Oil-producing green alga Botryococcus braunii is a potential biological source of petroleum substitutes. However, the identities of genes associated with oil biosynthesis, and thus the detailed biosynthesis pathways, still remain largely unknown. In order to gain such fundamental insight, we used representative high-oil-producing strains of two major races of B. braunii, namely, Race A and Race B strains known to biosynthesize hydrocarbons derived from unsaturated very long-chain fatty acids (VLCFAs) and triterpenes, respectively. We first generated novel datasets of cDNA reads and retrieved candidate genes associated with oil biosynthesis. We then examined the race-dependant expression patterns of the oil biosynthesis-related genes by comparison of the EST counts and the real-time PCR quantification. Regarding the unsaturated VLCFA biosynthesis, our results suggested (i) acyl-acp elongation is the major pathway for fatty acid elongation and (ii) both acyl-acp desaturases and acyl-CoA desaturases participate in the fatty acid desaturation. Regarding the triterpene biosynthesis, it was inferred that two entry routes into the 2-C-methyl-D-erythritol-4-phosphate pathway are active.

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Transcription factors (TFs), which regulate the first step of the gene expression, affect various biofunction. Therefore, elucidation of the function of each TF will lead to identify the relationship between genes and the biofunction that is regulated by a gene. However, gene redundancy of TFs often interferes such efforts. We have developed a novel gene silencing technology (CRES-T), which can overcome the gene redundancy and induces loss-of-function phenotype of TF by expressing chimeric repressor of the TF. Using CRES-T, we have clarified function of various TFs in Arabidopsis thaliana, a model plant in dicot.
In this study, we are applying the CRES-T to rice, a model plant in monocot, using rice full-length TF cDNAs. We have created 314 kinds of transgenic rice that express chimeric repressor of nine TF families including bZIP, Myb, NAC and others. We here report intriguing phenotypes and comparative analysis of TFs between rice and Arabidopsis.

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Oryza glaberrima, which is a distinct species from Asian rice O. sativa, was domesticated in West Africa, and is important for the global food problem. We determined the genome sequence of O. glaberrima IRGC104038 by the whole-genome shotgun methods after gene-enrichment by the methylation filtration method. As a result, 69,083,576 bp (~18%) of the O. sativa (cv. Nipponbare) genome were found to correspond to our sequence reads, while a total of ~12 Mb were unique to O. glaberrima. The sequences could cover (part of) 6,443 genes of O. sativa. Genome-wide comparisons between African and Asian rice revealed lineage-specific amino acid substitutions, splicing variants, etc. In addition, among 2,451 SSRs detected, the lengths of 1,568 were different. The SSR information found in this study will be a useful resource for development of new cultivars. We constructed a database in which the sequences we determined and other related information are available.

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We have developed fifty thousands insertion lines using the endogenous retrotransposon Tos17. Large scale phenotyping for mutant lines shows that half of mutant lines have at least one phenotypic change. However, co-segregation analysis between genotype of Tos17 and phenotype indicates relatively low efficient for tagging by Tos17. We have been analyzing the whole genome sequence of regenerated rice plants to detect cause of the somaclonal variation. We sequenced two lines of regenerated rice at M₄ stage using a next-generation sequencer (Solexa). At least, 1 mutation / Mb in the regenerated plant at M₁ stage is estimated. Estimated mutations were validated by Dyedeoxy terminator method. The mutation spectrum of somaclonal variation is analogous to the spectrum of spontaneous mutation of Arabidopsis except the relatively low level of transition from C to T. The C to T transition is caused by deamination of methyl-cytosine. Because no enzymatic repairing process in the deamination of methyl-cytosine, it is suggested that the high frequency of mutations in somatic cells is caused by suppression of mutation repairs.

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NBRP Tomato focuses on the development of bio-resources using Micro-Tom, a model cultivar of tomato genomics. Micro-Tom is a member of Solanum lycopersicum whose genome draft sequence has been obtained using cultivar Heinz 1706. Analysis of EST- and BAC sequences demonstrated that a genome sequence of Micro-Tom has considerable number of polymorphisms compared to that of Heinz 1706 in single-nucleotide level. Thus, genome sequencing of Micro-Tom will facilitate the development of a number of DNA markers and accelerate the elucidation of genes responsible for the phenotypic changes by using Micro-Tom mutagenized lines.In this study, Kazusa DNA Research Institute and the National Institute of Genetics (NIG) collaborate in sequencing by using Roche GS-FLX and Illumina HiSeq, respectively. Since the end of Nov. 2010, GS-FLX reads have accumulated to 10-fold coverage, and HiSeq Reads have accumulated to 120-fold coverage of tomato genome (0.9 Gb). We will report an up-to-date situation of read mapping and contig assembly, and data opening schedule as well. This work was supported by NBRP genome upgrading program Micro-Tom Genome Sequencing.

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Since plants cannot move to avoid stress, they have sophisticated acclimation mechanisms against various stress factors. The phytohormone abscisic acid (ABA) plays essential roles in such stress tolerances in land plants. Therefore, it is interesting to address the evolutionary origins of ABA metabolism and signaling pathways in land plants. Here, we focused on 48 Arabidopsis thaliana genes with ten key functions (ABA-related genes), and generated ten orthologous clusters of ABA-related genes from A. thaliana, Arabidopsis lyrata, Populus trichocarpa, Oryza sativa, Selaginella moellendorffii, and Physcomitrella patens. Phylogenetic analyses suggested that the common ancestor of these six species had most of the key functions of ABA-related genes. In the lineage of A. thaliana, gene expansion of ABA-related genes led to the expression divergences. This result indicates that in the evolution of A. thaliana, ABA-related genes have their functions in distinct organs and in response to various environmental cues. Taken together, these results suggest that the expansion of ABA-related genes may have contributed to the sophisticated stress tolerance mechanisms of higher land plants.

