The plant-tumorigenic 6b (AK-6b) gene of Agrobacterium tumefaciens AKE10 induces morphological alterations of tobacco plants. AK-6b-seedlings featured their cotyledons with protrusions, and short, thick roots. These morphological alterations were accompanied with reduced auxin transport activity by the AK-6b gene. To understand the relationship between auxin distribution and morphological alterations in more detail, we applied the well-established auxin influx carrier inhibitor, 1-Naphthoxyacetic acid (NOA) to AK-6b-tobacco seedlings. Resulting auxin distribution was monitored by auxin-specific reporter gene construct DR5:GUS expression. In roots, both wild-type and AK-6b seedlings showed reduced number of lateral roots. In contrast, wild-type shoots were unaltered with NOA, but AK-6b shoots showed reduction of protrusions and newly emergence of outgrowths developed from hypocotyls. Histochemical GUS assay revealed that the staining of AK-6b shoots was localized to the protrusions and altered tissues. These results show that redistribution of auxin was accomplished by combination of AK-6b gene expression and NOA, and the AK-6b gene may act as an auxin efflux carrier inhibitor.

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We had identified brassinosteroid biosynthesis inhibitor Brz insensitive mutant bil1 from EMS-mutation lines, and bil5 from fast neutron-mutation lines. To identify additional components of the brassinosteroid signaling, trial to screen new mutants as Brz-insensitive-long hypocotyls in the dark was applied to Arabidopsis activation-tag lines. We screened Arabidopsis activation-tag lines and isolated bil2 that showed long hypocotyls on the medium containing Brz in the dark. Light-grown bil2 mutants had long petiole phenotype similar to wild-type plants treated with BR or BRI1-OX mutants. These phenotypes suggested that bil2 mutants enhanced the BR signaling. Identification of over expression the bil2 candidate gene is progress.
We are starting to screen new bpg (Brz-insensitive pale green) mutant that showed pale green leaves on the Brz medium in the light. In this study, we used FOX (Full-length cDNA over expresser gene) lines, which were over expression of a normalized Arabidopsis full-length cDNA library.

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There is a substantial degree of genetic variation among Arabidopsis wild-type accessions. It is known that accessions vary in many phenotypes, such as seed dormancy and flowering time. However, little is known about variation of endogenous phytohormone levels among accessions. In this study, we performed semi-comprehensive hormone analysis in dry seed in over 150 accessions. In this hormone analysis, endogenous levels of abscisic acid (ABA), indole-3-acetic acid, jasmonic acid, isoleucine-conjugated JA, and salycilic acid were determined. Although any hormones did not show correlation with seed dormancy, there were significant variations in hormone levels among accessions. For example, ABA levels ranged from 47.0 to 506.3 ng/g fresh weight. To identify genetic factors that determine seed hormone levels and seed dormancy, we performed QTL analysis using a hyper-ABA accumulating accession. Several QTLs associating with hormone levels and seed dormancy have been identified so far.

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Abscisic acid (ABA), known to regulate seed dormancy, stomata closure, and tolerance to environmental stresses of higher plants, has been shown to function in other groups of land plants including ferns, lycophytes and mosses. However, the role of ABA in liverworts, comprising the earliest diverging group of land plants has not been clearly understood. We examined molecular responses of ABA in liverworts, using the model organism Marchantia polymorpha. Exogenous ABA was found to inhibit growth of gemmae of M. polymorpha and increased desiccation and freezing tolerance. Assays using the GUS reporter gene fused to the ABA-inducible wheat Em promoter (Em-GUS) in both cultured cells and transgenic plants revealed that exogenous ABA increases Em-GUS expression. In contrast, lunularic acid, which had been suspected to be functioning as an ABA-like substance, had no effect on the Em-GUS expression. The ABA-induced Em-GUS expression was dependent on the conserved ABA-responsive element (ABRE) and RY sequences These results indicate that the liverworts possess a distinct molecular mechanism for ABA-induced gene expression, which is not affected by lunularic acid.

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A cell cycle inhibitor group, KRPs (Kip-related proteins) from Arabidopsis consist of 7 molecular species. They may be divided into two groups based on their motif composition. Namely, AtKRP 3, 4, 5 have more than 7 motifs in each peptide. On the other hand, AtKRP1, 2, 6 and 7 have 4 or 5 motifs. Role of these motifs are not yet clear except 2-motifs that make CDK-cyclin binding domain at C-terminal. We have been interesting in roles of other motifs that might be contributed the binding to other functional protein(s).
To clarify the possibility, we have been tried to get binding protein(s) by yeast two-hybrid method. In this time, we mentioned to AtKRP3 with 7 motifs, because 1) which has many motifs, and 2) gene-expression pattern is quite unique. Namely, it is expressed in culture cells, but not in other tissues.
We screened 15 candidates from 30,000 yeast-colonies that have different cDNA from Arabidopsis young seedlings. These candidates include CDKA, unknown proteins and plastid-related proteins such as Rubisco small subunit. Now, we are checking on in vitro binding capacity between AtKRP3 and each candidate.

