A Histone Methyltransferase ESET Is Critical for T Cell Development.

ESET/SETDB1, one of the major histone methyltransferases, catalyzes histone 3 lysine 9 (H3K9) trimethylation. ESET is critical for suppressing expression of retroviral elements in embryonic stem cells; however, its role in the immune system is not known. We found that thymocyte-specific deletion of ESET caused impaired T cell development, with CD8 lineage cells being most severely affected. Increased apoptosis of CD8 single-positive cells was observed, and TCR-induced ERK activation was severely inhibited in ESET(-/-) thymocytes. Genome-wide comprehensive analysis of mRNA expression and H3K9 trimethylation revealed that ESET regulates expression of numerous genes in thymocytes. Among them, FcγRIIB, whose signaling can inhibit ERK activation, was strongly and ectopically expressed in ESET(-/-) thymocytes. Indeed, genetic depletion of FcγRIIB in ESET(-/-) thymocytes rescued impaired ERK activation and partially restored defective positive selection in ESET(-/-) mice. Therefore, impaired T cell development in ESET(-/-) mice is partly due to the aberrant expression of FcγRIIB. Collectively, to our knowledge, we identify ESET as the first trimethylated H3K9 histone methyltransferase playing a crucial role in T cell development.

Small open reading frames associated with morphogenesis are hidden in plant genomes.

It is likely that many small ORFs (sORFs; 30-100 amino acids) are missed when genomes are annotated. To overcome this limitation, we identified ∼8,000 sORFs with high coding potential in intergenic regions of the Arabidopsis thaliana genome. However, the question remains as to whether these coding sORFs play functional roles. Using a designed array, we generated an expression atlas for 16 organs and 17 environmental conditions among 7,901 identified coding sORFs. A total of 2,099 coding sORFs were highly expressed under at least one experimental condition, and 571 were significantly conserved in other land plants. A total of 473 coding sORFs were overexpressed; ∼10% (49/473) induced visible phenotypic effects, a proportion that is approximately seven times higher than that of randomly chosen known genes. These results indicate that many coding sORFs hidden in plant genomes are associated with morphogenesis. We believe that the expression atlas will contribute to further study of the roles of sORFs in plants.

MOROKOSHI: transcriptome database in Sorghum bicolor.

In transcriptome analysis, accurate annotation of each transcriptional unit and its expression profile is essential. A full-length cDNA (FL-cDNA) collection facilitates the refinement of transcriptional annotation, and accurate transcription start sites help to unravel transcriptional regulation. We constructed a normalized FL-cDNA library from eight growth stages of aerial tissues in Sorghum bicolor and isolated 37,607 clones. These clones were Sanger sequenced from the 5' and/or 3' ends and in total 38,981 high-quality expressed sequence tags (ESTs) were obtained. About one-third of the transcripts of known genes were captured as FL-cDNA clone resources. In addition to these, we also annotated 272 novel genes, 323 antisense transcripts and 1,672 candidate isoforms. These clones are available from the RIKEN Bioresource Center. After obtaining accurate annotation of transcriptional units, we performed expression profile analysis. We carried out spikelet-, seed- and stem-specific RNA sequencing (RNA-Seq) analysis and confirmed the expression of 70.6% of the newly identified genes. We also downloaded 23 sorghum RNA-Seq samples that are publicly available and these are shown on a genome browser together with our original FL-cDNA and RNA-Seq data. Using our original and publicly available data, we made an expression profile of each gene and identified the top 20 genes with the most similar expression. In addition, we visualized their relationships in gene co-expression networks. Users can access and compare various transcriptome data from S, bicolor at http://sorghum.riken.jp.

