The Plant Journal (2007) doi: 10.1111/j.1365-313X.2007.03217.

Differential gene expression in Arabidopsis wild-type and

mutant anthers: insights into anther cell differentiation and
regulatory networks
Asela J. Wijeratne1,2,†, Wei Zhang2,‡, Yujin Sun2, Wenlei Liu3,–, Reka Albert4, Zhengui Zheng5,§, David G. Oppenheimer5,
Dazhong Zhao2,6 and Hong Ma1,2,*
1
Intercollege Graduate Program in Plant Biology, Pennsylvania State University, University Park, PA 16802, USA,
2
Department of Biology and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park,
PA 16802, USA,
3
Department of Health Evaluation Sciences, Pennsylvania State University, College of Medicine, Hershey, PA 17033, USA,
4
Department of Physics, Pennsylvania State University, University Park, PA 16802, USA,
5
Department of Botany, UF Genetics Institute, University of Florida, 220 Bartram Hall, PO Box 118526, Gainesville, FL 32611,
USA, and
6
Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA

Received 31 January 2007; revised 14 May 2007; accepted 24 May 2007.

*
For correspondence (fax +1 814 863 1357; e-mail hxm16@psu.edu).
†
Present address: Department of Plant Cellular and Molecular Biology, Plant Biotechnology Center, Ohio State University, 206 Rightmire Hall, 1060 Carmack Road,
Columbus, OH 43210, USA.
‡
Present address: School of Life Sciences, Shanghai University, Shanghai 200444, China.
–
Present address: Global Discovery and Development Stats, Eli Lilly & Company, Indianapolis, IN 46285, USA.
§
Present address: Department of Zoology, 425 Cancer and Genetics Research Complex. University of Florida, 220 Bartram Hall, PO Box 118526, Gainesville,
FL 32611, USA.

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.

Keywords: anther development, Arabidopsis, cis-regulatory elements, EXCESS MICROSPOROCYTES1

(EMS1)/EXTRASPOROGENOUS CELL (EXS), microarray, regulatory network, SPOROCYTELESS (SPL)/
NOZZLE (NZZ).

ª 2007 The Authors 1

Journal compilation ª 2007 Blackwell Publishing Ltd
2 Asela J. Wijeratne et al.
Introduction
In flowering plants, male meiosis and male gametophyte
development occur in the anther (Goldberg et al., 1993;
Ma, 2005; McCormick, 1993). The anther contains both
reproductive and non-reproductive (somatic) cells and its
development has been divided into 14 stages according to
morphological features (Sanders et al., 1999). At stage 1,
the anther contains three cell layers, L1, L2 and L3. The L1
and L2 layers form the epidermis and archesporial cells,
respectively, and the L3 layer gives rise to the vascular
and connective tissues. At stage 2, the archesporial cells
further divide into primary parietal and primary sporo-
genous cells to form the stage 3 anther. The primary
parietal cell layer divides again to form two layers of
secondary parietal cells. At stage 4, further division and
differentiation of secondary parietal cells generate the
endothecium, middle layer and tapetum. The primary
sporogenous tissue gives rise to microsporocytes at
stage 5, and these undergo meiosis to form microspores
during stages 6–8. In addition to the importance of the
anther in plant reproduction and in understanding plant
evolution, the anther represents an excellent model sys-
tem to study the molecular mechanisms of cell differen-
tiation in higher plants.
According to the ABC model for floral organ identity, the
Arabidopsis B-function genes APETALA3 (AP3) and PISTIL-
LATA (PI), the C-function gene AGAMOUS (AG) and the
SEPALLATA (SEP)1/2/3 genes are required for stamen
identity (Ma, 2005). Additional genes that act downstream
of the ABC function genes are required for the later stages of
anther differentiation. One of the earliest genes acting
downstream is SPOROCYTELESS (SPL)/NOZZLE (NZZ; Schi-
efthaler et al., 1999; Yang et al., 1999). In spl/nzz mutants,
primary parietal cells and primary sporogenous cells are
formed, but the primary sporogenous cells fail to differen-
tiate into microsporocytes (Yang et al., 1999). Also, the
primary parietal cells fail to form the tapetum and endothe-
cium layers. It has been shown that AG regulates the
expression of SPL/NZZ to induce microsporogenesis (Ito
et al., 2004). Furthermore, SPL/NZZ expression is found
initially throughout the anther primordium (at reduced
levels in the epidermis), and becomes restricted to the
tapetum and microsporocytes/meiocytes during stage 5
(Schiefthaler et al., 1999; Yang et al., 1999). The SPL/NZZ
gene encodes a putative transcription factor (Schiefthaler
et al., 1999; Yang et al., 1999). However, the genes that act
downstream of SPL/NZZ during anther development have
not been identified.
Genes encoding putative MYB and basic helix-loop-helix
(bHLH) transcription factors also play important roles
during early anther development. In anthers of the
myb33 myb65 double mutant and dysfunctional tapetum1
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), doi: 10.1111/j.1365-313X.2007.03217.x
(dyt1) single mutants, the tapetum shows abnormalities at
the time of meiosis (Millar and Gubler, 2005; Zhang et al.,
2006). These results, and other data, indicate that DYT1,
which encodes a bHLH protein, and MYB33/MYB65 are
necessary but not sufficient for proper tapetum develop-
ment (Millar and Gubler, 2005; Zhang et al., 2006). Fur-
thermore, the genes MALE STERILE1 (MS1) and ABORTED
MICROSPORES (AMS) are also required for normal tape-
tum function and encode putative transcription factors
with a PHD finger motif and a bHLH domain, respectively
(Ito and Shinozaki, 2002; Sorensen et al., 2003; Wilson
et al., 2001).
In addition to transcription factors, several leucine-rich
repeat receptor-like protein kinases (LRR-RLKs) are also
important for early anther development, including BARELY
ANY MERISTEM1 (BAM1) and BAM2, which are functionally
redundant (DeYoung et al., 2006; Hord et al., 2006). In bam1
bam2 double mutant anthers, all L2-derived cells exhibit
properties of microsporocytes/meiocytes (Hord et al., 2006).
Additional genetic evidence for the role of LRR-RLKs in early
anther development comes from analysis of the excess
microsporocytes1 (ems1)/extrasporogenous cell (exs) and
tapetum determinant1 (tpd1) single mutants, and the soma-
tic embryogenesis receptor kinase1 (serk1) serk2 double
mutant (Albrecht et al., 2005; Canales et al., 2002; Colcombet
et al., 2005; Yang et al., 2003; Zhao et al., 2002). These
mutants are unable to form the tapetal layer and produce
excess microsporocytes, indicating that the corresponding
genes are important for tapetal cell fate determination.
EMS1/EXS, SERK1 and SERK2 encode putative LRR-RLKs
(Albrecht et al., 2005; Canales et al., 2002; Colcombet et al.,
2005; Zhao et al., 2002), and TPD1 encodes a putative ligand
(Yang et al., 2003).
Previous studies have indicated that a large number of
genes are expressed in the anther (reviewed by Ma, 2005).
In addition, large-scale screens have identified several
hundred mutants with anther developmental defects
(Sanders et al., 1999). However, many genes that may
contribute to the establishment and maintenance of
differentiated anther cell types have yet to be identified
by forward genetic studies, partly because defects in a
single gene may only result in subtle phenotypes that
might be overlooked in large-scale mutant screens. Alter-
natively, loss of function of one gene could be masked by
the function of redundant genes (Cutler and McCourt,
2005), as supported by recent functional studies (Albrecht
et al., 2005; Colcombet et al., 2005; Hord et al., 2006)
and large-scale segmental or whole-genome duplication
events in plant genomes (Maere et al., 2005; Moore and
Purugganan, 2005). In addition, some genes important for
anther development also may play essential roles at earlier
developmental stages, thereby preventing their identifica-
tion as male reproductive genes.
ª 2007 The Authors