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Monotropastrum humile belongs to Monotropeae, Monotropoideae in Ericaceae and is an achlorophyllous plant due to loss of chlorophyll. In addition, because M.humile has no photosynthetic capacity and is an obligate myco-heterotrophic plant, the life of this plant is completely dependent on the photosynthates supplied by symbiont mycorrhizal fungi. Since proplastid does not seem to differentiate to chloroplast in M.humile, we tried to figure out the molecular mechanism of shifting to achlorophylly in the M.humile by focusing on plastid differentiation. The analysis using DAPI staining and transmission electron microscope showed that M.humile has undifferentiated proplastid-like organelle. Western blot analysis demonstrated that M.humile does not have any RuBisCO subunit proteins (rbcS,rbcL). The analysis of genome DNA and RT-PCR analysis demonstrated that rbcS is transcribed, but rbcL is not. Because rbcL is transcribed by bacterial RNA polymerase (PEP) concerned with transcriptional control for differentiation of plastid to chloroplast, we suggest that proplastid in M.humile does not differentiate to chloroplast due to lacking the function of PEP.

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Although mesophyll chloroplasts have generally been used for studies on plastid replication, recent studies have suggested that there is tissue-dependent regulation of plastid replication, and that stromule formation may be involved in this process. Here, we report the detailed morphology of immature chloroplasts and their stromules in the leaf epidermis, the intraplastidic localization of chloroplast division regulators AtMinE1 and AtFtsZ1-1, and their associations with plastid replication and development in Arabidopsis. Using a functional AtMinE1-YFP fusion, we demonstrate that AtMinE1 concentrates at the constricting plane of the dividing plastids, and simultaneously diffuses throughout the stroma, in the epidermis of leaves and petals. We also show that the localization of AtMinE1-YFP is highly dependent on its expression level. In the knockdown atminE1 mutant expressing the plastid-targeted CFP, the size of epidermal chloroplasts varied widely, and chloroplasts emanated many stromules. Further examination of plastid morphology and visualization of AtFtsZ1-1 showed that stromule fission mediated by the FtsZ1 ring contributed to plastid heterogeneity in the atminE1 epidermis.

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Plastids and mitochondria have their own genomes descended from their ancestors, and the organelle DNA replications (ODR) of plant cells are coordinated with the nuclear DNA replication (NDR) as ODR precedes NDR during a cell cycle progression. Recently, we identified that a tetrapyrrole compound, Mg-Protoporphyrin IX (Mg-ProtoIX), is a cell-cycle coordinator from organelle to NDR in plant cells. Mg-ProtoIX somehow activates CDKA to direct the G1/S transition¹. However, the molecular mechanism for CDKA activation remained elusive. Here we identified an F-box protein Fbx3, which inhibits CDKA by ubiquitinating the cyclin 1 and inducing the degradation. Mg-ProtoIX binds to Fbx3 and inhibits the cyclin 1 ubiquitination. We will provide a model of molecular mechanisms for coordination among organelles and nuclear DNA replication.
1 Kobayashi, Y. et al. Proc. Natl. Acad. Sci. USA 106, 803 (2009).

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Chloroplasts have their own DNA and gene expression systems that are derived from endosymbiosis of ancestral cyanobacteria. However, during long history of evolution, chloroplasts lost their autonomy and most of regulatory system became under control of the nucleus. In this work, we used the primitive, unicellular red alga Cyanidioschyzon merolae, which shows ancestral characteristics on many aspects including chloroplast genome and transcription systems. Therefore, gene expression in C. merolae chloroplasts can be regulated more autonomously than that in higher plants.
We previously found that the chloroplast two-component system, which is composed of the unique histidine kinase (HIK) and one of chloroplast-encoded response regulators (Ycf27), is involved in this regulation. In this work, we further analyzed function of HIK. Expression level of HIK was constant in various light conditions, suggesting that activity of HIK could be regulated post-translationally. Including other data as well as structural characteristics of HIK, the system for light-responsive transcription regulation in C. merolae chloroplasts will be discussed.

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Cytoplasmic male sterility (CMS), a trait of failure to produce functional pollen caused by the aberrant mitochondrial genes, has been focused to study plant mitochondrial-nuclear communication. The sterility is repressed by nuclear genes designated as restorer of fertility (Rf) genes, which mostly encode pentatricopeptide repeat (PPR) proteins. Actual molecular mechanisms of Rfs are largely unknown, although it has been speculated the Rf protein directly suppresses the expression of CMS gene at RNA level.
The aberrant mitochondrial orf125 is associated with Kosena CMS radish, and the fertility is restored by a PPR protein, Raphanus sativus Rf (rsRf). To elucidate the molecular mechanism of reduction of ORF125 by rsRf, the RNA binding properties of recombinant rsRf with orf125 mRNA was analyzed. The rsRf was shown to interact with the downstream of start codon of orf125 mRNA.
It has been identified rsRf-like genes from various radish species with several amino acid polymorphisms, which could influence the restoration of fertility. We would also present our current studies involving the RNA binding activity of the polymorphic rsRf genes to discuss their functions as the restorers.