Loss of Arabidopsis 5'-3' Exoribonuclease AtXRN4 Function Enhances Heat Stress Tolerance of Plants Subjected to Severe Heat Stress.

mRNA degradation plays an important role in the rapid and dynamic alteration of gene expression in response to environmental stimuli. Arabidopsis 5'-3' exoribonuclease (AtXRN4), a homolog of yeast Xrn1p, functions after a de-capping step in the degradation of uncapped RNAs. While Xrn1p-dependent degradation of mRNA is the main process of mRNA decay in yeast, information pertaining to the targets of XRN4-based degradation in plants is limited. In order to better understand the biological function of AtXRN4, the current study examined the survivability of atxrn4 mutants subjected to heat stress. The results indicated that atxrn4 mutants, compared with wild-type plants, exhibited an increased survival rate when subjected to a short-term severe heat stress. A microarray and mRNA decay assay showed that loss of AtXRN4 function caused a reduction in the degradation of heat shock factor A2 (HSFA2) and ethylene response factor 1 (ERF1) mRNA. The heat stress tolerance phenotype of atxrn4 mutants was significantly reduced or lost by mutation of HSFA2, a known key regulator of heat acclimation, thus indicating that HSFA2 is a target gene of AtXRN4-mediated mRNA degradation both under non-stress conditions and during heat acclimation. These results demonstrate that AtXRN4-mediated mRNA degradation is linked to the suppression of heat acclimation.

Role of SOX17 in hematopoietic development from human embryonic stem cells.

To search for genes that promote hematopoietic development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), we overexpressed several known hematopoietic regulator genes in hESC/iPSC-derived CD34(+)CD43(-) endothelial cells (ECs) enriched in hemogenic endothelium (HE). Among the genes tested, only Sox17, a gene encoding a transcription factor of the SOX family, promoted cell growth and supported expansion of CD34(+)CD43(+)CD45(-/low) cells expressing the HE marker VE-cadherin. SOX17 was expressed at high levels in CD34(+)CD43(-) ECs compared with low levels in CD34(+)CD43(+)CD45(-) pre-hematopoietic progenitor cells (pre-HPCs) and CD34(+)CD43(+)CD45(+) HPCs. Sox17-overexpressing cells formed semiadherent cell aggregates and generated few hematopoietic progenies. However, they retained hemogenic potential and gave rise to hematopoietic progenies on inactivation of Sox17. Global gene-expression analyses revealed that the CD34(+)CD43(+)CD45(-/low) cells expanded on overexpression of Sox17 are HE-like cells developmentally placed between ECs and pre-HPCs. Sox17 overexpression also reprogrammed both pre-HPCs and HPCs into HE-like cells. Genome-wide mapping of Sox17-binding sites revealed that Sox17 activates the transcription of key regulator genes for vasculogenesis, hematopoiesis, and erythrocyte differentiation directly. Depletion of SOX17 in CD34(+)CD43(-) ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a key role in priming hemogenic potential in ECs, thereby regulating hematopoietic development from hESCs/iPSCs.

PromoterCAD: Data-driven design of plant regulatory DNA.

Synthetic promoters can control the timing, location and amount of gene expression for any organism. PromoterCAD is a web application for designing synthetic promoters with altered transcriptional regulation. We use a data-first approach, using published high-throughput expression and motif data from for Arabidopsis thaliana to guide DNA design. We demonstrate data mining tools for finding motifs related to circadian oscillations and tissue-specific expression patterns. PromoterCAD is built on the LinkData open platform for data publication and rapid web application development, allowing new data to be easily added, and the source code modified to add new functionality. PromoterCAD URL: http://promotercad.org. LinkData URL: http://linkdata.org.

PosMed: Ranking genes and bioresources based on Semantic Web Association Study.

Positional MEDLINE (PosMed; http://biolod.org/PosMed) is a powerful Semantic Web Association Study engine that ranks biomedical resources such as genes, metabolites, diseases and drugs, based on the statistical significance of associations between user-specified phenotypic keywords and resources connected directly or inferentially through a Semantic Web of biological databases such as MEDLINE, OMIM, pathways, co-expressions, molecular interactions and ontology terms. Since 2005, PosMed has long been used for in silico positional cloning studies to infer candidate disease-responsible genes existing within chromosomal intervals. PosMed is redesigned as a workbench to discover possible functional interpretations for numerous genetic variants found from exome sequencing of human disease samples. We also show that the association search engine enhances the value of mouse bioresources because most knockout mouse resources have no phenotypic annotation, but can be associated inferentially to phenotypes via genes and biomedical documents. For this purpose, we established text-mining rules to the biomedical documents by careful human curation work, and created a huge amount of correct linking between genes and documents. PosMed associates any phenotypic keyword to mouse resources with 20 public databases and four original data sets as of May 2013.

