The functions of floral meristem identity genes, such as AP1, have already been identified in model plants with whorled flowers. However, little is known about the floral evocation in plants with spirally arranged parts.
Ranunculus scleratus(Ranunculaceae) is an annual plant whose flowers with spirally arranged stamens and carpels. It can be readily cultivated under laboratory conditions. Therefore, it is a very good target to investigate the early phases of flower development concerning spirally arrangement and numerous floral organs.
In this study, we isolate the and AP1 orthologs from R.scleratus and analyze their expression patterns during the vegetative and reproductive development of R.scleratus to elucidate differences in characteristics of the two genes between spiral and whorled flowers. Additionally, we mention the importance to establish a transformation system for R.scleratus in terms of functional genomics.

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Cucumber plants produce male and female flowers on the same plant. All flower buds contain both stamens and pistils at early bisexual stage, and sexual differences are established by the selective arrest of sexual organ primordia. Arrest of stamen primordia involves programmed cell death (PCD) in female flowers. In animals, extracellular cell matrix (ECM) plays an important role for PCD, morphogenesis, and cell-fate specification. ECM is degraded by matrix metalloproteinase (MMP). Recently, Cs1-MMP which encodes MMP was isolated from cucumber cotyledon, and was demonstrated to be involved in PCD. In the present study, expression of Cs1-MMP in cucumber flower organs was analysed. Expression of Cs1-MMP was great at both sepals and the area where pistil primordia arrested through male flower development. Thus, it is possible that Cs1-MMP at the area where pistil primordia arrested might play an important role for sex differentiation and male flower development in cucumber.

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Sugi (Cryptomeria japonica) is one of the most important economical tree species in Japan. However pollen allergy caused by a large number of pollen released from sugi has become a serious social problem. Recently, a male sterile sugi clone (named as Soshun) was found, and has been multiplied by tissue culture and cuttings. Moreover, if male sterile genes or its related genes are isolated, it is easy to attempt breeding of other many various sugi male sterile clones.
Male flowers from Soshun were gathered every week from late August. Its mature process was observed by an electron microscope, and a subtraction library was constructed following pollen development. About 200 candidate genes was isolated, of which, 17% was corresponded to PrMale gene from Pinus radiata. Similar, 6.8% was chalcone synthase-like protein and 6.3% was dihydroflavonol 4-reductase. More genes were screened and expression patter was investigated.

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The pollen coat is made up of substances secreted or released in the locule when tapetum cells disintegrate, and it fills the cavities between the baculae of the exine. The tapetum is characterized by the accumulation of lipid deposits in tapetosomes and elaioplasts. We exhibited in detail the appearance and behavior of elaioplasts and tapetosomes before and after the tapetum cells breakdown, using ultrastructural observation of wild type Arabidopsis. Furthermore, we observed the tapetum of mutants known as pollen coat less phenotype, exhibiting sterility. The cer1 mutants, a mutational loss of alkanes by impaired in the VLCFAs pathway, fused tapetosomes and elaioplasts in intact tapetam cells at the late dicellular pollen stage, although wild type fused them after tapetum breakdown. Furthermore, we indicated that the main lipid components of tapetosomes and elaioplasts are originated in MVA biosynthesis upstream of squalene.

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Pollen coat is a surface structure of Arabidopsis pollen grains, which consists mainly of proteins and lipids. These components are synthesized in tapetal cells and are deposited on the pollen surface after the tapetal cells are ruptured. Pollen coat is significant in fertilization. The EXTRACELLULAR LIPASE4 (EXL4) and EXL6 are abundant proteins in Arabidopsis pollen coat. Here we examined the EXL4 and EXL6 function. By the TEM and SEM observations, it was appeared that the pollen grains of exl4-RNAi exl6 plant lacked normal pollen coat and thus they showed the difficulty in water absorption from stigma. Proteomic analysis revealed that the pollen coat lacked most major proteins. The EXL4-GFP and EXL6-GFP were localized in unidentified granules in the tapetal cells. These results suggested that the EXL4 and EXL6 are required for the formation of pollen coat components in tapetal cells.

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Although jasmonic acid (JA) has been shown to be required for the elongation of stamens and petals and dehiscence of anthers, little is known how the biosynthesis of JA is turned on. We found that the level of DAD1 mRNA was decreased in the inflorescences of arf6 arf8 double mutant, which showed the depletion of JA. This depletion was partially rescued by the introduction of P_PISTILLATA:DAD1 into the mutant. The arf6 arf8 double mutant shows several developmental defects such as aberration of vascular and epidermal cell differentiation. We found that the expressions of class I KNOX genes are upregulated in the mutant and that the arf6 arf8 stm^+/- plants partially rescued the DAD1 expression and JA production. These results suggested that the repression of KNOX genes by ARF6/8 is required for the development of floral organs, and subsequently for the DAD1 expression and JA production, which induces the flower opening.

