Plant genome contains a lot of Myb genes, but most of which encode for Myb proteins that contain only two repeats in the Myb domain. In contrast, much fewer number of plant genes, like mammalian c-Myb gene, encode for Myb proteins with three repeats. We previously reported that, in tobacco, the three-repeat Myb proteins bind to the cell cycle-regulated cis element called MSA in cyclin B promoter, and regulate G2/M phase-specific transcription. Arabidopsis genome contains 5 genes encoding for such three-repeat Myb proteins. Here we isolated and analyzed knockout mutants for these Myb genes in Arabidopsis. We found that a double knockout mutation for two related three-repeat Myb genes caused severe defects in embryogenesis, inflorescence architecture, and cytokinesis in epidermal cells. Some of these defects are consistent with our previous finding that a group of genes involved in cell plate formation during cytokinesis are the targets of three-repeat Myb factors.

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D-type cyclins (CycDs) act as the mediators linking extracellular and developmental signals to the cell division cycle. CycDs associate with CDKs (cyclin-dependent kinases) to phosphorylate the retinoblastoma protein (pRb) that plays a pivotal role in regulating the G1/S transition. We have isolated two cDNA clones of tobacco CycD3 (CycD3;1a, CycD3;1b) in which these genes expressed predominantly during G2 to M phase. We previously showed that these CycD3s bind to both of A-type and B1-type CDKs, and CycD3/CDKA purified from insect cells exhibited histone H1 kinase activity. In this study, we prepared the peptide antibodies against CycD3;1a and CycD3;1b. Using the antibodies, immunoprecipitations were conducted to analyze their kinase activity. Although CycD3;1a and CycD3;1b associated kinases phosphorylate both histone H1 and the tobacco Rb, histone H1 kinase activity remained through the cell cycle and Rb kinase activity increased at middle of G1 phase and declined rapidly during S phase.

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Hd6 gene encodes a casein kinaseII (CK II) alpha subunit and suppresses flowering in long-days (LD) in rice, a short-day plant. Since CKII has been shown to be a key component of the circadian clocks in several model organisms, we analyzed mRNA expression pattern of circadian clock-related genes of rice, such as OsLHY, OsGI and Hd1, between Nipponbare and a near isogenic line [NIL(Hd6)], which have a functional Hd6 allele. However, we did not detect any significant differences in those mRNA patterns between them. In addition, rhythmic CAB1R::LUC expression was not affected in rice seedlings by Hd6. These suggest that the floral inhibition by Hd6 is not mediated by phase shifts of circadian clocks. Furthermore, FT-like floral inducer genes are repressed by Hd6 at 50-days ~ 71-days old plants grown under LD, but OsGI and Hd1mRNA are not. Therefore, Hd6 controls flowering in rice by repressing FT-like genes.

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The rice (SD plant) orthologs of the three key regulators of flowering time in Arabidopsis (LD plant), GI, CO and FT have been shown to play important roles in the photoperiodic control of flowering in rice. We have previously obtained results suggesting that the GI - CO - FT pathway is conserved in rice and Arabidopsis. Under LD conditions, Hd1 suppresses Hd3a expression in rice. To further study suppression of Hd3a expression by Hd1, we examined the effect of Hd1 on Hd3a gene expression by co-transfection in rice protoplasts. The results indicated that co-transfection of Ubq :: Hd1 cDNA with Hd3a :: gus decreased GUS activity. These results suggest that Hd1 can suppress the promoter activity of the Hd3a gene. We obtained transgenic rice expressing Hd3a :: gus and found that GUS activity was detected in an area surrounding the vascular tissue.

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To investigate regulation of the photoperiodic control of flowering in rice, we employed the night break (NB) experiment. Although effects of NB have been studied in a few short day plants, little is known about the molecular basis of NB.
We investigated mRNA expression patterns of OsGI, Hd1 and Hd3a after a single NB. We found that Hd3a was dramatically suppressed by NB. However, strong Hd3a suppression was not observed in plants exposed to the light early or late in darkness. No difference in CAB1R expression suggests that NB does not cause the phase shift of circadian rhythm. Next, NB on the se5 mutants, which has the defect in the phytochrome pathway, exhibited no influence on Hd3a expression, indicating Se5 is essential for Hd3a suppression by NB in wild type. These results suggest that flowering in rice is mediated by the interaction with the internal rhythm and direct light signal.

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The photoperiodic control of flowering is one of the important responses of plants because it is directly related to success of reproduction of its progeny.
We have isolated a mutant that exhibited late flowering phenotype under natural light conditions by the screening of T-DNA inserted plants. T-DNA was integrated into the K-region (4^th intron) of OsMADS50, which is homolog of Arabidopsis SOC1/AGL20. Under short day conditions (10h / 14h day / night), the mutant shows heading at the same time as wild type (around 80 days after sowing), but heading is very late (50 days later than wild type) under long day (14h / 10h day / night) compared to wild type. We are investigating expression of key genes involved in flowering in rice at several developing stages to understand the role of the OsMADS50/OsSOC1 in the photoperiodic control of flowering in rice.

