Various spatiotemporal expression profiles of anther-expressed genes in rice.

Abstract

The male gametophyte and tapetum play different roles during anther development although they are differentiated from the same cell lineage, the L2 layer. Until now, it has not been possible to delineate their transcriptomes due to technical difficulties in separating the two cell types. In the present study, we characterized the separated transcriptomes of the rice microspore/pollen and tapetum using laser microdissection (LM)-mediated microarray. Spatiotemporal expression patterns of 28,141 anther-expressed genes were classified into 20 clusters, which contained 3,468 (12.3%) anther-enriched genes. In some clusters, synchronous gene expression in the microspore and tapetum at the same developmental stage was observed as a novel characteristic of the anther transcriptome. Noteworthy expression patterns are discussed in connection with gene ontology (GO) categories and gene annotations, which are related to important biological events in anther development, such as pollen maturation, pollen germination, pollen tube elongation and pollen wall formation.

Full text

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Various Spatiotemporal Expression Profi les of
Anther-Expressed Genes in Rice
Tokunori Hobo 1,11 , Keita Suwabe 2,11 , Koichiro Aya 1 , Go Suzuki 3 , Kentaro Yano 4 , Takeshi Ishimizu 5 ,
Masahiro Fujita 6 , Shunsuke Kikuchi 4 , Kazuki Hamada 4 , Masumi Miyano 2,7 , Tomoaki Fujioka 2 ,
Fumi Kaneko 2 , Tomohiko Kazama 2,7 , Yoko Mizuta 6 , Hirokazu Takahashi 8 , Katsuhiro Shiono 8 ,
Mikio Nakazono 8 , Nobuhiro Tsutsumi 8 , Yoshiaki Nagamura 9 , Nori Kurata 6 , Masao Watanabe 2,7,10,*
and Makoto Matsuoka 1,*
1 Bioscience and Biotechnology Center, Nagoya University, Furocho, Chikusa, Nagoya, 464-8601 Japan
2 Graduate School of Life Sciences, Tohoku University, Sendai, 980-8577 Japan
3 Division of Natural Science, Osaka Kyoiku University, Kashiwara, 582-8582 Japan
4 Faculty of Agriculture, Meiji University, Kawasaki, 214-8571 Japan
5 Department of Chemistry, Graduate School of Science, Osaka University, Osaka, 560-0043 Japan
6 Plant Genetics Laboratory, National Institute of Genetics, Mishima, 411-8540 Japan
7 The 21st Century Center of Excellence Program, Iwate University, Morioka, 020-8550 Japan
8 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, 113-8657 Japan
9 Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Tsukuba, 305-8602 Japan
10 Faculty of Science, Tohoku University, Sendai, 980-8587 Japan
The male gametophyte and tapetum play different
roles during anther development although they are
differentiated from the same cell lineage, the L2 layer.
Until now, it has not been possible to delineate their
transcriptomes due to technical diffi culties in separating
the two cell types. In the present study, we characterized
the separated transcriptomes of the rice microspore/
pollen and tapetum using laser microdissection (LM)-
mediated microarray. Spatiotemporal expression patterns
of 28,141 anther-expressed genes were classifi ed into 20
clusters, which contained 3,468 (12.3%) anther-enriched
genes. In some clusters, synchronous gene expression in
the microspore and tapetum at the same developmental
stage was observed as a novel characteristic of the
anther transcriptome. Noteworthy expression patterns
are discussed in connection with gene ontology (GO)
categories and gene annotations, which are related to
important biological events in anther development, such
as pollen maturation, pollen germination, pollen tube
elongation and pollen wall formation.
Keywords: Anther • Gene ontology • Laser microdissection •
Microarray • Oryza sativa L. • Synchronous gene expression.
Abbreviations: BC , bicellular ; GO , gene ontology; LM , laser
microdissection; MEI , meiosis ; miRNA , microRNA ; TC , tri-
cellular ; TET , tetrad ; UN , uninuclear ; VSN , variance stabiliza-
tion normalization.
Introduction
Anther tissues in fl owering plants are important for
microsporogenesis and dispersing the male gamete, pollen.