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Vacuolar processing enzyme (VPE) is involved in the pathogen-, the toxin-induced cell death and the cell death during seed coat formation. Arabidopsis has four VPE homologues, and two VPEs play the role in vegetative organs. Both the mRNA level and activity of the vegetative VPE were increased in the leaves during senescence. The VPE activity was suppressed in extract from ethylene-insensitive plant and salicylic acid-deficient plant. This result indicates that two phytohormones are involeved in the VPE activity during senescence. Leaf senescence delayed slightly in the Arabidopsis VPE-null mutant, which lacks all four VPE genes. Some proteinases that localize in the vacuoles accumulated in the leaves of the VPE-null mutant compared with wild-type leaves. Our results suggest that vegetative VPE regulates the activation of some functional proteins in the vacuoles during leaf senescence.

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We have been employing the Full-length cDNA Over-eXpresser gene (FOX) hunting system with transgenic rice plants for the genome-wide elucidation of rice gene functions. Among many FOX-rice plants, two lines showed promotion of internode elongation, early flowering and increase in endosperm size. Microscopic observation revealed that the growth promotion was caused by enhancement of cell division in the tissues, not by cell elongation. Both the lines had an identical transgene encoding a protein with TIFY/ZIM motif, which was designated FR29. Arabidopsis JAZ (JASMONATE ZIM-DOMAIN) proteins are reported to be transcriptional repressor(s) of the JA-responsive genes and the targets of SCF^COI1 ubiquitin ligase in the jasmonate (JA) signaling (Thines et al. 2007). The FR29-FOX rice plants exhibited reduced responses to JA and inhibition of JA-inducible gene expression. Our results suggest that the TIFY-motif-containing protein of rice regulates plant growth through negative regulation of the JA signaling.

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Excess amounts of reactive oxygen species are toxic to plant cells, while it has been shown that adequate degree of oxidative states sometimes promote growth and development of many plat species. Here we examined the effect of H₂O₂ treatment on germination and post-germinative growth of soybean.
We observed promotion of soybean germination by imbibing seeds in H₂O₂ solution. This promotion was observed on various soybean cultivars with different seed characteristics. No inhibition of germination was observed in our experimental conditions. H₂O₂-treated seeds were more vigorously germinated than those imbibed in oxygen-bubbled water. Thus, H₂O₂ treatment promoted soybean seed germination through positive effect other than O₂ formation by catalse(-like) reaction(s).
Post-germinative growth of soybean is retarded in anaerobic conditions. In low oxygen environment (4 to 5% O₂), H₂O₂-treated seeds were grown better than those imbibed in water, suggesting that H₂O₂ treatment also promotes post-germinative growth of soybean under anaerobic conditions.

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In Arabidopsis, the root of a light-grown plant has a higher density of root hairs than that of a dark-grown plant. Root hair formation is regulated by various factors such as phytochrome and hormone. To understand the molecular mechanism of light promoted root hair formation, we screened Arabidopsis activation tagging lines and isolated a new light-promoted root-hair development mutant, lrh1. By comparing root hair densities of the wild type and the lrh1 mutant under continuous light conditions, it was suspected that a mutated gene might induce root hair formation. Mutant seedlings also had shorter roots than wild type, and reduced numbers of lateral roots. A database search revealed that an 11-kilobase sequence adjacent to the location of T-DNA insertion contained two repeated sequences, hundreds of small RNAs, and several proproteins. We expect that lrh1 will be a useful tool for discovering novel factors to regulate root hair formation.

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Arabidopsis acaulis1 (acl1) mutants show growth defects such as curled leaves and short inflorescence stems at 22°C. The severe acl1-1 plants have tiny curly leaves, greatly reduced rosettes in size, and short inflorescences, which are only slightly longer than the height of the rosettes. A cluster of two or three flowers was produced at the top of the inflorescence stem. The weak allele acl1-3 plants also have small leaves, small rosettes and short inflorescence stems. High temperature is effective to restore acl1 phenotype and makes acl1 plants almost indistinguishable from wild type at 28°C.
We have treated acl1 seeds with EMS to screen enhancers and suppressors. Successfully, we have obtained mutants that could enhance or suppress acl1 phenotypes at 22°C. We also found that there is an inversion on chromosome 4 of the original acl1-1 genome. Although it is not the cause of acl1 phenotype, it enhances acl1-1 phenotype.

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In leaves of Arabidopsis thaliana, decreased cell proliferation activity often triggers excessive cell enlargement, a phenomenon that we named compensation. We have isolated and characterized five mutants (fugu1-fugu5) that exhibit compensation. The cotyledon of fugu5 is oblong on rockwool and exhibits strong compensation. However, we found that morphological phenotype of fugu5 is recovered in vitro cultures. Histological analysis showed that in fugu5 cotyledons grown in vitro, the number of cells recovered to wild type levels. On the other hand, compensated cell enlargement in fugu5 was completely cancelled. Analysis of fugu5 phenotype on MS-medium with different supplements revealed that sucrose was necessary and sufficient for fugu5 phenotype recovery. Similar concentrations of sorbitol did not affect fugu5 phenotype. Analysis of the effect of other sugars, such as glucose, fructose, and non-metabolizable analog on fugu5 phenotype is now underway. Based on these results, the mode of action of sucrose will be discussed.

