Lateral root (LR) formation is essential for the construction of plant root system. Auxin promotes LR formation which is initiated from asymmetric cell division of the root pericycle cells in most dicots. In Arabidopsis, several Aux/IAAs and two Auxin Response Factors (ARF7 and ARF19) regulate LR formation through auxin-mediated gene expression. It has been shown that Lateral Organ Boundaries-domain 16/Asymmetric Leaves2-like 18 (LBD16/ASL18) and LBD29/ASL16 genes are responsible for LR formation as direct targets of ARF7/19 (Okushima et al., 2007). In addition to LBD16 and LBD29, the other three LBD/ASL members (LBD17, 18 and 33) expressed around LR initiation sites and no obvious phenotype was observed in the knockout mutant of LBD16. These observations suggested the functional redundancy of the auxin-inducible LBD/ASL members in LR formation. This study is concerned with the detailed expression analysis of LBD16 during LR initiation and phenotypic analysis of the dominant negative mutant of LBD16.

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In vascular plants, the maintenance of activity in the root apical meristem is essential for the development of root architecture. To get insight into the mechanisms of root meristem maintenance, we isolated the recessive mutant LR11-4 that showed aberrant root growth and development in Arabidopsis. The length of the primary root in this mutant was about 50% of that in the wild type, whereas the frequency of lateral root formation remains unaffected. The lateral root growth was strongly inhibited in LR11-4. Fine mapping and sequence analysis revealed LR11-4 mutant genome to have a non-sense mutation in the FBA1 gene, encoding a fructose-1,6-bisphosphate aldolase1 that functions in Calvin cycle. This was confirmed by the complementation test of LR1-4 mutant with the transgene expressing FBA1 under the native FBA1 promoter. We also observed that FBA1-GFP fusion protein expressed under the control of FBA1 genomic region was localized in the plastids of the root cap columella and leaves. These results strongly suggest the involvement of FBA1 in root meristem maintenance in Arabidopsis.

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Amplification of genomic DNA by endoreduplication often marks the initiation of cell differentiation in both animals and plants. The transition from the mitotic cycle to the endocycle should be developmentally programmed but how this process is regulated remains largely unknown. Here we show that the TIR1/AFB-AUX/IAA-ARF dependent auxin signal modulates the switch from the mitotic cycle to the endocycle in Arabidopsis; high levels of auxin signalling are required to repress the endocycle, thus maintaining cells in the mitotic cycle whereas lower levels of auxin signalling trigger exit from the mitotic cycle and entry into the endocycle. We further demonstrate that this auxin-mediated modulation of the mitotic-to-endocycle switch serves to retain the meristematic state in root meristem cells through the expression of core cell cycle regulators. We propose that the dose-dependent control of the two alternative DNA replication cycles is part of the mechanisms that translate the developmental gradient into the cellular differentiation.

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Meristems in aerial parts of plants (shoot apical meristems and axillary meristems) are indeterminate structures and the sources of stem cells from which all post-embryonic aerial organs are derived. The regulatory network which functions within the meristems to control their activity has been well studied, while some reports suggested that the signals for the meristem regulation are provided also from outside of the meristems. However, the molecular mechanisms which are involved in the regulation of such signals are largely unknown. We previously isolated the Arabidopsis uni-1D mutant harboring a semi-dominant and gain-of-function allele of the UNI gene, a CC-NB-LRR member, and interestingly uni-1D plants show morphological phenotypes; a defect of meristem maintenance and ectopic formation of axillary meristems. During analysis of this mutant, we noticed that ERECTA family function outside the meristems affects their activities both in uni-1D mutant background and in wild-type background.

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Polar auxin transport is mainly dependent on the activity of auxin efflux carriers, PIN-FORMED (PIN) proteins, localized in the plasma membrane with polarity. However, molecular mechanisms for the establishment of sequential PIN polarity remain to be uncovered. Recently, a NPH3-like protein MACCHI-BOU (MAB) 4 has been reported to regulate polar auxin transport through the control of PIN localization in organ formation. This time, we analyzed the role of MAB4/ENP-LIKE (MEL) 1-4, homologous to MAB4, in the establishment of PIN polarity. mel1 and mel2 mutations enhanced mab4 mutant phenotypes as mab4 mel1 mel2 triple mutants developed pin-like inflorescences. In addition, mel1 mel2 mel3 mel4 quadruple mutants displayed severe defects in the root gravitropism. In these mutants, the abundance of polar localized PIN protein was severely reduced with weakening in PIN polarity. Furthermore, MAB4 and all MEL proteins were mainly localized close to the plasma membrane with polarity. MAB4 and MEL polarity was almost identical to PIN polarity. These results suggest that MEL genes regulate auxin-related morphogenesis through the control of PIN localization redundantly with MAB4.

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We have previously described the Arabidopsis semi-dominant mutant uni-1D. UNI encodes a CC-NBS-LRR-type protein belonging to the R gene family. Its mutation results in constitutive PR-1 gene expression and interesting morphological phenotypes: heterozygotes display bushy, dwarf phenotypes, forming many narrow leaves and inducing many ectopic axillary meristems, whereas homozygotes die in the early stage of true leaf formation after germination. The molecular basis of how uni-1D can induce these phenotypes is unknown. In this study, we focused on a UNI-interacting protein, isolated by Y2H screening, which was identified as the RPT2a subunit of the 26S proteasome. The phenotype of an rpt2a single mutant displayed pleiotropic defects such as the formation of abnormally shaped leaves, disrupted phyllotaxy and retarded root growth. The effect of the uni-1D mutation on morphology, particularly on initial ectopic axillary meristem formation, was suppressed in this rpt2a mutant. Moreover, lethality of the uni-1D homozygote was also suppressed in rpt2aand its phenotype became heterozygote-like. These results suggest that the expression of uni-1D phenotypes may be mediated by RPT2a.