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AtC401 is an Arabidopsis homolog of PnC401 which relates to photoperiodic induction of flowering in Pharbitis nil. C401 genes encode proteins containing a highly conserved pentatricopeptide repeat (PPR) domain. We previously reported that AtC401 expression was regulated by the circadian oscillator. The promoter analysis using the firefly luciferase reporter indicated that the 5’UTR sequence of AtC401 relates to the circadian regulation. To analyze the regulation mechanism in detail, we performed the promoter analysis using an Arabidopsis cultured cells. These analyses revealed that the 86-nt fragment containg 73-nt of 5’UTR is sufficient for the circadian regulation. Because there is no TATA box in the fragment, we suggested that AtC401 requires no TATA box for the transcription. The fragment contains an initiator consensus (Inr) and the tandem repeat of the GAKAA (K=A/T). Therefore, we propose that the Inr and the GAKAA repeat are required for the transcription and the circadian regulation, respectively.

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Cryptochromes (CRYs) are blue-light receptors found in both plants and animals. In Arabidopsis, CRY1 and CRY2 are major blue-light receptors known to regulate flowering time. We had previously isolated PnCRY1 (Pharbitis nil Cryptochrome 1) whose transcripts transiently increased during the 16-h flower inductive dark period. In this study, we cloned P. nil homolog of CRY2 (PnCRY2), which encoded a putative polypeptide of 653 amino acids with a molecular mass of 74 kD. The deduced amino acid sequence of PnCRY2 showed 72% and 67% amino acid identity with those of tomato and Arabidopsis, respectively. PnCRY2 expression showed circadian rhythm both under continuous dark (DD), and continuous light conditions (LL). We will show the results of its expression analysis in various conditions (different photoperiodic conditions, effects of exposure to blue and red light, etc), and discuss the participation of PnCRYs in the photoperiodic induction of flowering in P. nil.

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LHY, CCA1, TOC1 and GI have been suggested to be essential clock components or closely related to clock function in Arabidopsis. Loss-of-function of both LHY and CCA1 causes acceleration of flowering under short days (SD). We demonstrated that the early flowering phenotype of lhy cca1 double mutant under SD condition would be mainly caused by 1) the phase shift of GI expression, 2) the phase shift of CO expression into the daytime and 3) drastic up-regulation of FT expression. FT expression is mainly regulated by GI-dependent pathway. On the other hand, SOC1 that may have partial redundant function with FT on floral promotion shows rather different expression features from those of FT in several multiple mutants. Here we show that a circadian clock may regulate SOC1 expression by GI-dependent and -independent manners.

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In many organisms, a wide variety of biological processes are controlled by circadian rhythms. Two Myb-related genes, LHY and CCA1, have been shown to be closely associated with clock function in Arabidopsis. lhy cca1 double mutant plants flower much earlier than wild-type plants under SD and lose free-running rhythms in constant light. Loss-of-function of either a blue light receptor gene, FHA/CRY2 or a floral inducer gene, CO, causes late flowering and decreased expression of a floral activator gene, FT, under LD. To understand molecular mechanisms on photoperiodic control of flowering, we have tested genetic interactions among lhy, cca1, GI-ox, co, fha/cry2. Our results suggest that both FHA/CRY2 and CO mediate a circadian clock and a photoperiodic control of flowering in Arabidopsis.

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We have recently proposed that LHY and CCA1 are essential components for circadian clock function in Arabidopsis. Gain-of-function mutation of lhy (lhy-1) causes late flowering under long days (LD) and elongated hypocotyl phenotypes. Loss-of-function mutations of lhy (lhy-11, 12 and 13) were isolated as intragenic suppressors and causes early flowering phenotype in short days (SD). We have screened for mutations that suppressed the early flowering phenotype of the lhy-12 mutant under SD. Here we demonstrate isolation of a new allele of lhy (lhy-2) as an intragenic suppressor of the lhy-12. The lhy-2 is quite similar to the lhy-1 in terms of late flowering under LD and long hypocotyl phenotypes. We also show the lhy-12 has a point mutation in the end of the 5th intron and this causes mis-splicing of LHY. The mis-splicing seems to be partially suppressed by the lhy-2. We will report details on characterization of the lhy-2.

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We have recently proposed that LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) are essential components for circadian clock function and flowering time regulation in Arabidopsis. Over-expression of clock-associated genes such as LHY, CCA1 and LKP2 causes a dominant late flowering phenotype under LD. To isolate novel mutations which closely associated with clock function, we are screening Arabidopsis mutants similar to the gain-of function mutants of LHY, CCA1 and LKP2. Recently we have isolated such a mutant from one of our T-DNA tagged transgenic populations. We named it dll1 (dominant late flowering under long day 1). Consistent with the late flowering phenotype, expression level of a floral activator gene, FT, is significantly lowered in this mutant under LD. Details on genetic interactions between the dll1 and other flowering mutations will be reported.

