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ABSTRACT: Comprehensive genome-wide gene expression profiles during plant male gametogenesis have been thoroughly analyzed over the last decade. In contrast, gene expression profiles during female gametogenesis have been studied quite little, and our knowledge concerning plant female gametogenesis is limited. We determined the genome-wide gene expression profiles of developing ovules containing female gametophytes from the megaspore mother cell at pre-meiotic stage to the mature embryo sac in rice (Oryza sativa) using microarrays. In order to separate ovules from scutellum, we used a laser microdissection (LM) technique. Dynamic gene expression was revealed in developing ovules, and a major transition of the transcriptome was observed between middle and late meiotic stages, where many genes were down-regulated more than ten-fold. Many potential players in female gametogenesis, that showed dynamic or enriched expression, were highlighted. We identified the temporal and dramatic up-regulation of a subset of transposable elements during female meiotic stages that were not observed in males. Transcription factor genes enriched in developing ovules were also uncovered, which may play crucial roles during female gametogenesis. This is the first report of comprehensive genome-wide gene expression profiles during female gametogenesis useful for plant reproductive studies. Combined with additional experiments, our data may provide important clues to understand female gametogenesis in plant.
Plant and Cell Physiology 02/2013; · 4.70 Impact Factor
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ABSTRACT: When rice seeds germinate under complete submergence, only the coleoptile elongates efficiently. It has been reported previously that coleoptile elongation is reduced in the rice alcohol dehydrogenase 1 (ADH1)-deficient mutant, reduced adh activity (rad). The aim of this study was to elucidate how expressions of genes responsible for coleoptile elongation are affected by the ADH1 deficiency in the rad mutant under submergence.
To identify genes whose expressions are changed in the rad coleoptile at an early stage in germination (i.e. 1 d after imbibition), coleoptiles of the rad mutant and its wild type (WT) were isolated by laser microdissection, and their mRNA levels were examined with a microarray.
The microarray analysis identified 431 genes whose transcript levels were different between rad and WT. Interestingly, among the down-regulated genes in the rad coleoptile, 17·5 % were cell division-related genes and 5·1 % were cell elongation-related genes. It was found that cell division started at 1 d after imbibition and then gradually ceased, whereas in the WT coleoptile cell elongation started between 1 d and 2 d after imbibition and then continued. However, neither cell division nor cell elongation actively occurred in the rad coleoptile, in which the amounts of ATP were reduced.
These results indicate that cell division, as well as cell elongation, occur during coleoptile elongation in rice under complete submergence, and that the reduced ATP levels caused by the ADH1 deficiency repress both of them, thereby impairing coleoptile elongation in the rad mutant under submerged conditions.
Annals of Botany 08/2011; 108(2):253-61. · 4.03 Impact Factor
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ABSTRACT: Double fertilization in flowering plants refers to a process in which two sperm cells, carried by the pollen tube, fertilize both the egg and the central cell after their release into a synergid cell of the female gametophyte. The molecular processes by which the female gametophytic cells express their unique functions during fertilization are not well understood. Genes expressed in egg and synergid cells might be important for multiple stages of the plant reproductive process. Here, we profiled genome-wide gene expression in egg and synergid cells in rice (Oryza sativa), a model monocot, using a nonenzymatic cell isolation technique. We found that the expression profiles of the egg and synergid cells were already specified at the micropylar end of the female gametophyte during the short developmental period that comprises the three consecutive mitotic nuclear divisions after megaspore generation. In addition, we identified a large number of genes expressed in the rice egg and synergid cells and characterized these genes using Gene Ontology analysis. The analysis suggested that epigenetic and posttranscriptional regulatory mechanisms are involved in the specification and/or maintenance of these cells. Comparisons between the rice profiles and reported Arabidopsis (Arabidopsis thaliana) profiles revealed that genes enriched in the egg/synergid cell of rice were distinct from those in Arabidopsis.
