Summary
In flowering plants, the anther contains highly specialized reproductive and somatic cells that are required
for male fertility. Genetic studies have uncovered several genes that are important for anther development.
However, little information is available regarding most genes active during anther development, including
possible relationships between these genes and genetically defined regulators. In Arabidopsis, two
previously isolated male-sterile mutants display dramatically altered anther cell differentiation patterns.
The sporocyteless (spl)/nozzle (nzz) mutant is defective in the differentiation of primary sporogenous cells
into microsporocytes, and does not properly form the anther wall. The excess microsporocytes1 (ems1)/
extrasporogenous cells (exs) mutants produce excess microsporocytes at the expense of the tapetum. To
gain additional insights into microsporocyte and tapetum differentiation and to uncover potential genetic
interactions, expression profiles were compared between wild-type anthers (stage 4–6) and those of the spl
or ems1 mutants. A total of 1954 genes were found to be differentially expressed in the ems1 and/or spl
anthers, and these were grouped into 14 co-expression clusters. The presence of genes with known and
predicted functions in specific clusters suggests potential functions for other genes in the same cluster. To
obtain clues about possible co-regulation within co-expression clusters, we searched for shared cis-
regulatory motifs in putative promoter regions. Our analyses were combined with data from previous
studies to develop a model of the anther gene regulatory network. This model includes hypotheses that
can be tested experimentally to gain further understanding of the mechanisms controlling anther
development.
Therefore, alternative approaches such as expression (Tusher et al., 2001). We found that the expression of 573
profiling are required to identify genes active during anther and 6434 genes in ems1 and spl anthers, respectively,
development. The use of microarray analysis to study differed significantly from that in wild-type anthers, with a
genome-wide gene expression has been a very powerful 0.05 false-discovery rate. To focus on genes with a substan-
tool to determine gene regulation for many physiological tial extent of differential expression, only genes with a
and developmental processes (e.g. Honys and Twell, 2003; twofold or more difference between the wild-type and either
Schmidt et al., 2005; Wellmer et al., 2004; Zhang et al., of the mutants were analyzed further. Because expression
2005). For example, microarray technology has been levels below 50 may be unreliable or represent background
employed to detect genes expressed in the anthers of maize, (Zhang et al., 2005), we focused on gene expression levels
rice and Lotus japonica (Endo et al., 2004; Lu et al., 2006; that were 50 or higher in at least one genotype. With these
Ma et al., 2006; Masuko et al., 2006; Wang et al., 2005c; constraints, we identified 1954 genes with substantial
Yamaguchi et al., 2004). Following expression profiling, differential expression in the ems1 and/or spl anthers
further computational analysis can identify putative compared to wild-type anthers (Table S1). Of these genes,
co-regulated genes to determine regulatory networks (Hu more than 99% (1940/1954) showed differential expression
and Ma, 2006; Nemhauser et al., 2004). between spl and wild-type anthers, whereas only about 27%
Here we report a comparison of gene expression in the (525/1954) showed differential expression between ems1
wild-type Arabidopsis anther at stages 4–6 with that in either and wild-type anthers. Therefore, SPL/NZZ affects the
spl or ems1 mutant anthers. Using co-expression patterns expression of many more genes than EMS1/EXS, providing
with known genes, we have identified candidate genes for molecular evidence supporting the observation that the spl
anther development. In addition, bioinformatic analysis mutant shows more severe defects in anther cell differen-
revealed that the binding sequences for several known tiation than the ems1 mutant.
transcription factors were enriched in a large number of We found that 8572 genes were expressed in wild-type
predicted promoters. The results from this study, combined and spl anthers at similar levels, suggesting that the
with information from other studies, were used to establish expression of a large number of genes is independent of
a model for a regulatory network that is active during anther SPL function. Of these, several have known functions in
development. The results and analyses presented here early anther development, including BAM1, BAM2, SERK1
generate hypotheses regarding gene functions that can be and SERK2. In addition, two of the genes that were
tested using reverse genetics, and provide insight into the SPL-independent and differentially expressed in ems1
possible gene regulatory mechanisms underlying male (according to SAM) encode putative transcription factors
fertility. with a MYB domain and a PHD finger, respectively.
Therefore, both SPL-dependent and SPL-independent
transcriptional programs are probably required for normal
Results
anther development.