Therefore, alternative approaches such as expression
profiling are required to identify genes active during anther
development. The use of microarray analysis to study
genome-wide gene expression has been a very powerful
tool to determine gene regulation for many physiological
and developmental processes (e.g. Honys and Twell, 2003;
Schmidt et al., 2005; Wellmer et al., 2004; Zhang et al.,
2005). For example, microarray technology has been
employed to detect genes expressed in the anthers of maize,
rice and Lotus japonica (Endo et al., 2004; Lu et al., 2006;
Ma et al., 2006; Masuko et al., 2006; Wang et al., 2005c;
Yamaguchi et al., 2004). Following expression profiling,
further computational analysis can identify putative
co-regulated genes to determine regulatory networks (Hu
and Ma, 2006; Nemhauser et al., 2004).
Here we report a comparison of gene expression in the
wild-type Arabidopsis anther at stages 4–6 with that in either
spl or ems1 mutant anthers. Using co-expression patterns
with known genes, we have identified candidate genes for
anther development. In addition, bioinformatic analysis
revealed that the binding sequences for several known
transcription factors were enriched in a large number of
predicted promoters. The results from this study, combined
with information from other studies, were used to establish
a model for a regulatory network that is active during anther
development. The results and analyses presented here
generate hypotheses regarding gene functions that can be
tested using reverse genetics, and provide insight into the
possible gene regulatory mechanisms underlying male
fertility.
Results
Microarray analysis reveals SPL-dependent and
-independent genes
To identify genes that are expressed in anthers near the
time of meiosis, we collected stage 4–6 anthers from wild-
type, ems1 and spl floral buds, with two replicates from
independently grown populations for each genotype.
Total RNA from these samples was used to synthesize
cRNA for hybridization with the Affymetrix Arabidopsis
ATH1 genome array (here after referred to as the
Affymetrix chip), which contains probe sets for 22 746
Arabidopsis genes, representing approximately 80% of the
genes in the whole genome (approximately 29 000). The
results for two replicates for each genotype were highly
reproducible (Pearson correlation coefficients: wild-type,
0.97; ems1, 0.98; spl, 0.99). Therefore, the mean expres-
sion value for each locus in each genotype is presented
here unless otherwise indicated.
To identify genes that were differentially expressed
between wild-type and one of the mutant anthers, the
significant analysis of microarray (SAM) method was used
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 3
(Tusher et al., 2001). We found that the expression of 573
and 6434 genes in ems1 and spl anthers, respectively,
differed significantly from that in wild-type anthers, with a
0.05 false-discovery rate. To focus on genes with a substan-
tial extent of differential expression, only genes with a
twofold or more difference between the wild-type and either
of the mutants were analyzed further. Because expression
levels below 50 may be unreliable or represent background
(Zhang et al., 2005), we focused on gene expression levels
that were 50 or higher in at least one genotype. With these
constraints, we identified 1954 genes with substantial
differential expression in the ems1 and/or spl anthers
compared to wild-type anthers (Table S1). Of these genes,
more than 99% (1940/1954) showed differential expression
between spl and wild-type anthers, whereas only about 27%
(525/1954) showed differential expression between ems1
and wild-type anthers. Therefore, SPL/NZZ affects the
expression of many more genes than EMS1/EXS, providing
molecular evidence supporting the observation that the spl
mutant shows more severe defects in anther cell differen-
tiation than the ems1 mutant.
We found that 8572 genes were expressed in wild-type
and spl anthers at similar levels, suggesting that the
expression of a large number of genes is independent of
SPL function. Of these, several have known functions in
early anther development, including BAM1, BAM2, SERK1
and SERK2. In addition, two of the genes that were
SPL-independent and differentially expressed in ems1
(according to SAM) encode putative transcription factors
with a MYB domain and a PHD finger, respectively.
Therefore, both SPL-dependent and SPL-independent
transcriptional programs are probably required for normal
anther development.
Relating co-expressed genes to known developmental
functions
To begin to identify genes that might function together, we
first used the hierarchical clustering method to obtain an
approximate number of clusters among the 1954 differen-
tially expressed genes, resulting in the identification of 14
major clusters. However, a drawback of hierarchal clustering
is that clusters are defined by the users by selecting sub-
trees that show similar expression patterns (Sturn et al.,
2002); therefore, specific membership in a cluster can be
somewhat subjective. As an alternative approach, we used
the K-mean clustering method (Han and Kamber, 2001) with
14 as the cluster number (Figure 1). Clusters A–C contained
genes that showed significantly increased mRNA levels in
spl compared with wild-type, and differed in expression only
slightly between ems1 and wild-type. Therefore, these
clusters were renamed SPL-H1, SPL-H2 and SPL-H3.
Cluster D contained genes whose expression was reduced
significantly in ems1 and spl; hence this cluster was named
4 Asela J. Wijeratne et al.

(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

Table 1 A subset of transcription factors affected by ems1 and/or spl mutations

Gene ID Description Cluster Wild-type ems1 spl Interaction

At5g03790 Homeobox (LMI1) SPL-H2 45.2 123.2 602.4 ATMLBG

At5g60890 MYB (ATR1) SPL-H1 22.9 33.9 55.3 SPLNG
At2g21650 MYB (MEE 3) SPL-H2 27.3 43.2 134.2 SPLNG
At4g21750 ATML1 SPL-H2 524.8 838.3 1314.3 SPLNG
At4g18960* AGAMOUS 838.1 882.3 1655.2 SPLNG
At5g65790a,b MYB (RAX) ES-RH2 227.9 23.5 12.7 DG
At4g37750 ANT SPL-H2 312.2 377.1 2112.4 SPLNG
At3g11440 MYB 65 SPL-L3 366.7 285.4 78.5 MYB33/65
At5g06100 MYB 33 SPL-L4 318.3 221.2 88.7 MYB33/65
At1g48620 ANT-LIKE 5 SPL-H1 489.3 923.1 1022.2 AGBG2
At5g10140b MADS (FLC) ES-RH2 285.3 31 28.3 MDG
At2g16910a,b bHLH (AMS) ES-RH2 454.8 14.3 15.8 MDG
At3g10040 Tri-helix SPL-H1 76.6 132.4 191.1 AGBG2
At3g29020 MYB SPL-L4 53.4 43.1 17.4 AGBG2
At5g57390 AP2/EREBP SPL-H2 37.1 40.9 227.1 ATMLBG
At2g24650 B3 SPL-H2 44.3 64.7 190 ATMLBG
At2g26580 INO-like (INOL) SPL-H2 20.9 18.9 90.9 ATMLBG
At1g68480 C2H2 zinc finger SPL-H2 39.2 53.5 168.7 ATMLBG
At4g36870 Homeobox (BLH2) SPL-H2 14.1 16 91.4 ATMLBG
At2g23760 Homeobox (BLH4) SPL-H2 47.1 44.1 214.6 ATMLBG
At2g35310 B3 SPL-H1 365.4 575.6 731.6 SPLNG
At3g06220 B3 SPL-H1 510.6 865.1 1331.2 SPLNG
At3g17010 B3 SPL-H1 345.2 576 718.5 SPLNG
At5g67110 bHLH SPL-H3 363.5 510.2 737.9 SPLNG
At2g27230 bHLH SPL-H3 436.5 630.2 1144.8 SPLNG
At3g61250 MYB SPL-H3 272.8 311.5 619.3 SPLNG
At5g49330 MYB SPL-H3 181.2 253.7 513.4 SPLNG
At2g31210b bHLH ES-RH2 177.8 14.6 12.5 bHLH
At1g06170a,b bHLH ES-RH1 1062 27.4 13.4 bHLH
At3g60580 C2H2 zinc finger ES-RH2 311.4 23 12.9 MDG
At4g36590b MADS (AGL40) ES-RH2 118.6 19.8 15.5 MDG
At4g09460b MYB ES-RH2 467.4 77 29.8 MDG
At5g39610 NAC domain ES-RH2 297.6 56.4 32.3 MDG
At1g19230 AP2/EREBP ES-RH2 422.4 17.3 18 DG
At1g68200 C3H zinc finger ES-RH2 780.7 93.3 64 DG
At3g28470a,b MYB ES-RH1 861.9 21.3 14 DG
At3g49690a,b MYB ES-RH2 223.9 34.9 28.8 DG
At1g54240 MYB ES-RH2 151.9 24.2 24 DG
At2g42940a DNA-binding ES-RH2 646.1 37.6 31.2 DG

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.

Interestingly, the majority of the genes encoding putative

Functional predictions in the gene clusters
To probe the functional relatedness of the co-expressed
genes, we examined the GO annotations for the genes in
each cluster. We found that genes encoding transcription
factors were over-represented in differentially expressed
genes (9.7%, compared with approximately 6% among
genes represented by the Affymetrix ATH1 chip; Table 1 and
Table S4; see below for more details). In addition, cluster
SPL-L3 is enriched with genes encoding putative DNA
binding proteins with possible transcription factor activity.
transcription factors had not been previously associated
with anther development. This suggests that the gene reg-
ulatory network underlying early anther development is far
more complex than indicated by previous genetic studies
(Ma, 2005). In addition, the GO category ‘other binding’ was
significantly over-represented in clusters ES-RH1–3. Most of
these genes appear to be involved in metabolism, transport
facilitation, energy and electron transport (Table 3 and
Table S3). This finding is consistent with the fact that the
tapetum is known to be metabolically very active, and that

ª 2007 The Authors

Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), doi: 10.1111/j.1365-313X.2007.03217.x
6 Asela J. Wijeratne et al.

Table 2 Subset of genes involved receptor like-kinase signal transduction

Gene ID Description Cluster Wild-type ems spl Interaction

AT4G39400 LRR X (BRI1) SPL-H3 337.8 455.1 1020.2 SPLNG

AT3G56100 LRR III (MRLK) SPL-H2 62.4 174.4 276.1 SPLNG
AT4G20270 LRR XI (BAM3) SPL-H3 172.9 184.2 451.1 SPLNG
AT2G26330 LRR XIII (ER) SPL-H3 1736.2 2397.5 3828.7 SPLNG
AT5G62230 LRR XIII (ERL1) SPL-H1 258.3 398.2 541.9 SPLNG
AT5G07180 LRR XIII (ERL2) SPL-H1 294.8 436.1 752.2 SPLNG
AT5G07280 LRR X (EMS1) ES-RM2 1298.3 164.6 623.2
AT5G45780 LRR II SPL-H3 53.4 63.5 145.8 SPLNG
AT5G16000 LRR II SPL-H3 98.2 115.8 217.2 SPLNG
AT5G67200 LRR III SPL-H1 128.4 222.2 301 SPLNG
AT3G17840 LRR III SPL-H1 680.1 1267.2 1451.9 SPLNG
AT2G01210 LRR III SPL-H3 109.2 113 328.8 SPLNG
AT2G26730 LRR III SPL-H3 301.4 334.6 781.7 SPLNG
AT5G43020 LRR III SPL-H3 339.8 445.4 950.4 SPLNG
AT5G51560 LRR IV SPL-H1 602.2 1007.8 1206.4 SPLNG
AT4G03390 LRR V SPL-H1 177 261.5 402.3 SPLNG
AT1G11140 LRR V SPL-H1 273.5 471.6 725.8 SPLNG
AT3G03770 LRR VI SPL-H3 101.4 86.8 227.2 SPLNG
AT5G01890 LRR VII SPL-H1 282.4 537.5 1018.7 SPLNG
AT4G36180 LRR VII SPL-H1 572.1 1033.4 1253.6 SPLNG
AT2G24230 LRR VII SPL-H3 139.1 126.6 296.7 SPLNG
AT3G28040 LRR VII SPL-H3 289 303.1 623.9 SPLNG
AT3G56370 LRR VII SPL-H3 998.2 1132.5 2132 SPLNG
AT5G56040 LRR XI SPL-H3 291 270.3 618.6 SPLNG
AT5G62710 LRR XIII SPL-H1 185.5 403.9 433.2 SPLNG
At1g68780 RLP SPL-H2 32.0 39.9 378.5 SPLNG
At5g23400 RLP SPL-H1 44.8 68.5 148.2 SPLNG
At3g25905 CLE27 SPL-H2 43.3 74.3 247.1 SPLNG
At3g24770 CLE 41 SPL-H3 122.0 149.1 392.8 SPLNG
At5g66080 PP2C SPL-L3 140.5 61.4 26.6 PPC
At5g27930 PP2C SPL-H3 187.9 239.1 480.1 PPC
AT5G60080a RLK ES-RH3 261.7 53.6 37.5 MDG
AT5G60090a RLK ES-RH2 375 40 25.8 DG
AT5G06820a LRR V ES-RH3 456 67.2 49.8 MG