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Recently, peptides encoded by short open reading frames (sORFs) are reported to play roles in various functions in many organisms. To investigate function of sORFs in plants, we identified sORFs ranging from 90 to 300 bp in the intergenic regions of Arabidopsis and rice. We developed custom microarrays including sORFs and annotated genes in Arabidopsis and rice to analyze gene expression profile. Microarray analysis was carried out in various tissue types. The expression of sORFs could be detected in various plant tissues. Full length cDNA of sORF was verified by RACE experiment. Next, promoter region and sORF without stop codon was fused to GUS gene and then expressed it in Arabidopsis. GUS signal was detected in transgenic plants, indicating that sORF was actually translated in plants. We selected sORFs based on expression, conservation, peptide hormone-like sequence to generate sORF overexpression Arabidopsis and rice plants. Some overexpression plants showed visible phenotypes. Thus, we conclude that sORFs have roles in growth and developmental processes.

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Plant cell contains a number of organelles. Cellular activities are supported by the cooperative functions of organelles. Analysis of microscopic images play an essential role in organelle research. Specially, recent technical advance of live imaging allow us to obtain large data sets of time-sequential images of each organelle. However, in contrast to biosequential data and gene expression data, quantitative analysis are rarely performed for organelle images because of the diversity of imaging-technique including purposes, targets and conditions. To develop a versatile technique for organelle image analysis, we have focused on endoplasmic reticulum (ER) flow as a model case of organelle dynamics and investigated the method of speed measurement from time-sequential images. As a result, we succeeded to develop a software tool for flow analysis using local and spatio-temporal image correlation spectrum. Furthermore, we have investigated to measure the movements for multiple organelles visualized in single fluorescence band. As part of the investigation, we are currently attempting to develop the machine learning based image classifier and distribution estimator of organelles.

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RNA metabolism is an important physiological process of organisms. However, almost RNA metabolisms are still unrevealed. The RNA metabolism can be dissected from variation of an expression and an exon structure pattern affected from a condition change. In this research, we present mathematical model to elucidate transcriptome dynamics from multiple RNA-seq data. Expressed mRNA is observed with tag counting after fragmentation in RNA-seq. Then we do not know to which RNA molecular the fragment corresponds. For this matter, correlation information based on multi-conditional RNA-seq data is suitable to model the exon structure and transcriptome dynamics of RNA. Consequently, we elucidate transcriptome dynamics by modeling correlations of multi RNA-seq data at several scales and dissecting the constructed hierarchical model. We adopt proposed model to RNA-seq data of Arabidopsis for 7 conditions. Then we show novel RNA metabolisms and functions of them extracted from the model.

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Oryza rufipogon, Asian wild rice, is believed to be the progenitor of the cultivated rice O. sativa, whose cultivars can be classified into several diverse groups including japonica and indica. In this study, to decipher the evolutionary processes of wild and cultivated rice, we used ~2000 full-length cDNAs of O. rufipogon W1943 as well as genomic sequences of japonica and indica cultivars. Our genome-wide analysis resulted in one major and two minor gene tree topologies, showing expected gene tree discordance. The major gene tree topology implied that O. rufipogon W1943 is closely related to japonica, which is consistent with previous studies. These gene tree topologies also suggested incomplete lineage sorting and hybridization between japonica and indica cultivars after their divergence. In addition, we identified genes that were possibly lost in the japonica cultivar. We found that fifteen O. rufipogon-specific genes were related to submergence and/or wounding conditions. We highlighted the importance of gene tree discordance analysis on unraveling complicated evolutionary histories of closely related species and suggested possible gene loss events during rice domestication.

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We have been developing a web-based analysis tool, KaPPA-View, which enables to view transcriptome data, metabolome data and gene-coexpression data on the metabolic pathway maps. In addition to the existing system that has pathway map data for Arabidopsis (KaPPA-View4 Classic), we developed another version, KaPPA-Veiw4 KEGG, that installed the genes and pathway maps data acquired from the Kyoto Encyclopedia of Genes and Genomes (KEGG). Users can use 290 sheets of well curated KEGG PATHWAY maps, and 380 sheets of gene family maps that are prepared by us from KEGG BRITE categorization. The set of the maps facilitates the researchers to analyze novel gene functions by utilizing gene-to-gene co-expression data. Utilization of KEGG map also enables the users to analyze not only plant species but also animals and microorganisms. Currently, pathway maps for 20 species and gene co-expression data for 12 species in total are available.
URL: http://kpv.kazusa.or.jp/kpv4-kegg/
Sakurai et al. (2011) Nuc. Acids Res. Database Issue, in press

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Metabolome analysis using mass spectrometry such as GC-MS or LC-MS has been developed to identify metabolites in plants with an improved efficiency. Recently, CE-MS has been reported to be a useful tool to identify plant metabolites, especially in charged species. We established the simultaneous quantification of plant hormones together with primary metabolites including amino acids, organic acids and phosphate compounds in A. thaliana plants by CE-ESI-QTOF.
In this study, we investigated metabolic changes in response to exogenously applied auxin (IAA) using CE-ESI-QTOF. When plants were treated with 10^-7 M IAA for 12 hours, levels of some organic acids decreased in shoots and increased in roots compared to the control. Also, basic amino acids both in shoots and roots increased compared to the control. These results suggest that some metabolites, which are related to nitrogen assimilation, respond to exogenous IAA. We are now measuring the effect of shorter IAA treatment on the metabolite levels in A. thaliana plants, too.

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The carnivorous plants of the genus Drosera grow on nitrogen-limited wetlands. Year-round measurements showed that the nitrate concentrations in the habitats of D.routundifolia (Dr), D.spathulata (Ds) and D.tokaiensis (Dt) were 0~3.3 μM (av.0.9 μM), 3.1~39.7 μM (av.15.0 μM) and 0 ~148 μM (av.43.0 μM), respectively, suggesting that Dt and Ds prefer higher nitrate concentrations than Dr. Since Dt has been shown to originate from a natural cross between Dr and a closely related species Ds, we compared the response of the three species to different nitrogen conditions in the greenhouse and found that Dt and Ds are more tolerant to nitrogen than Dr is (2009 annual meeting). In an effort to analyze the cause of the different tolerance to nitrogen, we isolated and characterized nitrate reductase (NR) and nitrite reductase (NiR) cDNAs from the Drosera species. The predicted amino acid sequences of NR and NiR molecular species were used to construct unrooted phylogenetic trees. The phylogenetic trees of NR and NiR were distinct from that of RbcL, suggesting ancient origin of the Drosera-Type NR and NiR proteins in the evolution of angiosperms.