Microarray analysis reveals SPL-dependent and
-independent genes Relating co-expressed genes to known developmental
functions
To identify genes that are expressed in anthers near the
time of meiosis, we collected stage 4–6 anthers from wild- To begin to identify genes that might function together, we
type, ems1 and spl floral buds, with two replicates from first used the hierarchical clustering method to obtain an
independently grown populations for each genotype. approximate number of clusters among the 1954 differen-
Total RNA from these samples was used to synthesize tially expressed genes, resulting in the identification of 14
cRNA for hybridization with the Affymetrix Arabidopsis major clusters. However, a drawback of hierarchal clustering
ATH1 genome array (here after referred to as the is that clusters are defined by the users by selecting sub-
Affymetrix chip), which contains probe sets for 22 746 trees that show similar expression patterns (Sturn et al.,
Arabidopsis genes, representing approximately 80% of the 2002); therefore, specific membership in a cluster can be
genes in the whole genome (approximately 29 000). The somewhat subjective. As an alternative approach, we used
results for two replicates for each genotype were highly the K-mean clustering method (Han and Kamber, 2001) with
reproducible (Pearson correlation coefficients: wild-type, 14 as the cluster number (Figure 1). Clusters A–C contained
0.97; ems1, 0.98; spl, 0.99). Therefore, the mean expres- genes that showed significantly increased mRNA levels in
sion value for each locus in each genotype is presented spl compared with wild-type, and differed in expression only
here unless otherwise indicated. slightly between ems1 and wild-type. Therefore, these
To identify genes that were differentially expressed clusters were renamed SPL-H1, SPL-H2 and SPL-H3.
between wild-type and one of the mutant anthers, the Cluster D contained genes whose expression was reduced
significant analysis of microarray (SAM) method was used significantly in ems1 and spl; hence this cluster was named
(a) (b) and were named ES-RH1 (EMS1_SPL-reduced highly 1), ES-
RH2 and ES-RH3, respectively.
To investigate possible functions of genes in the SPL-H1,
SPL-H2 and SPL-H3 clusters, with higher expression in spl
than wild-type, we compared their expression in wild-type,
(A) (B)
spl, and ems1 anthers, as well as their expression in wild-
type young inflorescences, stage 12 flowers (just before
anthesis), root, stem, leaf and siliques (Zhang et al., 2005).
We found that genes in clusters SPL-H1–3 showed similar
(C) (D) expression levels in spl anthers and wild-type inflores-
cences. About 28% (234/824) of these genes had expression
levels below 50 in wild-type anthers, with 80% (187/234)
showing expression in either the inflorescence or stage 12
(E) (F) flower (Zhang et al., 2005). This result suggests that SPL
represses these genes in the wild-type anther. It is worth
noting that clusters SPL-H1, SPL-H2 and SPL-H3 contain
genes that are involved in various aspects of flower devel-
(G) (H) opment (Table 1 and Table S2), including ANT, which
interacts with SPL in ovule development, the floral regula-
tors LEAFY (LFY), AP2 and AP3, and the L1-specific genes,
PROTODERMAL FACTOR2 (PDF2) and Arabidopsis thaliana
(I) (J) MERISTEM LAYER1 (ATML1).
Similarly, the ES-RH1–3 clusters were found to contain
genes that were previously shown to be preferentially
expressed in the tapetum (Table S2), including MALE
(K) (L) STERILE2 (MS2, At3g11980) and AMS (At2g16910)
(Figure 1). As discussed above, most genes in these three
clusters showed significantly reduced expression in both spl
and ems1 anthers (more than sixfold in both genotypes),
consistent with the lack of a tapetum in both ems1 and spl
(M) (N)
mutant anthers (Yang et al., 1999; Zhao et al., 2002). There-
fore, genes in these clusters may be preferentially expressed
in the tapetum or regulated by the SPL- and EMS1-depend-
ent regulatory pathways. A number of genes in the SPL-H1–3
and ES-RH1–3 clusters encode putative transcription factors
Figure 1. Clusters of co-expressed genes. and receptor-like protein kinases (Tables 1 and 2, and Table
(a) Heat map for the mRNA expression profiles of 1954 differentially S3; see below for more details).
expressed genes. K-mean clustering was performed on transcript ratios of
wild-type anthers versus ems1 (log2 ems1/wt) and spl (log 2 spl/wt) anthers.
In addition, known meiotic genes were found in clusters
Genes were grouped into 14 co-expression clusters. Magenta indicates higher SPL-L3–5 and ES-RM1 and 2, including MS5 (At4g20900),
expression in mutant anthers compared to wild-type anthers, whereas green MMD1 (At1g66170), SPO11 (At3g13170), SDS (At1g14750),
indicates lower expression in mutant anthers compared to wild-type anthers.
(b) Centroid views of each cluster. Pink lines indicate the mean expression
ASY1 (At1g67370) and AtDMC1 (At3g22880) (Table S2).
profiles of each cluster. The y-axis is in log2 scale, and the x-axis shows the Both wild-type anthers and ems1 anthers contained micro-
transcript ratios of wild-type anthers versus ems1 (log 2 ems1/wt) and spl (log2 sporocytes, suggesting the possibility that some of the
spl/wt) anthers.
genes in clusters SPL-L3–5 and ES-RM1 and 2 are
expressed in microsporocytes. Furthermore, two genes
found in these clusters, PARTING DANCERS (PTD) (ES-
ES-L (EMS1-SPL-low). Genes in clusters E–I showed reduced RM1) and ROCK-N-ROLLERS (RCK)/AtMER3 (SPL-L5), have
expression in spl, and these clusters were named SPL-L1–5. recently been shown by genetic studies to be important for
Clusters J and K contained genes whose expression meiosis (Chen et al., 2005; Wijeratne et al., 2006). In
was reduced in both ems1 and spl, but less than sixfold. addition, the SPL-L1 cluster contained the homeobox gene
Therefore, clusters J and K were named ES-RM1 (EMS1-SPL- WUS, which is expressed in the stomium region of the
reduced moderately 1) and ES-RM2, respectively. Clusters anther wall (Deyhle et al., 2006). This suggests that some
L–N contained genes that showed a dramatic decrease in of these genes are expressed in the cells of the anther
expression in both ems1 and spl, often greater than sixfold, walls.