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Roughly 470 million years ago, the land colonization of plants was one of important events of biological evolution, and initiated the establishment of the modern earth's environment. The process of the land colonization of plants is a multifaceted topic, paleontology, biology, geology and so on. To elucidate biological determining factors for the land colonization of plants, we investigated organelle DNA of klebsormidium flaccidum (Klebsormidiales, Charophyceae).
In previous studies, phylogenetic and cell-biological analyses supported that land plants ancestor derived from Charophyte. The morphology of Klebsormidium is extremely simple in Charophyte. However Klebsormidium is widely distributed in both fresh water and land, and adapt to dry and oligotrophic environments. Thus, to know how klebsormidium adapt to land environment is a key to clarify biological determining factors for the land colonization of plants.
In this presentation, we report comparative analysis of organelle DNA sequences of klebsormidium and other plants, and discuss causal biological factors for the land colonization by plants.

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Mature plant cells contain large central vacuoles that occupy up to 95% of the total cell volume. It is still unclear how these vacuoles are generated. To investigate this process, we examined Arabidopsis and Tobacco root tip tissues that contain vacuoles of various sizes during different stages of cell development. To observe the dynamic membrane structure, we used the high-pressure freezing/freeze-substitution method (HPF/FS). The anti-vacuolar H⁺-pyrophosphatase (V-PPase) antibody was used for immunogold labeling of Arabidopsis and Tobacco root tip cells, and positive signals in the vacuolar membrane and a novel ring-like structure were detected. This ring-like structure was a flat and/or double-membrane structure that surrounded a cytoplasmic component. Further, we observed some ring-like structures fused with one another or fused with the central vacuoles. These ring-like structures were expected to develop into vacuoles. In addition, we found that the complex structures that fused with several ring-like structures were mainly present in the stele and not in meristematic cells. These ring-like structures containing V-PPase may play an important role in vacuole formation.

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In higher plants, peroxisomes are known to differentiate into several classes. Glyoxysomes that are abundant in cotyledons, contain enzymes of β-oxidation and glyoxylate cycle to degrade storage lipids for postgerminative growth of oil-seed plants. Leaf peroxisomes that are present in green leaves, participate in photorespiration with chloroplasts and mitochondria. Peroxisomal malate dehydrogenase(PMDH) is an enzyme of glyoxylate cycle, and exists as two isoforms, PMDH1 and PMDH2 in Arabidopsis.
We previously investigated transcription levels of the PMDH genes by RT-PCR and phenotypes of Arabidopsis pmdh mutants. These data showed that PMDH1 is mainly accumulated in glyoxysomes and function not only in glyoxylate cycle but also in β-oxidation, whereas PMDH2 is mostly accumulated in leaf peroxisomes and involved in photorespiration.
To reveal developmental and tissue-specific expression of PMDH genes, we generated transgenic plants expressing PMDH-GFP fusion genes driven by their own promoters. In this study we report the expression profiles of PMDH1 and PMDH2 based on information from fluorescent microscopic analysis.

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Root hair formation is induced in lettuce seedlings when they are transferred from pH 6 to pH 4 medium. In future hair-forming cells, transverse cortical microtubule (CMT) arrays randomize before root hairs form. CMT randomization is necessary for root hair initiation and it is induced by ethylene and auxin. Recently, it became clear that abscisic acid (ABA) also induced root hair initiation. Here, we examined the relationship between ABA and CMT randomization, and the crosstalk between ABA and auxin or ethylene during CMT randomization.
Addition of ABA induced CMT randomization and root hairs formed at pH 6. However, when ABA was applied together with inhibitors of ethylene or auxin, neither CMT randomization nor root hair initiation occurred. Addition of ABA biosynthesis inhibitor abamine inhibited CMT randomization and root hair initiation at pH 4. These results suggest that although ABA promotes CMT randomization, ABA itself can not induce it. ABA may induce CMT randomization in cooperation with ethylene and auxin. To make their relationship clearer, we are now observing CMTs in seedlings treated with abamine plus ethylene or auxin at pH 4. The results will also be reported.

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In growing plant cell, cortical microtubules play an important role in regulating the direction of cell expansion. The phs2 is recessive mutant, which shows slightly left-skewing of root growth under a usual MS condition. But in the presence of 3uM propyzamide, the root growth shows right-skewing. The phs2 mutant had a frame-shift mutation in a novel gene. The genomic fragments including the loci rescued the phs2 drug responses, resulting that the loci was identified as the PHS2 gene. The PHS2 is encoded a TPR motif protein. The four homologous genes exist in Arabidopsis. We constructed GFP-PHS2 transgenic plants using genomic PHS2 clone. Their plants rescued the phs2 phenotype, showing that the GFP-PHS2 protein is functional. The fluorescent were detected on cortical microtubule fibers in the root, hypocotyl and leaf epidermal cells. These results were suggested that PHS2 is a novel microtubule associate protein, which regulates cortical microtubule array in plant.

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Boron is an essential micronutrient required for plant growth and development. A mutant with inhibited root elongation under boron deficient and normal conditions was isolated from EMS treated Arabidopsis M2 populations. Map-based cloning revealed it is a new allele of mur1 mutants, mur1-9. Arabidopsis mur1-1 is a dwarf mutant with altered cell wall structures, in which L-fucose is partially substituted by L-galactose. In boron deficient condition, root elongation of mur1-9 was inhibited to ~70% and rhamnogalacturonan II (RG-II) borate diester cross-links decreased to ~50% that of wild-type. Interestingly, inhibited root elongation of mur1-9 was recovered by supply the excess boron, but the ratio of cross-liked RG-II did not change significantly. Furthermore, application of L-fucose was also induced root elongation of mur1-9 under low boron condition. These results imply that boron performs important roles in Arabidopsis root elongation through an RGII-independent pathway.