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Plants have mechanisms to regulate C and N metabolism and growth in response to the C/N ratio in cells and/or environments. Although a few proteins involved in this mechanism was identified in recent studies, the mechanism is largely unknown yet. BT proteins are plant-specific proteins that possess two protein interaction domains, the BTB and TAZ domains, and they are proposed to function as scaffold proteins in protein complexes. In Arabidopsis thaliana, there are 5 genes encoding BT proteins. The expressions of BT1 and BT2 are repressed by sugar but induced by nitrate. Here we show nuclear localization of BT1 and BT2 proteins. We also show the severer glucose inhibition of greening and the weaker antagonistic action of nitrate to glucose in the bt1 and bt2 mutants. Furthermore, we found that the phenotype was much severer in the bt1 bt2 double mutant, implying roles of BT1 and BT2 in the mechanism underlying C/N balance responses. We are currently attempting to identify proteins that interact with BT proteins and proteins of the complexes including BT proteins by a proteomic approach.

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Assimilation pathways of CO₂ and nitrogen are closely related to each other. It has been observed that elevated CO₂ leads to stimulation of growth under nitrogen-sufficient conditions. However, effects of elevated CO₂ on the growth under nitrogen-limited conditions are still unclear because of difficulties in maintaining such conditions in experiments. Here, we kept Arabidopsis thaliana under nitrogen-limited conditions by using the mutant with reduced nitrate uptake activities and examined the effects of elevated CO₂. Under the nitrogen-sufficient conditions (i.e., in the presence of ammonium), both the wild type and the mutant exhibited ~2-fold increase in biomass when CO₂ concentration was increased from 280 to 780 ppm. Similar effects of elevated CO₂ were observed for the wild type growing on the medium containing 15 mM nitrate as sole nitrogen source. In contrast, the mutant growing on the nitrate-containing medium showed unchanged shoot biomass regardless of elevated CO₂, whereas root biomass was increased in response to elevated CO₂. This symptom is considered to be a specific response to elevated CO₂ in plants kept under the nitrogen-limited condition.

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For assimilation of nitrate, marine cyanobacteria of the Synechococcus group have an MFS-type nitrate/nitrite transporter. Most marine cyanobacteria of the Prochlorococcus group do not assimilate nitrate or nitrite, but some retain nitrite reductase (NiR). Some marine Synechococcus species and the NiR-containing Prochlorococcus strains have focA, a gene encoding a putative nitrite transporter. Expression of focA from Synechococcus sp. PCC7002 conferred nitrite uptake activity on the double mutant (NA4) of the freshwater cyanobacterium Synechococcus elongatus that lacks the ABC-type nitrate/nitrite- and cyanate/nitrite transporters. However, the focA genes from other two marine cyanobacteria did not confer nitrite uptake activity, supposing that the FocA transporters of marine cyanobacteria have different structure and function in different species. The latter FocA transporters have conserved hydrophilic region at the C-terminus and expression of truncated forms of the latter focA genes lacking the 3'-terminal part conferred nitrite uptake activity on NA4, suggesting that the C-terminal regions of FocA inhibit nitrite uptake activity of the transporters.

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In cyanobacteria, the nitrate transporter (NRT) and/or nitrate reductase (NR) are inhibited upon addition of NH₄⁺ to the medium. Cyanobacteria has either an ABC-type NRT ecoded by the nrtABCD genes or an MFS-type transporter ecoded by nrtP, the former of which is sensitive to NH₄⁺ treatment, while the latter is not. Although cyanobacteria has only one type of NR encoded by narB, its activity is sensitive to NH₄⁺ treatment in some strains but not in others. In this study, we used a Synechococcus elongatus PCC7942 mutant (NP1), in which nrtABCD had been replaced with nrtP from Nostoc punctiforme to construct an NR-deficient mutant NP2. The NP2 mutant, which have NH₄⁺-resistant NRT activity, was used as the host for expression of various NR proteins. Examination of the effects of NH₄⁺ on these strains and comparison of the structure of their NR proteins suggested that the NH₄⁺-sensitive NRs commonly have a regulatory loop in the N-terminal [4Fe-4S]-binding domain. Site-specific replacement of one of the conserved residues in the loop (a Pro residue), rendered NR resistant to ammonium treatment, confirming its importance in the regulation of the NR activity.

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Plants have evolved a set of responses to adapt to nitrogen (N)-limited conditions. However, little is known about the molecular basis of these responses. Here we characterized AtNRT2.4, which encodes a putative high-affinity nitrate transporter, as a candidate gene involved in plant adaptation to N-limitation. AtNRT2.4 is preferentially expressed in lateral root epidermal cells of N-limited plants. Furthermore, transgenic plants expressing AtNRT2.4-GFP fusion protein demonstrated that AtNRT2.4-GFP is localized in distal side of the epidermal cell, implying that AtNRT2.4 plays a role in nitrate-acquisition under N-limiting conditions. Thus, to evaluate whether AtNRT2.4 can transport nitrate, the Xenopus laevis oocyte system was employed. AtNRT2.4 mRNA-injected oocytes imported significantly more nitrate than water-injected controls, showing that AtNRT2.4 functions as a nitrate transporter. Furthermore, we characterized T-DNA insertional mutants. The mutant showed reduction in nitrate uptake compared with WT at low nitrate concentration (< 50 μM). Together, these results indicate that AtNRT2.4 is involved in adaptation to low-N conditions in Arabidopsis.

