Xyloglucan endotransglucosylases (XETs) in the cell wall catalyze the transfer of a portion from a donor xyloglucan to acceptor xyloglucan molecule, and are considered to contribute to plant cell growth. We purified XET isoenzymes from growing pea stems. Kinetic studies using whole xyloglucans as a donor and xyloglucan heptasaccharide (XXXG) as an acceptor support a sequential mechanism for the enzymes. We suggest that XETs occur as an enzyme-acceptor complex as well as an enzyme-donor complex in the cell wall. In the transverse sections of pea stems, the incorporation of fluorescent XXXG was observed at the epidermis of the third internode, and that of fluorescent whole xyloglucan was in the vascular bundles of first and second internodes. The results showed that XET-donor and XET-acceptor complexes were differently localized in pea stems.

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There are many unknown enzymes related to metabolism of plant cell wall polysaccharides. Here, we report a novel α-1,2-fucosidase acting on xyloglucan.
We have discovered endo-β-mannosidase (EBM) as a plant specific endoglycosidase acting on N-glycans of glycoproteins (J. Biol. Chem. 279, 38555 (2004)). EBM located in vacuole is thought to play a role in the metabolism of N-glycans. We found a protein complex consisted of EBM and another protein, and purified this protein complex from lily flowers. This associated-protein is highly homologous to a functionally unknown-protein from plants, and slightly homologous to the fucosidase domain of the bifidus AfcA protein. Substrate specificity analysis of this complex protein revealed that it hydrolyzed the α-1,2-fucosyl linkages at the non-reducing end of the sugar chains including a sugar chain derived from xyloglucan. It is interesting to examine physiological role of this novel α-1,2-fucosidase and the significance of its association with EBM.

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Nucleotide sugars are generated from monosaccharide 1-phosphates and nucleotides in the salvage pathway in higher plants. The salvage system of L-fucose is completely independent from that of glucose, galactose, xylose, L-arabinose, and glucuronic acid, which are activated as UDP-sugars. Using genomic database of Arabidopsis and rice, we identified a novel gene, designated AtFKGP (Arabidopsis thaliana L-FucoKinase/GDP-L-fucose Pyrophosphorylase), with similarity to both L-fucokinase and GDP-L-fucose pyrophosphorylase genes. The recombinant AtFKGP expressed in Escherichia coli catalyzed formation of GDP-L-fucose in the presence of ATP, GTP, and L-fucose 1-phosphate. Relatively high expression of AtFKGP was detected in flower buds of Arabidopsis. Loss-of-function mutants with T-DNA insertion in AtFKGP showed accumulation of free L-fucose in the cytoplasmic fraction. These results indicate that AtFKGP catalyzes both conversion of L-fucose 1-phosphote from fucose and ATP, and GDP-L-fucose from L-fucose 1-phosphote and GTP, suggesting that AtFKGP acts for the salvage reaction of L-fucose in Arabidopsis.

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  Arabinogalactan-proteins (AGPs) are proteoglycans found in plasma membrane and/or cell wall of plants, and involved in various functions such as cell adhesion, morphogenesis, and cell death. The carbohydrate moieties of AGPs account for ~90% of weight and their structures are modified depending on organs or growth stage. Radish AGPs are known to contain Gal, L-Araf, GlcA and 4-O-methyl-GlcA (4-Me-GlcA), however, the glycosidic linkages and physiological functions of (4-Me-)GlcAs remain uncleared.
  To elucidate the structure and functions of the carbohydrate moieties of AGPs, we cloned two β-glucuronidases from Aspergillus niger and Neurospora crassa. Radish native AGPs were not hydrolyzed by β-glucuronidases, whereas α-L-arabinosidase-treated AGPs were susceptible to the enzymes. These enzymes released higher amounts of uronic acids from root AGP than L-fucose-containing leaf AGP, suggesting that the L-Araf and L-fucose residues in radish AGPs affect the action of β-glucuronidases.

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The functions of floral meristem identity genes, such as LFY and 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.
In this study, we isolate the LFY 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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In general, growth of axillary meristem is inhibited when the apical bud is present. Although this phenomenon, called apical dominance, has been known for long time, the exact mechanism is poorly understood. We are studying the apical dominance of rice. We observed that excision of the apical bud stimulated the growth of axillary tillers, suggesting that the apical dominance works in rice.
Loss of function mutants of D3, D10 genes of rice show striking increase of tillers. We have shown that D10 is an ortholog of Arabidopsis MAX4, which is implicated in the synthesis of SMS (Beveridge 2006). In d10 mutants, growth of tillers are not suppressed even in the presence of the apical bud, and the application of auxin induce transcription of D10. Base on these results, we will discuss the possibility that D10 and SMS act as second messengers of the auxin in the apical dominance.