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It is known that vessel and fiber cells form thick secondary cell walls (SCWs). TED6/7 genes, encoding membrane proteins, have been isolated as genes strongly expressed during xylem vessel cells differentiation. Suppression of expression of both TED6/7 genes induced aberrant SCW formation, and TED6 protein formed protein complexes with a subunit of the SCW-related cellulose synthase. These data suggested that TED6/7 play important roles in SCW formation.
To elucidate the role of TED6/7, here we attempt to purify and identify protein complexes which bind to TED6 protein. We constructed vectors for the expression of TED6 protein fused with the tag sequence driven by TED6 promoter (TED6pro:TED6-tag), which were introduced into a transgenic plant overexpressing VND7, a key regulator of xylem vessel differentiation, under the control of the DEX induction system. We extracted crude protein and purified protein complexes by antibodies against the tag sequences. By using mass spectrometry, we identified a number of the candidate proteins that form complexes with TED6 protein. We are currently investigating the binding between TED6 and the identified proteins in vivo by BiFC analysis.

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Fistulifera sp. JPCC DA0580 is an oleaginous marine diatom and hence promising strain for application to biodiesel production. In this study we investigated growth characteristics of this alga in ca. 1-liter culture scale. Culture medium was first modified and algal biomass productivity increased more than 10-fold in comparison with previous result. This alga showed optimum growth temperature between 25-30^οC and tolerance against higher temperature up to 35^οC. The algal growth was hardly affected by pH of culture medium between 7 to 9. The cells could grow in 2 to 8-fold diluted seawater. Optimum CO₂ concentration for growth was 0.5 % (v/v). Under optimized culture conditions, 6.6 g dry weight/liter/week was finally obtained and thus project's target productivity (4.0 g dry weight/liter) was achieved at laboratory culture scale. This alga seems to be applicable for cultivation with using alkaline wastewater-seawater mixture. High-temperature tolerance of this strain is advantageous in outdoor cultivation. Requirement of relatively low CO₂ concentration for growth can bring about reduction of CO₂ cost. [This study is supported by JST/CREST]

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To create novel floral traits in horticultural flowers efficiently, we have introduced many kinds of Arabidopsis chimeric repressors with a transcriptional repression domain (SRDX) driven by CaMV 35S promoter into torenia. While various flowers with characteristic traits have been obtained so far, some of them were accompanied by unfavorable effects such as phenotypic changes in leaves and defects in opening flower buds. We therefore utilized floral organ specific promoters instead of CaMV 35S promoter not only to avoid these problems but also to create novel floral phenotypes. In this study, we produced novel floral traits with the chimeric repressors of Arabidopsis MYB24 and TCP3 using floral organ specific promoters in torenia. While a 35S:MYB24-SRDX torenia formed unopened flower buds and exhibited phenotypic change in leaves, expression of MYB24-SRDX driven by floral organ specific promoter leaded to blooming and induced distinctive petal phenotype without phenotypic change in leaves. Furthermore, the expression of TCP3-SRDX driven by five types of floral organ specific promoters enabled to create characteristic floral traits in each promoter.

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Bolting of lettuce induced by high temperatures is an agriculturally undesirable event, so it is important to reveal the mechanism of the transition from vegetative to reproductive growth in lettuce. The FLOWERING LOCUS T (FT) genes play important role in inducing flowering in several plant species. To understand the floral induction of lettuce, we isolated FT gene (LsFT) from lettuce. Sequence analysis and phylogenetic relationships of LsFT revealed considerable homology to FT genes of arabidopsis, tomato, and other species. LsFT had the potential to induce early flowering in transgenic arabidopsis, but did not affect completely compared with FT of arabidopsis. LsFT mRNA was abundant in the largest leaves in flowering-inducible condition (higher temperature). These gene expressions showed good correlation to the flower differentiation of the shoot apical meristem. Our results indicated that LsFT is putative FT homolog gene in lettuce that regulates flower transition similar to its homolog in arabidopsis. This is the first information of the lettuce floral integrator gene for understanding the regulation of the flower transition in lettuce.

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We reported at the former meeting that Arabidopsis FT (AtFT) under rolC promoter set flowers in vitro in 3 to 5 months after the transformation. In this experiment, Arabidopsis TWIN SISTER OF FT (TSF) was introduced to apple under 35S or rolC promoter. The 35S::AtFT transformed apple showed early flowering in vitro, but the flowers on the shoot tips were 5 mm long and died without blossom. The 35S::TSF and rolC::TSF transformed apple set flowers in vitro, respectively. The precocious effect by FT gene was supposed the following order, AtFT >TSF>MdFT. The 35S promoter was more effective than rolC. These results meant that the precociousness was highly affected by the FT gene species and promoters. In the case of AtFT fused GFP, 35S::AtFT/GFP or rolC::AtFT/GFP transformed apple could be detected with the fluorescence from GFP and the fused proteins. But both transformants did not set flowers in vitro at all. These results suggested that the promotion of apple flowering by each FT gene depended on their protein sequences and structures. Then we tried to compare with the sequence between MdFT1, MdFT2, AtFT, TSF and Hd3a and analyzed the domains for the precocious effects.