Anther tissues consist of three layers, L1, L2 and L3. The epi-
dermis and stomium are differentiated at the fi nal devel-
opmental stage in the L1 layer. The microspore/pollen,
endothecium and outer tapetum are formed in the L2 layer,
while the L3 layer differentiates into vascular bundle and
inner tapetum ( Goldberg et al. 1993 ). Although the sporo-
genous cells and parietal cells originate from the same L2 layer,
their fi nal fates are different. In the case of sporogenous cells,
following meiosis, microspores are released into the anther
locule, and each microspore divides into vegetative and gen-
erative cells. In rice, the generative cell further divides into
two sperm cells. Desiccated mature pollen grains are dis-
persed from anthers onto the stigma surface ready
for fertilization. In contrast, parietal cells differentiate into
Plant Cell Physiol. 49(10): 1417–1428 (2008) doi:10.1093/pcp/pcn128, available online at www.pcp.oxfordjournals.org
© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.
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please contact journals.permissions@oxfordjournals.org
11 These authors contributed equally to this work.
*Corresponding authors: Masao Watanabe, E-mail, nabe@ige.tohoku.ac.jp; Fax, +81-22-217-5683 ;
Makoto Matsuoka, E-mail, makoto@nuagr1.agr.nagoya-u.ac.jp; Fax, +81-52-789-5226 .
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Special Issue –Regular Paper

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endothecium and outer tapetum. Tapetum cells function as
nurse cells for pollen development; the tapetum supports
pollen development by secreting nutrients and other
secondary metabolites. The tapetum constitutes the pollen
outer surface, but degrades before anther dehiscence ( Gold-
berg et al. 1993 ). Therefore, while microspore/pollen and
outer tapetum originate from the same cell lineage, the
L2 layer, their fetal functions are completely different.
In order to understand the molecular mechanisms of
anther development, particularly microspore/pollen differ-
entiation and tapetum development, transcriptome analy-
ses have been conducted in anthers of rice and Lotus
japonicus ( Endo et al. 2002 , Endo et al. 2004 ). Some anther-
specifi c genes were further characterized by RNA in situ
hybridization analysis, which is extremely laborious and not
comprehensive. These studies demonstrated the existence
of microspore/pollen-specifi c and tapetum-specifi c genes
( Endo et al. 2004 , Masuko et al. 2006 ). Briefl y, in the mature
pollen grain, coordinated expression of pollen-specifi c tran-
scripts was detected. These transcripts are related to the cell
cytoskeleton, cell wall re-organization, methionine metabo-
lism, proton pumps and sugar transporters, and are presum-
ably important for pollen germination and pollen tube
elongation ( Endo et al. 2002 , Endo et al. 2004 , Masuko et al.
2006 ). In contrast, tapetum-specifi c transcripts were
detected relating to secondary metabolism, fatty acid bio-
synthesis, protein secretion and the gibberellin signaling
cascade ( Endo et al. 2004 , Masuko et al. 2006 ). The different
characteristics of the two transcriptomes suggested that
gene functions in pollen and tapetum are distinct. However,
further comprehensive analysis of microspore/pollen-
specifi c and tapetum-specifi c genes is necessary for a com-
plete functional understanding of these cell types.
Because laser microdissection (LM) is a useful technique
to separate cell types within complex plant tissues ( Asano
et al. 2002 , Kerk et al. 2003 , Nakazono et al. 2003 , Day et al.
2005 , Nelson et al. 2006 , Ohtsu et al. 2007 ), we performed
a 44K microarray with RNAs derived from LM-separated
microspore/pollen and tapetum cells in rice. The reliability
of the data was confi rmed by comparison with the published
gene expression profi le of 156 previously identifi ed anther-
specifi c genes in rice (refer to Suwabe et al. 2008 in this
special issue). Having validated the technique, the genome-
wide expression profi les of microspore/pollen and tapetum
in rice were determined in this study according to the
developmental stages. From the cluster analysis, 28,141
genes were categorized into 20 clusters according to their
characteristic expression patterns, and gene ontology (GO)
was applied to each cluster. We discuss noteworthy character-
istics of the microspore/pollen and tapetum transcriptomes
in rice.
Results and Discussion
Various spatiotemporal expression patterns of genes
in rice anthers
We conducted a 44K LM-microarray analysis of microspore/
pollen and tapetum cells of japonica rice, Oryza sativa cv.