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Salicornia herbacea is one of halophyte, growing under high salt conditions. In this study we investigated growth and development, and parameters regulating the growth of Salicornia. Germination rate was not affected by different concentrations of NaCl, although germination delayed under high concentration of NaCl. Growth of the seedlings was promoted at 100-200 mM NaCl and water contents of the seedlings did not change in these conditions. Viscosity in cell walls of hypocotyls was decreased under 100 mM NaCl condition. Osmotic concentrations of cell sap increased as higher concentrations of NaCl. As described above, NaCl (100 mM) promoted the growth and increased the fresh weight of hypocotyls compared to control, but KCl (100 mM) did not. On the other hand, mannitol (200 mM) inhibited the growth. These results suggest that Na⁺ is required for normal growth and development of Salicornia, depending on water uptake and promotion of cell wall extensibility.

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Plant cells surrounded with rigid cell walls cannot move during development. Cell shape plays an important role for organ shaping and depends on the directions of cell elongation. However, the genetic regulation of anisotropic cell expansion is not well known.
We isolated an Arabidopsis mutant, itosugi (itg), defective in elongation of hypocotyls and roots. Cytological analyses of itg mutants revealed that longitudinal cell elongation was suppressed and that radial cell expansion was promoted in hypocotyls and roots. ITG:GFP fusion protein was localized around the cell periphery. These suggest that ITG function around the cell periphery is required for anisotropic cell growth.

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To elucidate the signal cascade that amplifies the nutrient signal and regulates plant growth, we are trying to establish a high throughput screening system to identify novel proteins that interact with the known regulatory factor based on protoplast transient assay. In plant cells, multiple signaling pathways build a complex network to response to the environmental and developmental signals. Consequently, extracting one signaling pathway from such a network can be difficult. In this purpose, to identify the protein interaction is a certain approach, since the signal cascade results from the interactions between signaling molecules.
A regulatory factor fused with the artificial tag is expressed in Arabidopsis protoplast transiently, and the complex including the factor is immunoprecipitated by using the antibody to the tag. The composition of the precipitated fraction is solved by LC/ MS/ MS analysis. This high throughput system should be a useful tool to elucidate a signal cascade efficiently

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Plants respond to environmental stresses in multiple ways including changing the hormonal responses. The plant hormone auxin controls every aspect of growth and development. However,little is known about the effect of temperature stress on auxin response. To understand the mechanistic basis of cold temperature stress and auxin response, we characterized the root growth of Arabidopsis thaliana at 23°C after pre-incubating the seedlings at 4°C. The time course assay revealed that 8-12 hr pre-incubation at 4°C inhibited the root growth and reduced the gravity response approximately 50% compared to that of untreated controls. The auxin-signaling mutant axr1-3, which shows a reduced gravity response, responded to cold treatment like wild-type indicating that auxin transport rather than auxin signaling mechanism is affected by cold stress. Consistently,the expression of the auxin responsive marker was found to be altered in cold treated plants; accumulating GUS staining in the outer layer cells of the root meristem.

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Jasmonic acid (JA) is a phytohormone which has roles in regulating stress response and development. COI1 (CORONATINE INSENSITIVE 1) is an F-BOX protein working in ubiquitin-proteasome system. Most of JA responsive genes (JRGs) are regarded to be regulated by the COI1 complex. 12-Oxo-phytodienoic acid (OPDA) is a precursor of JA and also has certain role inducing COI1-independent gene expression in contrast to JA. Some OPDA responsive genes (ORGs) are confirmed to be induced without the COI1 complex.We analyzed gene expression responses to JA and OPDA under coi1 background using GeneChip and confirmed COI1-dependent and COI1-independent expression for JRG and ORGs. However, from 342 JRGs, two gene groups which show similarity in expression profiles between WT and coi1 mutant were obtained. More detailed analysis for COI1-independent JA responsive genes will be introduced in this presentation.

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The study of rice lamina has long history and always correlates to that of plant hormone. Two plant hormones, brassinosteroid (BR) and auxin are well-known regulator of lamina joint (LJ) bending. In this work, we analyzed the regulation of LJ bending by BR and auxin using BR-deficient and -insensitive rice mutant.
In wild-type rice, the response to BR and auxin was highest 3.5 to 4 days after sowing, namely, after the second leaf appears and before it fully expands. In the 4-day-old LJ, the expression of BRI1, the BR receptor, was observed both the adaxial and abaxial sides of leaf however, in the 5-day-old LJ, it was restricted to the adaxial side. The elongation of LJ was enhanced solely by the cell elongation. Interestingly, in BRI1-deficient d61 mutant, the response to auxin in LJ was decreased while, in BR-deficient d2 of brd1, it was increased than in WT.

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The formation and degradation of indole acetic acid-amino acid conjugates (IAA-aa) play an important role in auxin homeostasis. GH3s, the early auxin-responsive genes, encode IAA-aa synthetases. Their functional redundancy has hampered the genetic approach to study auxin homeostasis. GH3s belong to the acyl-activating enzyme superfamily that activates IAA by adenylation. According to this catalytic mechanism, we designed and synthesized N-acylsulfamoyladenosine derivatives (sulfamates) of auxins as the intermediate analog inhibitors of GH3s. We previously presented that these compounds served as potent in vitro inhibitors of GH3s, and also exhibited in vivo activities.
In this study, we have designed and synthesized a series of N-acylsulfamoylaminoadenosine derivatives (sulfamides, acyl = IAA, PAA, NAA and 2,4-D) to improve chemical stability and hydrophobicity of the inhibitors. The sulfamides were found to exhibit potent in vitro inhibitory activities toward GH3 and were stable under acidic and basic conditions.