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-
ity (Ma, 2005).
Differential expression of genes encoding transcription
regulators
Among the genes differentially expressed in the spl and/
or ems1 mutants, 254 genes (13%) coding for putative
transcription factors or other transcriptional regulators
(TF) are enriched relative to the gene set on the Affyme-
trix chip (approximately 9%) (Table 1 and Tables S3 and
S4). We found that many members of the 26 TF families
defined by Riechmann et al. (2000) and the Sheen Lab
website (see Experimental procedures) are affected by the
spl and/or ems1 mutations. In particular, seven of nine
major transcription factor families (MYB, bHLH, AP2/ERE-
BP, homeobox, C2H2, NAC and MADS) had the highest
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), doi: 10.1111/j.1365-313X.2007.03217.x
numbers of genes affected by the two mutations (see
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
gene). Most of these genes have not been identified by
genetic studies.
Nearly 11% (27) of the 254 differentially expressed TF
genes coded for MYB proteins, making them the most
numerous type in this group. In particular, MYB genes
were statistically over-represented among TF genes in
clusters ES-RM1 (33%), SPL-L3 (27%) and SPL-L4 (21%),
compared to 10% (198/1990) (MYB among all TFs) on the
Affymetrix chip (Table S4). Among these, MYB33 (SPL-L4)
and MYB65 (SPL-L3) have redundant functions in promo-
ting tapetum development and function (Gocal et al., 2001;
Millar and Gubler, 2005). In addition, four members of one
MYB clade are also differentially expressed: AtMYB87/
ª 2007 The Authors
 Gene functions in the early Arabidopsis anther 7

Table 3 Subset of other genes discussed in the network

Gene ID Gene family Cluster Wild-type ems1 spl Interaction

a
AT5G07230 Acyl lipid metabolism (A9) ES-RH1 6301.2 124.6 28.5 MDG
AT1G06520a Acyl lipid metabolism (AtGPA1) ES-RH1 1084.3 40.4 22.7 DG
AT4G28395a Acyl lipid metabolism (ATA7) ES-RH3 488 58.5 41.1 DMG
AT3G15400a ATA20 ES-RH1 2487.3 115.1 43.5 MDG
AT3G42960a ATA1 ES-RH1 2379 38.8 22.3 DG
AT1G01280a Acyl lipid metabolism ES-RH1 549.3 11.5 12.6 MDG
AT3G52130a Acyl lipid metabolism ES-RH1 8650.6 123 37.5 MDG
AT5G52160a Acyl lipid metabolism ES-RH1 1295.1 21.2 18.8 MDG
AT5G07550a Acyl lipid metabolism ES-RH2 304.4 35.5 16 MDG
AT3G22620a Acyl lipid metabolism ES-RH3 116.3 18.8 16.4 MDG
AT3G22600a Acyl lipid metabolism ES-RH3 702.2 92.6 52.6 MDG
AT4G10850a MTN3-like protein ES-RH1 1033.5 24.2 18 MDG
AT5G61320a Cytochrome P450 ES-RH2 293.8 36.4 18.1 MDG
AT5G09970a Acyl lipid metabolism ES-RH1 1509.9 35.2 23.9 DG
AT5G62080a Acyl lipid metabolism ES-RH1 8737.2 290.7 69.9 DG
AT1G66850a Acyl lipid metabolism ES-RH1 3843.8 121 38.9 DG
AT3G07450a Acyl lipid metabolism ES-RH1 8258.9 162.5 41 DG
AT1G06250a Acyl lipid metabolism ES-RH2 229 19.3 16.6 DG
AT5G53190a MTN3-like protein ES-RH1 2006.9 53.9 37 DG
AT5G13170a MTN3-like protein ES-RH3 1109.8 164.7 125.7 DG
AT4G19230a Cytochrome P450 ES-RH3 447 73.5 52.8 DG
AT5G23190a Cytochrome P450 ES-RH2 379.8 20.3 16 DG
AT1G71160a Acyl lipid metabolism ES-RH1 1024.9 26.2 20.7 DMG
AT3G51590a Acyl lipid metabolism ES-RH2 572 50 18.5 DMG
AT3G52160a Acyl lipid metabolism ES-RH2 288.5 18.8 15.9 DMG
AT1G02813 Endomembrane ES-RH1 584.6 17.6 16.7 DMG
AT1G47980 Endomembrane ES-RH2 1032.8 52.1 59.1 DMG
AT1G68875 Endomembrane ES-RH2 418.2 27.1 14.1 DMG
AT1G76470 Endomembrane ES-RH2 577.8 30.9 32 MMDG
AT5G48210 Endomembrane ES-RH3 186.2 35.5 27.6 MMDG
AT1G13140a Cytochrome P450 ES-RH2 540.2 37.6 23.2 MMDG

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).

At4g37780 (ES-RM1), AtMYB38/At2g36890 (SPL-L3), At-

MYB84/At3g49690 (ES-RH3) and AtMYB68/At5g65790 (ES-
RH2) (Stracke et al., 2001). The four predicted proteins
shared 29–58% sequence identity and 42–67% similarity.
The differential expression data suggest that these four
MYB genes might function downstream of SPL and/or
EMS1.
In addition to members of the MYB family, 21 (8%) of
the 254 differentially expressed TF genes encode bHLH
proteins (Table S4). Of these 21 genes, 14 had one or
more close homologs, suggesting functional redundancy.
Two of them, AtbHLH091/At2g31210 (ES-RH2 cluster) and
AtbHLH089/At1g06170 (ES-RH1 cluster), share 40% amino
acid sequence identity and are found in a distinct clade in
the bHLH phylogenetic tree (Heim et al., 2003; Li et al.,
2006b), along with another member (AtbHLH010/
At2g31220) that is not present on the Affymetrix
chip. These findings suggest that additional bHLH
genes are candidate regulators of early anther
development.
Expression of several LRR-RLK genes is affected by ems1
and spl mutations
Because several LRR-RLKs play vital roles during anther
development, we were interested in determining whether
the expression of additional genes encoding LRR-RLKs was
affected by the ems1 and spl mutations. There were 37 LRR-
RLKs among the 1954 differentially expressed genes (Ta-
ble 2 and Table S3), of which 10 (27%) and 16 (43%) were in
clusters SPL-H1 and SPL-H3, respectively (more frequently
than other clusters). EMS1 is among the genes reduced in
the spl mutant (in cluster ES-RM2). Moreover, one of the
differentially expressed LRR-RLK genes, At2g41820 (in
cluster ES-L), belongs to the same sub-family as EMS1.
Another pair of LRR-RLKs, At2g23950 and At4g30520 (con-
taining the LRRII domain, also present in SERK1 and SERK2),
are present in cluster SPL-L4 and are closely related phylo-
genetically (Shiu and Bleecker, 2001, 2003; Shiu et al., 2004).
Within the ES-RH3 cluster, At5g06820 is one of the most
highly expressed LRR-RLK genes in the anther; in addition,

ª 2007 The Authors

Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), doi: 10.1111/j.1365-313X.2007.03217.x
8 Asela J. Wijeratne et al.

its closest homolog has only 38% amino acid sequence
identity and 57% similarity, suggesting a distinct role for this
LRR-RLK in the developing anther.
In addition to LRR-RLKs, two other differentially ex-
pressed genes (At5g60080 and At5g60090) encode putative
receptor-like kinases, with 47% amino acid sequence identity
(Table 2). Both show moderate levels of expression in the
wild-type anther and may function in the tapetum and/or
microsporocytes. Of the 1954 differentially expressed genes,
two encode putative receptor-like proteins (RLP) and two
others code for CLAVATA3 (CLV3)-like proteins (CLE)
(Table 2). These genes may also function in RLK signaling
pathways involved in anther development, similar to the
functional interactions between CLV1 (RLK), CLV2 (RLP) and
CLV3 (CLE) (Clark, 2001).
Differentially expressed genes encoding metabolic proteins
Among the differentially expressed genes in the clusters
ES-RH1–3, a number are predicted to encode metabolic
enzymes (Table 3 and Table S3). Noteworthy are genes for
acyl lipid metabolism, including ones that were previously
shown to be preferentially expressed in the tapetum,
consistent with the fact that the tapetum is absent in both the
spl and ems1 mutants. In addition, several cytochrome P450
genes were found, suggesting that they may also function in
the tapetum. Genes coding for lipid transfer proteins and
endomembrane proteins were also detected.
In situ RNA hybridization reveals expression in microspo-
rocytes and the tapetum
To assess expression patterns in the anther of a small subset
of the genes with differential expression between wild-type
and spl and/or ems1 anthers, we performed RNA in situ
hybridization experiments. Although the microarray experi-
ments used RNAs from stage 4–6 anthers, the in situ
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
that these genes are involved in early anther differentiation,
as is SPL. The clusters SPL-L1, SPL-L3 and SPL-L4 include
genes that show significantly reduced expression in spl
anthers, but not in ems1 anthers, suggesting that these
genes might be preferentially expressed in microsporocytes
or meiocytes because these cells are absent in spl anthers,

(a1) (a2) (b1) (b2) (b3)

At5g67110 At2g16210

(c1) (c2) (d1) (d2) (d3)

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.