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In higher plants, nearly all above ground part is derived from the shoot apical meristem (SAM). We isolated the recessive mutant gorgon (gor), which exhibits drastic expansion of the SAM, in Arabidopsis thaliana. In gor, the expansion of the SAM is already apparent in young seedlings. As the plant starts bolting, the SAM of gor continues to expand and produces numerous pin-shaped protrusions from its periphery but no branches or flowers. Shortly after, the SAM terminates by producing numerous flowers or branches from its entire surface. Moreover, some of these flowers produce extra flowers or floral organs from an additional whorl inside carpels. These observations show that gor is defective in the regulation of SAM size and floral organ development. We are now characterizing the gor phenotype in detail using molecular markers and map-based cloning of the mutated gene is in progress. Results of these experiments will also be presented.

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The Arabidopsis CUP-SHAPED COTYLEDON genes CUC1, CUC2 and CUC3 encode transcriptional activators of the NAC family and are redundantly required for shoot meristem formation and organ separation. The cuc1 cuc2 double mutation causes severely fused cotyledons and lack of an embryonic shoot meristem. Conversely, ectopic overexpression of CUC1 or CUC2 induces ectopic shoot meristem formation on the surface of cotyledons. To identify downstream genes of CUC genes, we carried out microarray-based screenings. We first compared expression profiles in wild-type and cuc1 cuc2 embryos and obtained ten candidate genes which are significantly downregulated in cuc1 cuc2. In addition, another set of microarray experiments using 35S-CUC1 seedlings identified eleven more genes activated by CUC1 overexpression. We are now analyzing expression patterns of these candidate downstream genes in wild type embryos and the effect of CUC1 and CUC2 on them. The results from these experiments will be presented.

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We analyzed two dominant mutants of Arabidopsis, crane-1 (cra-1) and cra-2, which are defective in lateral root formation. Although these mutants were isolated independently, they share several common phenotypes such as up-curled true leaves and dwarfism. We found out a missense mutation in domain II of IAA18 gene (At1g51950), a member of Aux/IAA transcriptional regulator family, in the genome of both cra-1 and cra-2, strongly suggesting that CRA encodes IAA18. These mutations are thought to stabilize IAA18 protein in crane mutants. Real-time RT-PCR analysis revealed that IAA18 is expressed in most organs including seedling roots. In yeast two-hybrid experiments, IAA18 protein has the ability to interact with ARF7 and ARF19, which are previously reported as key transcriptional factors required for lateral root formation. These results suggest that CRA/IAA18 is a novel regulator of lateral root formation in Arabidopsis.

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Lateral root (LR) formation is important for the establishment of root architecture in higher plants. To understand the molecular mechanism of LR formation, we isolated the novel recessive mutant rlr50 (reduced lateral root formation 50) in Arabidopsis thaliana. This mutant shows the decreased LR primordium density (about 60% of the wild-type density). Exogenous auxins promoted LR formation and inhibited primary root growth in rlr50, suggesting the mutation does not directly affect the auxin responsibility in roots. Map-based cloning revealed that RLR50 gene encodes a novel unknown protein. Expression analysis indicated that RLR50 is expressed in almost all organs of the plant. In addition, functional GFP-tagged RLR50 proteins are localized in the cytosol both in cultured cells and in planta, suggesting that RLR50 functions in the cytosol. Taken together, we conclude that RLR50 is a novel factor that controls LR primordium formation.

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Phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P₂) is synthesized by phosphatidylinositol4-phosphate5-kinase (PIP5K) and regulates activity and intracellular localization of various proteins. The PIP5K plays a pivotal role in phospholipid-mediated signal transduction with G-proteins and phospholipaseDs. In plant cell morphogenesis, involvements of low molecular weight G-protein(ROP1,2), phospholipaseDζ1(PLDζ1), and PtdIns(4,5)P₂ to tip-growth of root-hair or pollen-tube have been reported. Thus, phospholipid-mediated signal transduction is believed to control plant cell tip-growth. However the molecular characteristics and physiological roles of PIP5Ks have not been elucidated well as far in plants. Previously we reported the involvement of AtPIP5K3 in root-hair elongation among eleven PIP5K genes in Arabidopsis. In this work, we revealed that AtPIP5K3 was preferentially localized to a growing root-hair tip region. Furthermore, we found that two or more root-hairs were generated from one root-hair cell overexpressing AtPIP5K3. These results suggest that AtPIP5K3 is involved in root-hair initiation as well as elongation by synthesizing PtdIns(4,5)P₂ in the tip region.

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CAPRICE (CPC) encodes a small protein with a R3 MYB motif and promotes root-hair cell differentiation in Arabidopsis (Wada et al., 1997). We found four additional CPC-like Myb sequences. Plant lines that overexpress these four genes had a reduction in the number of trichomes and an increase in the number of root-hairs, which is similar to an overexpressor of CPC. We also made double and triple mutants to clarify the function of each gene. These results suggest that the CPC-like R3 MYB genes cooperatively regulate epidermal cell differentiation. Unlike CPC, CPC-like Myb GFP fusion proteins did not move from cell-to-cell. Promoter-GUS analyses indicated that these CPC homolog were specifically expressed in epidermal cells, some of them were in stomate guard cells. Notably, the CPL3 gene alone has pleiotropic effects on stomata guard cell distribution, flowering development, and epidermal cell size through the regulation of endoreduplication.