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Photoperiodic control of flowering is regulated by light and a circadian clock. TOC1, LHY and CCA1 consist both of positive and negative feedback loops and are clock components in Arabidopsis. PIF3, a bHLH transcription factor binds promoter regions of the LHY and CCA1 genes, affects the light induction of these genes and interacts with TOC1 protein. Although PIF3 has been assumed to be involved in the positive feedback regulation of clock components, the molecular nature has not been elucidated. Here we demonstrate that the antisense suppression of the PIF3 gene causes higher levels of mRNA of floral activator genes CO and FT and results in early flowering. Neither the circadian rhythms nor the diurnal rhythms of the clock-controlled genes are affected by the reduction in PIF3 gene expression. We will discuss a possibility that PIF3 may regulate CO and FT expressions without affecting circadian rhythms.

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Recent intensive studies on Arabidopsis thaliana have begun to shed light on the molecular mechanisms underlying the biological clock. We have been characterizing a small famiry of protein, designated as Arabidopsis pseudo response regulators, including APRR1 that is identical to TOC1, which is believed to be a component of central clock. Through our recent studies, several lines of evidence have already been provided to support the view that, not only APRR1/TOC1, but also other APRR1/TOC1 members are crucial for a better understanding of the molecular links between circadian rhythm, control of flowering time, and also photomorphogenesis. Amongst the APRR1/TOC1 family members, nonetheless, the least characterized one is APRR3 in the sense that no genetic study has been done to see if whether APRR3 also plays an important role in the circadian associated biological events. Here we provide such evidence from the intensive characterization of a transgenic line aberrantly overexpressing APRR3.

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Recent studies on Arabidopsis thaliana have begun to shed light on the mechanisms underlying the plant biological clock. We have been characterizing a small family of proteins, designated as APRRs, including APRR1 that is identical to TOC1, which is believed to be a component of central clock. Nevertheless, the molecular function of APRR1 remains to be fully elucidated. In this respect, we previously showed that APRR1 interacts with certain bHLH family members that include PIF3 and homologs (designated the PIL family). Among them, PIF4, PIL6, and PIL8 are particularly intriguing, because the transcription of each gene is clearly under the control of circadian rhythms. Here we constructed PIF4-overexpressing (-ox), PIL6-ox, and PIL8-ox plants. They were extensively characterized in terms of their light sensitivity, flowering time, and circadian rhythm. The functions of these novel bHLH family members will be discussed with reference to photosensory signal transduction and circadian clock.

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Diverse organisms possess a circadian clock system that arrows them to coordinate physiological events with environmental day/night cycles. In plants too, the circadian clock has recently been characterized at the molecular level. To extend such studies further, here we employed an established Arabidopsis cell line (named T87). When T87 cells were grown in an appropriate light and dark (LD) cycle, cell autonomous diurnal oscillations of the APRR1/TOC1 quintet genes were observed at the level of transcription, as are in intact plants. These phenomena were verified by monitoring CCA1::LUC and APRRs::LUC bioluminescence activity in transgenic cell lines. We showed that the properties of rhythms in cultured cells consistently fulfilled the diagnostic criteria of the circadian clock, namely, 'free-running', 'entrainment', and 'temperature compensation'. This is the first indication of cell autonomous circadian rhythms in cultured cells, which will provide us with an alternative and advantageous means to characterize the plant biological clock.clock.

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Microarray analysis was performed to identify genes diurnally expressed under continuous dark conditions in leaf blades or leaf sheaths of rice (Oryza sativa L.). Three diurnally expressed genes were selected to examine their functions. Here, we reported analysis of a gene which had a similarity to a transcription factor gene, Dof. Accumulation of the transcript for endogenous Dof-like gene in each organ was different between sense and antisense cDNA-overexpressing transgenic plants. Transcripts for its transgene in each organ also showed the accumulation patterns similar to those of the endogenous gene. Although the Dof-like gene exhibited diurnal oscillations of its transcript with the peak early in the light period in transgenic plants the same as in wild-type plants, it showed slightly longer peak in transgenic plants. The results of expression analysis of known circadian clock-regulated genes in transgenic plants will be also reported.

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Circadian clocks control the expression of Lhcb genes encoding the chlorophyll a/b-binding proteins broadly in seed plants. We show here that this regulation is also conserved in the primitive moss Physcomitrella patens. Northern blotting analyses revealed a robust daily oscillation of Lhcb mRNA levels in protonema cells in 12-hr:12-hr light-dark cycles (12:12LD) that damped rapidly in continuous darkness (DD). In continuous light (LL), by contrast with typical profiles in higher plants, Lhcb mRNA levels only peaked during the first day and thereafter it showed constant levels. Reverse transcription (RT)-PCR analyses showed similar patterns of expression in LL for three distinct Lhcb genes. At a higher concentration (4.5%) of glucose in the medium, transgenic reporter strains expressing luciferase under the control of the PpLhcb2 promoter showed self-sustained bioluminescence rhythms in DD, which was entrained to a differently phased 12:12LD, revealing a circadian regulation on the transcription.

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The pex (period extender) gene is a negative regulator of kaiA gene, an essential gene for circadian clock. The expression of pex gene was induced by light-dark transition. This suggested that pex gene had a certain role in light-dark cycle. Previously we compared the phase of circadian rhythm in wild-type and pex-inactivated mutant cultivated in the continuous light condition, but the remarkable difference was not observed. In this time, we report that pex-inactivated mutant cultivated in light-dark cycles notably advanced in the phase for 3 hours compared with the one in wild type. Under this light-dark condition, the growth rate and the color of colonies were the same between the wild-type and the pex-inactivated mutant. It seemed to be concluded that pex gene was a gene in the input pathway which modulate the circadian phase.