Plant physiology 02/2011; 155(2):881-91. · 6.53 Impact Factor
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Koichiro Aya,
Go Suzuki,
Keita Suwabe,
Tokunori Hobo, Hirokazu Takahashi,
Katsuhiro Shiono,
Kentaro Yano,
Nobuhiro Tsutsumi,
Mikio Nakazono,
Yoshiaki Nagamura,
Makoto Matsuoka,
Masao Watanabe
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ABSTRACT: Co-expression networks systematically constructed from large-scale transcriptome data reflect the interactions and functions of genes with similar expression patterns and are a powerful tool for the comprehensive understanding of biological events and mining of novel genes. In Arabidopsis (a model dicot plant), high-resolution co-expression networks have been constructed from very large microarray datasets and these are publicly available as online information resources. However, the available transcriptome data of rice (a model monocot plant) have been limited so far, making it difficult for rice researchers to achieve reliable co-expression analysis. In this study, we performed co-expression network analysis by using combined 44 K agilent microarray datasets of rice, which consisted of 33 laser microdissection (LM)-microarray datasets of anthers, and 143 spatiotemporal transcriptome datasets deposited in RicexPro. The entire data of the rice co-expression network, which was generated from the 176 microarray datasets by the Pearson correlation coefficient (PCC) method with the mutual rank (MR)-based cut-off, contained 24,258 genes and 60,441 genes pairs. Using these datasets, we constructed high-resolution co-expression subnetworks of two specific biological events in the anther, "meiosis" and "pollen wall synthesis". The meiosis network contained many known or putative meiotic genes, including genes related to meiosis initiation and recombination. In the pollen wall synthesis network, several candidate genes involved in the sporopollenin biosynthesis pathway were efficiently identified. Hence, these two subnetworks are important demonstrations of the efficiency of co-expression network analysis in rice. Our co-expression analysis included the separated transcriptomes of pollen and tapetum cells in the anther, which are able to provide precise information on transcriptional regulation during male gametophyte development in rice. The co-expression network data presented here is a useful resource for rice researchers to elucidate important and complex biological events.
PLoS ONE 01/2011; 6(10):e26162. · 4.09 Impact Factor
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Imene Rajhi,
Takaki Yamauchi, Hirokazu Takahashi,
Shunsaku Nishiuchi,
Katsuhiro Shiono,
Ryosuke Watanabe,
Ahmed Mliki,
Yoshiaki Nagamura,
Nobuhiro Tsutsumi,
Naoko K Nishizawa,
Mikio Nakazono
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ABSTRACT: • To adapt to waterlogging in soil, some gramineous plants, such as maize (Zea mays), form lysigenous aerenchyma in the root cortex. Ethylene, which is accumulated during waterlogging, promotes aerenchyma formation. However, the molecular mechanism of aerenchyma formation is not understood. • The aim of this study was to identify aerenchyma formation-associated genes expressed in maize roots as a basis for understanding the molecular mechanism of aerenchyma formation. Maize plants were grown under waterlogged conditions, with or without pretreatment with an ethylene perception inhibitor 1-methylcyclopropene (1-MCP), or under aerobic conditions. Cortical cells were isolated by laser microdissection and their mRNA levels were examined with a microarray. • The microarray analysis revealed 575 genes in the cortical cells, whose expression was either up-regulated or down-regulated under waterlogged conditions and whose induction or repression was suppressed by pretreatment with 1-MCP. • The differentially expressed genes included genes related to the generation or scavenging of reactive oxygen species, Ca(2+) signaling, and cell wall loosening and degradation. The results of this study should lead to a better understanding of the mechanism of root lysigenous aerenchyma formation.
New Phytologist 11/2010; 190(2):351-68. · 6.64 Impact Factor
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ABSTRACT: Laser microdissection (LM) combined with microarray analysis or next-generation sequencing of cDNA is a powerful tool for understanding molecular events in individual cell types of plants as well as animals. Obtaining high quality RNA is essential for this approach. For plant tissues, paraffin-embedded sections better preserve cell structure than do frozen sections. However, the conventional method for preparing paraffin sections is a lengthy process involving embedding the tissue and floating and drying the sections, during which time RNA degradation occurs. Here, we describe a method for preparing serial sections that greatly reduces RNA degradation: we reduced (1) the embedding time from 4-6 days to about 5 h by using a recently developed microwave method; (2) the time of floating sections from ~10 min to less than 5 min, (3) the drying time from ~12 to 1 h; and (4) the drying temperature from 42 to 4°C. With this method, we were able to isolate higher integrity RNA from many kinds of plant tissues than is typically obtained by the conventional paraffin preparation method. The improvement in RNA quality and yield removes a major obstacle to the widespread use of LM with high-throughput technologies for plants.