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Cell proliferation and differentiation occur in spatially distinct regions of the same leaf primordia. A defect in cell proliferation in a leaf primordium often triggers enhanced cell expansion. This phenomenon is called compensation. Compensation strongly suggests the existence of a system coordinating these two cellular processes. So far, we showed that compensation induced by the overexpression of a cyclin-dependent kinase inhibitor, KRP2, occurred in a cell-autonomous manner by using a heat-shock-inducible Cre/Lox system. Then, is this a common property of the coordination system?
To answer this question, genetic chimeric leaves for AN3 expression were generated. In these chimeric leaves, even AN3 overexpressing cells showed compensation in the presence of an3 cells. The size of AN3-overexpressing cells was comparable to that of an3 cells. This fact indicated that compensation induced by an3 mutation is mediated by cell-to-cell communication. Thus, there are two qualitatively distinct systems linking cell proliferation and differentiation in cell-autonomous manner as seen in KRP2 overexpressor and non-cell-autonomous manner in an3 mutant.

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Compensation is a phenomenon observed in mutants and transgenics that have a severe defect in cell proliferation in leaf primordia, where the leaf-cell expansion is aberrantly enhanced. This suggests that there are some intrinsic mechanisms which coordinates cell proliferation and cell expansion during leaf development. To elucidate the mechanism of compensation, we isolated several xs mutants that have a specific defect in cell expansion. Through the genetical analyses between xs and a compensation-exhibiting mutant, angustifolia3 (an3), we found that some of xs mutations clearly suppress aberrant cell enlargement in an3. For further analysis, we focused on xs2 that has the most significant defect in cell expansion among them. Positional cloning revealed that XS2 encodes a member of cation calcium exchanger proteins. Interestingly, genes involved salicylic-acid (SA) signaling were expressed much higher in xs2 compared to WT. In addition to it, exogenous SA supply clearly suppressed aberrant cell enlargement in an3. These indicated that the excessive SA-mediated response caused by xs2 mutation is somehow involved in suppression of compensated cell enlargement in an3.

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In the development of multicellular organisms, there is believed to be a system that integrates cell proliferation and cell expansion. Compensation is a phenomenon in which a decrease in cell number seems to trigger an increase in cell size, and this suggests existence of such a system. We have reported on our analyses of five fugu mutants that exhibit compensation. Here, we report about reverse genetic analysis on fugu2 mutant. Microarray data followed by RT-PCR conformation indicated that the expression of 46 genes was enhanced and that of 39 was repressed more than three fold in fugu2 leaves. To analyze the function of these genes in leaf organogenesis, we isolated 27 homozygous T-DNA insertional lines for these genes, and analyzed their leaf phenotype. As a result, we found several lines that showed defects in cell proliferation. Now, we are constructing double mutants between fugu2 and these lines to analyze the function of these genes in compensation. In this presentation, integrating these results we will discuss the mechanism of compensation in fugu2 mutant.

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Fertilization is a key process for plant sexual reproduction. After pollination and germination of pollen grains on the stigma, the pollen tube, which carries two male sperm cells, are guided by and growing toward the female ovule. This pollen tube guidance is highly species specific and is thought to be one of a mechanism to generate interspecies barrier. 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. Because sequence diversity is a general feature of Defensin-like proteins, we hypothesized that diversity of LUREs may contribute species-specific pollen tube attraction and speciation. To clarify their molecular features, we isolated LURE homologues from related species of T. fournieri. We found one LURE homologous gene that was not expressed in the ovule. Some key amino acids were substituted in its product, suggesting that this gene might be a pseudo-gene in the species. We also found several LURE homologues expressed predominantly in the ovule. We are currently purifying these LURE proteins and testing their activities for pollen tube attraction.

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It is essential for fertilization of angiosperms that a pollen tube grows in a pistil and reaches an embryo sac. There must be various mechanisms that guide pollen tubes to embryo sacs in the fertilization process. However, most of molecules underlying the mechanisms are unclear. Last year, we have identified small, secreted, cysteine-rich polypeptides (CRPs; LURE1, LURE2) as pollen tube attractants derived from the synergid cell of Torenia fournieri.
Our aim of this study is to identify pollen tube attractants of Arabidopsis thaliana that are derived from the synergid cell. As previously suggested by physiological experiments using closely relating species, the candidate genes rapidly evolved, which made difficult to search LUREs homologs in Arabidopsis by conventional molecular phylogenetic analysis. Thus candidates were inveitigated by genome-wide searching used expression data. We performed immunostaining and semi in vitro pollen tube attraction assay for the candidates. Here we report our trial to identify attractants of Arabidopsis, including knock-down analyses to discuss their functions in the pistil.

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Chemo-attractants from the ovule have been thought to be key molecules in pollen tube guidance of flowering plants for more than 140 years. However, there had been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Recently, we have identified that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins (DEFLs) are attractants derived from the synergid cells (Okuda, Tsutsui et al., Nature, 2009).
To elucidate how the pollen tube accepts the signal of LUREs and determines its growth direction, we aimed to identify the receptor for LUREs. First, to identify which port of the pollen tube interacts with LUREs, pollen tubes that were treated with recombinant LURE proteins were immunostained with anti-LURE antibodies. Next, we tried to detect the pollen tube protein directly interacted with LUREs. We will demonstrate the biochemical analysis of the interaction between LUREs and pollen tubes.