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We isolated a novel ABA hypersensitive mutant named ahg12 (ABA hypersensitive germination 12) in Arabidopsis. Using a map-based cloning, we identified a missense mutation in the gene encoding a subunit of 26S proteasome. The ahg12 mutant also showed some pleiotropic phenotypes other than ABA hypersensitivity. The combination of phenotypes observed in ahg12 is distinct from those of other subunit mutants reported so far, suggesting that the ahg12 mutation affects the selectivity of protein degradation depending on the subunit. The ahg12 mutation, which is an unreported dominant mutation, is expected to provide new information about the functions of the subunit in the protein degradation and ABA response. Furthermore, since the 26S proteasome is highly conserved among eukaryotes, the findings from this study can be applied to other species. Detailed analyses of this mutation in plant and yeast are now ongoing.

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Degradation of the Aux/IAA proteins has been recently found to be the primary reaction of auxin response that is mediated through the auxin receptors. To better understand the degradation of Aux/IAA in the context of physiological responses of auxin, we have conducted measurements of half lives of luciferase (LUC) fused with IAA17/AXR3 or IAA19/MSG2 expressed constitutively by 35S promoter in Arabidopsis. When cycloheximide was added to the transgenic seedlings grown hydroponically for 4 - 5 days in the dark, luminescence decreased rapidly, from whose kinetics half life was determined. When LUC was not fused to any Aux/IAA proteins, its half life was longer than 60 min. In contrast, half life of IAA17- and IAA19-LUC was about 10 and 15 min respectively. Rapid degradation of IAA17 has been reported by Dreher et al. (2006). Our results showed that IAA19 was also degraded as readily as IAA17.

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Plant hormones play an important role as signaling molecules in the regulation of almost all phases of plant development. They regulate expression of genes via each signal transduction systems, and in most cases they mutually regulate the signaling and metabolic systems between the hormones. Such complicated regulatory systems enable plant to grow and develop normally. To understand the fine regulatory systems at molecular and hormonal levels, we have established a method for high-throughput analysis of major plant hormones namely, cytokinins, auxins, abscisic acid, and gibberellins. Our method needs 10 to 100 mg fresh weight of plant tissues for determination and enables us to analyze 180 samples at a time. Using our method, we analyzed FOX hunting Arabidopsis lines overexpressing rice full-length cDNA and a variety of rice and Arabidopsis mutants. We will present the analyzed data and discuss the correlation between the morphological phenotypes of the mutants and hormone profiles.

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Oxylipin, 9-hydroxy-10-oxo-12(Z), 15(Z)-octadecadienoic acid (KODA) is one of signal compounds which was mediated from the linolenic acid. It was found that KODA has a function to give stress tolerance and to activate inflorescence development in some species. In KODA's biosynthesis pathway, it has been suggested that the precursor dehydrated by two kinds of enzymes (LOX, AOS) like Jasmonate (JA). And it was also suggested that there were some differences in enzyme activity of biosynthesis between KODA and JA. Namely 9-specific oxidation enzymes were necessary in KODA biosynthesis instead of 13-specific enzyme. According to the current study, 9-specific enzyme identified that tissue-specific occurrence in vascular bundle in Lycopersicon esculentum and Solanum tuberosum. However there was little information of 9-specific enzyme in Arabidopsis thaliana. To detect 9-specific enzyme activity in Arabidopsis, We have done the functional analysis using by KODA.

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The filamentous cyanobacterium Anabaena sp. PCC 7120 develops heterocysts in response to deprivation of fixed nitrogen. Heterocyst is capable of fixing nitrogen. Various genes encoding the transcription factors regulating the differentiation and the enzymes involved in heterocyst envelope formation have been identified. A regular pattern has been observed for heterosyst spacing. However, no continuous observation of a single filament was attempted during the heterocyst diffentiation. In the present study, we observed single filaments of Anabaena continuously, and recorded the cell shape and fluorescence of cells with intervals of four hours. Moreover, we analyzed relationship of cell division and fluorescence intensity over the filaments of cells. The results showed that the cell division was not synchronized. We also found a periodicity in the fluorescence intensity of the cells over a single filament, which changed as a function of time after nitrogen depletion. The spatial periodicity may be related to heterocyst pattern.

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Asymmetric cell division in a stem cell generates two different daughter cells; one is a self-renewed stem cell daughter and the other is a differentiated, non-stem cell daughter. Although unequal distribution of mRNA or proteins has been known to play a pivotal role to specialize each daughter cell, such molecules in plants remain largely unknown. Protoplasts and apical stem cells of protonemata of P. patens divide asymmetrically to generate two different daughter cells. We have previously reported 58 genes as candidates involved in the asymmetric cell division. For those candidates, we made cDNA-citrine knock-in transgenic plants by using gene targeting technique to investigate protein localization under a control of their native promoters. We found some proteins accumulated preferentially in the stem cells but not in differentiated protonemal cells. We will report time-lapse imaging of these proteins during asymmetric cell division.