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NADH-glutamate synthase (NADH-GOGAT) catalyzes the transfer of the amido-residue of glutamine to 2-oxoglutarate, resulting generation of two glutamate molecules. There are two genes encode two isoenzymes of NADH-GOGAT, i.e. NADH-GOGAT1 and NADH-GOGAT2 in rice. We have showed that NADH-GOGAT1 protein accumulated in vascular tissues of developing organs, such as unexpanded leaves and young grains. On the other hand, NADH-GOGAT2 promoter activity was mainly detected in vascular tissues of senescing leaves. However, their physiological functions are unknown. We isolated Tos17-insertion mutants lacking either NADH-GOGAT1 or NADH-GOGAT2. These mutants were cultivated till harvest in paddy field. Top dry weight and grain yield were decreased in both of NADH-GOGAT1 and NADH-GOGAT2 mutants. The NADH-GOGAT1 mutants decreased panicle number per plants, while spikelet number per panicle was mainly reduced in NADH-GOGAT2 mutants. These phenotypic characteristics suggest that NADH-GOGAT1 and NADH-GOGAT2 probably possess distinct functions on nitrogen utilization, respectively in rice.

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The Gln-signaling system to regulate expression of certain genes for NH₄⁺ absorption and assimilation in rice roots after a supply of NH₄⁺ has been proposed. OsACTPKs, rice homologs of Arabidopsis Ser/Thr/Tyr protein kinase (AtSTYPK), have putative amino acid-binding ACT domains similar to those of bacterial Gln sensor GlnD. OsACTPK1 mRNA was cumulatively expressed in roots by daily applications of NH₄⁺. As a first step to examine whether OsACTPK1 may be concerned with the Gln-signaling, OsACTPK1-knockout rice mutants (KO lines) by Tos17 insertion were isolated and phenotypes of their seedlings grown under the supply of low to sufficient NH₄⁺ were investigated. Null lines, in which Tos17 had segregated out, were used as controls. Under sufficient NH₄⁺ treatments, inhibition of root elongation, promotion of shoot growth, and increases in total nitrogen and free amino acids contents were observed in KO lines. Analysis of ¹⁵NH₄⁺ uptake into roots revealed Vmax values of high affinity transport systems in KO lines were ca. 2-fold increased. These results suggest that OsACTPK1 may negatively regulate at least NH₄⁺ absorption in seedling roots under the supply of sufficient NH₄⁺.

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We recently identified an Arabidopsis gain-of-function mutant of plastidic invertase (INV-E), sicy-192, as sucrose sensitive mutant. On treatment with sucrose, the expression of photosynthesis- and nitrogen assimilation-related genes was respective lower and higher in the mutants, suggesting that INV-E participates in regulating the carbon/nitrogen (C/N) balance. Expression of photosynthesis-related genes encoded by the nuclear genome is under control of the plastid signaling derived from plastid gene expression (PGE). To clarify the involvement of INV-E in the plastid signaling, we analyzed PGE in sicy-192 mutants on treatment with sucrose.
The transcript levels of plastid genes downstream of plastid-encoded RNA polymerase (PEP), but not of nuclear-encoded plastid RNA polymerase (NEP), were significantly reduced in the sicy-192 mutants on treatment with sucrose. Moreover, the expression of GLK1 and GLK2, components of plastid signaling, was down-regulated in the sicy-192 mutants. Now, we are analyzing the effect of treatment with sucrose on the PGE and nuclear gene expression in the wild-type, sicy-192, and knockout mutants lacking INV-E.

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Cyanobacteria are representative photoautotrophic organisms, which must regulate metabolic activities on various pathways under fluctuant environments. To investigate responses to nutrient starvation, we quantified amino acids, which are located at the center of carbon, nitrogen and sulfur metabolism, with gas chromatography - mass spectrometry and analyzed free amino acids pool of Synechocystis sp. PCC 6803 under nitrogen starvation. Unexpectedly, most of proteinogenic amino acids increased temporary in 24 h after transfer to nitrogen poor condition. More remarkably tyrosine and lysine accumulated compared to other amino acids. These two amino acids level returned to the original in 6 h of nitrate re-supply after 24 h starvation. Since protein biosynthesis and degradation depend on nitrogen availability, cyanobacteria degrade the abundant phycbiliproteins in response to the nitrogen depletion. To estimate contribution of phycobiliproteins degradation to the amino acids pool, we studied fluctuation of amino acids in a mutant that lacks nblA1/A2 genes, which are indispensable for degradation. We will also report amount of newly synthesized amino acids under nitrogen poor condition.

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Effects of light on expression of one of the glycolytic enzymes, fructose-1.6-bisphosphate aldolase (fbaA) were investigated in Synechocystis sp. PCC6803. The fbaA mRNA was induced by light pulse of 5 min in the presence of glucose, while glucose addition in the dark was not effective for induction of fbaA. Therefore, both light and glucose were required for the full induction of fbaA transcription. The induction of fbaA gene expression by light pulse and glucose were inhibited by deletion of gene encoding one of the response regulators, sll1330, suggesting that sll1330 is involved in regulation of fbaA. Investigation of the effects of disruption of the genes encoding other regulatory genes indicated that there was histidine kinase whose disruption promotes fbaA expression in the absence of pulse light under glucose. These results suggest that gene expression of glycolytic enzymes are controlled through complex signal pathways interacting to each other.

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To accomplish fertilization in angiosperms, pollen tube guidance is an essential mechanism. The mechanism precisely guides pollen tubes to embryo sacs in the fertilization process. However, most of molecules including pollen tube attractants have remained unclear for more than a century. Recently, our research group has been identified pollen tube attractants, Torenia fournieri LUREs, derived from the synergid cell. Even though the attractants of T. fournieri have been identified, universality in angiosperms, in vivo functions and molecular evolution of the attractants are still unclear.
Our aim of this study is to identify pollen tube attractants of a model plant Arabidopsis thaliana for which many experimental tool and resources are easily available. We have identified pollen tube attractants in A. thaliana which have the same properties as TfLUREs and shown in vivo function of the attractants (Takeuchi and Higashiyama, the 51st Annual Meeting of JSPP). Here we report our trial to undertake toward further development of study for the attractant using A. thaliana, and discuss gene regulatory network and molecular evolution of the attractants.