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In higher plants, the synergid cells play essential roles in sexual reproduction including pollen tube guidance and sperm cell release. We showed that both LURE1 and LURE2, which are identified by EST analysis of Torenia synergid cells, have the ability to attract pollen tubes in vitro. When Morpholino antisense oligo (MO) against each LURE was injected into the Torenia embryo sacs by Laser Micro Injection, the attraction was significantly impaired (Okuda, Tsutsui et al., Nature, 2009). However, it is not known that LUREs act coordinately or independently, and the functions of other genes expressed in the synergid cells are still unclear.
To investigate whether LUREs act solely or not, we injected MOs against both LUREs and observed pollen tube attraction. To confirm the gene-specific down-regulation by MO, we developed the method for RNA extraction from a single ovule, and LURE transcripts in an MO-injected ovule were analyzed by qRT-PCR. Additionally, we identified a family of genes that are highly expressed in synergid cells and at least one of them was synergid-specific.
Based on these results, we will discuss the function of synergid cells and the genes.

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Female and male gametophytes play a critical role in essentially every step of the reproductive process. Although the cellular functions of the gametophytic cells have been understood, little is known for the molecular-level function of those cells. However, Kasahara et al. identified a key gene, MYB98. MYB98 is essential for the interaction between female and male gametophytes. MYB98 is expressed exclusively in the synergid cells and myb98 mutant phenotype showed pollen tube guidance defect. To investigate genes essential for the development and function of the gametophytic cells, we started forward genetics approach by using MYB98::GFP visible screening. This screening method is very powerful to identify the important genes for female or male gametophyte. In addition, this method enables us to identify gametophytic mutants which have defects in fertilization after formation of the embryo sac. We identified a male gametophytic mutant, G21, which is required to form two sperm cells in one pollen grain. In this meeting, we discuss about the newly isolated mutant G21 and the future research plans for this project.

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Boron is an essential micronutrient for the plants. The male sterility is one of the B deficiency symptoms. BOR6 and BOR7 are boron transporters specifically expressed in pollen and pollen tubes. We investigated roles of BOR6 and BOR7 in reproduction. When plants are grown under B deficient condition and pollen tubes in pistils were observed, pollen tubes of double mutant for the BOR6 and BOR7 genes were shorter than the wild type. WT pollen tubes reached to the lower part of the pistil, while those of the double mutant did not. Pollen tubes of the double mutant tend to extend toward ovules in the upper part of the pistil rather than reaching to the lower part of pistils. T-DNA insertions in BOR6, BOR7 did not segregate in the 1:2:1 ratio and wild type plants appeared in a higher frequency. Our results suggest that BOR6 and BOR7 play an important role in the pollen tube elongation and reproduction.

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Female gametophytes of angiosperms have a central cell containing two polar nuclei. In many species including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We have shown that BiP, an Hsp70 in the endoplasmic reticulum (ER), is essential for the membrane fusion step of this process. Hsp70 requires partner proteins to complete its functions. Hsp40 co-chaperones are the functional partners for Hsp70. The ER of Arabidopsis has three soluble Hsp40, AtP58^IPK, AtERdj3A and AtERdj3B. We found that double mutant ovules lacking AtERdj3A and AtP58^IPK (3a p58) or AtERdj3B and AtP58^IPK (3b p58) were defective in the fusion of polar nuclei. Electron microscopy showed that polar nuclei were in close contact but no membrane fusion was observed in the 3a p58 ovule. In contrast, the outer nuclear membrane of the polar nuclei was appeared to be connected via the ER in the 3b p58 remaining the inner membrane unfused. These results suggest that different sets of ER-resident Hsp40 function as partners for BiP to promote different steps of the fusion of polar nuclei.

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The angiosperm female gametophyte most often consists of one egg cell, two synergid cells, one central cell and three antipodal cells. It is considered that each of these four cell type performs unique functions that are essential for the reproductive process. However, detailed functions of these cells are largely unclear because few genes expressed in these cells have been identified. To reveal detailed function of component cells in rice female gametophyte, we planned to do gene expression analysis of these four cells using microarray. At first, we isolated live rice egg and synergid cells and their extracted RNA were analyzed by Agilent rice 44k oligo microarray. Several rice homologues of the Arabidopsis genes which were expressed in egg or synergid cell were also highly expressed in corresponding rice cells. We validated the microarray data using transgenic rice harboring promoter::GFP fusions. These results indicated that our microarray analysis were highly reliable. Now we are analyzing rice central and antipodal cell in similar manner. Future functional analysis of genes revealed from our study may figure out detailed function of each component cell of female gametophyte.

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Oryza sativa is classified into two main subspecies, indica and japonica. Various reproductive isolations that are supposed to act as important factors in evolution have been observed between them. However attempts to identify the causal genes have been discouraged by their complexity. Here, we isolated DOPPELGANGER (DPL) 1 and 2 located on chromosome 1 and 6. They were revealed to be paralogous genes and to act as a pair of reproductive isolation genes between indica cv. Kasalath and japonica cv. Nipponbare. The hybrid pollen having both Kasalath DPL1 allele (DPL1-K) and Nipponbare DPL2 allele (DPL2-N) failed in pollen germination. Expression and sequence analyses suggested that DPL1-K and DPL2-N have lost their function. DPL structure is highly conserved among angiosperms, suggesting an essential role in pollen germination. Evolutionary analysis revealed multiple duplications and losses of DPL in angiosperm evolution.

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During sexual reproduction of Closterium psl. complex, the mt- cells secrete a pheromone PR-IP Inducer and then mt+ cells secrete PR-IP in response to the Inducer. Several reproduction-related and sex-specific genes have also been identified. To investigate function of such genes, we have developed a gene introduction system using particle bombardment. Here, we report the evaluation of stable transformants, as a first step of construction for over-expression system in C. psl. complex.
A novel vector, both for the selection by phleomycin and for the over-expression of a gene encoding PR-IP Inducer (CpPI), was constructed and introduced into the mt+ cells, in which expression of CpPI hardly occurred irrespective of the culture condition. Several transformants could be obtained and showed ~18-fold expression of CpPI compared with WT mt+ cells. The expression level of PR-IP gene (Cp19KSU) was also elevated ~80-fold in these cells. These results suggest that the active PR-IP Inducer can be produced in mt+ cells and can induce the expression of PR-IP gene in the same cells. This method would be applicable to the studies based on reverse genetics of unknown genes in the C. psl. complex.