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Most of G2/M phase-specific genes contain a common cis-acting element (MSA element) in their upstream regions, which binds with the R1R2R3-Myb proteins that act as transcriptional activators or repressors. Two R1R2R3-Myb genes, MYB3R1 and MYB3R4, encode structurally closely related transcriptional activators in Arabidopsis thaliana. The myb3r1 myb3r4 double mutant shows decreased expression of the G2/M-phase-sepcific genes and various developmental abnormalities that are caused by incomplete cytokinesis. To identify the novel factors that are involved in transcriptional activation of G2/M phase-specific genes, we performed a phenotypical enhancer screen of the loss-of-function myb3r4 mutant that shows weak cytokenetic defects, and obtained several enhancer mutants. In addition, we found that a polymorphism in the wild-type Landsberg erecta accession significantly enhances the defective cytokinesis of the myb3r4 null mutant. We are currently isolating the gene whose Landsberg allele is responsible for the phenotypical enhancement by a positional cloning approach.

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The eukaryotic cell cycle is controlled by the activities of evolutionarily conserved protein kinase complexes, which consist of cyclins and cyclin-dependent kinases (CDKs). In Arabidopsis, the A- and B-type CDKs and the A-, B-, and D-type cyclins are assumed to be crucial for cell cycle progression. Among these regulators, we have focused on a plant-specific B2-type CDK (CDKB2) and its regulatory subunit cyclin D4 (CYCD4).
We have recently proposed that the abundance of CDKB2 protein is regulated not only transcriptionally, but also through proteasome-mediated protein degradation. We have also found that CYCD4s are expressed in the upper region of hypocotyls, and this result coincides with our observation that the CYCD4 overexpression promoted cell division in the stomatal lineage in hypocotyls. For more detailed functional analysis, we are currently crossing T-DNA knockouts and CYCD4-overexpressing plants with mutants which are known to have defects in stomatal development.

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26S proteasome plays an essential housekeeping role to eliminate the proteins which are damaged or misfolded. It is also essential for most, if not all, aspects of cellular regulation by removing rate-limiting enzymes and dismantling existing regulatory networks as a way to fine-tune homeostasis, adapt to new environments, and redirect growth and development. The 26S proteasome consists of two multisubunit complexes, 20S core particle (CP) and 19S regulatory particle (RP). The RP presumably functions to identify appropriate substrates which are degraded, to release the attached Ubs, to open the CP gate, and to unfold the substrate proteins leading into the CP. The RP contains three non-ATPase subunits (RPN) and a ring of six AAA-ATPase subunits (RPT).
Comprehensive studies on deficient mutant for RPT subunits for 26S proteasome in Arabidopsis were examined. Completed views about the functions of Arabidopsis RPTs will be discussed.

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Ubiquitin(Ub)/26S proteasome pathway plays an essential housekeeping role to eliminate the proteins which are damaged or misfolded. It is also essential for most, if not all, aspects of cellular regulation by removing rate-limiting enzymes and desmantling existing regulatory networks as a way to fine-tune homeostasis, adapt to new environments, and redirect growth and development.
We examined the isolation and characterization of the RPT2a deficient mutants in Arabidopsis, and the rpt2a mutant showed enlarged leaves, which are caused by accelerated endoreduplication. The endoreduplication is a type of cell cycle where chromosomal DNA replication occurs without cell division. In general 3-branched trichomes undergo four endoreduplication cycles resulting in a DNA content of 32C. The rpt2a showed increased numbers of trichome. We generated double mutants between rpt2a and several under-branched trichome mutants. The function of RPT2a in terms of trichome formation will be discussed.

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Progression of cell division cycle is exposed to a variety of internal or external stresses. Failure of proper adaptation to these stresses leads to interruption of DNA replication or to abnormal chromosomal segregation. The cell-cycle checkpoint system monitors the progression of cell cycle, detects a problem such as DNA damage, and arrests the cycle until the problem is solved.
We previously isolated a UV-sensitive mutant suv2 in Arabidopsis. The suv2 was also sensitive to γ-ray, MMC, and HU, which is reminiscent of the phenotype of AtATR-disrupted mutant in Arabidopsis (Culligan et al., 2004). The SUV2 encodes a 646 aa protein containing a coiled-coil domain and two putative target sequences of PI3K-related protein kinase. The SUV2 protein could form a homodimer in the yeast cell and was phosphorylated with the Arabidopsis nuclear extract. These results strongly suggest that the SUV2 is an Arabidopsis homologue of ATRIP/Rad26.

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Endoreduplication is a special cell cycle containing successive DNA replication in the absence of mitosis. But, little is known about the molecular mechanism of endreduplication.
To reveal this mechanism, we analyzed a Arabidopsis mutant, increased level of polyploidy4-D (ilp4-D), which was isolated from activation tagging lines. ilp4-D showed increased polyploidy in the seedlings. ILP4 gene encodes a protein that contains a kinase domain. Histochemical analysis using a ILP4promoter-GUS reporter gene showed ILP4 was expressed in part of root, cotyledon and hypocotyls, suggesting ILP4 expression has cell specificity. ILP4 protein localized in nuclear analyzed with GFP fusion protein.
T-DNA insertion lines of ILP4 were did not show any obvious phenotype. ILP4 and ILP4L1 (ILP4 homologue) double mutant showed slightly decreased polyploidy level in dark seedlings.
Overexpression of ILP4 gene which was deleted kinase domain led to increase polyploidy level. These results suggested ILP4 kinase domain is not related to endocycle.