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), doi: 10.1111/j.1365-313X.2007.03217.x
Gene functions in the early Arabidopsis anther 5
Gene ID, gene identification number; Cluster, expression cluster according to K-mean clustering. The values are the mean mRNA expression levels
of two replicates for wild-type anther, ems1 anther and spl anther. Interaction, possible interaction of this gene with other genes (Figure 3).
a
mRNA expression of these genes was tested using mRNA in situ hybridization (Figure 2).
b
These genes contained putative cis-regulatory elements (Table S4).
*This gene is not included in the 1954 genes as the differences between wild-type and the mutant anthers are less than twofold.
Gene ID, gene identification number; Cluster, expression cluster according to K-mean clustering. The values are the mean mRNA expression levels
of two replicates for wild-type anther, ems1 anther and spl anther. Interaction, possible interaction of this gene with other genes (Figure 3).
a
These genes contained putative cis-regulatory elements (Table S4).
mitochondrial defects are often associated with male steril- numbers of genes affected by the two mutations (see
ity (Ma, 2005). Table S1 for detailed expression information for these
genes in wild-type anthers, and for ratios of expression
levels between wild-type and spl or ems1 anthers for each
Differential expression of genes encoding transcription
gene). Most of these genes have not been identified by
regulators
genetic studies.
Among the genes differentially expressed in the spl and/ Nearly 11% (27) of the 254 differentially expressed TF
or ems1 mutants, 254 genes (13%) coding for putative genes coded for MYB proteins, making them the most
transcription factors or other transcriptional regulators numerous type in this group. In particular, MYB genes
(TF) are enriched relative to the gene set on the Affyme- were statistically over-represented among TF genes in
trix chip (approximately 9%) (Table 1 and Tables S3 and clusters ES-RM1 (33%), SPL-L3 (27%) and SPL-L4 (21%),
S4). We found that many members of the 26 TF families compared to 10% (198/1990) (MYB among all TFs) on the
defined by Riechmann et al. (2000) and the Sheen Lab Affymetrix chip (Table S4). Among these, MYB33 (SPL-L4)
website (see Experimental procedures) are affected by the and MYB65 (SPL-L3) have redundant functions in promo-
spl and/or ems1 mutations. In particular, seven of nine ting tapetum development and function (Gocal et al., 2001;
major transcription factor families (MYB, bHLH, AP2/ERE- Millar and Gubler, 2005). In addition, four members of one
BP, homeobox, C2H2, NAC and MADS) had the highest MYB clade are also differentially expressed: AtMYB87/
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), doi: 10.1111/j.1365-313X.2007.03217.x
Gene functions in the early Arabidopsis anther 7
Gene ID, gene identification number; Cluster, expression cluster according to K-mean clustering. The values are the mean mRNA expression levels
of two replicates for wild-type anther, ems1 anther and spl anther. Interaction, possible interaction of this gene with other genes (Figure 3).
a
These genes contained putative cis-regulatory elements (Table S4).
its closest homolog has only 38% amino acid sequence consistent with the fact that the tapetum is absent in both the
identity and 57% similarity, suggesting a distinct role for this spl and ems1 mutants. In addition, several cytochrome P450
LRR-RLK in the developing anther. genes were found, suggesting that they may also function in
In addition to LRR-RLKs, two other differentially ex- the tapetum. Genes coding for lipid transfer proteins and
pressed genes (At5g60080 and At5g60090) encode putative endomembrane proteins were also detected.
receptor-like kinases, with 47% amino acid sequence identity
(Table 2). Both show moderate levels of expression in the
In situ RNA hybridization reveals expression in microspo-
wild-type anther and may function in the tapetum and/or
rocytes and the tapetum
microsporocytes. Of the 1954 differentially expressed genes,
two encode putative receptor-like proteins (RLP) and two To assess expression patterns in the anther of a small subset
others code for CLAVATA3 (CLV3)-like proteins (CLE) of the genes with differential expression between wild-type
(Table 2). These genes may also function in RLK signaling and spl and/or ems1 anthers, we performed RNA in situ
pathways involved in anther development, similar to the hybridization experiments. Although the microarray experi-
functional interactions between CLV1 (RLK), CLV2 (RLP) and ments used RNAs from stage 4–6 anthers, the in situ
CLV3 (CLE) (Clark, 2001). hybridization experiments revealed that some of the genes
showed expression at early stages of anther development
(at or before stage 3) (Figure 2 and Figure S1). It is possible
Differentially expressed genes encoding metabolic proteins
that these genes are involved in early anther differentiation,
Among the differentially expressed genes in the clusters as is SPL. The clusters SPL-L1, SPL-L3 and SPL-L4 include
ES-RH1–3, a number are predicted to encode metabolic genes that show significantly reduced expression in spl
enzymes (Table 3 and Table S3). Noteworthy are genes for anthers, but not in ems1 anthers, suggesting that these
acyl lipid metabolism, including ones that were previously genes might be preferentially expressed in microsporocytes
shown to be preferentially expressed in the tapetum, or meiocytes because these cells are absent in spl anthers,
At5g67110 At2g16210
At1g06170 At3g28470
Figure 2. Spatial expression patterns of selected genes using mRNA in situ hybridization.