ª 2007 The Authors

Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), doi: 10.1111/j.1365-313X.2007.03217.x

but present in ems1 anthers. We tested this hypothesis for
several genes and found that they are indeed expressed
predominantly in the microsporocytes in stage 5 anthers
(Figure 2 and Figure S1). Specifically, a B3 gene, At2g16210
(in cluster SPL-L3), exhibited preferential expression in the
microsporocytes, with moderate to low levels of expression
in the tapetum (Figure 2).
Expression of the genes in cluster ES-RH1–3 was dramat-
ically reduced in both spl and ems1 mutants, usually by
sixfold or more (Figure 1). One possibility is that these genes
are preferentially expressed in the tapetum, which is absent
in both mutants. This idea is supported by the presence of
known tapetum-specific/preferential genes in these clusters.
Surprisingly, the in situ hybridization results showed that
several of these genes are expressed in both the tapetum
and microsporocytes, perhaps because the ems1 meiocytes
are abnormal and cannot complete meiosis. Among the 27
differentially expressed MYB genes, AtMYB35/At3g28470
had the highest level of expression in wild-type anthers, and
showed a significant reduction in transcript levels in both
ems1 and spl anthers. The in situ hybridization results
(Figure 2) showed that this gene was expressed in both the
tapetum and microsporocytes during anther stages 4 and 5.
In addition, AtbHLH089/At1g06170 was expressed in the
tapetum and microsporocytes, a pattern similar to that of
AtMYB35/At3g28470.
Detection of putative cis-regulatory elements in promoters
of differentially expressed genes
To obtain clues for possible mechanisms of co-regulation,
we analyzed the presumed promoter regions of the co-ex-
pressed genes for the presence of known cis-regulatory
elements (see Experimental procedures for details). We
found that a number of cis-regulatory elements were en-
riched in the putative regulatory regions of the differentially
expressed genes, with frequencies greater than those found
in random samples of putative regulatory sequences
(Table S5). Among the genes with increased expression in
the spl mutant, cluster SPL-H1 was enriched for genes
whose promoter contained a MYB binding site (YAACKG;
Abe et al., 2003), and SPL-H2 was enriched for genes with a
promoter that contained a binding site for the L1-specific
protein ATML1 (TAAATGYA; Abe et al., 2001). In addition,
genes in clusters SPL-H2 and H3 were enriched for two Myc
(a specific type of bHLH) binding sites, CATGTG and CA-
CATG (Abe et al., 1997; Simpson et al., 2003). We also
detected enrichment of genes that contained binding sites
for MADS domain proteins, including AG in the SPL-H1 and
SPL-L4 clusters, AGAMOUS-like2 (AGL2) in the SPL-L2
cluster, and AGL15 in the ES-RH3 cluster (Huang et al., 1996;
Tang and Perry, 2003). In addition, genes containing several
other MYB and bHLH binding sites and hormone-responsive
elements were enriched in other clusters (Table S5).
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 9
The analysis for over-represented motifs in clusters ES-
RH1–3 (with greatly reduced expression in both spl and
ems1 anthers) identified putative binding sites for known
types of transcription factors, including bHLH and MYB
proteins (Table S6). In the three clusters, 198 genes con-
tained an E-box for binding to bHLH proteins in their
promoters, and 103 of the 198 genes also contained a
TAACAAA box for binding MYB proteins. This result
suggests that bHLH and MYB factors might regulate the
expression of these genes. Several of the 198 genes encode
putative transcription factors or other regulatory proteins
(Tables 1 and 2). Specifically, the promoters of the bHLH
gene AMS/At2g16910 and a gene encoding a serine/threon-
ine protein kinase (At5g60080) contained both an E-box and
a TAACAAA box. Other genes with promoters that have
E-boxes include the bHLH gene AtbHLH091/At2g31210 and
the MYB genes AtMYB35/At3g28470, AtMYB84/At3g49690
and AtMYB68/At5g65790, as well as the putative LRR-RLK
gene, At5g06820, and a protein kinase gene, At5g60090. The
promoter of another bHLH gene, AtbHLH089/At1g06170,
contained a TAACAAA box, but not an E-box. Other over-
represented cis-regulatory sequences include two Myc
binding sites, CATGTG and CACATG (cluster ES-RH1) (Abe
et al., 1997; Simpson et al., 2003). These sites might be
targets for Myc and MYB proteins expressed in the anther.
Discussion
The Arabidopsis SPL/NZZ and EMS1/EXS genes are key
regulators of anther cell differentiation (Canales et al.,
2002; Schiefthaler et al., 1999; Yang et al., 1999; Zhao et al.,
2002). We have identified 1954 genes whose expression is
affected by the ems1 and/or spl mutations. The expression
patterns of these genes fall into several major groups,
including (i) elevated expression in the spl mutant, (ii) re-
duced expression in spl, (iii) moderately reduced expression
in both spl and ems1, and (iv) greatly reduced expression in
both mutants. Specifically, expression of 1940 of the 1954
genes is affected by the spl mutation, whereas the expres-
sion of only 525 genes is affected by the ems1 mutation,
indicating that SPL function is more general than that of
EMS1, as supported by the mutant phenotypes. The pres-
ence of genes with known functions in our expression pat-
tern clusters provides clues regarding the possible functions
of other genes in the same clusters. In addition, genes
encoding transcription factors and other types of regulators
were enriched in our differentially expressed gene clusters
in comparison to the frequencies of these types of genes on
the Affymetrix chip. These results strongly suggest that SPL
is near the top of an anther regulatory hierarchy that
includes other regulatory genes, whereas EMS1 is involved
in the regulation of a smaller subset of genes.
Our analysis has uncovered potential binding sites for
MADS box, MYB and bHLH proteins in predicted promoter
10 Asela J. Wijeratne et al.

regions of co-expressed genes. The information on differ-
ential gene expression and putative cis-regulatory elements,
as well as known gene functions, has allowed the formula-
tion of a number of hypotheses about anther gene regula-
tion, leading to a model of gene regulatory networks during
anther development (see below for more discussion;
Figure 3). Clearly, the genetic interactions proposed here
may be either direct or indirect, and will require functional
verification using molecular genetic studies.
Expression profiling reveals molecular characteristics of
anther cells
Little is known about the molecular nature of wild-type
anther cell types and abnormal cells in the spl and ems1
mutants. Our microarray analysis provides a genome-scale
description of anther cells. In wild-type, spl and ems1
anthers, approximately 14 000 genes were detected as
expressed at or above the conservative cut-off value of 50
(Zhang et al., 2005). The fact that a large subset (clusters
SPL-H1–3) of differentially expressed genes showed abnor-
mally high levels of expression in spl anthers suggests that
SPL normally restricts the expression of these genes, inclu-
ding several floral homeotic genes. In the absence of SPL,
mis-expression of these genes may lead to the formation of
cell types not typically found in wild-type anthers.
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,
both PDF2 and ATML1 encode homeodomain transcription
factors and have been shown to be involved in maintaining
the identity of the L1 layer, presumably by regulating the
expression of L1-specific genes (Abe et al., 2001). In addi-
tion, the presumptive promoters of some of the differentially
expressed genes, such as INOL and genes in box ATML1BG
(Figure 3 and Table S7), contain ATML1-binding sites, sug-

Figure 3. Gene regulatory network for early

AG anther development.
Ovals represent genes (nodes) with known func-
tions in anther development, whereas genes
represented by boxes are proposed to have
anther functions based on this and other studies.
BAM1/2 SPL Interactions represented by black lines are based
AGBG* on information derived from past studies (see
text for references). Interactions with dotted lines
MYB110 are based on previous studies, but there is no
direct evidence. Interactions represented by
dashed lines are information derived from micro-
array and cis-regulatory element analysis. Lines
ATML1 ANT BAM3 terminating in arrows represent positive regula-
SPLNG* tion, lines ending with diamonds indicate nega-
BRI1
ERECTA tive interactions, and lines without an arrowhead
or diamond denote binding. The nodes marked
INOL ATML1BG* with an asterisk (boxes) represent more than one
BLH2 gene. These genes are listed in Table S7 (AGBG,
BLH4 TPD1 AG binding gene set; ATML1BG, ATML1 binding
gene set; SPLNG, SPL negatively regulated
genes) and Table S8 (MG, MYB33/65 binding
EMS1 SERK1/2
genes; MDG, MYB33/65 and DYT1 binding
genes; DG, DYT1 binding genes; MMDG,
MYB33/65 and DYT1 binding and genes regula-
PPC
ted by MS1; MSG, genes regulated by MS1;
DMG, DYT1 binding genes and genes regulated
by MS1). A subset of these genes is listed in
miR159 MYB33/65 DYT1 bHLH* Tables 1–3.

AtMYB103
RLK1

RLK2 MG* MDG* RLK3 DG*

AMS MS1

* Represents more
than one gene AtCP1L MMDG* MSG* DMG*

ª 2007 The Authors

Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), doi: 10.1111/j.1365-313X.2007.03217.x