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In RIKEN PSC, 18,000 transposon insertion mutants of Arabidopsis were established. From these transposon tagging lines, several seedling lethal mutants were identified and we named segregation distortion (sd). One of them, sd5 did not develop mature leaves and this phenotype was caused by transposon inserting in At3g24730.
The protein encoded by At3g24730 is homologous to DIM2, a component of spliosome. Animal and plants have a DIM family comprised of DIM1 and DIM2, but yeast has only DIM1. Loss of DIM1 caused lethal as a result of defect of cell cycle in S.pombe. SD5 protein did not complement to S.pombe dim1-35 mutation when SD5 was expressed at limited temperature although Arabidopsis DIM1 complemented to this. This result show SD5 has defferent functions of DIM1 protein.
Here we show the detail of sd5 phenotypes and the SD5 functions.

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B-type cyclins are known as mitotic cyclins. We have isolated and sequenced a cDNA clone encoding a cyclin B gene from Nicotiana tabacum. The full-length cDNA had an open reading frame of 1422 bp with a deduced amino acid sequence of 473 residues. In order to investigate the role of cyclin B gene in the plant growth and development, transgenic tobacco overexpressing cyclin B gene was constructed. Cyclin B transgenic plants showed longer root hairs than the wild type. RT-PCR analysis revealed that expression of both cyclin B and CDK genes in transgenic plants were enhanced. Also, flow cytometry analysis showed that the isolated cells from root hair elongation zone in transgenic plants had increased G2-M phase of cell cycle compared with wild type.

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Chloroplast differentiation in cotyledons is distinct from true leaves because of organization formed during embryo biogenesis. We isolated a novel Arabidopsis thaliana mutant by insertion of Ac/Ds transposon that has albino cotyledon and green true leaves, called apg7. Recently, apg7 was reported as cyo1, and it was shown that CYO1 has a chaperon-like activity in cotyledons (Shimada et al., 2007). We observed in detail the plastids in cotyledons and shoot apical meristems (SAM) during embryogenesis and seed germination. In wild-type, the plastids in cotyledons contained the prolamellar body, although the plastids in SAM contained little membraneous structure. In apg7/cyo1, the cotyledon plastids exhibited abnormal development of thyrakoid membrane from prolamellar body. Furthermore, albino cotyledons of mutant turned green under continuous light through seed germination. These results may suggest APG7/CYO1 plays a role in formation of thyrakoid membrane from prolamellar body.

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Polyamines (PA) are small polycations observed in various organisms such as bacteria, plants and animals. Putrescine, spermidine and spermine are the most abundant polyamines in plants. Genetic analyses using series of Arabidopsis mutants of PA biosynthetic genes have helped to clarify the PA function for a variety of physiological processes such as stress response, seed development and stem elongation. In this study, we analyzed a candidate protein involved in Arabidopsis Polyamine Transport system (AtPTP) and characterized the atptp mutant. The atptp leaf showed albino phenotype and the atptp seed was dark brown and shriveled. We are performing the domain analysis of the AtPTP protein, and discuss the physiological roles of AtPTP for plant development.

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Plant FtsH protease is a membrane-associated protease localized on the thylakoid membrane. Its function is to remove a damaged protein from large protein complex. Major physiological substrate of this protease is D1 protein whose subunit plays a key role in the photosystem II. Knockout mutants of this protease shows variegated phenotype in Arabidopsis, therefore physiological function of this protease is to maintenance the chloroplast proteins. In this study, we constructed over-expression system of protease domain in FtsH protease from tobacco. Protein was produced as inclusion body, though these were possible to be activated by urea mediated refolding. Expressed protein showed protease activity on FITC (fluorescein isothiocyanate)-labeled casein. FITC-BSA (bovine serum albumin) did not digested by this protease, thus substrate specificity was detected on this protease. Biochemical study revealed that protease activity was enhanced by higher concentration of magnesium ion. Optimum pH was determined as alkalescent condition.

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D1 protein, localized in the reaction center of photosystem II, is easily damaged, degraded, and removed by FtsH protease. In this study, we examined characteristics of ATPase domain in DS9, FtsH protease from Tobacco. We constructed over-expression system of ATPase domain in DS9 in E.coli. ATPase domain in DS9 was amplified by PCR, and was subcloned into pET21b. Finally, the vector was transformed into BL21-CodonPlus (DE3)-RIL. This system produced the recombinant protein in the soluble fraction. The protein was purified by Ni2+ affinity chromatography. The eluted fraction was verified as ATPase domain in DS9 by western blotting and peptide sequencing. ATPase activity was obviously detected. Using this system, we analyzed the dependencies of pH, divalent cations, ATP concentration, and the effect of detergents. In this study, we discussed biochemical properties of ATPase domain in DS9.