Corresponding gene names are given below each set of panels. Arrowheads indicate microsporocytes/meiocytes; arrows indicate the tapetum layer.
(a1) An anther section at approximately anther stage 5, with strong expression in the tapetum and microsporocytes.
(a2) An anther section at approximately anther stage 6; expression is stronger in microsporocytes compared to the tapetum.
(b1) An anther section at approximately anther stage 2 (marked A); expression was detected throughout the anther.
(b2) A section of the anther at approximately anther stage 5 showing expression in the tapetum and microsporocytes.
(b3) A section of an anther at approximately anther stage 6 showing stronger expression in microsporocytes than in the tapetum.
(c1) An anther section at approximately anther stage 5 showing expression in the tapetum and microsporocytes.
(c2) A section of an anther at approximately anther stage 6 showing stronger expression in microsporocytes than in the tapetum.
(d1) A section of an anther at approximately anther stage 3 expression was detected throughout the anther.
(d2) A section of an anther at approximately anther stage 5 showing expression in the tapetum and microsporocytes.
(d3) An anther section at approximately anther stage 6 showing expression in the tapetum and microsporocytes. All the panels have the same magnification.
Bar = 50 lm.
but present in ems1 anthers. We tested this hypothesis for The analysis for over-represented motifs in clusters ES-
several genes and found that they are indeed expressed RH1–3 (with greatly reduced expression in both spl and
predominantly in the microsporocytes in stage 5 anthers ems1 anthers) identified putative binding sites for known
(Figure 2 and Figure S1). Specifically, a B3 gene, At2g16210 types of transcription factors, including bHLH and MYB
(in cluster SPL-L3), exhibited preferential expression in the proteins (Table S6). In the three clusters, 198 genes con-
microsporocytes, with moderate to low levels of expression tained an E-box for binding to bHLH proteins in their
in the tapetum (Figure 2). promoters, and 103 of the 198 genes also contained a
Expression of the genes in cluster ES-RH1–3 was dramat- TAACAAA box for binding MYB proteins. This result
ically reduced in both spl and ems1 mutants, usually by suggests that bHLH and MYB factors might regulate the
sixfold or more (Figure 1). One possibility is that these genes expression of these genes. Several of the 198 genes encode
are preferentially expressed in the tapetum, which is absent putative transcription factors or other regulatory proteins
in both mutants. This idea is supported by the presence of (Tables 1 and 2). Specifically, the promoters of the bHLH
known tapetum-specific/preferential genes in these clusters. gene AMS/At2g16910 and a gene encoding a serine/threon-
Surprisingly, the in situ hybridization results showed that ine protein kinase (At5g60080) contained both an E-box and
several of these genes are expressed in both the tapetum a TAACAAA box. Other genes with promoters that have
and microsporocytes, perhaps because the ems1 meiocytes E-boxes include the bHLH gene AtbHLH091/At2g31210 and
are abnormal and cannot complete meiosis. Among the 27 the MYB genes AtMYB35/At3g28470, AtMYB84/At3g49690
differentially expressed MYB genes, AtMYB35/At3g28470 and AtMYB68/At5g65790, as well as the putative LRR-RLK
had the highest level of expression in wild-type anthers, and gene, At5g06820, and a protein kinase gene, At5g60090. The
showed a significant reduction in transcript levels in both promoter of another bHLH gene, AtbHLH089/At1g06170,
ems1 and spl anthers. The in situ hybridization results contained a TAACAAA box, but not an E-box. Other over-
(Figure 2) showed that this gene was expressed in both the represented cis-regulatory sequences include two Myc
tapetum and microsporocytes during anther stages 4 and 5. binding sites, CATGTG and CACATG (cluster ES-RH1) (Abe
In addition, AtbHLH089/At1g06170 was expressed in the et al., 1997; Simpson et al., 2003). These sites might be
tapetum and microsporocytes, a pattern similar to that of targets for Myc and MYB proteins expressed in the anther.
AtMYB35/At3g28470.