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Arabidopsis thaliana PROTODERMAL FACTOR 2 (PDF2) encodes a HD-ZIP type IV transcription factor and is indicated to play a critical role in maintaining the identity of the L1 cells. PDF2 overexpressing plants (35S::PDF2) exhibited normal shoot development. However, it showed late flowering phenotype only under long day conditions. This flowering phenotype indicates that in 35S::PDF2 there is some defects in the photoperiodic pathway not like fwa dominant mutant. Then the circadian expression of photoperiodic pathway genes were analyzed. In 35S::PDF2 the day time peak of CO expression was lost.
Using yeast-two-hybrid screening, 14 candidates which interact with PDF2 and are not related with L1 layer development were obtained. Two of these candidates, SHOOT GRAVITROPISM 5 (SGR5) and PLASTID TRANSCRIPTION FACTOR 1 (PTF1) are assumed to be related with flowering. SGR5 has a high similarity to maize ID1, a defect in ID1 causes late flowering. ptf1 mutant causes late flowering only under short day conditions.

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We identified those mutants, related with two genes, FLORAISON TARDIVE1 (FLA1) and FLA2, that showed a late flowering phenotype (about three more leaves than wild type).
FLA1 is described as a C2 domain-containing protein with an enzymatic domain. FLA1 is expressed in leaves and flowers. fla1 mutants flowered late only in long day conditions (LD). This phenotype indicates that FLA1 belongs to the photoperiodic pathway. However, FLA1 expression is constant over the time. CO expression was not affected by mutation of FLA1 suggesting that FLA1 acts downstream of CO or in a CO-independant pathway inside the photoperiodic pathway.
FLA2 seems to have a transcriptional factor activity. FLA2 expression has been detected only in flowers. In both long and short day conditions (LD and SD), fla2-1 and fla2-4 were late-flowering. These observations indicate that FLA2 belongs to the autonomous pathway.
Moreover, fla2-1 mutation negatively regulates most of the autonomous pathway genes. fla2-1/flc double mutant flowered as early as flc mutant suggesting that FLA2 is upstream of FLC.

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Circadian clock in higher plants regulates various phenomena such as flowering, stomatal opening, and organ elongation. Two related myb proteins, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) play key roles in the clock-controlled processes such as regulation of flowering time and organ elongation in Arabidopsis thaliana. Recently, we have found that mutations in these two genes (lhy;cca1) affected both chlorophyll content and chlorophyll a/b ratio under different photoperiodic conditions. To understand a molecular mechanism underlying the clock-controlled regulation of chlorophyll, genetic analysis was performed and chlorophyll contents and a/b ratio of lhy;cca1 and control plants were compared.
Under different photoperiods, the amount of chlorophyll was almost constant in wild-type plants. By contrast, the chlorophyll content of lhy;cca1 was highly affected by length of light period. In addition, the ratio of chlorophyll a/b was altered in lhy;cca1 under the different photoperiods. We identified several mutations that suppressed the phenotypes of lhy;cca1. Hypothetical models to explain the phenotypes of lhy;cca1 will be discussed.

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Some flowers open in the morning. And some ones do in the evening. Linne proposed the idea of the floral clock which was made up of various kinds of flowers opening at different time each other. Recently, variation of the timing among closely related plant species in the field has been studied on aspect of the differentiation of reproductive timing. Here, we present chronobiological features of flower opening in Arabidopsis thaliana. The plant opens flower in the morning, but the detail is unknown. Then, we examined it under diurnal light/dark cycle. The petal began the expansion when lamp started illumination. Then, it opened completely for 3 hours in the light. We examined whether the rhythm continued in light without the daily dark periods. Observation of four days in light showed the rhythm with 23 hours period. The period was not changed in low or high temperature. In addition, the rhythm was entrained by external light/dark cycles. These properties are essential to circadian rhythm. Thus, Arabidopsis petal movement is under circadian clock. The chronobiological properties will arrow us to know signal transduction of the movement under circadian clock.

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An interlocking transcriptional-translational 24-h-feedback loop of clock-associated genes is thought to be the clock central oscillator in plants. Genetic studies have suggested that PSEUDO-RESPONSE REGULATOR 9 (PRR9), PRR7, and PRR5 act within or close to the loop, however their molecular functions remain unknown. Here we demonstrate that PRR9, PRR7, and PRR5 act as transcriptional repressors of CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Each PRR9, PRR7, and PRR5 suppresses CCA1 and LHY promoter activities, and can give transcriptional repressor activity to a heterologous DNA binding protein in a transient reporter assay. Using a glucocorticoid-induced PRR5-GR construct, we found that PRR5 directly down-regulates CCA1 and LHY expression. Furthermore, PRR9, PRR7, and PRR5 associate with the CCA1 and LHY promoters in vivo from early daytime to midnight, coinciding with the time these genes are repressed. These results suggest that the repressor activities of PRR9, PRR7 and PRR5 on the CCA1 and LHY promoter regions constitute the molecular mechanism that accounts for the role of these proteins in the feedback loop of the circadian clock.