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FtsH protease degrades the damaged D1 protein which locates in the reaction center of photosystem II. FtsH protease is an ATP dependent metalloprotease and is composed from the following three domains; transmembrane, ATPase and protease domain. In this study, we focused on the molecular mechanism of the ATPase domain. We transformed this ATPase domain into BL21-CodonPlus (DE3)-RIL and over-expressed the protein in soluble fraction. Purification was performed using Ni-NTA column. The quality of this protein was verified by western blotting and peptide sequencing. We examined biochemical analysis on the purified protein. And we revealed pH optimum and dependency of Mg2+ concentration. We also investigated effect of inhibitors. Among them, EDTA was the highest inhibitory effect. The results suggest that ATPase domain in the FtsH protease depends on divalent cations. We are trying to determine Km and Vmax, optimum temperature, and effective substrate except for ATP.

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A mutation in CRL of Arabidopsis thaliana causes a severe defect in plastid division. Besides the inhibition of plastid division, the mutation causes distortion of cell division planes and abnormality in cell differentiation (Plant J., 38, 448-459, 2004). Our previous data indicated that CRL protein localized in the outer envelope membrane of plastids. In this study, we have reexamined the subcellular localization of the CRL protein using the transgenic lines expressing a CRL-GFP fusion protein that can complement crl mutation. Our results indicated that the CRL-GFP protein localized plastid periphery and the intensity of the signal was not uniform. The pattern of the CRL-GFP localization in dividing chloroplasts was the same as that of non-dividing ones. We are now investigating the subcellular localization of the CRL-GFP in roots and developing leaves under the normal condition and stress conditions. These results will be presented.

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The apg4 mutant was shown to have a disrupted gene for a ribosome binding factor RBFA homologue. The phenotype of the apg4 mutant had white cotyledons and yellow or green variegated true leaves. In Escherichia coli the RBFA was shown to be involved in processing for the pre-rRNA to form the mature 16S rRNA. We found that the APG4 is involved in processing of the pre-rRNA to form the mature 23S and 4.5S rRNA and related accumulation of rrn16 transcripts. RimM and Era have similar functions of RBFA in E.coli. We identified the RimM and Era homologous genes in Arabidopsis thaliana. The mutant phenotype of apg4 was strongly abserved in cotyledons but week in true leaves, which may be due to high expression of the APG4 gene in cotyledon but not in true leaves. We report the function and expression of these genes closely related to APG4.

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To understand the basic mechanism of plastid differentiation, we prepared and analyzed proteins from various types of plastid using proteomic approaches. First, we succeeded in isolating intact not only chloroplasts from wild type Arabidopsis but also plastids with abnormal thylakoids from the two apg (albino or pale green mutant 2) mutants, apg2, with a disrupted gene for TatC homologue of the ΔpH-dependant protein transport system in thylakoid membrane and apg3 with a disrupted gene for chloroplast ribosomal releasing factor I in translation system. Protein profiles were markedly different between apg2 and apg3 mutants. Next, to understand the differentiation of chlomoplast from chloroplast, we obtained plastid proteomics date of Micro-Tom fruits at four developmental stages (Mature Green, Yellow, Orange, Red). ATPase and endoribonuclease/protein kinase like protein are increased in Mature Green fruits. In contrast, plastid lipid associated protein and lipocalin are accumulated in Red fruits.

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Many proteins required for chloroplast biogenesis and function are encoded nuclear genome. PPR (Pentatricopeptide Repeat)protein family is one large family of these proteins, which have a degenerate 35-amino acid motif that is typically found in tandem arrays of 5-15 copies. They are considered to be important RNA-binding proteins involved in post-transcriptional regulation of organelle genes expression such as RNA processing, splicing and editing. We have isolated knockout mutants of PPR proteins from Arabidopsis tagged lines and mainly analyzed apg(albino or pale-green)14, 15 and 16 mutants to elucidate these proteins function in chloroplast. APG14 is a member of pTAC(plastid transcriptionally active chromosome)family. APG15 is a member of PPR-PLS subfamily characterized by DYW motif. And APG16 is a PPR-P subfamily characterized by only PPR motif. We found that 4.5srrn and 5srrn have processing defects in apg15. We report that these proteins related in processing of chloroplast transcripts.

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To study the functions of novel nuclear encoded genes involved in chloroplast development and photosynthesis, we systematically analyzed a series of albino or pale green (apg) mutants screened from Ds-transposon tagged lines in Arabidopsis thaliana. One of the apg mutants apg9 showed albino phenotype. APG9 has a SAP domain and PPR domains. The SAP domain function as a DNA binding domain and the PPR domains are involved in RNA maturing process in higher plant. In apg9, the chloroplast proteins involved in photosynthesis were not detected because of deficient in chloroplast development. We found that chloroplast encoded genes such as rpoA, rpoB, rpoC1, rpoC2, 4.5srrn and 5srrn have processing defect in apg9 mutants, while the nuclear encoded genes, cab and rbcS have no defect in these processing. Those results suggest that the APG9 function related to RNA maturing process in many chloroplast encoded genes.