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Double fertilization is unique reproductive system to flowering plants. One of two sperm cells delivered by the pollen tube fertilizes the egg cell to produce the embryo and the other fertilizes the central cell to produce a nutritional tissue, the endosperm. Genes involved in gamete fusion have been identified. However, dynamics of two sperm cells deep in the female tissue, including the long lasting issue of preferentiality fertilization, remains unknown. In this study, we observed 132 examples of double fertilization. Migration of sperm cells during pollen tube discharge is finished within 20 sec and the maximum velocity was 10 μm/sec. Fertilization in egg and central cells occurred in 8.4 ± 4.1 min, 8.5 ± 3.1 min respectively without preference of order. Furthermore, when we marked two sperm cells the position in pollen tube by photo-conversion of mKikGR, front sperm cell fertilized egg and central cell equal frequency. This result suggests that the target of isomorphic Arabidopsis sperm cells is not determined in pollen tube. Actual behavior of gametophytic cells provide us insights in to the mechanism of double fertilization.

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During fertilization in flowering plants, chemo-attractants from the ovule have been thought to be key molecules in pollen tube guidance. We have identified defensin-like peptide LUREs as attractant peptides (Okuda, Tsutsui et al., Nature, 2009). Recently, we established a method enabling the direct visualization of a LURE peptide by conjugating it with the Alexa Fluor 488 fluorescent dye (Goto et al., Plant Cell Physiol., 2011).
These recent findings allow us to analyze the mechanism of pollen tube guidance directly at a molecule level. Elucidation of the receptor for LUREs is essential for understanding the mechanism of pollen tube-LUREs interaction. We, then, aimed to identify the receptor to reveal how the pollen tube accepts the signal of LUREs and determines its growth direction. First, to investigate which part of the pollen tube interacts with LUREs, pollen tubes treated with recombinant LUREs were immunostained with anti-LURE antibodies. Next, we analyzed how the pollen tube is affected by maternal tissues. Here, we show maternal effects on the pollen tube when the pollen tube receives LUREs by demonstrating physiological analyses.

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Pollen tube guidance, a mechanism to guide pollen tubs to the ovule, is highly species specific and is thought to be one of a mechanism to prevent interspecies crosses. We have recently identified that a group of Defensin-like Cysteine-Rich Polypeptides, LUREs, are the pollen tube attractants secreted from the synergid cells of Torenia fournieri. We further tried to isolate LURE homologues from related species of T. fournieri and compare their sequences and functions for pollen tube guidance. We found one LURE-like protein, named TcCRP1, which is expressed exclusively in the synergid cell of T. concolor, had strong pollen tube attractant activity against T. concolor pollen tubes. TcCRP1 also showed weak attractant activity against T. fournieri pollen tubes. We also found a LURE-like gene that was not expressed in the ovule of T. concolor. Some key amino acids were substituted in its product, suggesting that this gene might be a pseudo-gene. These results suggested that diversification of Cysteine-Rich Polypeptides contribute pollen tube attraction activities. We are currently trying to identify key structure of LUREs for pollen tube attraction.

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During sexual reproduction in higher plants, a pollen tube grows through a pistil to deliver sperm cells to a female gametophyte. In the process, controlling direction of the pollen tube growth is essential for successful fertilization. Physiological studies indicated that an ovule emits guidance signals to attract or repel a pollen tube. Recently, we showed LUREs, the polypeptides secreted from synergid cells in the female gametophyte of Trenia fournieri, have an activity that attracts pollen tubes. However, these molecules identified so far are insufficient to understand the mechanism of pollen tube guidance. For example, if the guidance signal were merely diffusible attractants like LUREs, many pollen tubes would rush into unfertilized ovules. But in reality, only single pollen tube accurately targets to an ovule. This suggests a mechanism of polyspermy block by which fertilized ovule prevents arrival of second pollen tube. In order to study the polyspermy block in higher plants, I performed a visual screening to find mutants containing ovules that attract more than one pollen tube in Arabidopsis thaliana.

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Female gametophyte plays a critical role in essentially every step of the reproductive process. One of the most important functions of the female gametophyte is the pollen tube guidance. Within the female gametophyte, synergid cells are required for pollen tube guidance (Higashiyama et al. 2002). Although the cellular functions of the synergid cells have been understood, little is known for the molecular-level function of the synergid cells. However, Kasahara et al. identified a key gene, MYB98. To investigate genes essential for the development and function of the synergid cell, we started forward genetics approach by using MYB98::GFP visible screening and identified a male gametophytic mutant, g21. Although a pollen grain has two sperm cells in wild type Arabidopsis, g21 mutant has only single sperm-like cell inside of the pollen grain. Because of the single sperm-like cell, g21 mutant shows fertilization defect even though the mutant pollen tubes get into the female gametophyte. In this meeting, we report that we discovered a remarkable function during plant fertilization. This newly identified function will deeply contribute to gain insight of the plant fertilization event.

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Flowering plants have evolved a unique and complex fertilization system known as double fertilization. This event that two sperm cells fuse an egg cell and a central cell respectively, was discovered more than a century ago. However, the mechanism of fertilization remains largely unclear because these gamete fusions take place in the embryo sac, which is deeply embedded in the ovule. In this study, to address the mechanism of fertilization, we carried out a comprehensive analysis of gene expression profiles of the egg cells, sperm cells, and zygotes that were isolated from unpollinated or pollinated ovaries of rice. Additionally, to reveal whether two sperm cells have distinct identities for fusion with egg cell and central cell or not, investigations for monitoring gene expression profiles of each two sperm cells derived from one pollen are underway. Based on the results from these transcriptome analyses, we will discuss the possible molecular mechanisms in gamete fusion underlying reproductive event.

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Gamete fusion activates the egg in animals and plants, and the gamete fusion site on the zygote might provide a possible cue for zygotic development and/or embryonic patterning. In angiosperms, a zygote generally divides into a two-celled proembryo consisting of an apical and a basal cell with different cell fates. This is a putative step in the formation of the apical-basal axis of the proembryo. We observed the positional relationship between the gamete fusion site and the division plane formed by zygotic cleavage using an in vitro fertilization system with rice gametes. There was no relationship between the gamete fusion site and the division plane leading to the two-celled proembryo. Thus, the gamete fusion site on the rice zygote does not appear to function as a determinant for positioning the zygote division plane, and the zygote apparently possesses autonomous potential to establish cell polarity along the apical-basal axis for its first cleavage. In addition, this observation indicates a possibility that there is no specific or restricted point on the egg cell surface for gamete fusion.