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The pex gene extends the circadian period and pex mRNA is abundant in the darkness. This implied signal transduction pathway to the pex gene expression. Then, by using bioluminescence reporter strain for pex expression, we identified a pex expression mutant. The causative transposon insertion was occurred in cmpR gene coding a LysR-type DNA binding factor, CmpR. This regulates gene expression for bicarbonate transporter operon (Omata et al., 2001). Bioluminescence rhythm in the mutant showed circadian phase-delay by 6-hours as compared with the one in a wild-type reporter for the expression of clock operon kaiBC. This delay was complemented by transformation of the cmpR trans-gene. The phase-delay phenotype was partially suppressed by disruption of pex, suggesting that pex gene expression is required to the abnormal rhythm. From these results, it was revealed that cmpR and pex functioned to modulate phase of the circadian oscillator.

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A double flower mutant (flora pleno; fp) of the common morning glory (Ipomoea purpurea) shows transformation of stamens to petals and they fused with original petals. The tip of pistil often converts to sepal like organ. The fp mutation was caused by inactivation of an ortholog of DUPLICATED (DP) which belongs to a C-function MADS-box gene in the Japanese morning glory (Ipomoea nil). Phenotypic and molecular analyses revealed that the fp mutant had an insertion of a novel Helitron-related transposable element (Helip). Although only a possible example of transposition of Helitron was reported in maize, Helip element in the fp gene excise frequently and the reverted region was completely repaired. Possible mechanisms for weak phenotype of fp mutation will be discussed.

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Flowering is regulated by endogenous factors and by environmental stimuli such as light and temperature.
COP1 in A. thaliana is a gene that is involved in photomorphogenesis. The cop1 mutants show light independent morphogenesis. The weak allele, cop1-6 mutation causes early flowering in LD and SD. To elucidate the mechanism of early flowering in cop1-6, the double mutants of cop1-6 with one of several late-flowering mutations (cry2, gi, co and ld) were constructed and their flowering characteristics were examined. All of the double mutants except cry2-1 cop1-6 flowered later than cop1-6 single, demonstrating that cop1-6 is epistatic to cry2-1 for early flowering. The relative increase in the number of leaves was higher in co-1 cop1-6 plants than in co-1 plants, whereas the delay in flowering of gi-2 cop1-6 plants was almost the same as that in gi-2 plants.

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We have shown that FD acts with FT in the floral transition during Arabidopsis development. FD gene encodes a bZIP protein and may act as a transcription factor. We examined the expression of meristem identity genes (AP1, CAL, and FUL), which are candidates for the FD targets. AP1, CAL, and FUL mRNA levels were greatly reduced in fd-1; lfy-26 inflorescences and were increased in 35S::FD seedlings, suggesting the role of FD in up-regulation of these genes. Expression of AP1(1.7 kb)::GUS in 35S::FD seedlings was restricted to the vascular tissues of cotyledons and leaves, suggesting the involvement of additional factor which is likely FT. In 35S::FD; ft-3 seedlings, the expression of AP1 was reduced to wild-type level. FD protein can interact with FT protein in yeast cells. Taken together, these results suggest that FD requires FT for up-regulation of its target, AP1 during the transition to flowering.

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Floral pathways are integrated at the transcriptional regulation of floral pathway integrators including FT. Arabidopsis has six homologous genes of FT, which include TSF with the highest homology to FT, and FT-antagonist of TFL1. Although certain aspects of expression and overexpression phenotype have been described, the function of TSF remains unknown.
We compared expression patterns of TSF and FT in detail. First, diurnal oscillation of mRNA levels in plants grown in long day or short day was examined. Subsequently, developmental regulation of expression was examined with RNA samples at a fixed zeitgeber time on variant dates from germination to maturation. Regulation of TSF expression by flowering time genes, especially CO, was analyzed. The results obtained from these analyses suggest that transcription of TSF is regulated in a similar manner with FT. Phenotype of a T-DNA insertion line with decreased TSF mRNA levels is also being examined.

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Late-flowering mutant fwa is a dominant epigenetic mutant that ectopically expresses a GL2-type HD-ZIP gene due to promoter hypomethylation. In wild type, FWA is not expressed during vegetative phase. This suggests that ectopically-expressed FWA somehow interferes the regulatory mechanisms of flowering. Genetic analysis suggests that FWA blocks the pathway at FT and/or downstream of FT. We envisage that FWA may provide a unique tool to dissect pathway from FT to flowering. We examined protein-interaction of FWA with FT and a bZIP protein FD. Our results showed that FWA can strongly bind to FT, but not to FD. This suggests that ectopically-expressed FWA inhibits floral transition by interfering with the interaction between FT and FD through binding to FT. Since interference of FT function alone cannot fully explain fwa phenotype, we are investigating other proteins that interact with FWA using yeast two-hybrid screening. Results of these analyses will be presented.