gesting that they might be regulated by ATML1. The
identification of many genes that show increased expression
in spl anthers supports the hypothesis that SPL functions as
a transcriptional repressor (Balasubramanian and Schneitz,
2002). Therefore, it is possible that SPL restricts the expres-
sion of L1-specific genes in L2-derived cells by negatively
regulating the expression of ATML1 in the L2-derived cells
(Figure 3).
A regulatory network for early anther development inclu-
ding SPL-dependent and independent components
Floral homeotic genes can control target genes with various
functions, including SPL, during the development of floral
organs (Ito et al., 2004; Sablowski and Meyerowitz, 1998).
Our expression analysis clearly indicated that the expression
of more than 5250 genes is affected by the spl mutation, with
over 1900 of them showing twofold or more changes from
normal levels. Furthermore, some of these (Figure 3,
MYB110 and box AGBG, Table S7) have predicted AG
binding sites in their putative promoter regions, suggesting
that they might be regulated by both AG and SPL. On the
other hand, the expression levels of approximately 8600
genes are not significantly altered in the spl mutant anthers,
indicating that these genes not under the transcriptional
control of SPL. Therefore, AG (and other MADS box pro-
teins) probably also regulate other genes to promote anther
development, as supported by the observation that some
SPL-independent genes contained putative AG binding sites
in their promoters (not shown). Furthermore, our data sug-
gest that SPL and AG are involved in a positive–negative
regulatory feedback loop, where AG positively regulates the
expression of SPL, and SPL in turn represses the expression
of AG (Table 1 and Figure 3).
SPL/NZZ is also required for normal ovule development
(Elliott et al., 1996; Klucher et al., 1996; Schiefthaler et al.,
1999), and knowledge on genetic interactions between SPL
and other ovule genes can facilitate the understanding of
interactions between SPL and other genes in anther devel-
opment. SPL interacts genetically with two other ovule
genes, INNER NO OUTER (INO) and AINTEGUMENTA
(ANT), which encode YABBY and AP2 domain proteins,
respectively (Villanueva, 1999; Balasubramanian and
Schneitz, 2002). SPL/NZZ negatively regulates ANT expres-
sion in the ovule (Balasubramanian and Schneitz, 2002).
Similarly, we found that ANT was up-regulated in spl mutant
anthers, suggesting that ANT and SPL have a similar
relationship in the anther (Figure 3). In the ovule, BELL1
(BEL1) is required for INO expression (Balasubramanian and
Schneitz, 2002). We found that expression of INO and BEL1
was low and not significantly different in wild-type and spl
and ems1 mutant anthers (data not shown). However, a
gene (INOL, At2g26580) encoding a protein that has 31%
amino acid sequence identity and 44% similarity to INO, and
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 11
two BEL1-like genes, BHL2 (At4g36870) and BHL4
(At2g23760), showed elevated expression levels in spl
anthers, and are among the genes enriched for putative
AtML1 binding sites (SPL-H2 cluster, Table 1 and Figure 3).
Therefore, these INO and BEL1 homologs are probably
regulated by SPL/NZZ, and are possibly downstream of
ATML1 during early anther development (Figure 3). Our
analysis has also identified additional candidate genes for
anther development downstream of SPL (Figure 3, box
SPLNG).
Function of LRR-RLKs in cell–cell communication during
anther cell differentiation
Genetic studies have uncovered critical roles for several
LRR-RLKs during anther cell differentiation, including BAM1
and BAM2 for the formation of parietal cells, which in turn
develop into endothecium, middle layer and tapetum (Hord
et al., 2006). Although our microarray data did not reveal
significant differences in BAM1 and BAM2 expression
between wild-type and spl anthers, in situ hybridizations
results support the hypothesis that SPL positively regulates
the expression of BAM1, and that BAM1/2 restrict the
domain of SPL expression (Figure 3; Hord et al., 2006).
BAM1/2 together with BAM3 and CLV1 form a monophyletic
group (DeYoung et al., 2006; Hord et al., 2006). Our results
indicate that SPL negatively regulates the expression of
BAM3 (Figure 3). In addition, members of the ERECTA clade
of LRR-RLK genes are important for position-dependent
guard cell differentiation (Shpak et al., 2004, 2005); we found
an increase in the expression of members of the ERECTA
sub-family in spl mutant anthers. Furthermore, a number of
other LRR-RLKs (Table 2 and Figure 3, box SPLNG) also
showed elevated expression in spl anthers. Our results
suggest that BAM3, ERECTA homologs and other LRR-RLKs
function antagonistically to SPL/NZZ during anther devel-
opment (Figure 3).
LRR-RLKs may interact with each other during signal
transduction (Shpak et al., 2003; Wang et al., 2005b). For
example, SERK1 and SERK3 (also named BRI1-associated
kinase1) can interact with BRI1 for brassinosteroid (BR)
signaling (Karlova et al., 2006; Li et al., 2002; Nam and Li,
2002; Wang et al., 2005a). Because SERK1/2 and EMS1/EXS
are required for specifying tapetum identity (Albrecht et al.,
2005; Canales et al., 2002; Colcombet et al., 2005; Zhao et al.,
2002), and BRI1 and EMS1 are both members of the LRR X
sub-family, it was postulated that SERKs interact with EMS1/
EXS (Figure 3; Albrecht et al., 2005; Colcombet et al., 2005).
We observed that the BRI1 mRNA level was significantly
greater, but EMS1 expression was reduced, in spl anthers as
compared to wild-type (Table 2 and Figure 3), suggesting
that BRI1 and EMS1 are differentially regulated by SPL.
Moreover, three other RLKs are predicted to function
downstream of MYB33/65 and/or DYT1, which are key
12 Asela J. Wijeratne et al.
regulators of tapetum function at the time of meiosis
(Figure 3). Therefore, receptor-mediated cell–cell communi-
cation may play a role in coordinating meiotic events and
processes in the surrounding somatic cells.
Previous studies have shown that the protein phospha-
tase 2C (PP2C) POLTERGEIST (POL) acts downstream of
CLV1 (Song and Clark, 2005; Song et al., 2006; Yu et al.,
2003). We found that two of the differentially expressed
genes, At5g66080 and At5g27930, encode PP2Cs (Figure 3,
box PPC); they are candidates for downstream components
of LRR-RLK-mediated signaling pathways during anther
development, and need to be tested by functional studies.
In part because of functional redundancy (Albrecht et al.,
2005; Colcombet et al., 2005; DeYoung et al., 2006; Hord
et al., 2006; Shpak et al., 2004, 2005), few LRR-RLKs have
known genetic functions. Our expression comparison and
cis-element analysis have identified LRR-RLKs that poten-
tially facilitate signaling events between anther cells.
Transcriptional regulation during tapetum development and
meiosis
Genetic studies have demonstrated that the bHLH putative
transcription factor DYT1 functions downstream of the LRR-
RLK EMS1/EXS to promote tapetum development and
function (Zhang et al., 2006) (Figure 3). This is analogous to
the function of another bHLH gene BRI1-EMS-SUPPRESSOR
1 (BES1) downstream of the LRR-RLK gene BRI1 (Vert et al.,
2005; Yin et al., 2002, 2005). In addition, AtMYB33 and At-
MYB65 are also needed for normal tapetum development
(Millar and Gubler, 2005). The importance of MYB and bHLH
proteins during anther development is strongly supported
by our observation that several MYB and MYC/bHLH binding
sites were over-represented in the clusters of differentially
expressed genes. In particular, the bHLH-binding E-box was
found in presumptive promoters of genes in clusters ES-
RH1–3. In addition, the putative promoters of many of these
genes have predicted binding sites for MYB proteins
(Table S5). Because the earliest known tapetum transcrip-
tional regulators are MYB33/65 and DYT1, we propose that
these genes are downstream of MYB33/65 (Figure 3, box
MG), DYT1 (box DG) or both (box MDG). It is possible that
some of the proposed DYT1 targets are regulated by close
homologs of DYT1 (box bHLH). The putative DYT1 targets
include several MYB genes (Figure 3, box DG).
The putative DYT1 target AMS is required for normal
tapetum function and microspore development (Sorensen
et al., 2003; Zhang et al., 2006) (Figure 3). In addition, the
rice ortholog of AMS, TDR1, was down-regulated in the
anthers of a rice mutant defective in the GAMYB gene (Li
et al., 2006a; Tsuji et al., 2006), which is closely related to the
AtMYB33 and AtMYB65 genes. Thus, it is possible that AMS
is also transcriptionally activated by AtMYB33/65, as sup-
ported by known instances of the regulation of a single gene
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), doi: 10.1111/j.1365-313X.2007.03217.x
by both MYB and MYC/bHLH factors (Abe et al., 1997;
Baudry et al., 2006; Hartmann et al., 2005). AMS and TDR1
encode bHLH proteins and are critical for tapetum functions
that support microsporogenesis (Li et al., 2006a; Sorensen
et al., 2003). In 75 of the 77 genes in the SPL-L3 cluster, the
putative promoter regions contained an E-box, suggesting
that these genes might be regulated by bHLH proteins, such
as DYT1 and AMS, that act downstream of SPL (Figure 3).
MS1 is required for relatively late tapetum function that is
critical for pollen development, and encodes a putative
transcriptional regulator with a PHD finger (Ito and Shinoz-
aki, 2002; Wilson et al., 2001). Recently, it was shown that the
MS1 gene is down-regulated in the dyt1 mutant (Figure 3)
(Zhang et al., 2006). Therefore, some of the genes regulated
by DYT1 and possibly by MYB33/65 might be targets of MS1.
To test this hypothesis, we compared genes that are down-
regulated in ms1 mutant flowers (based on publicly avail-
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
in ms1 flowers than wild-type flowers, 15 were also present
in clusters ES-RH1–3. Furthermore, all 15 genes contained an
E-box in their putative promoters, and five also contained a
TAACAAA (MYB) box, suggesting that they are also regula-
ted by Myc/bHLH proteins or by both bHLH and MYB factors
(Figure 3, boxes DMG and MMDG). The expression of two of
the genes, At3g51590 and AT3G23770, was found to be
down-regulated in dyt1 anthers (Zhang et al., 2006).
Although a number of genes important for meiosis have
been identified (Hamant et al., 2006; Li and Ma, 2006; Ma,
2005), little is known about the transcriptional regulation of
meiotic genes. Our findings that genes encoding putative
transcriptional regulators, including MYB and bHLH pro-
teins, are co-expressed with known meiotic genes (in
clusters SPL-L3 and 4 and ES-RM1 and 2, Table 1) suggest
that these transcription factors might regulate meiotic gene
expression. It is interesting to note that in mice, MYB genes
are known to be required for meiosis (Latham et al., 1996;
Toscani et al., 1997). The presumptive promoters of some of
the genes encoding transcription factor have putative E-box
and/or MYB binding sites, suggesting that they might be
targets of DYT1 and/or MYB33/65 (Figure 3, boxes DG and
MDG). For example, AtMYB35/At3g28470 is expressed in
meiocytes/microsporocytes (Figure 2), and its expression is
reduced in the dyt1 mutant (Figure 3, box DG) (Zhang et al.,
2006). Because DYT1 is also expressed in meiocytes (Zhang
et al., 2006), it is possible that it directly regulates meiotic
gene expression. Alternatively, other meiotic bHLH proteins
might serve as regulators of meiotic gene expression, such
as AtbHLH091/At2g31210 (ES-RH2) and AtbHLH089/
At1g06170 (ES-RH1). Our microarray results, together with
our in situ hybridization results, strongly suggest that these
two genes (and possibly also At2g31220/AtbHLH010) func-
tion downstream of EMS1/EXS (Figure 3). Because EMS1/
ª 2007 The Authors