Histone H2A mono-ubiquitination is a crucial step to mediate PRC1-dependent repression of developmental genes to maintain ES cell identity.

Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3), respectively. Compared to H3K27me3, localization and the role of H2AK119u1 are not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation.

Transduction of RNA-directed DNA methylation signals to repressive histone marks in Arabidopsis thaliana.

RNA-directed modification of histones is essential for the maintenance of heterochromatin in higher eukaryotes. In plants, cytosine methylation is an additional factor regulating inactive chromatin, but the mechanisms regulating the coexistence of cytosine methylation and repressive histone modification remain obscure. In this study, we analysed the mechanism of gene silencing mediated by MORPHEUS' MOLECULE1 (MOM1) of Arabidopsis thaliana. Transcript profiling revealed that the majority of up-regulated loci in mom1 carry sequences related to transposons and homologous to the 24-nt siRNAs accumulated in wild-type plants that are the hallmarks of RNA-directed DNA methylation (RdDM). Analysis of a single-copy gene, SUPPRESSOR OF drm1 drm2 cmt3 (SDC), revealed that mom1 activates SDC with concomitant reduction of di-methylated histone H3 lysine 9 (H3K9me2) at the tandem repeats in the promoter region without changes in siRNA accumulation and cytosine methylation. The reduction of H3K9me2 is not observed in regions flanking the tandem repeats. The results suggest that MOM1 transduces RdDM signals to repressive histone modification in the core region of RdDM.

Arabidopsis HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1.

Heterochromatin silencing is pivotal for genome stability in eukaryotes. In Arabidopsis, a plant-specific mechanism called RNA-directed DNA methylation (RdDM) is involved in heterochromatin silencing. Histone deacetylase HDA6 has been identified as a component of such machineries; however, its endogenous targets and the silencing mechanisms have not been analyzed globally. In this study, we investigated the silencing mechanism mediated by HDA6. Genome-wide transcript profiling revealed that the loci silenced by HDA6 carried sequences corresponding to the RDR2-dependent 24-nt siRNAs, however their transcript levels were mostly unaffected in the rdr2 mutant. Strikingly, we observed significant overlap of genes silenced by HDA6 to those by the CG DNA methyltransferase MET1. Furthermore, regardless of dependence on RdDM pathway, HDA6 deficiency resulted in loss of heterochromatic epigenetic marks and aberrant enrichment for euchromatic marks at HDA6 direct targets, along with ectopic expression of these loci. Acetylation levels increased significantly in the hda6 mutant at all of the lysine residues in the H3 and H4 N-tails, except H4K16. Interestingly, we observed two different CG methylation statuses in the hda6 mutant. CG methylation was sustained in the hda6 mutant at some HDA6 target loci that were surrounded by flanking DNA-methylated regions. In contrast, complete loss of CG methylation occurred in the hda6 mutant at the HDA6 target loci that were isolated from flanking DNA methylation. Regardless of CG methylation status, CHG and CHH methylation were lost and transcriptional derepression occurred in the hda6 mutant. Furthermore, we show that HDA6 binds only to its target loci, not the flanking methylated DNA, indicating the profound target specificity of HDA6. We propose that HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1, possibly recruiting MET1 to specific loci, thus forming the foundation of silent chromatin structure for subsequent non-CG methylation.

Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis.

Genome integrity is continuously threatened by external stresses and endogenous hazards such as DNA replication errors and reactive oxygen species. The DNA damage checkpoint in metazoans ensures genome integrity by delaying cell-cycle progression to repair damaged DNA or by inducing apoptosis. ATM and ATR (ataxia-telangiectasia-mutated and -Rad3-related) are sensor kinases that relay the damage signal to transducer kinases Chk1 and Chk2 and to downstream cell-cycle regulators. Plants also possess ATM and ATR orthologs but lack obvious counterparts of downstream regulators. Instead, the plant-specific transcription factor SOG1 (suppressor of gamma response 1) plays a central role in the transmission of signals from both ATM and ATR kinases. Here we show that in Arabidopsis, endoreduplication is induced by DNA double-strand breaks (DSBs), but not directly by DNA replication stress. When root or sepal cells, or undifferentiated suspension cells, were treated with DSB inducers, they displayed increased cell size and DNA ploidy. We found that the ATM-SOG1 and ATR-SOG1 pathways both transmit DSB-derived signals and that either one suffices for endocycle induction. These signaling pathways govern the expression of distinct sets of cell-cycle regulators, such as cyclin-dependent kinases and their suppressors. Our results demonstrate that Arabidopsis undergoes a programmed endoreduplicative response to DSBs, suggesting that plants have evolved a distinct strategy to sustain growth under genotoxic stress.