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Flowering state lasts long in Perilla frutescens var. crispa, a short-day plant. This reminds us of vernalization where the low temperature effect lasts long suggesting the existence of a common mechanism between them.
DNA methylation/demethylation is involved in the regulation of vernalization. Accordingly, we treated Perilla with 5-azacytidine in long-day condition. The treatment caused flowering.
It is known that rDNA intergenic spacer region is methylated. Accordingly, the genomic DNAs were isolated from leaves of the 5-azacytidine-treated and untreated Perilla, digested with a restriction enzyme, Msp I (methylation-insensitive) or Hpa II (methylation-sensitive), and then Southern hybridization analysis was performed with 25S-18S rDNA intergenic spacer region as a probe. The ladder signals shifted to lower molecular weight region in the treated plants, indicating that the 5-azacytidine treatment caused demethylation of the genomic DNA. The result suggests that DNA demethylation is involved in the flowering of Perilla.

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Imprinted genes are expressed differently on the maternal and paternal chromosomes. In mammal, such differential gene expression is controlled by DNA methylation, which is erased and re-established in each generation. In contrast, molecular mechanism underling genomic imprinting in flowering plants had not been elucidated.
FWA, an Arabidopsis homeobox gene, displays maternal-origin-specific-expression in endosperm. The expression of FWA gene is always associated with hypomethylation of the tandem repeats around transcription starting site. We will report that the FWA imprinting is established by allele-specific activation rather than allele-specific silencing that is found in mammalian imprinting. Since endosperm is a terminally differentiated tissue, the activated FWA gene needs not to be reprogrammed again. We will also discuss how this simple controlling mechanism of FWA imprinting fits to the relatively complex sexual reproduction in flowering plants.

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We have studied on an Arabidopsis MADS-box gene, AGL24 which function in the promotion of flowering. Interestingly, SVP which represses flowering, is phylogenetically very close to AGL24. Recent works suggest that MADS-box proteins form complexes with other MADS-box proteins. To reveal whether AGL24 protein form complexes with other MADS-box proteins involved in flowering, proteins interacting with AGL24 were screened by yeast two-hybrid method. As a result, several MADS-box genes such as SOC1, AP1 and FUL were isolated. Studies with yeast experiments indicated that SVP also interacted with all of them. The interactions among these proteins were confirmed by in vitro binding assays. Based on the expression patterns of these genes, AGL24 may interact with SOC1 in shoot apical meristems during vegetative development and with AP1 and FUL in early floral meristems.

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MADS-box genes are known as floral homeotic genes and are also involved in various developmental processes in flowering plants. Our researches on a moss, a fern and a green algae suggest that MADS-box genes function in the differentiation of reproductive cells in non-flowering plants. We also reported that some MADS-box genes are specifically expressed in pollen (Kofuji et al., Mol. Biol. Evol., in press), which bears reproductive cells, suggesting that the involvement of MADS-box genes in the differentiation of reproductive cells are evolutionally conserved among land plants. To investigate the detailed function of MADS-box genes in reproductive cell differentiation, functional analyses of three pollen specific MADS-box genes in Arabidopsis are in progress. We found that the in vitro germination rate of triple disruptant pollen was much reduced compared to that of wild type pollen, although mutant pollen normally grow in vivo. Results on microarray and morphological analyses will be presented.

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Proper pollen tube guidance to ovules is one of crucial events in plant reproduction. Several molecules such as Rho-GTPase, actin and calcium ion have been reported to have roles on pollen germination and/or elongation. However, attractants and receptors for pollen tube guidance are still unclear. As receptor-like kinases (RLKs) were shown to be involved in various signal transduction pathways in plants, RLKs dominantly expressed in pollen tubes were searched using the microarray analyses. Total RNA was extracted from pollen grains, germinated pollen grains, and inflorescences respectively. Expression profiles were compared to each other and 45 genes were found as putative receptors dominantly expressed in pollen tubes. Characterization of pollen tube phenotype in pistils of each gene disruption lines by T-DNA insertion and/or RNA interference were compared to those of wild types, and the results will be presented.

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To understand the program of sexual reproduction of Closterium, cDNA microarrays containing a total of 2801 cDNAs were prepared. A Cy-5-labeled cDNA population was prepared from cells proceeding sexual reproduction (2, 8, 24, 72 h after the mixing) and cells treated with purified sex pheromones. As a control, labeled cDNA probes prepared individually from mt⁺ and mt^- cells were also used for the analysis. As a result, 35 genes that showed a 5-fold increase in expression over the time-course were identified. Among them, expression levels of 8 genes including leucine-rich repeat protein were elevated only in mt^- cells by the addition of PR-IP, whereas that of 14 genes including a receptor-like protein kinase were elevated only in mt⁺ cells by the addition of PR-IP inducer. Up-regulation of two genes in both cells in response to the respective pheromones was also confirmed, whereas remaining 11 genes were not regulated by pheromones.

