang et al.,
2003; Zhao et al.,
2002). In addition, phenotypic changes caused by TPD1
overexpression are dependent on EMS1/EXS
(Yang et al., 2005). We show
here that SPL/NZZ and EMS1/EXS positively regulate
expression of DYT1. The AtMYB33 and AtMYB65 genes
are expressed in the tapetum and their expression does not require the
SPL/NZZ, EMS1/EXS or DYT1 gene.
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|
;
Hernould et al., 1998;
Liu and Dickinson, 1989;
Rubinelli et al., 1998;
Scott et al., 2004;
Taylor et al., 1998;
Zheng et al., 2003). Among the
genes regulated by DYT1 are AMS and MS1, which also encode
transcription factors that probably regulate late tapetum genes. Analysis of
DYT1 overexpression transgenic plants and expression studies in
various mutants suggest strongly that other genes are needed for normal
tapetum development. Phenotypic similarities suggest that AtMYB33 and
AtMYB65, as well as potentially others (such as GNE1)
(Sorensen et al., 2002), may
act at approximately the same step as DYT1. Therefore, DYT1
is a crucial component at a key step in the regulatory network responsible for
tapetum development and function (Fig.
8).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/16/3085/DC1
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ACKNOWLEDGMENTS |
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