Discussion
Detection of putative cis-regulatory elements in promoters
The Arabidopsis SPL/NZZ and EMS1/EXS genes are key
of differentially expressed genes
regulators of anther cell differentiation (Canales et al.,
To obtain clues for possible mechanisms of co-regulation, 2002; Schiefthaler et al., 1999; Yang et al., 1999; Zhao et al.,
we analyzed the presumed promoter regions of the co-ex- 2002). We have identified 1954 genes whose expression is
pressed genes for the presence of known cis-regulatory affected by the ems1 and/or spl mutations. The expression
elements (see Experimental procedures for details). We patterns of these genes fall into several major groups,
found that a number of cis-regulatory elements were en- including (i) elevated expression in the spl mutant, (ii) re-
riched in the putative regulatory regions of the differentially duced expression in spl, (iii) moderately reduced expression
expressed genes, with frequencies greater than those found in both spl and ems1, and (iv) greatly reduced expression in
in random samples of putative regulatory sequences both mutants. Specifically, expression of 1940 of the 1954
(Table S5). Among the genes with increased expression in genes is affected by the spl mutation, whereas the expres-
the spl mutant, cluster SPL-H1 was enriched for genes sion of only 525 genes is affected by the ems1 mutation,
whose promoter contained a MYB binding site (YAACKG; indicating that SPL function is more general than that of
Abe et al., 2003), and SPL-H2 was enriched for genes with a EMS1, as supported by the mutant phenotypes. The pres-
promoter that contained a binding site for the L1-specific ence of genes with known functions in our expression pat-
protein ATML1 (TAAATGYA; Abe et al., 2001). In addition, tern clusters provides clues regarding the possible functions
genes in clusters SPL-H2 and H3 were enriched for two Myc of other genes in the same clusters. In addition, genes
(a specific type of bHLH) binding sites, CATGTG and CA- encoding transcription factors and other types of regulators
CATG (Abe et al., 1997; Simpson et al., 2003). We also were enriched in our differentially expressed gene clusters
detected enrichment of genes that contained binding sites in comparison to the frequencies of these types of genes on
for MADS domain proteins, including AG in the SPL-H1 and the Affymetrix chip. These results strongly suggest that SPL
SPL-L4 clusters, AGAMOUS-like2 (AGL2) in the SPL-L2 is near the top of an anther regulatory hierarchy that
cluster, and AGL15 in the ES-RH3 cluster (Huang et al., 1996; includes other regulatory genes, whereas EMS1 is involved
Tang and Perry, 2003). In addition, genes containing several in the regulation of a smaller subset of genes.
other MYB and bHLH binding sites and hormone-responsive Our analysis has uncovered potential binding sites for
elements were enriched in other clusters (Table S5). MADS box, MYB and bHLH proteins in predicted promoter
regions of co-expressed genes. The information on differ- expressed at or above the conservative cut-off value of 50
ential gene expression and putative cis-regulatory elements, (Zhang et al., 2005). The fact that a large subset (clusters
as well as known gene functions, has allowed the formula- SPL-H1–3) of differentially expressed genes showed abnor-
tion of a number of hypotheses about anther gene regula- mally high levels of expression in spl anthers suggests that
tion, leading to a model of gene regulatory networks during SPL normally restricts the expression of these genes, inclu-
anther development (see below for more discussion; ding several floral homeotic genes. In the absence of SPL,
Figure 3). Clearly, the genetic interactions proposed here mis-expression of these genes may lead to the formation of
may be either direct or indirect, and will require functional cell types not typically found in wild-type anthers.
verification using molecular genetic studies. Clues about the effect of the spl mutation on the molecular
properties of anther cells may be obtained by examining
some of the differentially expressed genes. For example,
Expression profiling reveals molecular characteristics of
both PDF2 and ATML1 encode homeodomain transcription
anther cells
factors and have been shown to be involved in maintaining
Little is known about the molecular nature of wild-type the identity of the L1 layer, presumably by regulating the
anther cell types and abnormal cells in the spl and ems1 expression of L1-specific genes (Abe et al., 2001). In addi-
mutants. Our microarray analysis provides a genome-scale tion, the presumptive promoters of some of the differentially
description of anther cells. In wild-type, spl and ems1 expressed genes, such as INOL and genes in box ATML1BG
anthers, approximately 14 000 genes were detected as (Figure 3 and Table S7), contain ATML1-binding sites, sug-
AtMYB103
RLK1
AMS MS1
* Represents more
than one gene AtCP1L MMDG* MSG* DMG*
gesting that they might be regulated by ATML1. The two BEL1-like genes, BHL2 (At4g36870) and BHL4
identification of many genes that show increased expression (At2g23760), showed elevated expression levels in spl
in spl anthers supports the hypothesis that SPL functions as anthers, and are among the genes enriched for putative
a transcriptional repressor (Balasubramanian and Schneitz, AtML1 binding sites (SPL-H2 cluster, Table 1 and Figure 3).
2002). Therefore, it is possible that SPL restricts the expres- Therefore, these INO and BEL1 homologs are probably
sion of L1-specific genes in L2-derived cells by negatively regulated by SPL/NZZ, and are possibly downstream of
regulating the expression of ATML1 in the L2-derived cells ATML1 during early anther development (Figure 3). Our
(Figure 3). analysis has also identified additional candidate genes for
anther development downstream of SPL (Figure 3, box
SPLNG).