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In lower land plants, genes controlling the transition from vegetative growth to sexual reproduction have not yet been identified. In the dioecious liverwort Marchantia polymorpha, the transition to sexual reproduction accompanied with the formation of sexual organs on the gametophytic thallus is initiated under long-day conditions. By particle bombardment-mediated mutagenesis, we generated 2,100 tagged lines of mutant and identified a mutant of M. polymorpha which constitutively forms sexual organs. This mutant is fully fertile, showing that the mutation does not affect formation of male or female sexual organs per se. Genetic analysis reveals that this phenotype is caused by mutation of a single autosomal locus, suggesting that this mutation defines or controls a gene regulating the transition to sexual reproduction in M. polymorpha. (Sex Plant Reprod, in press)

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Pollen development is a fundamental and essential biological process in flowering plants. Although several sporophytic and gametophytic mutations affecting male gametogenesis have been isolated and analyzed, little is known about the precise molecular mechanism. We identified two mutational alleles of the TCP16 gene. Genetic analysis revealed that the mutational alleles are hardly transmitted to the progeny specifically through male gametophytes. The mature pollen grains generated in the heterozygous tcp16 mutant comprised equal amounts of normal and abnormal, non-viable, pollen grains. Expression analysis of the promoter::GUS fusion gene suggested that the TCP16 transcription is activated at early stage from the tetrad to the unicellular stage, at which the defective phenotype is observed. Because the TCP16 gene encodes a putative transcription factor carrying a TCP domain, our results strongly suggested that TCP16 plays an essential and crucial role in early processes in pollen development as a transcriptional regulator.

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Pistillody has been reported in the cytoplasmic substitution (alloplasmic) lines of common wheat (Triticum aestivum) with Aegilops crassa cytoplasm. The induction of pistillody is suppressed by the Rfd1 gene located on the long arm of chromosome 7B (7BL) in wheat cultivar Chinese Spring (CS). Because of the absence of Rfd1, the alloplasmic line of CS ditelosomic 7BS ((cr)-CSdt7BS) without 7BL exhibits pistillody, whereas the euplasmic CS ditelosomic 7BS (CSdt7BS) having normal cytoplasm forms normal stamens. The class B MADS box genes, WPI (Wheat PISTILLATA) and WAP3 (Wheat APETALA3), were expressed in the normal stamen primordia of CSdt7BS, but not in the pistil-like stamen primordia of (cr)-CSdt7BS. On the other hand, a YABBY gene, TaDL, was expressed in the pistil-like stamen primordia of (cr)-CSdt7BS. The results indicate that the pistillody in the alloplasmi wheat is associated with alterations to expression pattern of floral organ identity genes.

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In rice one particular CMS has been obtained by combining the cytoplasm of Chinsurah Boro II (indica rice) with the nuclear genome of Taichung 65 (japonica rice). The possibility has been reported that a unique sequence (orf79) located downstream from mitochondrial atp6 causes male sterility. In CMS lines, the B-atp6 transcript is a 2.0 kb RNA consisting of normal atp6 and a unique sequence containing orf79, whereas two discontinuous RNAs of 1.5 and 0.45 kb are generated from the 2.0 kb RNA by processing in presence of the Rf1. By means of map-based cloning and complementation test, we demonstrated that one of the PPR gene (PPR8-1) promotes the processing of B-atp6 RNA and restores pollen fertility. Sequence analysis around the Rf1 locus revealed there are two other PPR genes that have highly homology to the Rf1 gene. We are now analyzing the post-transcriptional regulation of B-atp6 RNA by the Rf1.

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A novel male-sterile mutant of Arabidopsis thaliana was isolated by means of T-DNA tagging. Pollen abortion of the mutant was evident after microspore release and resulted in complete disappearance of pollen grains at anthesis. A transmission electron microscope analysis revealed that primexine formation was significantly reduced and that sporopollenin was not deposited onto the microspore plasma membrane. The sporopollenin that failed to deposit aggregated and accumulated within the locule and on the locule wall. Finally, no exine formation was observed, thus, the mutant was named nef1(no exine formatioin 1). Cloning of the full-length nef1 gene indicated that the gene encodes a novel plant protein with limited sequence similarities to membrane proteins or transporter-like proteins. Expression analysis revealed that the nef1 gene was strongly expressed in flower-buds, and weakly in leaf tissues. A considerable role of the NEF1 protein is discussed.

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Leaves develop as lateral organs from shoot apical meristem (SAM). And the cells of the SAM resemble stem cells in that they have the capacity for self-regulation and remain in an undifferentiated state. The genome sequence of Arabidopsis has revealed 4 members of class 1 knox genes, STM, KNAT1/BP and KNAT2 and KNAT6. Though it is proposed that these genes have important functions for the maintenance of the SAM, the details are not clarified. No loss-of-function mutation in KNAT6 has been described. To elucidate the function of KNAT2 and KNAT6, we identified the T-DNA insertion allele of KNAT2 and KNAT6, and analyzed these phenotypes and the expression pattern of KNAT6 at the inflorescence stem using KNAT6 promoter::GUS transgenic plants. Genetic interaction among class 1 knox genes will be discussed.

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The axial growth of plant organs changes under genetic and environmental influences, which can be observed as alternations in cell proliferation and elongation. These aspects, however, are not mutually independent and each aspect consists of various factors. Therefore, it is necessary for comprehensive growth analysis to dissect such multiplex relationship quantitatively. We developed a simple mathematical model for this purpose and demonstrated that this model roughly fits the real growth of primary roots of Arabidopsis thaliana. In this work, we have improved the analytical procedure by incorporating a non-parametric smoothing method. With this improved analysis, we prove that the measured data of root growth in the Columbia strain agree well with the model estimates. We also show here that the model-assisted analysis is applicable for speculating aspects and factors responsible for inter-ecotype differences in root growth.