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Japanese larch (Larix leptrepis)is a deciduous conifer that grows under subarctic regions. This species normally flowers once every few years. However, a mutant of this species, designated "HONMA FL", produces flowers annually. Comparative analysis of the wild-type and mutant variant of, L.leptrepis might help us to characterize the molecular mechanism of flowering in this woody plant. In this study, we are focusing on the expression of the four flowering-related genes, namely, FLOWERING LOCUS T (FT),LEAFY,NEEDLY,CONSTANS. Previous studies have revealed that they have significant rule in flowering in many higher plants. Semi-quantitative PCR and Real-time PCR were performed in attempt to relate the expression of these genes and flower development in this species. Interestingly, FT mRNA expressed generally higher in FL mutant than wild-type and its expression was detected most in leafy organs. We hope that our analysis will shed some light on the mechanism of tree flowering and will also contribute, eventually, to the cloning and traditional breeding of fast-growing trees.

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Day length plays an essential role in the transition from vegetative phase to reproductive phase in long-day (LD) plants (LDP) and short day (SD) plants (SDP). Inductive LD or SD signal induces the differentiation from shoot apical meristem to floral meristem. Chrysanthemum is a typical SDP and responds to shorting of day length in the phase transition. Night break (NB) treatment is sufficient for the repression of flowering in SDP and commonly applied to manipulate the production of chrysanthemum. After short day signal induced floral meristem identity in chrysanthemum, their development was inhibited by NB. The length of SD exposure was sufficient for the activation of floral activator gene homologues and repression of floral repressor gene homologues in dosage manner. Therefore, continuous SD signal can be sufficient for the development of floral organs in chrysanthemum.

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To elucidate the role of light signaling on photoperiodic flowering in Chrysanthemum, flowering response under various photoperiodic treatments were tested with different light qualities. When white light was given during the main photoperiod, short irradiation (Night Break; NB) with red light has the strongest effect on inhibition of flowering, whereas NB with blue (NB-B) or far-red light has little inhibitory effect. However, with main photoperiod of blue light, NB-B strongly suppressed flowering. This inhibitory effect of NB-B was cancelled when red light was supplemented with blue light during the main photoperiod. These results suggest that phytochrome(s) play a major role in NB-induced inhibition of flowering, and the light quality during main photoperiod affect the sensitivity to light irradiation at middle of the flower-inductive dark period.

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The short-day plant Pharbitis nil, var. Violet can be induced to flower by responding to poor-nutrition stress under long days, whereas var. Tendan was not induced to flower by the stress conditions. The Violet plants induced to flower by poor-nutrition stress produced fertile seeds and the progeny developed normally. We grafted Violet and Tendan in various combinations, and indicated that a transmissible flowering stimulus is involved in the induction of flowering by poor-nutrition stress. The poor-nutrition stress-induced flowering was inhibited by aminooxyacetic acid, a phenylalanine ammonia-lyase inhibitor, and this inhibition was almost completely reversed by salicylic acid(SA). However, exogenously applied SA did not induce flowering under non-stress conditions, suggesting that SA may be necessary but not sufficient to induce flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the Violet plants were induced to flower under poor-nutrition stress conditions, but expression of PnFT1, another ortholog of FT, was not induced, suggesting the involvement of PnFT2 in stress-induced flowering.

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In case of closely related bacterial genomes, segments of conserved gene order that are referred to synteny blocks are observed by mapping orthologs to each genome. In many cases, they share a stable structure consisting of synteny blocks, which are often called 'genome core'. This structure is a marker for the structural evolution in these genomes. They also possess variable genomic islands consisting of laterally transferred genes. Bacterial genomes are, therefore, envisaged as a mosaic of genomic core interspersed with genomic islands.
Existing software to find a genome core is based on combinatorial search of neighboring relationship of genes, but it is difficult to find a genome core and genomic islands simultaneously. We have previously reported positional profile method to detect both of them and the result of application to 16 cyanobacterial genomes. Here we report results of enhanced set of 36 cyanobacterial genomes and nodulating bacterial genomes.

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To understand the plant physiology at the genome levels, it is important to obtain genomic and transcriptomic data on green algae. We focus a biflagellate green alga, Chlamydomonas reinhardtii as a model system. To collect the genomic resources, full-length cDNA enriched libraries and genomic BAC/Fosmid libraries were constructed using mt(-) strain C-9. Full-length cDNA libraries were generated by an oligo-capping method using mRNAs isolated from cells in various environment stress conditions. Mapping analysis of the end-sequences of BAC and Fosmid clones showed that twenty-two scaffolds, of which were not integrated into the chromosomes derived from the mt(+) strain, suggested to be integrated into the 17 chromosomes. Furthermore, to analyze cellular responses to nutrient-limiting stress response including photosynthetic substrate CO₂-limiting stress on the Chlamydomoans genome, end sequences of mRNAs from the cells that suffered to various stress conditions were analyzed by Illumina genome analyzer IIx. The genomic and transcriptome resources were integrated into expression database, the Kyoto Chlamydomonas Genome Database (KCGD; http://chlamy.pmb.lif.kyoto-u.ac.jp/)

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Knocking out a gene from a genome often causes no phenotypic effect. This phenomenon has been explained in part by the existence of duplicate genes. Here, we study whether it is also true for the knockout data in Arabidopsis. From the knockout data in Arabidopsis thaliana obtained in our study and in the literature, we find that duplicate genes show a significantly lower proportion of knockout effects than singleton genes. Because the persistence of duplicate genes in evolution tends to be dependent on their phenotypic effect, we compared the ages of duplicate genes whose knockout mutants showed less severe phenotypic effects with those with more severe effects. Interestingly, the latter group of genes tends to be more anciently duplicated than the former group of genes. Moreover, using multiple-gene knockout data, we find that functional compensation by duplicate genes for a more severe phenotypic effect tends to be preserved by natural selection for a longer time than that for a less severe effect. Taken together, we conclude that duplicate genes contribute to genetic robustness mainly by preserving compensation for severe phenotypic effects in A. thaliana.