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Sugi (Cryptomeria japonica) pollinosis has become serious a social problem. Biotechnological control of flowering is expected as a means for regulating pollen dispersal of C. japonica. We have reported that the twelve MADS-box genes were isolated from C. japonica, and they were classified into six clades. To determine the effect of MADS-box genes of C. japonica on flowering time, six kinds of MADS-box genes were transformed into Arabidopsis. Two kinds of six transgenic Arabidopsis lines flowered earlier than wild type plants. They were transformed by CjSOC1and CjAGL6 belonged to the SOC1 clade and AGL6 clade, respectively. Expression analysis of theses two genes in transgenic Arabidopsis indicated that transformants expressing exogenous genes at high level flowered earlier than those at low level. We examined the change of expression of CjSOC1 and CjAGL6 in shoots of C. japonica treated with gibberellin to induce bud formation artificially. Expression of CjSOC1 increased within a week after treatment, while that of CjAGL6 increased after one month of treatment. Our results suggested that CjSOC1 and CjAGL6 could function to promote flowering in C. japonica.

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Promoter behaviors at the single cell level are of great interest. Variance of promoter activity between cells and its fluctuation in single cells should be unveiled for the clarity of mechanism and meanings of biological systems. We succeeded in monitoring the promoter activities in single cells in the plant body by using a bioluminescence reporter imaging technology. We used Lemnaceae plants (duckweeds) that are suitable for imaging of the entire body due to the flatness and tininess. We introduced luciferase reporter genes under control of promoters into cells of plant surface by using a particle bombardment method, and then those plants were monitored for the bioluminescence of transfected cells with an EM-CCD camera system. We succeeded in obtaining time-series data of bioluminescence under the growth conditions with various light-dark schedules. We applied promoters with constitutive or circadian expression to duckweeds (Lemna and Spirodela genera) and also Arabidopsis.

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Circadian clocks allow plants to adapt their daily physiological activities to the daily changes in the environment. The Lemnaceae (duckweed) family are monocot plants. Two species in the Lemna genus, L. gibba and L. paucicostata are a long-day and a short-day plant respectively. There are many physiological studies of their circadian rhythms. We developed a real-time monitoring system of circadian gene expression in Lemnaceae using the combination of the particle-bombardment method and bioluminescent reporter genes.
To study the functions of the endogenous clock genes in Lemnaceae in detail, we have been trying to develop a stable-transformation method. By using an Agrobacterium-mediated transformation of Lemna calli, the reporter-gene expression was detected in the callused cells. Through the analyses of Lemna calli, it was suggested that the circadian rhythm of them appeared to be different from that of Lemna plants. We will report the progress of the gene introduction method for Lemna calli, and discuss the properties of promoter activities and circadian rhythms in them.

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In cyanobacterium Syncehococcus elongatus PCC7942, self-sustainable oscillation of KaiC phosphorylation has been reconstituted in vitro, demonstrating that this cycle is the basic time generator of the circadian clock. The ATPase activity of KaiC satisfies the characteristics of the circadian oscillation, the period length, and the temperature compensation. KaiC possesses extremely weak but stable ATPase activity in vitro. Moreover, the activities of wild-type KaiC and period-mutant proteins are directly proportional to their in vivo circadian frequencies, indicating that the ATPase activity defines the circadian period. Thus, we attempt to demonstrate that the energy released upon ATP hydrolysis is stored in KaiC as structural tension which downregulates its own ATPase activity.

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Cyanobacteria are the simplest organisms to exhibit circadian rhythms, which have been exclusively studied in the unicellular strain, Synechococcus. Since Anabaena is one of the simplest multicellular organisms harboring both regular pattern formation with cell differentiation and presumably circadian rhythms, it provides an excellent model to address following questions: 1) difference in the kaiABC functions and output pathways between Synechococcus and Anabaena; 2) clock systems in vegetative cells and heterocysts; 3) synchronization of neighboring clock cells; and 4) circadian modulation of heterocyst differentiation/patterning. Surprisingly, in contrast to the highest amplitude cycle of Synechococcus kaiBC mRNA, none of Anabaena kai genes showed significant expression rhythms, while we found ~200 clock-controlled genes. It suggests striking differences in clock and clock-controlled gene expression mechanism between the two species. Nevertheless, by using bioluminescence reporters to monitor rhythmic gene expression, we confirmed that both strains have similar circadian properties in terms of temperature compensation of the period and phase-response curves against dark-pulses.

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Physiological rhythms with a cycle of about 24 hour period are called circadian rhythms. The rhythms share "robustness of period" against environmental fluctuations. Specifically, the robustness for temperature perturbations is called "temperature compensation".
An empirical fact about temperature compensation is known; the range of temperature where temperature compensation works is identical to the range of physiological temperature. If ambient temperature is out of the range, circadian rhythms can no longer be observed. Although many reports have mentioned this phenomenon, it is unclear if temperature stimulus affect circadian clock directly or through indirect paths, e.g. metabolic changes.
In this presentation, we will focus on why circadian rhythms can not be observed at low temperature. The circadian clock of cyanobacteria, the simplest organism that shows circadian rhythms, has been well examined. KaiC phosphorylation rhythms had been specified as the central oscillator generating rhythms and even reconstituted in vitro. Combining behavior of the chemical oscillator and knowledge of bifurcation theory, we will present a scenario of circadian arrhythmia at low temperature.