EXS promotes differentiation of the tapetum, its proposed
role in activating (indirectly) transcriptional regulators in the
meiocytes provides a possible mechanism for interaction
between the two cell types.
Putative targets of transcriptional regulators are candidate
genes for tapetum function
Among the putative targets of DYT1 and MYB33/65 (Fig-
ure 3, boxes DG and MDG, Table 3), several encode meta-
bolic enzymes. These genes might be important for tapetum
function to support pollen development. In addition, one of
the putative targets of AMS is AtCP1, a homolog of the rice
gene OsCP1 encoding a cysteine protease that is important
for microsporogenesis (Li et al., 2006a). OsCP1 expression is
regulated by TDR1, the rice ortholog of AMS, suggesting
that this regulatory relationship for tapetum function is
conserved between eudicots and monocots. Moreover,
some putative downstream genes of MS1, DYT1 and/or
MYB33/65 (Figure 3, boxes MMDG and DMG, Table 3) en-
code lipid transfer proteins and ER proteins, consistent with
known functions of such proteins in the tapetum to provide
materials for pollen wall formation.
Conclusion
Here we report differential expression for approximately
1900 genes in spl and/or ems1 mutant anthers, compared
with their expression levels in wild-type anthers. These
genes might have important functions during anther devel-
opment and meiosis. Our attempt to integrate microarray
data, bioinformatic analysis of cis-regulatory sequences and
results from previous studies has resulted in a set of hypo-
theses about genetic interactions in a complex regulatory
network. These ideas will stimulate further experiments to
expand our understanding of anther development. Our
finding of a large number of candidate anther regulatory
genes and other genes with unknown functions indicates
that much remains to be discovered concerning this
important floral organ.
Experimental procedures
Plant material, plant growth and anther collection
All the plants used in this study were of the ecotype Landsberg
erecta. The two male-sterile mutants ems1 (Zhao et al., 2002) and
spl (Yang et al., 1999) have been described previously. Plants were
grown in Metro-Mix 200 soil (Scotts-Sierra Horticultural Product
Co.) at 22C with 16 h of light and 8 h of darkness for 28–35 days.
Stage 4–6 anthers were collected from wild-type, spl and ems1
mutant plants under the dissecting microscope and immediately
frozen in liquid nitrogen. The collected anthers were stored at )80C
until the extraction of total RNA. For each replicates, approximately
50 000 anthers were collected.
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
Microarray experiment and analysis
The microarray experiment was performed according to the method
described in the Affymetrix GeneChip Expression Analysis Over-
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
State University DNA Microarray Facility. The hybridization signals
for all the experiments were scaled to the same target intensity of
150 to compare expression profiles between samples.
Statistical analysis for the expression values was carried out at
the Department of Health Evaluation Sciences at Penn State
College of Medicine. Before statistical tests were performed,
scanned data were converted to expression data. If a probe set
represented more than one gene, the corresponding data were
excluded to avoid any misinterpretations. We used the quantile
normalization method to normalize the data (Bolstad et al., 2003).
To remove outliers and to summarize probe intensities within
one probe set into a single expression value, the Li–Wong
method was applied to background-adjusted, normalized perfect-
match (PM) probe intensities (Chu et al., 2004). PM and mismatch
(MM) probe intensities were highly correlated, and MM intensi-
ties composed of background noise as well as probe-specific
signal expression values were obtained based on PM intensities
rather than PM–MM intensities. To identify genes with statisti-
cally significant changes between wild-type and a mutant, a
significance analysis of microarray method (SAM) was used with
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
ms1 young and old flowers compared to wild-type young flowers
(http://affymetrix.arabidopsis.info/narrays/experimentpage.pl?
experimentid=23).
Clustering of co-expressed genes were carried out using the
GENESIS program (release 1.6.0 beta 1; Sturn et al., 2002). For the
identification of functionally related genes and of genes involved
in the same biological process, GO annotations were obtained
from the Arabidopsis Information Resource (TAIR). Gene family
information and gene annotations from the Sheen lab database
were also used (Arabidopsis transcription factors: Riechmann
et al., 2000, http://www.sciencemag.org/cgi/content/full/sci;290/
5499/2105/DC1/11; Sheen lab website: http://genetics.mgh.harvard.
edu/sheenweb/Ara_gene_families.html). Identification of closely
related homologs in the same gene family in the dataset was
performed using BLASTP (Altschul, 1990). Protein sequences were
compared with Arabidopsis proteins. A protein sequence in the
same family with an e-value cut-off of <1 · 10)20 was considered a
homolog.
cis-regulatory element analysis
Putative regulatory sequences consisting of 1000 bp upstream of
the start codon were downloaded from the TAIR database (http://
arabidopsis.org). The sequences that were in reverse orientation
were reverse-complemented using GENEDOC (Nicholas et al.,
1997). The sequences were submitted to the Plant cis-regulatory-
acting Regulatory DNA Elements Database server (http://
www.dna.affrc.go.jp/PLACE/) to identify known regulatory motifs
(Higo et al., 1999), and the output results were analyzed using Perl
scripts as described previously (Hu and Ma, 2006). To determine
whether a particular motif was enriched in the genes in a partic-
ular cluster, the statistical methods described by Hu and Ma
(2006) and Nemhauser et al. (2004) were used. Using the differ-
ence between the expected and the observed value, one-tailed
14 Asela J. Wijeratne et al.
P-values were calculated using the Z-score. A motif was consid-
ered enriched if the P-value was <0.01 and if the motif was found
in the regulatory regions for more than 10% of the genes in a
particular cluster.
In situ RNA hybridization
In situ hybridization to mRNA was carried out essentially as des-
cribed previously (Larkin et al., 1993). Digoxigenin (DIG)-labeled,
single-stranded RNA probes were synthesized from a PCR-derived
template containing an appropriately positioned T7 promoter to
produce a sense or antisense probe. A DIG-labeled, single-stranded
sense strand of a Ceratopteris richardii gene (accession number
CV735270) was used as a negative control. The sequences that were
used to derive antisense and sense strand DIG-labeled probes are
shown in Table S9. These sequences had the lowest sequence
identity to any other related genes.
Acknowledgements
We greatly appreciate the assistance provided by Saranga Wijeratne
(University of Peradeniya, Sri Lanka) and Dr Wei Hu (University of
California at Davis, USA) in producing Perl and Java scripts. We
thank Craig Praul (The Pennsylvania State University, USA) for
performing microarray hybridizations, Jiong Wang and Anthony
Omeis for plant care, Dr Zoe Wilson (University of Nottingham, UK)
for microarray data for the ms1 flower versus wild-type flowers,
Damitha Wickramasinghe (The Pennsylvania State University, USA)
and Peony Jena (The Pennsylvania State University, USA) for the
help with manuscript preparation, and Dr Naomi Altman (The
Pennsylvania State University, USA) for helpful discussion about
the microarray procedure. A.J.W. is partially supported by the Bio-
logy Department and the Intercollege Graduate Program in Plant
Biology at Pennsylvania State University. This work was supported
by a grant from the US Department of Energy (DE-FG02-02ER15332)
to H.M. and a grant from the National Science Foundation (DBI
0115684) to D.G.O. H.M. gratefully acknowledges the support of the
John Simon Guggenheim Memorial Foundation. R.A. acknowled-
ges NSF CAREER grant CCF-0643529.
Supplementary Material
The following supplementary material is available for this article
online:
Table S1. List of 1954 genes that were differentially expressed in
wild-type anthers and ems1 and/or spl anthers.
Table S2. Differentially expressed genes with known genetic func-
tions.
Table S3. Additional differentially expressed genes encoding tran-
scription factors, LRR-RLKs, and other proteins.
Table S4. Over-representation of genes encoding transcription
factors.
Table S5. cis-regulatory elements enriched in various clusters.
Table S6. Distribution of cis-regulatory elements in specific genes.
Table S7. Genes with elevated expression in the spl mutant.
Table S8. Comparison of genes present in clusters ES-RH1–3 with
genes down-regulated in ms1 flower buds.
Table S9. Primers for synthesis of probes for mRNA in situ
hybridization.
Figure S1. Expression patterns of additional selected genes using
mRNA in situ hybridization. Most of the genes showed at least some
level of expression in both the tapetum and microsporocytes.
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), doi: 10.1111/j.1365-313X.2007.03217.x
This material is available as part of the online article from http://
www.blackwell-synergy.com.
References
Abe, H., Yamaguchi-Shinozaki, K., Urao, T., Iwasaki, T., Hosokawa,
D. and Shinozaki, K. (1997) Role of Arabidopsis MYC and MYB
homologs in drought- and abscisic acid-regulated gene expres-
sion. Plant Cell, 9, 1859–1868.
Abe, M., Takahashi, T. and Komeda, Y. (2001) Identification of a cis-
regulatory element for L1 layer-specific gene expression, which is
targeted by an L1-specific homeodomain protein. Plant J. 26, 487–
494.
Abe, H., Urao, T., Ito, T., Seki, M., Shinozaki, K. and Yamaguchi-
Shinozaki, K. (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2
(MYB) function as transcriptional activators in abscisic acid
signaling. Plant Cell, 15, 63–78.
Albrecht, C., Russinova, E., Hecht, V., Baaijens, E. and de Vries, S.
(2005) The Arabidopsis thaliana SOMATIC EMBRYOGENESIS
RECEPTOR-LIKE KINASES1 and 2 control male sporogenesis.
Plant Cell, 17, 3337–3349.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J.
(1990) Basic local alignment search tool. J. Mol. Biol., 215, 403–
410.
Balasubramanian, S. and Schneitz, K. (2002) NOZZLE links prox-
imal–distal and adaxial–abaxial pattern formation during ovule
development in Arabidopsis thaliana. Development, 129, 4291–
4300.
Baudry, A., Caboche, M. and Lepiniec, L. (2006) TT8 controls its own
expression in a feedback regulation involving TTG1 and homol-
ogous MYB and bHLH factors, allowing a strong and cell-specific
accumulation of flavonoids in Arabidopsis thaliana. Plant J. 46,
768–779.
Bolstad, B., Irizarry, R.A., Astrand, M. and Speed, T.P. (2003) A
comparison of normalization methods for high density oligonu-
cleotide array data based on variance and bias. Bioinformatics,
19, 185–193.
Canales, C., Bhatt, A.M., Scott, R. and Dickinson, H. (2002) EXS, a
putative LRR receptor kinase, regulates male germline cell num-
ber and tapetal identity and promotes seed development in Ara-
bidopsis. Curr. Biol. 12, 1718–1727.
Chen, C., Zhang, W., Timofejeva, L., Gerardin, Y. and Ma, H.
(2005) The Arabidopsis ROCK-N-ROLLERS gene encodes a
homolog of the yeast ATP-dependent DNA helicase MER3 and
is required for normal meiotic crossover formation. Plant J. 43,
321–334.
Chu, T.M., Weir, B.S. and Wolfinger, R.D. (2004) Comparison of
Li–Wong and loglinear mixed models for the statistical analysis of
oligonucleotide arrays. Bioinformatics, 20, 500–506.
Clark, S.E. (2001) Cell signalling at the shoot meristem. Nat. Rev.
Mol. Cell Biol. 2, 276–284.
Colcombet, J., Boisson-Dernier, A., Ros-Palau, R., Vera, C.E. and
Schroeder, J.I. (2005) Arabidopsis SOMATIC EMBRYOGENESIS
RECEPTOR KINASES1 and 2 are essential for tapetum develop-
ment and microspore maturation. Plant Cell, 17, 3350–3361.
Cutler, S. and McCourt, P. (2005) Dude, where’s my phenotype?
Dealing with redundancy in signaling networks. Plant Physiol.
138, 558–559.
Deyhle, F., Sarkar, A., Tucker, E.J. and Laux, T. (2006) WUSCHEL
regulates cell differentiation during anther development. Dev.
Biol. 302, 154–159.
DeYoung, B.J., Bickle, K.L., Schrage, K.J., Muskett, P., Patel, K. and
Clark, S.E. (2006) The CLAVATA1-related BAM1, BAM2 and BAM3
ª 2007 The Authors