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Plastid transcription plays an important role in plastid differentiation. Proteome analysis of pTAC, plastid transcriptional active chromosome, revealed several plant-specific putative gene regulatory proteins that have no homologue in cyanobacteria, in addition to the bacterial-type RNA polymerase (PEP) subunits. Thus it is likely that plastid transcription is regulated by non-cyanobacterial factors during plastid differentiation. pTAC3 is one of the plant-specific pTAC proteins, and has a sap domain that is involved in DNA binding in eukaryotic matrix attachment region binding proteins. Gel shift assay suggested that the sap domain possesses non-specific DNA binding activity. To examine the role of pTAC3 in plastid differentiation, we isolated a T-DNA insertion mutant of pTAC3. The ptac3 mutant shows an albino phenotype, and a significant decrease in the expression of all PEP-dependent photosynthesis and rRNA genes. These results suggest the possible involvement of pTAC3 in the regulation of plastid gene expression during chloroplast development.

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Calcium plays an important role as a second messenger in biotic and abiotic stress responses in plants. We recently found that chloroplasts are involved in the generation of cytosolic Ca⁺² signals and subsequent stomatal closure (Nomura et al., 2007 Plant J). However, little is known about stromal Ca⁺² responses. Here we provide direct evidence that stromal Ca⁺² levels are up-regulated by H₂O₂ or elicitor treatments. Stromal free Ca⁺² concentration is usually maintained at sub-μM. H₂O₂ treatment caused a rapid elevation of cytosolic Ca⁺² concentration within 1-2 min. Subsequently, slower increases in stromal Ca⁺² levels were observed within 20-30 min. Similarly, elicitor treatments caused a slow increase in stromal Ca⁺² concentration, preceded by a rapid cytosolic Ca⁺² elevation. These results suggest that Ca⁺² communication between the cytosol and chloroplasts plays important roles in stresses and pathogen responses.

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To reveal functions of a large population of rice genes efficiently, we are taking advantage of Full-length cDNA Over-eXpresser gene (FOX) hunting system. During generation of FOX-rice lines, we obtained two green calli on a 2,4-D-containing medium, on which wild-type rice calli are ivory yellow. OsGLK1 gene, which encodes a GARP-transcription factor, was ectopically overexpressed in the two calli in which chloroplasts with well-developed grana stacks were differentiated [Nakamura et al. (2007) PCP 48(Suppl): s117]. OsGLK1-FOX calli produced oxygen and were able to grow on sucrose-depleted media. These results suggested that developed chloroplasts in the OsGLK1 rice calli are functionally active, and that OsGLK1 is a key regulator of chloroplast development. To dissect OsGLK1-regulated genes, we performed microarray analysis of the OsGLK1-FOX line using the rice 44k oligo microarray. Based on the detailed analysis of microarray data, we will discuss the role of OsGLK1 in chloroplast development.

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The precursors to LHCPII are large polyproteins containing multiple copies of LHCPII, and photocontrol of their formation is largely translational. Under conditions favoring LHCPII accumulation in the thylakoids, a reaction with anti-LHCPII antibody can be observed in the Golgi apparatus by immuno-electron microscopy. The timing of the immunoreaction in the Golgi apparatus in synchronous cells and in cells undergoing normal light-induced chloroplast development suggests that the nascent LHCPII passes through the Golgi on the way to the thylakoids. In addition, the same phenomenon has been reported not only for Euglena but also for dinoflagellate cells and the higher plants (2006). The phenomenon of the photosynthetic proteins synthesized in the cytoplasm goes through the Golgi is considered to be a common phenomenon observed in plant cells.

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In plants and algae, it is thought that distant homologs of Escherichia coli DNA polymerase I (Pol I) is involved in the replication of organellar genomes. So far, such enzymes have been identified in Arabidopsis, rice, and tobacco. The genome of the red alga Cyanidioschyzon merolae contains the gene encoding a homolog of plant enzymes PolB, and the gene encoding PolA that has a higher homology to Pol I than to plant enzymes. We purified PolB from the cells of C. merolae and characterized the enzymatic properties.

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Plastids are organelles such as chloroplasts, amyloplasts and leucoplasts, and are responsible for photosynthesis and other metabolic functions. Although it is well known that plastids were generated due to endosymbiosis and have own DNA, relatively little is known about the mechanism of plastidial DNA replication and repair. Recently, we identified a novel DNA polymerase in Oryza Sativa L.(OsPolI-like) that is homologous to prokaryotic DNA polymerase Is (PolIs), and suggested that this polymerase might be involved in plastidial DNA replication and repair. Here, we propose to rename the plant PolI homologs as DNA polymerase π (POLP), and investigate the biochemical properties of full-length OsPOLP1. In addition, we would like to discuss the mode of plastidial DNA replication and repair from these results.

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We have identified two recA homologue genes in the cDNA database of the moss Physcomitrella patens and named it PprecA1 and PprecA2 (Physcomitrella patens recA). The PpRecA2 protein is targeted to chloroplast. PprecA2 complements a recA deletion in E.coli and is upregulated by DNA-damaging reagents. PprecA2 null mutant exhibits slow growth rate, a reduction of chloroplast DNA copy number and a low photosynthetic rate under genotoxic stress. The efficiency of gene targeting into the chloroplast genome in PprecA2 null mutant was lower than that in the wild type strain. These results suggest an important role of PprecA2 in homologous recombination and repair in P.patens chloroplast DNA.

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