Nipponbare, at each of the fi ve stages of anther develop-
ment: meiosis (MEI), tetrad (TET), uninuclear (UN) microspore,
bicellular (BC) pollen and tricellular (TC) pollen ( Suwabe
et al. 2008 ). Because tapetum degradation occurs at the BC
stage, the tapetum transcriptome was only characterized at
the MEI, TET and UN stages. After normalization of Z-scored
44K array data, a cluster analysis was performed with TIGR
MeV software ( Fig. 1 , Supplementary Figs. S1–S20,
Tables S1–S20). It should be noted that the microspore/
pollen and tapetum transcriptomes were successfully sepa-
rated without contamination in the present LM-microarray,
as described by Suwabe et al. (2008) in this issue. This
conclusion was further supported by the existence of the
anther-specifi c and microspore-specifi c clades in the heat
map ( Fig. 2 ). In total, 28,141 anther-expressed genes were
categorized into 20 clusters, and the average number of
genes in each cluster was 1,407. Expression levels of genes in
six clusters (Nos. 4, 5, 6, 8, 10 and 19) were higher in the
microspore/pollen than in the tapetum, indicating that
these clusters were microspore/pollen dominant. On the
other hand, no obvious tapetum-dominant profi les were
observed in the 20 clusters, mainly because only a small
proportion of genes showed the strictly tapetum-dominant
expression pattern, and these were distributed among sev-
eral clusters in this analysis.
To discriminate genes preferentially expressed in anthers,
we compared the LM-microarray data of microspore/pollen
and tapetum with microarray data sets obtained for rice roots
and leaves (Y. Nagamura, unpublished results). In the present
study, anther-expressed genes with a vegetative expression
signal score <50 were defi ned as ‘anther-specifi c’ genes.
According to this defi nition, 3,468 anther-specifi c genes were
identifi ed, accounting for 12.3% of the total 28,141 anther-
expressed genes. The percentage of anther-specifi c genes was
then calculated in each cluster ( Fig. 1 ), and relatively lower
ratios of anther-specifi c genes (∼2–4%) were observed for the
clusters of constitutively tapetum-expressed genes (Nos. 13,
14, 15 and 18). In contrast, in the clusters of genes expressed in
MEI microspore (clusters 12 and 17) and in late developmen-
tal stages of pollen (clusters 5 and 10), >20% of genes were
anther specifi c. The increased proportion of anther-specifi c
transcripts in clusters 5 and 10 is consistent with analysis of
the Arabidopsis male gametophyte-specifi c transcriptome
( Honys and Twell 2004 ), which showed that the transition
from BC to TC pollen was accompanied by an increase in the
proportion of male gamete-specifi c transcripts.
Transcriptome analysis in male gamete and tapetum
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Fig. 1 Gene expression patterns of 20 clusters in microspore/pollen and tapetum. In each cluster, Z-scored data with a standard error bar were plotted according to developmental
stages. The number of genes in the cluster is indicated at the upper left side. The calculated percentage of anther-enriched genes in each cluster is shown at the lower right side. The high
synchronous gene expression pattern between microspore/pollen and tapetum is shown by red circles in clusters 2 and 20. The low synchronous pattern is shown by blue circles in
clusters 3, 7, 12, 13, 16 and 17.
30
−3
Relative signal intensity
30
−330
−330
−3
Relative signal intensityRelative signal intensityRelative signal intensity
TET
MEI
BC
UN
MEI
TC
UN
Microspore/pollen
Tapetum
TET
TET
MEI
BC
UN
MEI
TC
UN
Microspore/pollen
Tapetum
TET
TET
MEI
BC
UN
MEI
TC
UN
Microspore/pollen
Tapetum
TET
TET
MEI
BC
UN
MEI
TC
UN
Microspore/pollen
Tapetum
TET
TET
MEI
BC
UN
MEI
TC
TET
UN
Microspore/pollen
Tapetum
7.8%
15.4%
28.8%
9.5%
26.8%
4.3%
11.8%
29.8%
3.4%
22.3%
2.2%
29.3%
3.9%
4.1%
2.7%
4.9%
22.3%
3.3%
6.6%
18.6%
#1: 1019 genes
#2: 1212 genes
#3: 1196 genes
#4: 1160 genes
#5: 2053 genes
#6: 1809 genes
#7: 1215 genes
#8: 904 genes
#9: 865 genes
#10: 2170 genes
#11: 1395 genes
#12: 1217 genes
#13: 2515 genes
#14: 2117 genes
#15: 1719 genes
#16: 1567 genes
#17: 1255 genes
#18: 1074 genes
#19: 1046 genes
#20: 633 genes
Transcriptome analysis in male gamete and tapetum
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When we focused on the tapetum transcriptome, genes
in clusters 12, 20 and 2 were preferentially expressed only at
one stage (MEI, TET and UN stages, respectively), suggesting
that the biological functions of tapetum genes differed in
the three stages. In other clusters, genes in cluster 7 were
predominantly expressed in both MEI and TET stages, while
those in clusters 13, 14, 15 and 18 were expressed continu-
ously in all the three stages.