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For a firm gene annotation, it is necessary to repeat experiments and obtain a robust result. With more stack observations of transcripts, the clearer gene structure would be seen. Then, we developed a statistical genome-wide gene prediction method ''ARTADE2.0'' from the discovery that the correlations matrix made with tiling array probes of many experiments shows transcription units clearly. However, the estimation result may be mixed gene model if an alternative splicing (AS) model exists since expressions of transcripts are observed from plural kinds of RNAs. We now proposed a method for a detection of AS part based on a factor analysis (as a statistical methods). The method can divide the gene region into part belonging to major AS isoform and part belonging to minor AS isoform or alternatively spliced region. For the method, we used tiling arrays results from eighteen conditions, including plants under various stresses or several organs. The method succeeded to elucidate some known ASs and other novel alternatively spliced regions in genes extracted through the ARTADE2.0.

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Small RNAs are 20-25nt non-coding RNAs as guides for posttranscriptional and epigenetic regulation. In plants, miRNAs, trans-acting (ta) siRNAs and siRNAs influence their targets through distinct regulation mechanism. Previous report indicated that some of miRNAs have the function in response to environmental stress. However, the profiles of whole-genome small RNAs under abiotic stress have not been well understood. In this study, we analyzed the expression profiles of the small RNAs under drought, cold, high-salinity stress and ABA treatments by 454 DNA sequencing technology and obtained the following results: I) Twelve small RNAs responded to the abiotic stress. II) ta-siRNAs and their precursors were down-regulated significantly under drought, high-salinity and ABA treatment. III) Large amount of siRNAs were generated from transposable elements in response to the abiotic stress. These results suggest that the various types of small RNAs may function in the regulation of the gene expression under abiotic stress.

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Cold and dehydration are adverse environmental conditions that affect plant growth and productivity. Many genes have been described that respond to both stresses at the transcriptional level, and their gene products are thought to function in stress tolerance and response even though these stresses are quite different. These genes include late embryogenesis abundant (LEA) proteins, detoxification enzymes, chaperones, protein kinases, and transcription factors. The cis-acting elements that function in stress-responsive gene expression were analyzed to elucidate the molecular mechanisms of gene expression in response to these stresses.
In this study, we searched for cold and/or dehydration inducible genes in Arabidopsis thaliana, Oryza sativa and Glycine max and analyzed conserved sequences in their promoters. The 1000-bp promoter regions upstream of putative transcriptional initiation sites located at the 5'ends of the full-length cDNA clones were used for this analysis. We found some highly conserved sequences including known cis-acting elements and novel conserved sequences in these promoter regions.

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We have developed and opened a genome-wide database, termed SALAD database (http://salad.dna.affrc.go.jp/salad/), based on similarity clustering and distribution diagrams of evolutionarily conserved peptide motifs. Recent version (ver. 3) contains genome information on ten species, rice, sorghum, Arabidopsis thaliana, grape, lycophyte, moss, two green algae, red alga, and yeast. Using this database we can compare any annotations of your interest and discuss on their similarity relationships efficiently and quickly. In addition, this database has a new-type microarray data viewer (termed SALAD on ARRAYs), with which users can compare gene expression of microarray data for paralogous (or related) genes using Agilent platform. The gene expression level is shown as a gray-scale gradient in the colored boxes. We here report new functions on this viewer such as creating graph, selecting microarray datasets and changing the order of microarray data. Moreover, we plan to add public AtGenExpress microarray data (Affymetrix platform) which contains 783 array samples (29 series) from GEO. To display both Agilent and Affymetrix platform data we are now comparing these platform data.

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The Rice Annotation Project Database (RAP-DB) is the annotation database for the genome sequence assembly of IRGSP. Since an updated japonica rice genome assembly, IRGSP build 5, was released in 2008, we have created new annotation data (IRGSP/RAP build 5). For better gene structure annotation that covers more rice genes, a system for cross-species FLcDNA mapping and protein mapping was developed. As a result, 34,902 loci with transcript evidence were identified. In addition, 9,975 loci with no transcript supports were predicted by ab initio gene prediction methods. We also provide rice gene family information based on MCL and BLAST searches. The gene family data contain sorghum and Arabidopsis proteins so that inter-specific gene family relationships are displayed. To further examine genome-wide conservation of genes, genomic sequences of indica rice and sorghum aligned to the IRGSP assembly are shown in GBrowse_syn. Here we present new functions and contents of the RAP-DB, which provides new data download system and manually curated functional annotation data as well as other useful genome information, such as organelle-derived regions and transposable elements.

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We present our pipeline for analysis of short reads generated by Illumina Genome Analyzer. The pipeline supports multiple mapping programs, and outputs mapping results in multiple formats. In addition, we developed a short-read viewer to browse a large number of DNA fragments that mapped to a reference genome. The viewer displays SNPs, indels, and possible sequencing errors on exons, introns and coding frames of the known gene structures. In parallel with the mapping results, the pipeline reports novel gene structures, which are predected by the Bowtie, TopHat and Cufflinks programs on the basis of a short-read profile. Short-read mapping results as well as the known and predicted gene structures were used to estimate normalized expression levels (number of Reads Per Kilobase of the exon models per Million of the mapped reads; RPKMs) of the gene structures as their gene expression levels. We further assessed that Illumina's mRNA-seq data showed high technical reproducibility, and investigated how many short-reads were required for estimation of accurate expression levels. Detection of differentially expressed rice genes under abiotic stress is discussed.