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Arabidopsis ENHANCER OF SHOOT REGENERATION (ESR) 1 and ESR2 are thought to play critical roles in in vitro shoot regeneration. In this study, we investigated the relationship between ESR1/ESR2 expression and shoot formation during in vitro shoot regeneration using ESR1::GFP and ESR2::CFP constructs. ESR1 initially expressed 1 day after trasfer onto shoot-inducing medium (SIM) whereas ESR2 initiated to express on day 4. ESR1 expression occurred prior to shoot formation and the expression continued during early stages of shoot formaion. ESR1 and ESR2 expressed in shoot apical meristem-like structures. These results suggest that ESR1 may act on commitment to shoot differentiation and ESR1 and ESR2 are involved in early events of shoot formation.
We also examined ESR2 transcript levels when translocation of ESR1 to nuclei was induced. Treatment of an inducer of expression increased ESR2 transcript levels approximately 2.9-fold higher. Howerever, application of cycloheximide inhibited upregulation of ESR2 transcripts. These results suggest that ESR1 upregulates ESR2 expression, although the upregulation is indirect.

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A rice proteinase REP-1 degrades seed storage proteins, and expression of the gene Rep1 is induced by gibberellin (GA). We found that two Myb transcription factors interact with a cis-element CARE in the Rep1 promoter, were encoded in the same gene and designated CTMyb1L (long type) and CTMyb1S (short type). CTMyb1S activates the Rep1 promoter with a GA responsible transcription factor OsGAMyb synergistically, but over-expression of CTMyb1L has no effect on Rep1 activation (Sutoh et al. 2007). In this study, bimolecular fluorescence complementation was carried out to analyze the complex formation between CTMyb1 and a DOF transcription factor RPBF involved in GA response and their localization. The complex between RPBF and CTMyb1S was detected in nucleus, although that between RPBF and CTMyb1L was found in cytoplasm. Co-expression of CTMyb1L and RPBF activated the Rep1 promoter, but CTMyb1S with RPBF did not. Taken together, it is probable that the RPBF CTMyb1L complex exports negative regulators outside nucleus, so that Rep1 expression is promoted.

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Rice (Oryza sativa ) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. The rice flo2 mutation resulted in reduced grain size and starch quality. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Map-based cloning identified FLOURY ENDOSPERM 2 (FLO2) , as the gene responsible for the rice flo2 mutation. The wild-type FLO2 restored the size of grains of the flo2 mutant. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves. FLO2 harbors a tetratricopeptide repeat (TPR) motif, considered to mediate protein-protein interactions. As the candidate proteins of interaction with FLO2, a bHLH protein and a LEA protein was detected. These results suggest that FLO2 plays a pivotal regulatory role in the rice grain size and starch quality, by affecting storage substances accumulation in the endosperm.

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In our previous study, we found that CLE1-7 encoding 12 amino acid signaling peptides repress lateral root (LR) elongation associated with the function of CLV1 receptor kinase. Regulatory components directly controlling LR elongation can be postulated downstream of the CLE-CLV1 signaling cascade, however, its function in roots has not been clarified yet. In this study, we surveyed candidates of the downstream components of CLE-CLV1 cascade, and analyzed their functions in root development.
We first conducted microarray analysis of mRNA from roots of CLE3 overexpressor plants or clv1 mutant, and compared with the wild-type plant roots. We found nine genes, which responded oppositely between CLE3 overexpressor and clv1 mutant. Among these genes, we focused particularly on three genes whose transcript levels were most significantly affected by CLE3 overexpression and/or clv1 mutation.
These three genes encoded a heat-shock protein, a putative transcription factor and a transporter family protein, respectively. Overexpressors and promoter-GFP lines for all three candidate genes were constructed. Localization and roles of these candidates in root development are being analyzed.

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Phosphatidylserine (PS) has many important biological roles, but little is known in plants. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6% pss1-1 pollen death in PSS1/pss1-1 plants. However, Alexander's staining indicated 79.9% dead pss1-1 pollen, suggesting that pss1-1 and PSS1 pollen must undergo sporophytic cell death. Analysis with the nuclear stain DAPI showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores, and nuclear localization of PS seems to be important for maturation of Arabidopsis microspores.

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Spring ephemerals are a group of herbaceous plants that fulfill their life cycle on the floor of deciduous forests in temperate and boreal regions during a short period of time between snowmelt and closure of canopy of trees. Near the closure, synchronous voluntary senescence of their aboveground parts occurs and underground storage organs lie dormant until the next spring. An endogenous factor must be involved in this rapid synchronous senescence. Strong senescence-promoting activity was detected in extracts of shoots of a spring ephemeral, Gagea lutea, by a bioassay. The active compound was identified as free a-linolenic acid. The level of linolenic acid was very low one week before flowering, increased rapidly with time and reached a maximum one week after flowering. Senescence became prominent thereafter. These results suggest that the senescence is triggered by an accumulation of linolenic acid. Maintenance of shoots on the dim forest floor must cause a deficit of storage compounds. To minimize the cost, spring ephemerals seem to have acquired voluntary active senescence thereby filling a narrow niche on the forest floor.