A regulatory network for early anther development inclu-
ding SPL-dependent and independent components
Function of LRR-RLKs in cell–cell communication during
Floral homeotic genes can control target genes with various
anther cell differentiation
functions, including SPL, during the development of floral
organs (Ito et al., 2004; Sablowski and Meyerowitz, 1998). Genetic studies have uncovered critical roles for several
Our expression analysis clearly indicated that the expression LRR-RLKs during anther cell differentiation, including BAM1
of more than 5250 genes is affected by the spl mutation, with and BAM2 for the formation of parietal cells, which in turn
over 1900 of them showing twofold or more changes from develop into endothecium, middle layer and tapetum (Hord
normal levels. Furthermore, some of these (Figure 3, et al., 2006). Although our microarray data did not reveal
MYB110 and box AGBG, Table S7) have predicted AG significant differences in BAM1 and BAM2 expression
binding sites in their putative promoter regions, suggesting between wild-type and spl anthers, in situ hybridizations
that they might be regulated by both AG and SPL. On the results support the hypothesis that SPL positively regulates
other hand, the expression levels of approximately 8600 the expression of BAM1, and that BAM1/2 restrict the
genes are not significantly altered in the spl mutant anthers, domain of SPL expression (Figure 3; Hord et al., 2006).
indicating that these genes not under the transcriptional BAM1/2 together with BAM3 and CLV1 form a monophyletic
control of SPL. Therefore, AG (and other MADS box pro- group (DeYoung et al., 2006; Hord et al., 2006). Our results
teins) probably also regulate other genes to promote anther indicate that SPL negatively regulates the expression of
development, as supported by the observation that some BAM3 (Figure 3). In addition, members of the ERECTA clade
SPL-independent genes contained putative AG binding sites of LRR-RLK genes are important for position-dependent
in their promoters (not shown). Furthermore, our data sug- guard cell differentiation (Shpak et al., 2004, 2005); we found
gest that SPL and AG are involved in a positive–negative an increase in the expression of members of the ERECTA
regulatory feedback loop, where AG positively regulates the sub-family in spl mutant anthers. Furthermore, a number of
expression of SPL, and SPL in turn represses the expression other LRR-RLKs (Table 2 and Figure 3, box SPLNG) also
of AG (Table 1 and Figure 3). showed elevated expression in spl anthers. Our results
SPL/NZZ is also required for normal ovule development suggest that BAM3, ERECTA homologs and other LRR-RLKs
(Elliott et al., 1996; Klucher et al., 1996; Schiefthaler et al., function antagonistically to SPL/NZZ during anther devel-
1999), and knowledge on genetic interactions between SPL opment (Figure 3).
and other ovule genes can facilitate the understanding of LRR-RLKs may interact with each other during signal
interactions between SPL and other genes in anther devel- transduction (Shpak et al., 2003; Wang et al., 2005b). For
opment. SPL interacts genetically with two other ovule example, SERK1 and SERK3 (also named BRI1-associated
genes, INNER NO OUTER (INO) and AINTEGUMENTA kinase1) can interact with BRI1 for brassinosteroid (BR)
(ANT), which encode YABBY and AP2 domain proteins, signaling (Karlova et al., 2006; Li et al., 2002; Nam and Li,
respectively (Villanueva, 1999; Balasubramanian and 2002; Wang et al., 2005a). Because SERK1/2 and EMS1/EXS
Schneitz, 2002). SPL/NZZ negatively regulates ANT expres- are required for specifying tapetum identity (Albrecht et al.,
sion in the ovule (Balasubramanian and Schneitz, 2002). 2005; Canales et al., 2002; Colcombet et al., 2005; Zhao et al.,
Similarly, we found that ANT was up-regulated in spl mutant 2002), and BRI1 and EMS1 are both members of the LRR X
anthers, suggesting that ANT and SPL have a similar sub-family, it was postulated that SERKs interact with EMS1/
relationship in the anther (Figure 3). In the ovule, BELL1 EXS (Figure 3; Albrecht et al., 2005; Colcombet et al., 2005).
(BEL1) is required for INO expression (Balasubramanian and We observed that the BRI1 mRNA level was significantly
Schneitz, 2002). We found that expression of INO and BEL1 greater, but EMS1 expression was reduced, in spl anthers as
was low and not significantly different in wild-type and spl compared to wild-type (Table 2 and Figure 3), suggesting
and ems1 mutant anthers (data not shown). However, a that BRI1 and EMS1 are differentially regulated by SPL.
gene (INOL, At2g26580) encoding a protein that has 31% Moreover, three other RLKs are predicted to function
amino acid sequence identity and 44% similarity to INO, and downstream of MYB33/65 and/or DYT1, which are key
regulators of tapetum function at the time of meiosis by both MYB and MYC/bHLH factors (Abe et al., 1997;
(Figure 3). Therefore, receptor-mediated cell–cell communi- Baudry et al., 2006; Hartmann et al., 2005). AMS and TDR1
cation may play a role in coordinating meiotic events and encode bHLH proteins and are critical for tapetum functions
processes in the surrounding somatic cells. that support microsporogenesis (Li et al., 2006a; Sorensen
Previous studies have shown that the protein phospha- et al., 2003). In 75 of the 77 genes in the SPL-L3 cluster, the
tase 2C (PP2C) POLTERGEIST (POL) acts downstream of putative promoter regions contained an E-box, suggesting
CLV1 (Song and Clark, 2005; Song et al., 2006; Yu et al., that these genes might be regulated by bHLH proteins, such
2003). We found that two of the differentially expressed as DYT1 and AMS, that act downstream of SPL (Figure 3).