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The ETO1 family is thought to be a conserved negative regulator of ethylene biosynthesis in higher plants, that interacts with a class of ACC synthases (type 2 ACS) and decreases their stability through the proteasome-dependent pathway. By Y2H analysis, we identified a C-terminal amino acid sequence of the type 2 ACS required for the interaction with ETO1, and named TOE (target of ETO1). When the TOE from LE-ACS3 was fused to the C-terminus of GFP and introduced into rice callus, fluorescence of GFP-TOE^LE-ACS3 was significantly reduced compared to the native GFP. Furthermore, Western blot analysis showed significant reduction of the fusion protein in rice callus. These results indicate TOE is sufficient to decrease the protein stability.

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Multiprotein bridging factor 1 (MBF1) is known to be a transcriptional co-activator that mediates transcriptional activation by bridging between an activator and a TATA-box binding protein (TBP). Although amino acid sequences of MBF1 are evolutionarily conserved among eucaryotes, the function of MBF1 in plant is poorly understood. Arabidopsis thaliana, which has three subtypes of MBF1 genes, is the first example among all organisms. We demonstrated that expression of every three AtMBF1 partially rescues the yeast mbf1 mutant phenotype and every AtMBF1 directly binds to TBP and GCN4, indicating that all of them function as co-activators for GCN4-dependent transcriptional activation. We also report that each of their subtypes shows distinct tissue-specific expression patterns and responses to phytohormones. These observations suggest that even though they share a similar biochemical function, each MBF1 has distinct roles in various tissues and conditions. We will discuss roles of MBF1 in Arabidopsis.

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The hormonal action of abscisic acid (ABA) depends on the endogenous level of ABA which are controlled by the precise balance between its biosynthesis and catabolism. ABA is mainly catabolized into phaseic acid through 8'-hydroxylation. The enzyme ABA 8'-hydroxylase was shown to be a member of cytochrome P450 (P450), however, its gene had not been identified. Based on the phylogenetic analysis, DNA microarray analysis, and comparison with the rice genome, the candidate genes for this enzyme were narrowed down from among 272 Arabidopsis P450 genes. Functional expression of these genes in yeast revealed that members of CYP707A family, CYP707A1 through CYP707A4, code for the target ABA 8'-hydroxylase. Expression analysis showed that CYP707A2 is responsible for rapid decrease in ABA level during seed imbibition. cyp707a2 mutant phenotype confirmed this result. Furthermore, strong correlation was seen between mRNA level of these genes and the level of ABA during dehydration and rehydration conditions.

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The hydroxylation at the 8'-position of abscisic acid (ABA) has been known as the key step of ABA catabolism, and this reaction is catalyzed by cytochromes P450 (CYPs or P450s). Here, we demonstrate CYP707A genes as the P450 responsible for the 8'-hydroxylation of (+)-ABA. CYP707A cDNAs were cloned from Arabidopsis. The insect cells expressing CYP707A3 metabolized (+)-ABA to phaseic acid (PA), the isomerized form of 8'-hydroxy-ABA. The micorsomes from the insect cells exhibited a K_m value for (+)-ABA of 1.3 μM and a k_cat value of 15 min^-1. The solubilized CYP707A3 protein bound (+)-ABA (K_s = 3.5 μM), but did not bind (-)-ABA. Analyses of the reaction products confirmed that CYP707A3 does not isomerize 8'-hydroxy-ABA to PA. The transcripts of CYP707A genes increased in response to salt, osmotic and dehydration stresses as well as ABA. These results establish that CYP707A genes play a key role in regulating the ABA level.

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ABA is known to have an important role in the water stress response. In this study, we investigated the site(s) of ABA synthesis and the ABA movement using wild type and aao3 mutant of Arabidopsis thaliana. In the wild type plants, ABA levels were rapidly increased both in leaves and roots after drought treatment. In contrast, no significant increase was observed in aao3 plants. When wild type plants divided to leaf and root parts were independently exposed to drought, increase of ABA levels was detected in leaf parts, but not in roots. Furthermore, when ¹³C-ABA was supplied to leaves of aao3 plants, ¹³C-ABA was detected from the roots after within 60 hr. These results indicate that ABA biosynthesis takes place mainly in leaves in response to drought stress and the accumulated leaf ABA is immediately transported to roots.

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Abscisic acid (ABA) plays important roles on dormancy. It is known that the abi5 mutant is defective in a bZIP transcription factor that binds to ABRE. However, only fragmentary information has been reported on how the expression profiles differ in abi5. We have performed molecular phenotyping of WT and abi5 seeds by microarray analysis. A statistical cis-element search in highly expressed genes in WT seed revealed ABRE. Combination of ABRE and another elements such as CE or RY showed stronger correlation to high expression in the seed. In abi5, the correlation between ABRE and high expression level is largely weakened. ABRE was also enriched in down-regulated genes in abi5. These results indicate that ABRE functions in ABA inducible expression during seed maturation, and ABI5 plays important roles on expression of those genes. The results were also consistent with the previous in vitro results, confirming our phenotyping to be effective.