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The balance between cellular proliferation and differentiation is a key aspect of development in multicellular organisms. Using high-resolution expression data from the Arabidopsis root we identified a transcription factor, UPBEAT1 (UPB1) that regulates this balance. Genome-wide expression profiling coupled with ChIP-chip analysis revealed that UPB1 directly regulates the expression of a set of peroxidases that modulate the balance of reactive oxygen species (ROS) between the zones of cell proliferation and the zone of cell elongation where differentiation begins. Disruption of UPB1 activity alters this ROS balance leading to a delay in the onset of differentiation. Modulation of either ROS balance or peroxidase activity through chemical reagents affects the onset of differentiation in a manner consistent with the postulated UPB1 function. This pathway functions independently of auxin and cytokinin plant hormonal signaling. Comparison to ROS-regulated growth control in animals suggests that a similar mechanism is used in plants and animals.

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MCA1 is identified candidates for the Ca²⁺-permeable mechanosensitive channel in Arabidopsis thaliana, and MCA2 is the only paralog of MCA1. In the roots, Ca²⁺ uptake activity is lower in the roots of mca2 than those of wild type, while there is no significant change in Ca²⁺ uptake activity between mca1 and wild type. MCA1 is expressed in the whole region of primary root, and MCA2 is expressed only in the elongation zone of primary root.
In this paper, we kinematically analyzed the root growth of mca1, mca2 and mca1mca2 double mutant. The analyses showed that there was no significant change in the root growth of mca1 compared to that of wild type plant, however the cell size and cell volume growth rate of mca2 is lower than those of wild type in root elongation zone. The region where the root growth of mca2 decreased is consistent with the expression pattern of MCA2. mca1mca2 also showed a decrease in cell size and cell volume growth rate in the same region, but the degree of decrease is less than that of mca2. These results suggest that the Ca²⁺ uptake by MCA2 directly affects the primary root growth, and MCA1 may mediate some signal by a slight Ca²⁺ uptake.

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Autophagy is a degradative pathway of cellular components. It occurs widely in the eukaryote. In plant autophagy, it has been thought that autophagosomes and/or autolysosomes, which are formed during the proceeding of autophagy, eventually fuse with vacuoles. We have reasoned that such fusions cause the expansion of vacuoles and the elongation of cells. In this study, we have examined whether autophagy is related to cell elongation or not.
We excised root tips of 5 mm long from Arabidopsis seedlings and incubated them for 2 to 3 days in a culture medium. The root tips elongated when sucrose was present in the medium, but hardly elongated when sucrose was absent. This result shows the elongation of root tips largely depends on sucrose in the medium in our experimental system. The elongation that occurs in the presence of sucrose was inhibited by addition of the protease inhibitor E-64d and of the autophagy inhibitor 3-methyladenine into the medium. Furthermore, mutants, in which the autophagy-related gene ATG5 is disrupted, exhibited slower elongation growth than the wild type. These results show that autophagy contributes to cell elongation.

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Plasma membrane (PM) H⁺-ATPase is an enzyme mainly related to the membrane transport in plants. PM H⁺-ATPase is involved in many physiological functions,mineral nutrition, metabolite translocation and cellular growth. In Nicotiana plumbaginifolia and Arabidopsis, PM H⁺-ATPases are encoded by a family of about 10 genes, organized into five subfamilies. It is suggested that each PM H⁺-ATPase isoforms in fact function in different cells and tissues or at different metabolic states and under different environmental conditions. Flowers of Portulaca generally open in response to a temperature rise and a light exporsure. We had reported that Vanadate-sensitive ATPase activity in crude microsome fraction of petal increased during the dark period just before the flower-opening and flower-opening process induced by light irradiation. One of the main factors of these changes was the increase of enzyme amount. In this study, we isolated 4 new PM H⁺-ATPase genes from petals. They were classified into the subfamily II and their time-course expression profiles and organ specific ones were investigated. We now examine about the effects of light and temperature condition on their expression.

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It is recently suggested that ZIP family, one of the most important transporter families involved in metal uptake into the cytoplasm, plays a critical role on metal accumulation or tolerance in metal hyperaccumulator plants, but the detail role remains unclear. Here, we report the function of TjZNT1, a ZIP transporter of a Ni hyperaccumulatorThlaspi japonicum, in Ni tolerance. A sequence similarity search and yeast assay showed that TjZNT1 is an ortholog of a Zn/Cu transporter of Arabidopsis thaliana, AtZIP4. As a result of TjZNT1 promoter-GUS reporter assay in T. japonicum, GUS stain was observed in the steles of roots and veins, suggesting that TjZNT1 is involved in the root-to-shoot translocation of Zn/Cu as well as AtZIP4. A qRT-PCR study showed that the transcriptional level of TjZNT1 in T. japonicum was 1000-fold higher than that of AtZIP4 in A. thaliana, indicating that T. japonicum constitutively expresses TjZNT1 at high level. Furthermore, the yeast strain over expressing TjZNT1 showed high level Ni tolerance, suggesting that the uptake of Zn/Cu contributes to excess Ni tolerance. We presently investigate the Ni tolerance of transgenic A. thaliana expressing TjZNT1.