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Gene targeting (GT) by homologous recombination is the most powerful technique available for specific modification of a chromosomal target gene. In angiosperms, the transferred DNA (T-DNA) is randomly integrated into the genome by non-homologous end-joining (NHEJ)-related mechanism, therefore GT efficiency is extremely low. Thus, it is expected that suppression of NHEJ pathway increase GT frequency. It has been recently reported that the lack of AtLigIV cause an increase in the efficiency of GT.
We generated rice plants (KD-Ku70, Ku80 and LigIV) transformed with an RNAi construct using 3' sequence of Ku 70, Ku80 and LigIV mRNA. To test the efficiency of T-DNA integration, KD calli were infected with Agrobacterium harboring reporter genes ; 35S pro+GFP and Eluc gene trap constructs. 3-day after infection, the transient expression of GFP in KD calli were comparable to that in WT callus. 9-day after infection, LUC signals, monitoring stable expression of the trans-gene, were suppressed in KD plants compared to control plants. We will investigate the effect of the suppression of NHEJ on the efficiency of HR using HR repair assay system.

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Recent advances in analyses of CLV3/ESR-related (CLE) peptides have revealed some of their functions in modulating cell proliferation and differentiation, but most of CLE peptide functions remain unknown. Using an Arabidopsis pollen tube culture, we previously found a novel activity of synthetic CLE peptides on promoting the in vitro pollen tube growth. Three receptor-like kinase (RLK) genes expressing in pollen were shown to be responsible for the CLE peptide perception, examining a collection of T-DNA insertion mutants for the in vitro assay. Here we report functional analyses of the selected CLE and RLK genes in Arabidopsis plants. One CLE gene promoter activity was observed in pistil where pollen tubes penetrated. A severe reduction in seed production efficiency was first observed in plants in which the CLE gene expression was down-regulated by RNA interference. A similar defect was found in plants harboring a kinase-dead version of the corresponding RLK gene instead of the wild-type gene. These results indicate the involvement of a novel CLE peptide-mediated signaling in Arabidopsis pollen-pistil interactions for maintaining seed production efficiency.

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The rice gene MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), which encodes an ARGONAUTE (AGO) family protein, is required for normal germ-cell development during premeiotic stages. AGO proteins are known to function with small RNAs (sRNA) in gene silencing and heterochromatinization, and play essential roles in various developmental processes in many organisms.
To reveal the molecular function of rice MEL1, the total RNAs were extracted from rice young panicles, and MEL1-associated sRNA were filtrated by RNA-immunoprecipitation (RIP). Both unfiltrated and filtrated sRNA were mega-sequenced by Illumina Genome Analyzer II. The majority of MEL1-associated sRNA was 21-nt in length, and the 5'end of which tended to be the cytocine residue. Most of these sRNA were derived from intergenic regions without any functional annotations. MEL1-associated sRNA made hundreds of clusters on the rice genome. In this presentation, we will discuss the putative function of these sRNA clusters during rice reproduction.This study is supported by the Grant-in-Aid for Young Scientists (S) (21678001) from JSPS and MEXT, Japan.

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Identification of genes responsible for barriers to gene flow between two species provides insight into molecular mechanisms of reproductive isolation. Asian cultivated rice, Oryza sativa has diverged subspecies, indica and japonica. Inter-subspecific crosses between them exhibit various hybrid incompatibilities, however the mechanisms are still largely unknown. We identified DOPPELGANGER (DPL) 1 and DPL2 that are paralogous genes, as a pair of causal genes of reproductive isolation between indica cv. Kasalath and japonica cv. Nipponbare. DPLs encode highly conserved plant specific proteins, and are strongly expressed in mature anther. Expression analysis and complementation test indicated that both DPL1 Kasalath allele and DPL2 Nipponbare allele lost their function. The pollen carrying these two defective alleles together did not germinate, suggesting an essential role of DPL in pollen germination. Evolutionary time points of DPL duplication and disruption of each allele were estimated by sequence comparison of DPL genes of 42 angiosperm species and 43 accessions/varieties of Oryza (Mizuta et al., 2010).

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CRES-T(Chimeric REpressor gene-Silencing Technology) system is a powerful tool for genetic engineering of floriculturel plants, and transcription factor changes into transcriptional repressor by connecting the transcription repression domain, SRDX. The chimeric repressor dominantly suppresses the target genes. However, in some cases, severe growth defects and decreases in ability of fertilization were observed. In this study, we developed the new technology to control the expression of the transgene. The transcription system of the GAL4DB transcription factor of yeast was used, and the vectors which GAL4SRDX expressed by inducible promoters (Heat-shock pro or Alc pro) were constructed, and GAL4SRDX represses the overexpression of transgene, under the control of the 35S promoter:GAL4 UAS. Transgenic Pharbitis plants overexpressing the DPSRDX, which is a homolog of AG in Pharbitis, formed a double flower bud, but its growth stopped during the in vitro redifferentiation. Whereas, Heat-shock or EtOH treatment induced the GAL4SRDX expression, thus double flower Pharbitis was generated. Furthermore, we succeeded to induce non-transformant(NT) like flower in the double flower Pharbitis.

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It is known that the common cultivated rice, Oryza sativa, was domesticated from Asian wild rice, O. rufipogon. Loss of seed-shattering is one of the most important traits during rice domestication. In order to better understand the genetic control of seed shattering, chromosome segment substitution lines were prepared by introgressing non-shattering qSH1 and sh4 alleles from cultivated rice, O. sativa cv. Nipponbare, in the genetic background of wild rice. The degree of shattering was examined to evaluate the genetic interaction of two loci. The results showed that the backcross plants individually having Nipponbare homozygous alleles at either shattering locus (qSH1 or sh4) shed all the seeds. These results indicated that two cultivated non-shattering alleles were not sufficient to express full non-shattering behavior in the genetic background of wild rice. Probably some other minor loci are still involved in the control of seed-shattering in wild rice.