Journal of Plant Research 03/2010; 123(6):807-13. · 1.75 Impact Factor
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Tokunori Hobo,
Keita Suwabe,
Koichiro Aya,
Go Suzuki,
Kentaro Yano,
Takeshi Ishimizu,
Masahiro Fujita,
Shunsuke Kikuchi,
Kazuki Hamada,
Masumi Miyano, [......],
Tomohiko Kazama,
Yoko Mizuta, Hirokazu Takahashi,
Katsuhiro Shiono,
Mikio Nakazono,
Nobuhiro Tsutsumi,
Yoshiaki Nagamura,
Nori Kurata,
Masao Watanabe,
Makoto Matsuoka
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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.
Plant and Cell Physiology 10/2008; 49(10):1417-28. · 4.70 Impact Factor
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Keita Suwabe,
Go Suzuki, Hirokazu Takahashi,
Katsuhiro Shiono,
Makoto Endo,
Kentaro Yano,
Masahiro Fujita,
Hiromi Masuko,
Hiroshi Saito,
Tomoaki Fujioka,
Fumi Kaneko,
Tomohiko Kazama,
Yoko Mizuta,
Makiko Kawagishi-Kobayashi,
Nobuhiro Tsutsumi,
Nori Kurata,
Mikio Nakazono,
Masao Watanabe
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ABSTRACT: In flowering plants, the male gametophyte, the pollen, develops in the anther. Complex patterns of gene expression in both the gametophytic and sporophytic tissues of the anther regulate this process. The gene expression profiles of the microspore/pollen and the sporophytic tapetum are of particular interest. In this study, a microarray technique combined with laser microdissection (44K LM-microarray) was developed and used to characterize separately the transcriptomes of the microspore/pollen and tapetum in rice. Expression profiles of 11 known tapetum specific-genes were consistent with previous reports. Based on their spatial and temporal expression patterns, 140 genes which had been previously defined as anther specific were further classified as male gametophyte specific (71 genes, 51%), tapetum-specific (seven genes, 5%) or expressed in both male gametophyte and tapetum (62 genes, 44%). These results indicate that the 44K LM-microarray is a reliable tool to analyze the gene expression profiles of two important cell types in the anther, the microspore/pollen and tapetum.
Plant and Cell Physiology 09/2008; 49(10):1407-16. · 4.70 Impact Factor
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Akira Endo,
Yoshiaki Sawada, Hirokazu Takahashi,
Masanori Okamoto,
Keiichi Ikegami,
Hanae Koiwai,
Mitsunori Seo,
Tomonobu Toyomasu,
Wataru Mitsuhashi,
Kazuo Shinozaki,
Mikio Nakazono,
Yuji Kamiya,
Tomokazu Koshiba,
Eiji Nambara
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ABSTRACT: The regulation of abscisic acid (ABA) biosynthesis is essential for plant responses to drought stress. In this study, we examined the tissue-specific localization of ABA biosynthetic enzymes in turgid and dehydrated Arabidopsis (Arabidopsis thaliana) plants using specific antibodies against 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), AtABA2, and Arabidopsis aldehyde oxidase 3 (AAO3). Immunohistochemical analysis revealed that in turgid plants, AtABA2 and AAO3 proteins were localized in vascular parenchyma cells most abundantly at the boundary between xylem and phloem bundles, but the AtNCED3 protein was undetectable in these tissues. In water-stressed plants, AtNCED3 was detected exclusively in the vascular parenchyma cells together with AtABA2 and AAO3. In situ hybridization using the antisense probe for AtNCED3 showed that the drought-induced expression of AtNCED3 was also restricted to the vascular tissues. Expression analysis of laser-microdissected cells revealed that, among nine drought-inducible genes examined, the early induction of most genes was spatially restricted to vascular cells at 1 h and then some spread to mesophyll cells at 3 h. The spatial constraint of AtNCED3 expression in vascular tissues provides a novel insight into plant systemic response to drought stresses.