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Arabidopsis thaliana metal tolerance protein 1 (AtMTP1) transports zinc into vacuoles by Zn²⁺/H⁺ exchanger mechanism. AtMTP1 has six transmembrane domains and a histidine-rich (His-rich) loop between the 4th and 5th transmembrane domains. Deletion of the most part of the loop decreased Km for Zn²⁺ and increased Vmax of AtMTP1.
In this study, we divided the His-rich loop (25 His residues) into 5 sections, which contain 5 His residues, and substituted His residues in each section with Ala. The AtMTP1 mutants were expressed in a zinc sensitive strain (Δzrc1cot1) of Saccharomyces cerevisiae. The growth of yeast varied with AtMTP mutants under excess Zn conditions. This indicates that each His residue in the loop has an individual role in transport of Zn. Furthermore, we examined the growth of yeast expressing mutant AtMTP1 in the presence of Co²⁺, Cd²⁺, Mn²⁺ or Ni²⁺. We will discuss the metal selectivity of these AtMTP1 mutants.

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Rice grains are matured in 40 days after flowering. During that time, intense changes can be seen such as storage protein and starch accumulation, decrease in water content etc. To identify aquaporins involved in these processes, mRNA expression levels were analyzed in a realtime-PCR method at each stage. As a result, many aquaporins were expressed in early ripening stage. In contrast, OsPIP2;1 and OsTIP3;1 accounted for the most part of aquaporin mRNA expression level in mid-ripening stage. Western blotting results showed that OsPIP2;1 and OsTIP3;1 proteins increased rapidly from mid-ripening stage. Furthermore, immunocytochemical analysis clarified that OsPIP2;1 was present throughout the organ, whereas OsTIP3;1 was present only in the aleurone layer. In conclusion, it is assumed that OsPIP2;1 and OsTIP3;1 showed correlation with the rapid decrease of water content in rice grains during mid-ripening stage. In particular, OsTIP3;1 was only expressed in rice grains and was not found to be expressed in other organs. These results suggest that OsTIP3;1 has a unique function related to the aleurone layer.

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Cold acclimation process plays a vital role in plants survival to cold temperature stress. To elucidate the cold acclimation process, we analyzed the osmotic root hydraulic conductivity under cold stress for prolonged time and showed that chilling sensitive rice roots respond to the cold stress by a gradual increase in its osmotic hydraulic conductivity. Compared to 25C control plants, low root temperature treatment (LRT) at 10C dramatically reduced the hydraulic conductivity. However it gradually increased during the 5 day treatment and reached 10 fold higher at day 5 compared to the plants whose LRT treatment was just started. The gradual increase of the root hydraulic conductivity was found to be linked with a gradual increase in the expression of root aquaporins, specifically the OsPIP2;5. Further, the contrasting expression pattern of aquaporins under LRT and whole plant chilling techniques suggests that shoot signal is a prerequisite for triggering the root aquaporin signal. Collectively, these results demonstrate that cold acclimation process is also functional in chilling sensitive plants and regulated by inter-cellular signaling of aquaporin expressions.

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Mimosa pudica responds to external stimuli, including touch and heat, and quickly bends its petioles. Water movement through cell membrane occurs during the bending movement, resulting in the rapid loss of turgor pressure of pulvinar extensor motor cells. Thus, aquaporin (AQP) was considered to play a role in this rapid water movement. We have isolated 7 isoforms of plasma membrane intrinsic proteins (PIPs) and examined their water permeability. In the present study, we examine the expression levels of each isoform by RT-PCR. As a result, PIP2;2 and PIP2;3 were expressed in the main pulvinus, and PIP2;2 was abundantly expressed in extensor cells. Change in water permeability of PIPs in response to the rapid movement is thought to be regulated by phosphorylation. Therefore, effects of inhibitors were examined. As results, water permeability of PIP2;2 was improved by the dephosphorylation inhibitor. PKC phosphorylation motif is conserved at the C terminus of PIP2;2, thus mutagenesis was also performed. The result showed that all 3 residues in the motifs of PIP2;2 appear to be important for the phosphorylation-mediated regulation of water permeability.

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Zostera marina is a kind of marine angiosperms (seagrasses), and lives under the sea throughout the life cycle stages. Here, we study plasma membrane aquaporins (PIPs) of Z. marina to reveal the water control mechanism of seagrass cells in seawater. Generally, aquaporins facilitate water flux across biomembranes as water channels, and more than 30 kinds of aquaporins exist in the plant genome. We successfully isolated two genes for PIPs (ZoPIP1 and ZoPIP2) from leaves of Z. marina. They may function as water channels because the functional domains (NPA motifs and transmembrane domains) were conserved in their predicted amino acid sequences. The expression of ZoPIPs was detected in leaves, inflorescence axes, roots, rhizomes, female flowers, male flowers, seeds and fruits, and seedlings. In leaves, the expression of ZoPIPs was increased during desiccation. The higher expression of ZoPIPs was also detected during seed germination than seed maturation. These suggest that ZoPIPs may be involved in unique physiological features of Z. marina to adapt to the marine environment.

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