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Angiosperm gametophytes are generated from germ-line cells established from somatic-line cells in the final phase of the plant life cycle. During the course of the study on the meristem disorganization1 (mdo1) mutation, affecting the genome maintenance, we found a quite intriguing phenomenon. While the mdo1 homozygous seeds generated from mdo1 heterozygotes germinated and grew normally, albeit with the morphological abnormality, almost the mdo1 homozygous seeds generated from mdo1 homozygotes did not. In other words, germination phenotype depends not on the zygotic genotype, but on the genotype of the parental sporophytes. Microscopic observations suggested that embryogenesis of the mdo1 homozygotes is affected in early stages. In contrast, any histological abnormalities in female and male gametophytes produced by the mdo1 homozygous plants were not detected. The result obtained from genetic cross excluded a possibility that the mdo1 phenotype on embryogenesis is due to the maternal effect. These results, taken together, suggested that the MDO1 gene is involved in a novel mechanism generating embryogenesis-competent gametophytes during gametogenesis in parental sporophytes.

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Arabidopsis pollen, the male gametophyte consists of three haploid cells that are a single vegetative cell and two enclosed sperm cells. Drastic degradation of organelle DNA during pollen maturation is observable by DNA-specific fluorescent dye such as 4',6-diamidino-2-phenylindole (DAPI). The regulatory mechanism and biological significance of organelle DNA degradation in pollen is not known so far. Our research group has screened for mutants defective in pollen organelle DNA degradation. We identified two mutant loci, dpd1 and dpd2, showing retention of DAPI-detectable organelle DNAs in the vegetative cell. DPD2 gene encodes an enzyme involved in nucleotide biosynthesis. We will present the characterization of dpd2, which points to an interplay between organelle DNA degradation and nucleotide biosynthesis in pollen.

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Reproductive stage of plant development is much more sensitive to various abiotic stresses than the vegetative stage. For this reason, changes in climate can cause abortion of plant reproductive development. In particular, the phase of pollen development is most susceptible to temperature fluctuation.In this presentation, we introduce the influence of low temperature upon the response to phytohormones and the cell proliferation in the inflorescence of Arabidopsis. In a recombinant line DR5-GUS, the anther signals at stage 10 were obviously repressed by increasing temperature. On the other hand, the signals oppositely increased by mild cold stress at 10-13C for 24h or more. We also analyzed the expression of cell cycle regulators, Cyclin, CycA and CycB genes. The strong expression of early floral buds prior to stage 8 was significantly repressed by the similar cold stress. These results indicate that the temporal and spatial expressions of phytohormone-response genes and cell cycle related genes were dynamically altered in reproductive cells by temperature fluctuation. In addition, we would like to discuss whether these responses are conserved in the low-temperature injury of rice.

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Lower or higher temperature stress reduces seed setting rate in several plant species. In this phenomenon, development of pollen is more sensitive to the stress than that of female gamate. In this study, we aimed to elucidate the role of gibberellin in low-temperature injury of rice. Rice plants were cultured in a growth cabinet under 27C day/ 22C night with a 12-h photoperiod prior initiating panicle development, and were transferred to deep cool water (20.5C) as low-temperature treatment until heading period. As a result, under low temperature conditions, the number of mature pollen and the seed setting rate were significantly reduced. In addition, certain gibberellins-insensitive mutants became much more sensitive to low-temperature stress. Moreover, in Koshihikari sd1 (Tsukuba 1) lacking SD1 gene, which encodes a gibberellin biosynthesis enzyme, the injury was much severe than that of parental line Koshihikari. On the other hand, we could not find any influence on the female development under these experimental conditions. These findings suggest that gibberellin and its signaling make an important and essential contribution to cold tolerance in anther development.

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The pollen coats are sticky substances composed of lipids and proteins, covering the surfaces of pollen grains of many higher plants. They are believed to have many roles in pollen function, such as adhesion to pollinators and stigmas, recognition by stigmas, and water transport for pollen germination. The EXTRACELLULAR LIPASE4/6 (EXL4/6) and GLYCINE-RICH PROTEIN17 (GRP17) are two major proteins in Arabidopsis pollen coat, and are necessary for complete fertility of pollen grains. To examine the mechanism how these proteins synthesized in tapetal cells are incorporated into pollen coat, we started to analyze the subcellular localization of EXL4-GFP fusion protein in Arabidopsis and Brassica tapetal cells. It has been established that the GRP17 is accumulated in tapetosomes, which are specific oil-accumulating organelles in tapetal cells. The developing tapetosomes in Brassica anthers were visualized by GRP17-GFP protein. We attempted to isolate the tapetosomes by affinity purification using GFP antibody.

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Besides the resistance to insect and pathogen attacks, the jasmonic acid (JA) has an important role to open flowers, i.e. to start the elongation of floral organs in fully developed flower buds, in Arabidopsis and related species. In flower buds, the JA is accumulated in stamens, in which most genes for JA biosynthetic enzymes are highly expressed. We have focused on the expression of DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) gene, which encodes a lipase responsible for the first step of JA biosynthesis. The DAD1 expression first appeared in stamens two days before flower opening, which was fitted to the initiation timing of JA accumulation. Therefore, the DAD1 expression may function as a switch to turn on the flower opening. In order to know the regulatory mechanism to activate the DAD1 expression, promoter analyses with many truncated promoters were carried out. We found that the cis-regulatory element responsible for the stamen expression is located within a 49-bp region in the DAD1 promoter.

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The exine, an outermost wall of pollen grains, is believed to play an important role for successful pollination. However, little is known about the exine formation mechanism based on molecular genetics. The pollen grains of Arabidopsis kaonashi4 (kns4) mutant isolated by our screening for exine-defective mutants displayed remarkably thin exine layer. They also showed low fertility and abnormal entire morphology. Histological analysis revealed that abnormal development of pollen mother cells (PMCs) and poor primexine formation in tetrads occurs in kns4. The KNS4 gene encodes a protein having high homology with animal β-1,3-galactosyltransferase. Biochemical analysis using Yariv reagents indicated that the amount of arabinogalactan proteins (AGPs) in tetrads is reduced in kns4. The co-localization analysis showed the probability that KNS4 protein is localized in the Golgi apparatus. We hypothesized that the KNS4 is required in the Golgi of PMCs and/or tapetal cells for the biosynthesis of AGPs, which are abundant in primexine. Experiments to determine the expression profile of KNS4 are in progress.