receptor kinase-like proteins are required for meristem function in
Arabidopsis. Plant J. 45, 1–16.
Elliott, R.C., Betzner, A.S., Huttner, E., Oakes, M.P., Tucker, W.Q.,
Gerentes, D., Perez, P. and Smyth, D.R. (1996) AINTEGUMENTA,
an APETALA2-like gene of Arabidopsis with pleiotropic roles in
ovule development and floral organ growth. Plant Cell, 8, 155–168.
Endo, M., Tsuchiya, T., Saito, H. et al. (2004) Identification and
molecular characterization of novel anther-specific genes in
Oryza sativa L. by using cDNA microarray. Genes Genet. Syst. 79,
213–226.
Gocal, G.F., Sheldon, C.C., Gubler, F. et al. (2001) GAMYB-like
genes, flowering, and gibberellin signaling in Arabidopsis. Plant
Physiol. 127, 1682–1693.
Goldberg, R., Beals, T.P. and Sanders, P.M. (1993) Anther develop-
ment: basic principles and practical applications. Plant Cell, 5,
1217–1229.
Hamant, O., Ma, H. and Cande, W.Z. (2006) Genetics of meiotic
prophase I in plants. Annu. Rev. Plant Biol. 57, 267–302.
Han, J. and Kamber, M. (2001) Data Mining: Concepts and Tech-
niques, 1st edn. San Francisco: Morgan Kaufmann Publishers.
Hartmann, U., Sagasser, M., Mehrtens, F., Stracke, R. and
Weisshaar, B. (2005) Differential combinatorial interactions of
cis-acting elements recognized by R2R3-MYB, BZIP, and BHLH
factors control light-responsive and tissue-specific activation
of phenylpropanoid biosynthesis genes. Plant Mol. Biol. 57, 155–
171.
Heim, M., Jakoby, M., Werber, M., Martin, C., Weisshaar, B. and
Bailey, P.C. (2003) The basic helix-loop-helix transcription factor
family in plants: a genome-wide study of protein structure and
functional diversity. Mol. Biol. Evol. 20, 735–747.
Higo, K., Ugawa, Y., Iwamoto, M. and Korenaga, T. (1999) Plant cis-
acting regulatory DNA elements (PLACE) database: 1999. Nucleic
Acids Res. 27, 297–300.
Honys, D. and Twell, D. (2003) Comparative analysis of the Ara-
bidopsis pollen transcriptome. Plant Physiol. 132, 640–652.
Hord, C.L., Chen, C., Deyoung, B.J., Clark, S.E. and Ma, H. (2006)
The BAM1/BAM2 receptor-like kinases are important regulators
of Arabidopsis early anther development. Plant Cell, 18, 1667–
1680.
Hu, W. and Ma, H. (2006) Characterization of a novel putative zinc
finger gene MIF1: involvement in multiple hormonal regulation of
Arabidopsis development. Plant J. 45, 399–422.
Huang, H., Tudor, M., Su, T., Zhang, Y., Hu, Y. and Ma, H. (1996) DNA
binding properties of two Arabidopsis MADS domain proteins:
binding consensus and dimer formation. Plant Cell, 8, 81–94.
Ito, T. and Shinozaki, K. (2002) The MALE STERILITY1 gene of
Arabidopsis, encoding a nuclear protein with a PHD-finger motif,
is expressed in tapetal cells and is required for pollen maturation.
Plant Cell Physiol. 43, 1285–1292.
Ito, T., Wellmer, F., Yu, H., Das, P., Ito, N., Alves-Ferreira, M.,
Riechmann, J.L. and Meyerowitz, E.M. (2004) The homeotic
protein AGAMOUS controls microsporogenesis by regulation of
SPOROCYTELESS. Nature, 430, 356–360.
Karlova, R., Boeren, S., Russinova, E., Aker, J., Vervoort, J. and de
Vries, S. (2006) The Arabidopsis SOMATIC EMBRYOGENESIS
RECEPTOR-LIKE KINASE1 protein complex includes BRASSINO-
STEROID-INSENSITIVE1. Plant Cell, 18, 626–638.
Klucher, K.M., Chow, H., Reiser, L. and Fischer, R.L. (1996) The
AINTEGUMENTA gene of Arabidopsis required for ovule and
female gametophyte development is related to the floral home-
otic gene APETALA2. Plant Cell, 8, 137–153.
Larkin, J.C., Oppenheimer, D.G., Pollock, S. and Marks, M.D. (1993)
Arabidopsis GLABROUS1 gene requires downstream sequences
for function. Plant Cell, 5, 1739–1748.
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 15
Latham, K.E., Litvin, J., Orth, J.M., Patel, B., Mettus, R. and Reddy,
E.P. (1996) Temporal patterns of A-myb and B-myb gene
expression during testis development. Oncogene, 13, 1161–1168.
Li, W. and Ma, H. (2006) Double-stranded DNA breaks and gene
functions in recombination and meiosis. Cell Res. 16, 402–412.
Li, J., Wen, J., Lease, K.A., Doke, J.T., Tax, F.E. and Walker, J.C.
(2002) BAK1, an Arabidopsis LRR receptor-like protein kinase,
interacts with BRI1 and modulates brassinosteroid signaling. Cell,
110, 213–222.
Li, N., Zhang, D.S., Liu, H.S. et al. (2006a) The rice tapetum degen-
eration retardation gene is required for tapetum degradation and
anther development. Plant Cell, 18, 2999–3014.
Li, X., Duan, X., Jiang, H. et al. (2006b) Genome-wide analysis of
basic/helix-loop-helix transcription factor family in rice and Ara-
bidopsis. Plant Physiol. 141, 1167–1184.
Lu, X.C., Gong, H.Q., Huang, M.L. et al. (2006) Molecular analysis of
early rice stamen development using organ-specific gene
expression profiling. Plant Mol. Biol. 61, 845–861.
Ma, H. (2005) Molecular genetic analyses of microsporogenesis and
microgametogenesis in flowering plants. Annu. Rev. Plant Biol.
56, 393–434.
Ma, J., Morrow, D.J., Fernandes, J. and Walbot, V. (2006) Com-
parative profiling of the sense and antisense transcriptome of
maize lines. Genome Biol. 7, R22, 1–18.
Maere, S., De Bodt, S., Raes, J., Casneuf, T., Van Montagu, M.,
Kuiper, M. and Van de Peer, Y. (2005) Modeling gene and genome
duplications in eukaryotes. Proc. Natl Acad. Sci. USA, 102, 5454–
5459.
Masuko, H., Endo, M., Saito, H. et al. (2006) Anther-specific genes,
which expressed through microsporogenesis, are temporally and
spatially regulated in model legume, Lotus japonicus. Genes
Genet. Syst. 81, 57–62.
McCormick, S. (1993) Male gametophyte development. Plant Cell, 5,
1265–1275.
Millar, A.A. and Gubler, F. (2005) The Arabidopsis GAMYB-like
genes, MYB33 and MYB65, are microRNA-regulated genes that
redundantly facilitate anther development. Plant Cell, 17, 705–721.
Moore, R.C. and Purugganan, M.D. (2005) The evolutionary dyna-
mics of plant duplicate genes. Curr. Opin. Plant Biol. 8, 122–128.
Nam, K.H. and Li, J. (2002) BRI1/BAK1, a receptor kinase pair
mediating brassinosteroid signaling. Cell, 110, 203–212.
Nemhauser, J.L., Mockler, T.C. and Chory, J. (2004) Interdepend-
ency of brassinosteroid and auxin signaling in Arabidopsis. PLoS
Biol. 2, E258. 1460–1671.
Nicholas, K.B., Nicholas, H.B. Jr and Deerfield, D.W. II. (1997)
GeneDoc: analysis and visualization of genetic variation.
EMBNEW.NEWS, 4, 14.
Riechmann, J.L., Heard, J., Martin, G. et al. (2000) Arabidopsis
transcription factors: genome-wide comparative analysis among
eukaryotes. Science, 290, 2105–2110.
Sablowski, R.W. and Meyerowitz, E.M. (1998) A homolog of NO
APICAL MERISTEM is an immediate target of the floral homeotic
genes APETALA3/PISTILLATA. Cell, 92, 93–103.
Sanders, P.M., Bui, A.Q., Weterings, K., McIntire, K.N., Hsu, Y.C.,
Lee, P.Y., Truong, M.T., Beals, T.P. and Goldberg, R.B. (1999)
Anther developmental defects in Arabidopsis thaliana male-
sterile mutants. Sex. Plant Reprod. 11, 297–322.
Schiefthaler, U., Balasubramanian, S., Sieber, P., Chevalier, D.,
Wisman, E. and Schneitz, K. (1999) Molecular analysis of
NOZZLE, a gene involved in pattern formation and early sporo-
genesis during sex organ development in Arabidopsis thaliana.
Proc. Natl Acad. Sci. USA, 96, 11664–11669.
Schmidt, D.D., Voelckel, C., Hartl, M., Schmidt, S. and Baldwin, I.T.
(2005) Specificity in ecological interactions: attack from the same