genes, At5g66080 and At5g27930, encode PP2Cs (Figure 3, MS1 is required for relatively late tapetum function that is
box PPC); they are candidates for downstream components critical for pollen development, and encodes a putative
of LRR-RLK-mediated signaling pathways during anther transcriptional regulator with a PHD finger (Ito and Shinoz-
development, and need to be tested by functional studies. aki, 2002; Wilson et al., 2001). Recently, it was shown that the
In part because of functional redundancy (Albrecht et al., MS1 gene is down-regulated in the dyt1 mutant (Figure 3)
2005; Colcombet et al., 2005; DeYoung et al., 2006; Hord (Zhang et al., 2006). Therefore, some of the genes regulated
et al., 2006; Shpak et al., 2004, 2005), few LRR-RLKs have by DYT1 and possibly by MYB33/65 might be targets of MS1.
known genetic functions. Our expression comparison and To test this hypothesis, we compared genes that are down-
cis-element analysis have identified LRR-RLKs that poten- regulated in ms1 mutant flowers (based on publicly avail-
tially facilitate signaling events between anther cells. able microarray data, see Experimental procedures and
Table S8 for details) with the differentially expressed genes
from this study. Of 56 genes that showed lower expression
Transcriptional regulation during tapetum development and
in ms1 flowers than wild-type flowers, 15 were also present
meiosis
in clusters ES-RH1–3. Furthermore, all 15 genes contained an
Genetic studies have demonstrated that the bHLH putative E-box in their putative promoters, and five also contained a
transcription factor DYT1 functions downstream of the LRR- TAACAAA (MYB) box, suggesting that they are also regula-
RLK EMS1/EXS to promote tapetum development and ted by Myc/bHLH proteins or by both bHLH and MYB factors
function (Zhang et al., 2006) (Figure 3). This is analogous to (Figure 3, boxes DMG and MMDG). The expression of two of
the function of another bHLH gene BRI1-EMS-SUPPRESSOR the genes, At3g51590 and AT3G23770, was found to be
1 (BES1) downstream of the LRR-RLK gene BRI1 (Vert et al., down-regulated in dyt1 anthers (Zhang et al., 2006).
2005; Yin et al., 2002, 2005). In addition, AtMYB33 and At- Although a number of genes important for meiosis have
MYB65 are also needed for normal tapetum development been identified (Hamant et al., 2006; Li and Ma, 2006; Ma,
(Millar and Gubler, 2005). The importance of MYB and bHLH 2005), little is known about the transcriptional regulation of
proteins during anther development is strongly supported meiotic genes. Our findings that genes encoding putative
by our observation that several MYB and MYC/bHLH binding transcriptional regulators, including MYB and bHLH pro-
sites were over-represented in the clusters of differentially teins, are co-expressed with known meiotic genes (in
expressed genes. In particular, the bHLH-binding E-box was clusters SPL-L3 and 4 and ES-RM1 and 2, Table 1) suggest
found in presumptive promoters of genes in clusters ES- that these transcription factors might regulate meiotic gene
RH1–3. In addition, the putative promoters of many of these expression. It is interesting to note that in mice, MYB genes
genes have predicted binding sites for MYB proteins are known to be required for meiosis (Latham et al., 1996;
(Table S5). Because the earliest known tapetum transcrip- Toscani et al., 1997). The presumptive promoters of some of
tional regulators are MYB33/65 and DYT1, we propose that the genes encoding transcription factor have putative E-box
these genes are downstream of MYB33/65 (Figure 3, box and/or MYB binding sites, suggesting that they might be
MG), DYT1 (box DG) or both (box MDG). It is possible that targets of DYT1 and/or MYB33/65 (Figure 3, boxes DG and
some of the proposed DYT1 targets are regulated by close MDG). For example, AtMYB35/At3g28470 is expressed in
homologs of DYT1 (box bHLH). The putative DYT1 targets meiocytes/microsporocytes (Figure 2), and its expression is
include several MYB genes (Figure 3, box DG). reduced in the dyt1 mutant (Figure 3, box DG) (Zhang et al.,
The putative DYT1 target AMS is required for normal 2006). Because DYT1 is also expressed in meiocytes (Zhang
tapetum function and microspore development (Sorensen et al., 2006), it is possible that it directly regulates meiotic
et al., 2003; Zhang et al., 2006) (Figure 3). In addition, the gene expression. Alternatively, other meiotic bHLH proteins
rice ortholog of AMS, TDR1, was down-regulated in the might serve as regulators of meiotic gene expression, such
anthers of a rice mutant defective in the GAMYB gene (Li as AtbHLH091/At2g31210 (ES-RH2) and AtbHLH089/
et al., 2006a; Tsuji et al., 2006), which is closely related to the At1g06170 (ES-RH1). Our microarray results, together with
AtMYB33 and AtMYB65 genes. Thus, it is possible that AMS our in situ hybridization results, strongly suggest that these
is also transcriptionally activated by AtMYB33/65, as sup- two genes (and possibly also At2g31220/AtbHLH010) func-
ported by known instances of the regulation of a single gene tion downstream of EMS1/EXS (Figure 3). Because EMS1/
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), doi: 10.1111/j.1365-313X.2007.03217.x
Gene functions in the early Arabidopsis anther 13
EXS promotes differentiation of the tapetum, its proposed Microarray experiment and analysis
role in activating (indirectly) transcriptional regulators in the The microarray experiment was performed according to the method
meiocytes provides a possible mechanism for interaction described in the Affymetrix GeneChip Expression Analysis Over-
between the two cell types. view (Affymetrix; http://www.affymetrix.com) as described previ-
ously (Zhang et al., 2005). Hybridization, washing, staining,
scanning and data collection were performed at the Pennsylvania
Putative targets of transcriptional regulators are candidate State University DNA Microarray Facility. The hybridization signals
genes for tapetum function for all the experiments were scaled to the same target intensity of
150 to compare expression profiles between samples.