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Recent advances in genomics and transcriptomics have yielded a vast amount of microarray data and have begun to deepen our understanding of biological systems. Gene coexpression across publicly available microarrays has demonstrated its usefulness for investigating transcriptome and for predicting unknown gene functions in different organisms from yeast to humans. In Oryza sativa, however, no overall coexpression-network module has been examined in detail. Here we present the coexpression clusters of rice genes based on unbiased graph clustering of the network of 4,495 genes. The coexpression network was constructed by using over 230 microarrays. The resultant network displayed several properties of complex networks such as the scale-free degree distribution. We detected 1,220 clusters using the DPClus algorithm that can extract densely connected clusters, and these were evaluated using the enrichment analysis of gene ontology. We conclude that this approach is important for generating experimentally testable hypotheses for uncharacterized gene functions in rice. The data are downloadable from the PRIMe website (http://prime.psc.riken.jp/rico/index.html).

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The information of gene coexpression is valuable to predict gene functions. Several useful databases are available for this purpose including our databases, ATTED-II (for Arabidopsis and rice, http://atted.jp) and COXPRESdb (for human, mouse, rat, chicken, zebrafish, Drosohila and C. elegans, http://coxpresdb.jp). Several hundreds or thousands of microarray data are generally used for gene coexpression database. However microarray data in non-model species are still limited. Although several studies reported the number of required microarrays to construct reliable gene coexpression data, meaning of number of samples is still unclear. One of the reasons is different status of target data source. To overcome this problem, we analysis four species (human, mouse, rat and Arabidopsis) actually used in our coexpression databases.

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Horizontal gene transfer (HGT) represents the incorporation of genetic material from one organism into another that is not its offspring. In plants, the majority of reported cases of HGT are limited to plant-microbe genetic exchanges, mitochondrial transfer, or the translocation of mobile elements among related species. Here we show clear evidence for recent HGT of a nuclear-encoded gene from a monocot host to a eudicot recipient, the parasitic "witchweed" Striga hermonthica.
S. hermonthica belongs to the eudicot Orobanchaceae, and infests to crop species, such as sorghum and rice, which belong to the monocot Poaceae. To identify the HGT events between hosts and the parasite, the S. hermonthica ESTs were searched for the genes conserved in monocot genomes but not in eudicot genomes. We found a strong candidate gene which encodes an unknown function protein. This candidate gene is most similar to homologues from sorghum, a common monocot host of the parasitic weed, with sequence similarity extending to the 5' and 3' untranslated regions of the open reading frame. Our results suggest that nuclear genes from crops can be captured by a parasitic weed in nature.

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Proteomics is one of the best available tools for studying posttranslational modifications (PTMs), and it has no limitation like those encountered with forward genetics. Therefore, it is well suited for the analysis of unknown signaling pathways. Among the several PTMs described thus far, phosphorylation is the most extensively studied, and they have been shown to play a role in plant immune signaling.
We have developed a phosphoproteomics platform, which enables monitoring phosphorylation events in plant cells at the cellular level ¹⁾. Using our approach, we have identified 5,143 and 6,919 unique phosphopeptides from unfractionated Arabidopsis and rice cell lysates, respectively.
Besides developing the phosphoproteomics platform, we have also established a method for the differential analysis of plant samples which enables proteome dynamics to be monitored. Currently, we are analyzing phosphorylation system which regulates plant immune signaling, by using the established differential phosphoproteomics platform.
1) Mol Syst Biol. 2008;4:193. Epub 2008 May 6.

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Land plants use two chlorophyll molecules, chlorophyll a and b, for photosynthesis. While both two chlorophylls work together in light harvesting antenna, only chlorophyll a is used in the photochemical reaction center. Previous studies showed that chlorophyll b-less mutants can survive under normal growth conditions, although they are susceptible to photo-oxidative damage by high light illumination. However, the detailed structure of photosynthetic apparatus in chlorophyll b-less plants remains unknown.
In this study, we analyzed the thylakoid proteins of chlorophyll b-excess and -less mutants by both BN-PAGE and the proteomic approaches.
BN-PAGE showed that neither PSII supercomplexes nor LHCII trimers were not formed in the chlorophyll b-less mutant, and the size of the PS I complex was greatly decreased. Two dimentional SDS gel electrophoresis further suggested that chlorophyll b was essential for integration of light harvesting proteins into PS I and PS II core complexes.
Further proteomic analysis showed that the complete loss of chlorophyll b caused the dynamic changes of thylakoid protein compositions.

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Commercialization of genetically-modified (GM) plants requires the achievement of public acceptance (PA) for the genetic engineering in addition to the reduction of various costs concerning legal regulations. We have produced torenia flowers with various novel morphological and physiological aspects by combining the latest technologies such as CRES-T and heavy-ion beam irradiation with traditional genetic engineering techniques. To instantize these GM flowers for public use, we have improved a production system for resin-embedded specimen of transgenic plants. The GMO-based material of which the body fluid has been replaced by resin is no longer a living organism and can be distributed irrespective of the legal regulation of Cartagena protocol domestic law. Colors of plant materials in the specimens are stable for years and can be seen from any angle regardless of location and season. To assess the applicability of the specimens for GMO education, we produced 20 sets of educational kits and provided to educational institutes including high schools, universities and museums. We will discuss how we should use and improve this specimen for the achievement of the PA.

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We quantify the transcripts, proteins, metabolites etc. as relative quantification. Northern hybridization analysis quantifies the transcripts based on total amounts of RNA or mRNA. RT-PCR including real-time RT-PCR quantifies the transcripts based on the level of reference gene. DNA array assay detects the relative levels of transcripts among the samples by normalization. CBB-stained SDS-PAGE, immunoblot analysis and ELISA usually quantify a particular protein level as relative quantification based on the weight of total protein, fresh weight, dry weight or culture volume. Quantify of metabolites is often based on the weight of total protein, fresh weight or dry weight. It is difficult to know which base is no difference among the samples before analysis such as novel mutant or treatment analysis. The normalization using total RNA, total mRNA total protein, fresh weight, dry weight or culture volume has potentially the problem that those are fluctuating. We report the quantificational method based on copy number of genomic DNA that is comparatively stable in a cell.