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In the female gametophyte of most angiosperms, the egg, central and synergid cells have unique structural features and functions that are essential for the reproductive process. However, how they develop and which genes underlie these mechanisms, have not been revealed. To address these questions, we had two approaches: one is using a unique plant having a protruding embryo sac, Torenia and the other is using a model plant of molecular genetics, Arabidopsis.
First, we showed by time-lapse analysis in vitro cultivated immature Torenia ovules developed to functional female gametophyte. Furthermore, by laser-ablating the immature egg or central cell, we revealed that these cells are required for the synergid cell to establish the function to attract pollen tubes.
Second, we established system to isolate each-type cells of Arabidopsis. Our RT-PCR analysis with the selected genes showed that the isolated cells retained their own gene expression. We are analyzing the gene expression profiling of egg and synergid cells from wild type and synergid cells from a mutant defective in attracting pollen tubes with the next generation sequencer.

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Rab GTPases regulate tethering and fusion of transport vesicles to the target membranes in membrane traffic by acting as a molecular switch through cycling between the GDP- and GTP-bound states. Rab5 has been shown in mammalian cells to perform various functions in the endocytic pathway such as regulation of fusion and dynamics of endosomes. Endocytosis has recently been found to play fundamental roles also in plant cells, which include regulation of PIN localization and brassinosteroid signaling. Molecular mechanisms and functions of endocytosis are now being investigated actively, which has also been revealing functions of plant Rab5s at molecular and cellular levels. However, biological importance of Rab5 at organ and whole-plant levels has hardly been examined thus far. VPS9a is the sole guanine nucleotide exchange factor for Rab5 in Arabidopsis thaliana, which activates all three Rab5 proteins. Using A. thaliana vps9a-2 mutant, we are studying physiological roles of RAB5 activation in plant development. Our results suggest that proper activation of RAB5 plays important roles in radial cell patterning, establishment of cell identity, and cell elongation.

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The tonsoku (tsk) was originally isolated as an Arabidopsis mutant showing short roots, disorganized phyllotaxy and fasciated stems. Further analysis found hypersensitivity to DNA-damaging agents and expression of mRNA from heterochromatic region in tsk. The tsk also showed increased ploidy and expression of CYCB1;1-GUS, suggesting that tsk has defects in cell cycle progression, especially in G2/M, which caused the disorganized cell division pattern and morphological phenotypes. It is known that cell cycle progression from G2 to M is regulated by checkpoint kinases, mainly by ATR. We hypothesized that the cell cycle in tsk mutant was temporally stopped by ATR, resulting in the disorganized cell division and structures of meristems. To probe this hypothesis, we generated the tsk/atr double mutant and found that developmental defects associated with tsk were almost fully recovered by atr. The increased expression of CYCB1;1-GUS in tsk was also repressed by atr. However, some phenotypes including hypersensitivity to DNA-damaging agents were not restored by atr. These results suggest that the developmental phenotypes observed in tsk is dependent on the activity of ATR.

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The plasma membrane H⁺-ATPase is a master enzyme that creates a driving force to uptake the many ions, and controls the cell expansion. It has been demonstrated that phosphorylation of a penultimate Thr in the C-terminus is required for activation of the H⁺-ATPase in planta. However, signaling components leading to activation of the H⁺-ATPase has yet to be identified. In order to identify the components, we screened and isolated the mutants exhibiting low pH-hypersensitive in root growth, since the H⁺-ATPase activity is prerequisite for the root growth. We further characterized one of them, E8-4 mutant. The E8-4 roots grew slower than wild type, especially in low pH condition. Interestingly, we found that both the H⁺-ATPase activity and phosphorylation level of the penultimate Thr of the H⁺-ATPase in roots of E8-4 were lower that those of wild type under normal pH condition. We are trying to identify the E8-4 locus by map-based cloning and characterize expression of the H⁺-ATPase isoforms in E8-4 mutant. We will repot these results.

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Auxin is a plant hormone that regulates diverse aspects such as tissue elongation, tropic growth, embryogenesis, apical dominance, lateral root initiation, vascular differentiation, and fruit development. Recently, the ubiquitin-ligase complex SCF^TIR1, including the TIR/ABF auxin-receptor family, was demonstrated to be critical for auxin-mediated transcriptional regulation. In contrast, early-phase auxin-induced cell expansion, as explained by the acid-growth theory, was observed to occur without this system. To clarify the molecular mechanism by which auxin induces cell expansion we developed the experimental system for analyzing auxin-induced elongation with hypocotyls of a model plant Arabidopsis. In our experimental system we used the hypocotyl segments excised from etiolated Arabidopsis seedlings to deplete the endogenous auxin. When applied to the isolated hypocotyl segments, exogenous auxin induced hypocotyl elongation after a lag phase of around 10 minutes. Furthermore we will discuss a possible mechanism of auxin-acid-growth demonstrated by the biochemical and pharmacological analyses of the auxin-induced hypocotyl elongation.

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Plant organ growth is regulated by two distinct processes, first, the cell number which is controlled by cell proliferation, and second, the size of individual cells, which is regulated by cell expansion and cell growth. In Arabidopsis, various studies have shown a strong positive correlation between cell size and cell ploidy. In this respect, we have previously identified GT-2-LIKE1 (GTL1), a member of the GT2 trihelix transcription factor family, as a repressor of ploidy-dependent cell growth in trichomes. GTL1 is a nuclear protein, and further expression analysis revealed that GTL1 is expressed during late developmental stages of cells throughout the plant, suggesting that GTL1 might function as a general transcriptional regulator of plant cell growth. To confirm this experimentally, we are now using ectopic cell- and tissue-specific promoters to assess the potential role of GTL1 in controlling growth, ploidy and size of plant cells.

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