In contrast, analysis of the microspore/pollen transcrip-
tome revealed complex profi les. Expression patterns in the
BC pollen were variable in some clusters, e.g. in cluster 1,
expression was high in the UN microspore, gradually down-
regulated in the late BC pollen, and low in the TC pollen.
Such gradual down-regulation within the BC stage was also
observed in clusters 15 and 16, whereas gradual up-regulation
was seen in clusters 5, 6, 10, 11 and 19. In view of these pat-
terns, it would be possible to divide the BC stage in the pollen
transcriptome into early BC and late BC stages.
Throughout the developmental stages from MEI to TC,
there are many expression patterns among the microspore/
pollen genes. For example, gradual up-regulation of genes
from MEI to TC was observed in clusters 5 and 6, while grad-
ual down-regulation occurred in cluster 16. In the case of
clusters 9 and 19, genes were gradually down-regulated from
MEI to UN, but then gradually up-regulated again from BC
to TC pollen. Another pattern was shown in cluster 3, with
constant expression in the microspore/pollen at the UN to
TC pollen stages. The variety of expression profi les in the
Fig. 2 Heat map view of the microspore/pollen-specifi c clade (A) and tapetum-specifi c clade (B). Microspore/pollen- and tapetum-specifi c
clades were extracted from clusters 3 and 2, respectively. Red indicates higher, while green represents lower expression.
Microspore/Pollen
Tapetum
MEI-
TET-
2
UN-
2
BC-
TC-
2
MEI-
2
TET-
2
UN-
2123456781234131313131313
AK068928
AK100524
AK108848
AK059587
AK061188
AK099072
AK099532
AK104095
CB964569
AK070734
AK068703
AK065235
AK104264
A
B
Os07g0430200
CI330756
Os04g0428300
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
CI330756
AK070393
Os10g0151700
Os01g0606200
AK107025
AK070508
Os04g0357700
CI330756
CI330756
AK064697
AK064291
CI330756
Os10g0423900
CI119405
CI119405
−3.00.03.0
Transcriptome analysis in male gamete and tapetum
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Page 5

microspore/pollen indicated that biological events in the
male gametophyte are developmentally regulated by many
anther-expressed genes with various expression patterns.
Interestingly, synchronous gene expression in both micro-
spore and tapetum was observed in eight clusters: Nos. 2 and
20 with a high degree of synchrony (indicated by the red
color in Fig. 1 ), and Nos. 3, 7, 12, 13, 16 and 17 with low syn-
chronization (indicated by the blue color). These synchro-
nous profi les cannot be attributed to contamination artifacts
during the LM procedure, because microspore-specifi c and
tapetum-specifi c clades could be identifi ed ( Fig. 2 , Suwabe
et al. 2008 ), and the expression patterns of microspore and tape-
tum were fundamentally independent in the other 12 clus-
ters. The number of genes classifi ed into the eight synchronous
clusters was 10,810 (38.4%). Synchronous expression in the
microspore/pollen and tapetum is one of the major features
of the expression profi les in anthers, and argues against the
conventional idea that tapetum cells function solely as nurse
cells for feeding microspores in anther locules, with genes in
different categories being expressed in the microspore and
tapetum. The fi nding that synchronous transcription of some
groups of genes occurs in both the micro spore and tapetum
suggests that these cells do share many developmental path-
ways as described in Scott et al. (2004) , and is also consistent
with the fact that both the microspore and tapetum are
derived from the same cell lineage, L2.