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Abiscisic acid (ABA) regulates many aspects of plant growth and development, including seed maturation, germination and adaptation to environmental stresses. To identify the components involved in the ABA signaling pathway, we had attempts to isolate novel ABA-related mutants. We screened for mutants that have an altered sensitivity to ABA analog, PBI-51 and isolated several candidates for ABA hypersensitive mutant. Here we will report the study of another ABA analog, #18. This analog more strongly inhibits the growth of stems or leaves than that of roots, whereas ABA usually inhibits the root elongation. We tested the response of known ABA mutants to this analog. All the tested ABA insensitive mutants showed insensitivity to this analog and ABA hypersensitive mutant, era1, showed an enhanced sensitivity, indicating that this analog affects ABA signaling pathway. We are now undertaking a genetic screen for mutants that are resistant to this analog.

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We isolated Arabidopsis mutants that have an altered ABA sensitivity using ABA analog. One of these putative ABA hypersensitive mutants, ahg3 (ABA hypersensitive germination 3) is analyzed in this study. This mutant exhibit less germination or growth activities at the presence of ABA analog as well as ABA. Characterization of ahg3 revealed that ahg3 have enhanced sensitivity to salinity and high osmotic stress. Northern blot analysis revealed that ahg3 shows delayed induction of ABA inducible genes. We conducted mapping of ahg3 and identified a point mutation in the AtPP2CA gene that encodes a protein phosphatase 2C (PP2C) protein. PP2C is predicted to be a negative regulator of ABA pathway. We want to prove this prediction through the identification of ahg3 loci and analysis of AHG3 protein.

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Abscisic acid-β-D-glucosyl ester (ABA-GE) was recently found in Citrus junos peel as a main allelopathic substance. Aqueous methanol extracts of fruit peel of C. junos, C. unshiu and C. hassaku inhibited the growth of the roots and hypocotyls of lettuce, cress and alfalfa seedlings, and there was a good correlation between ABA-GE concentrations in fruit peel of C. junos, C. unshiu and C. hassaku and the inhibitory activities of their aqueous methanol extracts. Annual changes in concentrations of ABA-GE were determined in C. junos, C. unshiu, C. sudachi fruits and leaves. The concentrations in fruits of C. junos increased rapidly from the end of October, but the concentrations in the others did not increase so. The inhibitory activities of aqueous methanol extracts obtain from fruits and leaves of C. junos, C. unshiu, C. sudachi also correlated well with their ABA-GE concentrations.

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In the rice seedling elongation inhibition assay, 5'α,8'-cyclo-ABA (CycloABA) exhibited stronger and longer-lasting activity than ABA. This may be explained in view of (1) uptake, (2) affinity for the ABA receptor, and (3) catabolic inactivation. In the light of structure-activity relationships of ABA, we focused the metabolism of CycloABA. In rice seedlings, CycloABA was accumulated seven times as much as ABA. In radish seedlings, the major metabolite of ABA was phaseic acid that is produced by the 8'-hydroxylation, whereas we found no significant oxidative products of CycloABA. The major metabolite of CycloABA was the C-1 glucose conjugate, which was the minor metabolite of ABA. In this study, we uncovered the complete resistance of CycloABA to 8'-hydroxylation. CycloABA is expected to be a useful tool for studying plant physiology in which ABA metabolism involved.

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In Arabidopsis, the dehydration-responsive expression of rd29B gene is mainly mediated by ABA, two ABA-Responsible Elements (ABREs) function as cis-acting elements, and bZIP transcription factors, AREBs bind ABRE and transactivate downstream gene expression. Transactivation of AREBs requires ABA dependent post-translational modification. As there are highly conserved target sites for putative protein kinases in the AREB proteins, we analyzed ABA-dependent phosphorylation of AREBs
In-gel kinase assay revealed that specific ABA-activated protein kinase phosphorylates the conserved regions in AREBs. These AREB fragments were also phosphorylated by ABA-activated SRK2 kinases.
Amino-acid substitution (Ser/Thr to Ala) of the target sites for protein kinase in the conserved region resulted in suppression of both ABA-dependent phosphorylation and transactivation. Substitution of the Ser/Thr to Asp causes the high transactivation activity without ABA. Transgenic plants expressing constitutive active AREB1 expressed rd29B without ABA treatment suggesting that the ABA-dependent phosphorylation of AREB1 is required for its activation in plants.

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The receptor-like kinases are important components in the signal transduction pathway of various environmental and developmental signals. The RPK1, an LRR receptor kinase has been isolated from Arabidopsis, and its expression was induced by abscisic acid (ABA). We analyzed the phenotypes of T-DNA insertion mutants and antisense-transgenic plants for RPK1 to determine the function of RPK1 in ABA signaling. Repression of RPK1 expression in Arabidopsis decreased plant sensitivity to ABA during germination, growth, and stomatal closure; microarray and RNA gel analysis showed that many ABA-inducible genes are down-regulated in these plants. RPK1 is involved in the main ABA signaling pathway and in early ABA perception in Arabidopsis.

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