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Temporal induction of gene expression is an important technique to examine cell lineage, gene function and intercellular communication. In Arabidopsis thaliana, several gene induction systems were reported already, such as steroid hormone, ethanol, and heat shock.
Recently, a new method of gene induction in a single cell of living organisms using an infrared laser-evoked gene operator (IR-LEGO) system was reported (Y. Kamei et al. 2009).
Here, we report the application of IR-LEGO to Arabidopsis. In transgenic line for HSP 18.2 promoter : GUS, we successfully induced gene expression in a single cell of a seedling. IR-LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in Arabidopsis thaliana.

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The color of biological materials provides an indicator of their physiological status and, combined with the morphology, serves as the major visible phenotype. In plants, composition of pigments such as chlorophylls, carotenoids and anthocyanins is the primary determinant of the leaf color. Conventionally, pigment analysis required multistep procedures of liquid-phase extraction followed by absorbance measurement of the extracts. Because of their invasive nature, however, leaf samples used for pigment analysis are no more applicable to additional biochemical assays and to consecutive analysis of pigment dynamics. In research areas of remote sensing, alternative solutions for pigment analysis with non-invasive optical methods have been developed. In this study, we show the advantage of hyperspectral imagery in quantitative analysis of leaf color phenotypes in Arabidopsis. As examples, two-dimensional monitoring system for leaf pigment parameters (concentrations and ratios) and application of the technique in genetic screen for aberrant color phenotypes are presented. Potential impact of such 'spectromics' approach in accelerating data integration from genome to phenome is discussed.

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In the legume-rhizobia symbiosis, rhizobia enter into the root tissues via tubular plant-derived structures termed infection threads. Between two accessions of Lotus japonicus, MG-20 Miyakojima and B-129 Gifu, the number of infection threads is different. The stem color is also distinct by different accumulation of anthocyanins: red (B-129) or green (MG-20). Composite interval mapping using recombinant inbred lines between B-129 and MG-20 revealed two quantitative trait loci, on the southern area of chromosome 5 (QTL1) and near the stem color locus (VIC6) on chromosome 2 (QTL2). We also found significant correlations among infection thread number, pod width, seeds per pod, and stem color. A mutant of the stem color locus (vic6-1) with B-129 background showed the characteristics of the four traits similar to MG-20. Comparison of the response to a precursor and an inhibitor of ethylene biosynthesis between B-129 and MG-20 suggested that there is each optimal concentration of the precursor or the inhibitor for the number of infection threads and that MG-20 has lower endogenous ethylene level than B-129.

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Legumes are capable of establishing root symbiosis with rhizobia and form root-derived organs, called nodules. In model legume Lotus japonicus, nodule number is controlled by long-distance signaling mediated by HAR1 that is a CLAVATA1-like LRR-RLK and thought to function in the shoot.
The Lotus mutant klavier (klv) exhibits a typical hypernodulation phenotype. KLV encodes leucine-rich repeat receptor-like kinase (LRR-RLK). Based on the grafting experiments, KLV is thought to function in the shoot to mediate the long-distance negative regulation of nodulation like HAR1.
Double mutant analysis indicates that KLV functions in the same genetic pathway as HAR1 to control the negative regulation of nodulation.
It has been shown that over-expression of either of the two CLE peptide genes LjCLE-RS1 or LjCLE-RS2 suppresses nodulation systemically via HAR1. The klv mutants with transgenic hairy roots where LjCLE-RS1 or -RS2 were over-expressed exhibited typical hypernodulating phenotypes. These results indicate that KLV is also required for the LjCLE-RS1/-RS2-mediated negative regulation of nodulation, as is the case with HAR1.

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Arbuscular mycorhiza (AM) is mutualistic plant-microbe interaction observed in more than 80% of land plants. In this symbiosis, mycorrhizal fungi provide inorganic materials to the host plant and in return, obtain photosynthetic products from the host. In addition, legume plants acquired the other form of symbiosis, root nodule symbiosis (RNS) by sharing some signaling factors with AM. Recent studies about RNS system revealed the signaling factors and mechanisms, and the knowledge was applied to the AM system through the shared mechanisms. However the AM specific system has been poorly understood because there are few tools for the molecular analysis of AM. Therefore, we established an AM gene maker 'SbtM1', which was specifically induced during AM and worked in the colonization of AM fungi in the host root. The fluorescent protein fusion with SbtM1 promoter visualized infected cells of AM fungi. In addition, the fusion with the secretion signal peptide of SbtM1 enabled visualization of the AM fungi in the host root. Using the AM specific response of the gene maker, we isolated two AM-response cis regions in the promoter.

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Legumes can establish symbiotic relationships with rhizobia and mycorrhizae. The two different symbiosis systems are regulated by host legume genes, which belong to common symbiosis pathway (CSP). CASTOR and POLLUX, both of which encode potassium channels, have been identified as CSP genes in Lotus japonicus. On the other hand, only DMI1 has been identified as a symbiotic gene in Medicago truncatula. Like DMI1, mutations of CASTOR or POLLUX lead to Nod- and Myc- phenotypes. Despite their high similarity, single mutation of CASTOR or POLLUX caused symbiotic defective phenotypes, suggesting functional differentiation of CASTOR and POLLUX in Lotus.
Recently, MtCASTOR was isolated from the EST library of M. truncatula. Here we examined whether MtCASTOR plays a role in legume endosymtioses like its homologs DMI1, CASTOR and POLLUX. Additionally, cross-complementation analyses by expressing LjCASTOR and LjPOLLUX in dmi1 mutant of M. truncatula and to introduce M. truncatula DMI1 in Ljcastor and Ljpollux mutants of L. japonicus raised a novel model for functional evolution of CASTOR/POLLUX/DMI1.

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