Characterization of specifi c gene clusters based on GO
Genes with functional annotation were classifi ed in each
cluster based on GO by using Gene Ontology Slim terms
( Fig. 3 , http://www.tigr.org/tdb/e2k1/osa1/batch_down
load.shtml ). We focused on fi ve categories with likely func-
tional signifi cance in anther development: transcription,
translation, lipid metabolism, secondary metabolism and
the cell cycle ( Fig. 4 ).
Genes related to transcription occupied 1.8–7.2% of genes
in each cluster, and were frequently observed in clusters 7,
13, 14 and 20 (6.0–7.2% in each cluster, Fig. 4 ). These clusters
were all characterized as tapetum expression, indicating that
transcriptional regulation might be signifi cantly active in the
tapetum. In particular, clusters 13 and 14, showing similar
expression profi les, included 312 genes (28.0%) out of the
1,114 transcription-related genes in all 20 clusters. Clusters
13 and 14 contained various transcription factor genes, with
annotations including bHLH, MYB, GRAS, MADS, HD-Zip
and Zn-fi nger-type transcription factors.
Translation-related genes were frequently observed in clus-
ters 1 and 4 (6.3 and 7.3% in each cluster, respectively); the two
clusters contained 149 genes (34.6%) out of the 431 transla-
tion-related genes in all the clusters. Common characteristics
of the expression profi les in clusters 1 and 4 were higher gene
expression in early BC pollen. The profi les in the clusters with a
high percentage of transcription-related genes (Nos. 7, 13, 14
and 20) and translation-related genes (Nos. 1 and 4) were con-
siderably different; in particular, the expression profi les of clus-
ters 4 and 14 were almost opposite ( Fig. 1 ). Thus, translation in
the BC pollen might be achieved using transcripts produced in
earlier stages of pollen (TET and UN microspores).
Lipid metabolism is important in pollen development,
and failure of lipid synthesis frequently causes male sterility
( Mariani and Wolters-Arts 2000 , Ariizumi et al. 2003 ). In our
GO analysis, lipid metabolism genes often appeared in cluster
2, whose profi les were characterized by high expression in the
UN microspores and tapetum. This indicates that regulation
of lipid metabolism-related genes might function at the UN
stage. Several fatty acid synthesis genes, such as genes for
β-ketoacyl-CoA synthase and β-keto acyl reductase, were also
observed in clusters 13, 14 and 15 (described further in the
following section), although the percentage coverage of lipid
metabolism genes in these clusters was not so high.
Genes related to secondary metabolism were the most
frequently observed in clusters 2 and 20 (3.9 and 3.2% in
each cluster), including chalcone synthase, chalcone
reductase and phytoene synthase. These two clusters were
categorized as showing synchronous expression in the TET
or UN microspore and tapetum.
A relatively small portion of cell cycle-related genes
(0.0–1.4%) was categorized in the GO analysis. However,
most of these (19.8%; 17 genes out of all the 86 cell cycle genes)
were included in cluster 17. This might be due to the exis-
tence of genes related to meiosis, because notable gene
expression was observed at the MEI stage in cluster 17. Genes
annotated as cyclin, cyclin–F-box and rad51/recA were
identifi ed in cluster 17.
Functional relationships between other
characteristic genes in each cluster and biological
events in the microspore/pollen and tapetum
During the maturation of anther tissues, many characteristic
biological events can be related to the microspore/pollen
and tapetum transcriptomes. For example, important bio-
logical events for the male gamete are pollen maturation,
pollen germination and pollen tube elongation. To date,
many pollen-specifi c genes, related to carbohydrate metab-
olism (glycosyltransferase, glycoside hydrolase, sugar trans-
porter, etc.), cytoskeleton re-organization (actin), Ca +
signaling (calmodulin) and tip growth (small GTPase), have
been identifi ed and characterized in terms of these biologi-
cal events ( Franklin-Tong 1999 , Endo et al. 2000 , Endo et al.
2002 , Golovkin and Reddy 2003 , Honys and Twell 2003 , Endo
et al.2004 , Jiang et al. 2005 , Huang and Yang 2006 , Malhó
et al. 2006 , Shimamura et al. 2007 , Cheung and Wu 2008 ).
These genes, whose transcripts are present in mature pollen
grains and are translated during pollen germination ( Hoekstra
and Bruinsma 1979 , Mascarenhas et al. 1984 , Schrauwen
et al. 1990 ), were confi rmed in our study to be predominantly
Transcriptome analysis in male gamete and tapetum
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