16 Asela J. Wijeratne et al.
lepidopteran herbivore results in species-specific transcriptional
responses in two solanaceous host plants. Plant Physiol. 138,
1763–1773.
Shiu, S.H. and Bleecker, A.B. (2001) Receptor-like kinases from
Arabidopsis form a monophyletic gene family related to animal
receptor kinases. Proc. Natl Acad. Sci. USA, 98, 10763–10768.
Shiu, S.H. and Bleecker, A.B. (2003) Expansion of the receptor-like
kinase/Pelle gene family and receptor-like proteins in Arabidop-
sis. Plant Physiol. 132, 530–543.
Shiu, S.H., Karlowski, W.M., Pan, R., Tzeng, Y.H., Mayer, K.F. and Li,
W.H. (2004) Comparative analysis of the receptor-like kinase
family in Arabidopsis and rice. Plant Cell, 16, 1220–1234.
Shpak, E.D., Lakeman, M.B. and Torii, K.U. (2003) Dominant-neg-
ative receptor uncovers redundancy in the Arabidopsis ERECTA
leucine-rich repeat receptor-like kinase signaling pathway that
regulates organ shape. Plant Cell, 15, 1095–1110.
Shpak, E.D., Berthiaume, C.T., Hill, E.J. and Torii, K.U. (2004)
Synergistic interaction of three ERECTA-family receptor-like kina-
ses controls Arabidopsis organ growth and flower development
by promoting cell proliferation. Development, 131, 1491–1501.
Shpak, E.D., McAbee, J.M., Pillitteri, L.J. and Torii, K.U. (2005)
Stomatal patterning and differentiation by synergistic interac-
tions of receptor kinases. Science, 309, 290–293.
Simpson, S.D., Nakashima, K., Narusaka, Y., Seki, M., Shinozaki, K.
and Yamaguchi-Shinozaki, K. (2003) Two different novel cis-act-
ing elements of erd1, a clpA homologous Arabidopsis gene
function in induction by dehydration stress and dark-induced
senescence. Plant J. 33, 259–270.
Song, S.K. and Clark, S.E. (2005) POL and related phosphatases are
dosage-sensitive regulators of meristem and organ development
in Arabidopsis. Dev. Biol. 285, 272–284.
Song, S.K., Lee, M.M. and Clark, S.E. (2006) POL and PLL1 phos-
phatases are CLAVATA1 signaling intermediates required for
Arabidopsis shoot and floral stem cells. Development, 133, 4691–
4698.
Sorensen, A., Krober, S., Unte, U.S., Huijser, P., Dekker, K. and
Saedler, H. (2003) The Arabidopsis ABORTED MICROSPORES
(AMS) gene encodes a MYC class transcription factor. Plant J. 33,
413–423.
Stracke, R., Werber, M. and Weisshaar, B. (2001) The R2R3-MYB
gene family in Arabidopsis thaliana. Curr. Opin. Plant Biol. 4, 447–
456.
Sturn, A., Quackenbush, J. and Trajanoski, Z. (2002) GENESIS:
cluster analysis of microarray data. Bioinformatics, 18, 207–208.
Tang, W. and Perry, S.E. (2003) Binding site selection for the plant
MADS domain protein AGL15: an in vitro and in vivo study. J.
Biol. Chem. 278, 28154–28159.
Toscani, A., Mettus, R.V., Coupland, R., Simpkins, H., Litvin, J.,
Orth, J., Hatton, K.S. and Reddy, E.P. (1997) Arrest of spermato-
genesis and defective breast development in mice lacking A-myb.
Nature, 386, 713–717.
Tsuji, H., Aya, K., Ueguchi-Tanaka, M. et al. (2006) GAMYB controls
different sets of genes and is differentially regulated by microRNA
in aleurone cells and anthers. Plant J. 47, 427–444.
Tusher, V.G., Tibshirani, R. and Chu, G. (2001) Significance analysis
of microarrays applied to the ionizing radiation response. Proc.
Natl Acad. Sci. USA, 98, 5116–5121.
Vert, G., Nemhauser, J.L., Geldner, N., Hong, F. and Chory, J. (2005)
Molecular mechanisms of steroid hormone signaling in plants.
Annu. Rev. Cell Dev. Biol. 21, 177–201.
Villanueva, J.M., Boradhvest, J., Hauser, B.A., Meister, R.J.,
Schneitz, K. and Gasser, C.S. (1999) INNER NO OUTER regulates
abaxial-adaxial patterning in Arabidopsis ovules. Genes Dev, 13,
3160–3169.
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), doi: 10.1111/j.1365-313X.2007.03217.x
Wang, X., Goshe, M.B., Soderblom, E.J., Phinney, B.S., Kuchar, J.A.,
Li, J., Asami, T., Yoshida, S., Huber, S.C. and Clouse, S.D. (2005a)
Identification and functional analysis of in vivo phosphorylation
sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1
receptor kinase. Plant Cell, 17, 1685–1703.
Wang, X., Li, X., Meisenhelder, J., Hunter, T., Yoshida, S., Asami, T.
and Chory, J. (2005b) Autoregulation and homodimerization are
involved in the activation of the plant steroid receptor BRI1. Dev.
Cell, 8, 855–865.
Wang, Z., Liang, Y., Li, C., Xu, Y., Lan, L., Zhao, D., Chen, C., Xu, Z.,
Xue, Y. and Chong, K. (2005c) Microarray analysis of gene
expression involved in anther development in rice (Oryza sativa
L.). Plant Mol. Biol. 58, 721–737.
Wellmer, F., Riechmann, J.L., Alves-Ferreira, M. and Meyerowitz,
E.M. (2004) Genome-wide analysis of spatial gene expression in
Arabidopsis flowers. Plant Cell, 16, 1314–1326.
Wijeratne, A.J., Chen, C., Zhang, W., Timofejeva, L. and Ma, H.
(2006) The Arabidopsis thaliana PARTING DANCERS gene enco-
ding a novel protein is required for normal meiotic homologous
recombination. Mol. Biol. Cell, 17, 1331–1343.
Wilson, Z.A., Morroll, S.M., Dawson, J., Swarup, R. and Tighe,
P.J. (2001) The Arabidopsis MALE STERILITY1 (MS1) gene is a
transcriptional regulator of male gametogenesis, with homol-
ogy to the PHD-finger family of transcription factors. Plant J. 28,
27–39.
Yamaguchi, T., Nakayama, K., Hayashi, T., Yazaki, J., Kishimoto, N.,
Kikuchi, S. and Koike, S. (2004) cDNA microarray analysis of rice
anther genes under chilling stress at the microsporogenesis stage
revealed two genes with DNA transposon Castaway in the 5¢-
flanking region. Biosci. Biotechnol. Biochem. 68, 1315–1323.
Yang, W.C., Ye, D., Xu, J. and Sundaresan, V. (1999) The SPORO-
CYTELESS gene of Arabidopsis is required for initiation of spor-
ogenesis and encodes a novel nuclear protein. Genes Dev. 13,
2108–2117.
Yang, S.L., Xie, L.F., Mao, H.Z., Puah, C.S., Yang, W.C., Jiang, L.,
Sundaresan, V. and Ye, D. (2003) Tapetum determinant1 is re-
quired for cell specialization in the Arabidopsis anther. Plant Cell,
15, 2792–2804.
Yin, Y., Wang, Z.-Y., Mora-Garcia, S., Li, J., Yoshida, S., Asami, T.
and Chory, J. (2002) BES1 accumulates in the nucleus in response
to brassinosteroids to regulate gene expression and promote
stem elongation. Cell, 109, 181–191.
Yin, Y., Vafeados, D., Tao, Y., Yoshida, S., Asami, T. and Chory, J.
(2005) A new class of transcription factors mediates brassino-
steroid-regulated gene expression in Arabidopsis. Cell, 120, 249–
259.
Yu, L.P., Miller, A.K. and Clark, S.E. (2003) POLTERGEIST encodes a
protein phosphatase 2C that regulates CLAVATA pathways con-
trolling stem cell identity at Arabidopsis shoot and flower meri-
stems. Curr. Biol. 13, 179–188.
Zhang, X., Feng, B., Zhang, Q., Zhang, D., Altman, N. and Ma, H.
(2005) Genome-wide expression profiling and identification of
gene activities during early flower development in Arabidopsis.
Plant Mol. Biol. 58, 401–419.
Zhang, W., Sun, Y., Timofejeva, L., Chen, C., Grossniklaus, U. and
Ma, H. (2006) Regulation of Arabidopsis tapetum development
and function by DYSFUNCTIONAL TAPETUM1 (DYT1) encoding
a putative bHLH transcription factor. Development, 133, 3085–
3095.
Zhao, D.Z., Wang, G.F., Speal, B. and Ma, H. (2002) The EXCESS
MICROSPOROCYTE1 gene encodes a putative leucine-rich
repeat receptor protein kinase that controls somatic and
reproductive cell fates in the Arabidopsis anther. Genes Dev. 16,
2021–2031.
ª 2007 The Authors