Plant physiology 07/2008; 147(4):1984-93. · 6.53 Impact Factor
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Hiroaki Saika,
Masanori Okamoto,
Kentaro Miyoshi,
Tetsuo Kushiro,
Shoko Shinoda,
Yusuke Jikumaru,
Masaru Fujimoto,
Taku Arikawa, Hirokazu Takahashi,
Miho Ando,
Shin-Ichi Arimura,
Akio Miyao,
Hirohiko Hirochika,
Yuji Kamiya,
Nobuhiro Tsutsumi,
Eiji Nambara,
Mikio Nakazono
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ABSTRACT: A rapid decrease of the plant hormone ABA under submergence is thought to be a prerequisite for the enhanced elongation of submerged shoots of rice (Oryza sativa L.). Here, we report that the level of phaseic acid (PA), an oxidized form of ABA, increased with decreasing ABA level during submergence. The oxidation of ABA to PA is catalyzed by ABA 8'-hydroxylase, which is possibly encoded by three genes (OsABA8ox1, -2 and -3) in rice. The ABA 8'-hydroxylase activity was confirmed in microsomes from yeast expressing OsABA8ox1. OsABA8ox1-green fluorescent protein (GFP) fusion protein in onion cells was localized to the endoplasmic reticulum. The mRNA level of OsABA8ox1, but not the mRNA levels of other OsABA8ox genes, increased dramatically within 1 h after submergence. On the other hand, the mRNA levels of genes involved in ABA biosynthesis (OsZEP and OsNCEDs) decreased after 1-2 h of submergence. Treatment of aerobic seedlings with ethylene and its precursor, 1-aminocyclopropane-1-carboxylate (ACC), rapidly induced the expression of OsABA8ox1, but the ethylene treatment did not strongly affect the expression of ABA biosynthetic genes. Moreover, pre-treatment with 1-methylcyclopropene (1-MCP), a potent inhibitor of ethylene action, partially suppressed induction of OsABA8ox1 expression under submergence. The ABA level was found to be negatively correlated with OsABA8ox1 expression under ACC or 1-MCP treatment. Together, these results indicate that the rapid decrease in ABA levels in submerged rice shoots is controlled partly by ethylene-induced expression of OsABA8ox1 and partly by ethylene-independent suppression of genes involved in the biosynthesis of ABA.
Plant and Cell Physiology 03/2007; 48(2):287-98. · 4.70 Impact Factor
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ABSTRACT: Laser microdissection (LM) allows for the isolation of specific cells of interest from heterogeneous tissues under direct microscopic visualization with the assistance of a laser beam. By permitting global analyses of gene expression and metabolites in the selected cells, it is a powerful tool for understanding the biological processes in individual cell types during development or in response to various stimuli. Recently, LM technology has been successfully applied to the separation of individual plant cell types. Here, we provide an overview of applications of LM combined with high-throughput technologies including transcript analyses [microarrays, serial analysis of gene expression (SAGE) and 454-sequencing], proteomic analyses and metabolomic profiling, for cell type-specific gene expression analyses in plants.
Plant and Cell Physiology 02/2007; 48(1):3-7. · 4.70 Impact Factor
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ABSTRACT: Rice (Oryza sativa L.), unlike other cereals, can grow well in paddy fields and is highly tolerant of excess water stress, from either submergence (in which part or all of the plant is under water) or waterlogging (in which excess water in soil limits gas diffusion). Rice handles submergence stress by internal aeration and growth controls. A quiescence strategy based on Submergence-1A (SUB1A) or an escape strategy based on SNORKEL1 (SK1) and SNORKEL2 (SK2) is used for the growth controls. On the other hand, rice handles waterlogging stress by forming lysigenous aerenchyma and a barrier to radial O 2 loss (ROL) in roots in order to supply O 2 to the root tip. In this article, we summarize recent advances in understanding the mechanisms of responding to excess water stresses (i.e., submergence and waterlogging) in rice and other gramineous plants.