Integrated database of information from structural genomics experiments.

Information from structural genomics experiments at the RIKEN SPring-8 Center, Japan has been compiled and published as an integrated database. The contents of the database are (i) experimental data from nine species of bacteria that cover a large variety of protein molecules in terms of both evolution and properties (http://database.riken.jp/db/bacpedia), (ii) experimental data from mutant proteins that were designed systematically to study the influence of mutations on the diffraction quality of protein crystals (http://database.riken.jp/db/bacpedia) and (iii) experimental data from heavy-atom-labelled proteins from the heavy-atom database HATODAS (http://database.riken.jp/db/hatodas). The database integration adopts the semantic web, which is suitable for data reuse and automatic processing, thereby allowing batch downloads of full data and data reconstruction to produce new databases. In addition, to enhance the use of data (i) and (ii) by general researchers in biosciences, a comprehensible user interface, Bacpedia (http://bacpedia.harima.riken.jp), has been developed.

Positional correlation analysis improves reconstruction of full-length transcripts and alternative isoforms from noisy array signals or short reads.

MOTIVATION: A reconstruction of full-length transcripts observed by next-generation sequencer or tiling arrays is an essential technique to know all phenomena of transcriptomes. Several techniques of the reconstruction have been developed. However, problems of high-level noises and biases still remain and interrupt the reconstruction. A method is required that is robust against noise and bias and correctly reconstructs transcripts regardless of equipment used. RESULTS: We propose a completely new statistical method that reconstructs full-length transcripts and can be applied on both next-generation sequencers and tiling arrays. The method called ARTADE2 analyzes 'positional correlation', meaning correlations of expression values for every combination on genomic positions of multiple transcriptional data. ARTADE2 then reconstructs full-length transcripts using a logistic model based on the positional correlation and the Markov model. ARTADE2 elucidated 17 591 full-length transcripts from 55 transcriptome datasets and showed notable performance compared with other recent prediction methods. Moreover, 1489 novel transcripts were discovered. We experimentally tested 16 novel transcripts, among which 14 were confirmed by reverse transcription-polymerase chain reaction and sequence mapping. The method also showed notable performance for reconstructing of mRNA observed by a next-generation sequencer. Moreover, the positional correlation and factor analysis embedded in ARTADE2 successfully detected regions at which alternative isoforms may exist, and thus are expected to be applied for discovering transcript biomarkers for a wide range of disciplines including preemptive medicine. AVAILABILITY: http://matome.base.riken.jp CONTACT: toyoda@base.riken.jp SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genome-wide analysis of target genes regulated by HoxB4 in hematopoietic stem and progenitor cells developing from embryonic stem cells.

Forced expression of the transcription factor HoxB4 has been shown to enhance the self-renewal capacity of mouse bone marrow hematopoietic stem cells (HSCs) and confer a long-term repopulating capacity to yolk sac and embryonic stem (ES) cell-derived hematopoietic precursors. The fact that ES cell-derived precursors do not repopulate bone marrow without HoxB4 underscores an important role for HoxB4 in the maturation of ES-derived hematopoietic precursors into long-term repopulating HSCs. However, the precise molecular mechanism underlying this process is barely understood. In this study, we performed a genome-wide analysis of HoxB4 using ES cell-derived hematopoietic stem/progenitor cells. The results revealed many of the genes essential for HSC development to be direct targets of HoxB4, such as Runx1, Scl/Tal1, Gata2, and Gfi1. The expression profiling also showed that HoxB4 indirectly affects the expression of several important genes, such as Lmo2, Erg, Meis1, Pbx1, Nov, AhR, and Hemgn. HoxB4 tended to activate the transcription, but the down-regulation of a significant portion of direct targets suggested its function to be context-dependent. These findings indicate that HoxB4 reprograms a set of key regulator genes to facilitate the maturation of developing HSCs into repopulating cells. Our list of HoxB4 targets also provides novel candidate regulators for HSCs.