Among the putative targets of DYT1 and MYB33/65 (Fig- Statistical analysis for the expression values was carried out at
ure 3, boxes DG and MDG, Table 3), several encode meta- the Department of Health Evaluation Sciences at Penn State
bolic enzymes. These genes might be important for tapetum College of Medicine. Before statistical tests were performed,
scanned data were converted to expression data. If a probe set
function to support pollen development. In addition, one of represented more than one gene, the corresponding data were
the putative targets of AMS is AtCP1, a homolog of the rice excluded to avoid any misinterpretations. We used the quantile
gene OsCP1 encoding a cysteine protease that is important normalization method to normalize the data (Bolstad et al., 2003).
for microsporogenesis (Li et al., 2006a). OsCP1 expression is To remove outliers and to summarize probe intensities within
regulated by TDR1, the rice ortholog of AMS, suggesting one probe set into a single expression value, the Li–Wong
method was applied to background-adjusted, normalized perfect-
that this regulatory relationship for tapetum function is match (PM) probe intensities (Chu et al., 2004). PM and mismatch
conserved between eudicots and monocots. Moreover, (MM) probe intensities were highly correlated, and MM intensi-
some putative downstream genes of MS1, DYT1 and/or ties composed of background noise as well as probe-specific
MYB33/65 (Figure 3, boxes MMDG and DMG, Table 3) en- signal expression values were obtained based on PM intensities
code lipid transfer proteins and ER proteins, consistent with rather than PM–MM intensities. To identify genes with statisti-
cally significant changes between wild-type and a mutant, a
known functions of such proteins in the tapetum to provide
significance analysis of microarray method (SAM) was used with
materials for pollen wall formation. a false-discovery rate of 0.05. For the microarray data comparing
ms1 flowers to wild-type flowers, we selected genes with more
than an eightfold decrease in expression levels in both
Conclusion ms1 young and old flowers compared to wild-type young flowers
(http://affymetrix.arabidopsis.info/narrays/experimentpage.pl?
Here we report differential expression for approximately experimentid=23).
1900 genes in spl and/or ems1 mutant anthers, compared Clustering of co-expressed genes were carried out using the
with their expression levels in wild-type anthers. These GENESIS program (release 1.6.0 beta 1; Sturn et al., 2002). For the
genes might have important functions during anther devel- identification of functionally related genes and of genes involved
opment and meiosis. Our attempt to integrate microarray in the same biological process, GO annotations were obtained
from the Arabidopsis Information Resource (TAIR). Gene family
data, bioinformatic analysis of cis-regulatory sequences and
information and gene annotations from the Sheen lab database
results from previous studies has resulted in a set of hypo- were also used (Arabidopsis transcription factors: Riechmann
theses about genetic interactions in a complex regulatory et al., 2000, http://www.sciencemag.org/cgi/content/full/sci;290/
network. These ideas will stimulate further experiments to 5499/2105/DC1/11; Sheen lab website: http://genetics.mgh.harvard.
expand our understanding of anther development. Our edu/sheenweb/Ara_gene_families.html). Identification of closely
related homologs in the same gene family in the dataset was
finding of a large number of candidate anther regulatory
performed using BLASTP (Altschul, 1990). Protein sequences were
genes and other genes with unknown functions indicates compared with Arabidopsis proteins. A protein sequence in the
that much remains to be discovered concerning this same family with an e-value cut-off of <1 · 10)20 was considered a
important floral organ. homolog.
P-values were calculated using the Z-score. A motif was consid- This material is available as part of the online article from http://
ered enriched if the P-value was <0.01 and if the motif was found www.blackwell-synergy.com.
in the regulatory regions for more than 10% of the genes in a
particular cluster.
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