The Hbo1-Brd1/Brpf2 complex is responsible for global acetylation of H3K14 and required for fetal liver erythropoiesis.

The histone acetyltransferases (HATs) of the MYST family include TIP60, HBO1, MOZ/MORF, and MOF and function in multisubunit protein complexes. Bromodomain-containing protein 1 (BRD1), also known as BRPF2, has been considered a subunit of the MOZ/MORF H3 HAT complex based on analogy with BRPF1 and BRPF3. However, its physiologic function remains obscure. Here we show that BRD1 forms a novel HAT complex with HBO1 and regulates erythropoiesis. Brd1-deficient embryos showed severe anemia because of impaired fetal liver erythropoiesis. Biochemical analyses revealed that BRD1 bridges HBO1 and its activator protein, ING4. Genome-wide mapping in erythroblasts demonstrated that BRD1 and HBO1 largely colocalize in the genome and target key developmental regulator genes. Of note, levels of global acetylation of histone H3 at lysine 14 (H3K14) were profoundly decreased in Brd1-deficient erythroblasts and depletion of Hbo1 similarly affected H3K14 acetylation. Impaired erythropoiesis in the absence of Brd1 accompanied reduced expression of key erythroid regulator genes, including Gata1, and was partially restored by forced expression of Gata1. Our findings suggest that the Hbo1-Brd1 complex is the major H3K14 HAT required for transcriptional activation of erythroid developmental regulator genes.

The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA.

DNA methyltransferase (cytosine-5) 1 (Dnmt1) is the principal enzyme responsible for maintenance of CpG methylation and is essential for the regulation of gene expression, silencing of parasitic DNA elements, genomic imprinting and embryogenesis. Dnmt1 is needed in S phase to methylate newly replicated CpGs occurring opposite methylated ones on the mother strand of the DNA, which is essential for the epigenetic inheritance of methylation patterns in the genome. Despite an intrinsic affinity of Dnmt1 for such hemi-methylated DNA, the molecular mechanisms that ensure the correct loading of Dnmt1 onto newly replicated DNA in vivo are not understood. The Np95 (also known as Uhrf1 and ICBP90) protein binds methylated CpG through its SET and RING finger-associated (SRA) domain. Here we show that localization of mouse Np95 to replicating heterochromatin is dependent on the presence of hemi-methylated DNA. Np95 forms complexes with Dnmt1 and mediates the loading of Dnmt1 to replicating heterochromatic regions. By using Np95-deficient embryonic stem cells and embryos, we show that Np95 is essential in vivo to maintain global and local DNA methylation and to repress transcription of retrotransposons and imprinted genes. The link between hemi-methylated DNA, Np95 and Dnmt1 thus establishes key steps of the mechanism for epigenetic inheritance of DNA methylation.

Semantic-JSON: a lightweight web service interface for Semantic Web contents integrating multiple life science databases.

Global cloud frameworks for bioinformatics research databases become huge and heterogeneous; solutions face various diametric challenges comprising cross-integration, retrieval, security and openness. To address this, as of March 2011 organizations including RIKEN published 192 mammalian, plant and protein life sciences databases having 8.2 million data records, integrated as Linked Open or Private Data (LOD/LPD) using SciNetS.org, the Scientists' Networking System. The huge quantity of linked data this database integration framework covers is based on the Semantic Web, where researchers collaborate by managing metadata across public and private databases in a secured data space. This outstripped the data query capacity of existing interface tools like SPARQL. Actual research also requires specialized tools for data analysis using raw original data. To solve these challenges, in December 2009 we developed the lightweight Semantic-JSON interface to access each fragment of linked and raw life sciences data securely under the control of programming languages popularly used by bioinformaticians such as Perl and Ruby. Researchers successfully used the interface across 28 million semantic relationships for biological applications including genome design, sequence processing, inference over phenotype databases, full-text search indexing and human-readable contents like ontology and LOD tree viewers. Semantic-JSON services of SciNetS.org are provided at http://semanticjson.org.

The RIKEN integrated database of mammals.

The RIKEN integrated database of mammals (http://scinets.org/db/mammal) is the official undertaking to integrate its mammalian databases produced from multiple large-scale programs that have been promoted by the institute. The database integrates not only RIKEN's original databases, such as FANTOM, the ENU mutagenesis program, the RIKEN Cerebellar Development Transcriptome Database and the Bioresource Database, but also imported data from public databases, such as Ensembl, MGI and biomedical ontologies. Our integrated database has been implemented on the infrastructure of publication medium for databases, termed SciNetS/SciNeS, or the Scientists' Networking System, where the data and metadata are structured as a semantic web and are downloadable in various standardized formats. The top-level ontology-based implementation of mammal-related data directly integrates the representative knowledge and individual data records in existing databases to ensure advanced cross-database searches and reduced unevenness of the data management operations. Through the development of this database, we propose a novel methodology for the development of standardized comprehensive management of heterogeneous data sets in multiple databases to improve the sustainability, accessibility, utility and publicity of the data of biomedical information.

Functional compensation of primary and secondary metabolites by duplicate genes in Arabidopsis thaliana.

It is well known that knocking out a gene in an organism often causes no phenotypic effect. One possible explanation is the existence of duplicate genes; that is, the effect of knocking out a gene is compensated by a duplicate copy. Another explanation is the existence of alternative pathways. In terms of metabolic products, the relative roles of the two mechanisms have been extensively studied in yeast but not in any multi-cellular organisms. Here, to address the functional compensation of metabolic products by duplicate genes, we quantified 35 metabolic products from 1,976 genes in knockout mutants of Arabidopsis thaliana by a high-throughput Liquid chromatography-Mass spectrometer (LC-MS) analysis. We found that knocking out either a singleton gene or a duplicate gene with distant paralogs in the genome tends to induce stronger metabolic effects than knocking out a duplicate gene with a close paralog in the genome, indicating that only duplicate genes with close paralogs play a significant role in functional compensation for metabolic products in A. thaliana. To extend the analysis, we examined metabolic products with either high or low connectivity in a metabolic network. We found that the compensatory role of duplicate genes is less important when the metabolite has a high connectivity, indicating that functional compensation by alternative pathways is common in the case of high connectivity. In conclusion, recently duplicated genes play an important role in the compensation of metabolic products only when the number of alternative pathways is small.

Tissue-specific transcriptome analysis reveals cell wall metabolism, flavonol biosynthesis and defense responses are activated in the endosperm of germinating Arabidopsis thaliana seeds.

Seed germination is a result of the competition of embryonic growth potential and mechanical constraint by surrounding tissues such as the endosperm. To understand the processes occurring in the endosperm during germination, we analyzed tiling array expression data on dissected endosperm and embryo from 6 and 24 h-imbibed Arabidopsis seeds. The genes preferentially expressed in the endosperm of both 6 and 24 h-imbibed seeds were enriched for those related to cell wall biosynthesis/modifications, flavonol biosynthesis, defense responses and cellular transport. Loss of function of AtXTH31/XTR8, an endosperm-specific gene for a putative xyloglucan endotransglycosylase/hydrolase, led to faster germination. This suggests that AtXTH31/XTR8 is involved in the reinforcement of the cell wall of the endosperm during germination. In vivo flavonol staining by diphenyl boric acid aminoethyl ester (DPBA) showed flavonols accumulated in the endosperm of both dormant and non-dormant seeds, suggesting that this event is independent of germination. Notably, DPBA fluorescence was also intense in the embryo, but the fluorescent region was diminished around the radicle and lower half of the hypocotyl during germination. DPBA fluorescence was localized in the vacuoles during germination. Vacuolation was not seen in imbibed dormant seeds, suggesting that vacuolation is associated with germination. A gene for δVPE (vacuolar processing enzyme), a caspase-1-like cysteine proteinase involved in cell death, is expressed specifically in endosperms of 24 h-imbibed seeds. The δvpe mutant showed retardation of vacuolation, but this mutation did not affect the kinetics of germination. This suggests that vacuolation is a consequence, and not a trigger, of germination.