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Root epidermal cells differentiate into either root hair cells or hair-less cells in Arabidopsis. In current model, a transcriptional complex consisting of WER (R2R3-MYB transcription factor), GL3/EGL3 (bHLH transcription factor) and TTG1 (WD40 repeat protein) promotes GL2 (encoding an HD-Zip transcription factor) expression, which results in differentiation into hair-less cell. CPC (R3-MYB protein) inhibits formation of the WER-GL3/EGL3-TTG1 complex to repress GL2 expression, resulting in differentiation into root hair cell.
TTG2, encoding a WRKY transcription factor, regulates trichome development. TTG2 is expressed in atrichoblasts of roots as well as in trichomes. We found that TTG2 expression in roots was directly promoted by the WER-GL3/EGL3-TTG1 complex and was repressed by CPC. Transgenic Arabidopsis expressing chimeric TTG2 protein fused with transcription repression domain (TTG2:SRDX) formed root hairs from all epidermal cells of roots. In TTG2:SRDX lines, GL2 expression was repressed in roots, suggesting a possibility that TTG2 promotes GL2 expression.

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We isolate the tip growth mutant on the root hair cells, antlers1-1 (atl1-1), and identify the ATL1 gene by a positional cloning method. atl1-1 is an allele of the MZH2 kinesin-related gene, and we elucidate the structure of the ATL1 gene and a tissue-specific activity of its promoter. The ATL1 kinesin-related gene shows two homologus genes, named ATL2 and ATL3, in the Arabidopsis genome, and we report an involvement of the ATL2 gene in the helical growth of roots. Furthermore, by yeast two-hybrid screening, we isolate a NIMA-related protein kinase (Nek) gene, AtNek6, which encode a protein kinase bound to the C-terminus of the ATL family proteins. We reveal the function of AtNek6 in the epidermal cell morphogenesis in Arabidopsis seedlings. Our results shed light on the functions of the ARM repeat-containing kinesins and a NIMA-related kinase in epidermal cell morphogenesis of plants.

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The consensus is that a cyanobacterium with cell wall phagocytosed by a host cell evolved into plastids. However, no wall-like structures including peptidoglycan (PG or murein) were found in plastids of all green plants. Ten Mur genes are associated with PG biosynthesis in bacteria. Recently, we found that the moss Physcomitrella patens has eight homologs related to PG biosynthesis. Gene knockout experiments for the PpMurE or PpPbp (penicillin-binding protein) genes in P. patens showed appearance of macrochloroplasts both in protonema and in leaf cells. Electron microscopic observations showed no obvious differences in the shape or stacking of thylakoid membranes in the transformants. We isolated two genes (PpMraY and PpMurG) that were not found previously by screening the P. patens genomic database. PpMraY and PpMurG encoded a protein of 487 and 400 amino-acids, respectively. Gene disruption of the PpMraY gene in P. patens resulted in the appearance of macrochloroplasts.

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It is now widely agreed that plastid is originated from a single cyanobacterial ancestor. Cyanobacterium contains peptidoglycan layer in its cell wall and peptidoglycan synthesis is essential for its division. Though no wall-like structure has been observed in chloroplasts of green plants, we found all of ten homologs to bacterial Mur genes for peptidoglycan synthesis pathway from Physcomitrella patens. Disruption of PpMurE in P. patens causes a small number of large chloroplasts in a cell by inhibition of chloroplast divisions. These results strongly suggest that P. patens Mur genes are still functional for peptidoglycan synthesis. To find out PpMurE function in P. patens, we carried out complementation test of P. patens PpMurE gene disruptant lines with Anabaena (Ana)MurE gene. PpMurE KO lines with expression of AnaMurE gene showed recovery of number and shape of chloroplasts. This result suggests that MurE genes in Anabaena and in P. patens are functionally identical.

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It is known that one plant-specific type of dynamin-related proteins (DRP5B) is associated with the late stage of chloroplast divisions in Arabidopsis thaliana and red alga Cyanidioshyzon merolae. In A. thaliana, AtDRP5B (arc5) mutants have enlarged, dumbbell-shaped chloroplasts. We identified three homologs of AtDRP5B (PpDRP5B-1, -2, and -3) from the moss Physcomitrella patens. The amino-acid identity between AtDRP5B and PpDRP5Bs was 54.7% for PpDRP5B-1, 54.9% for -2 and 55.0% for -3. Each PpDRP5B-1 or -2 single knockout (KO) lines was generated by a P. patens gene-targeting technique. The average chloroplast number of PpDRP5B-1 and -2 single KO line is 46.4 ± 5.5 and 46.2 ± 5.3, respectively, and is the statistically same to that of wild type cells (49.3 ± 5.5). Therefore, PpDRP5B-1 and -2 double KO mutants were created. In the double KO lines, cells contained fewer and larger chloroplasts (30.03 ± 6.4 chloroplasts per cell).

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Genes harbored on the plastid genome are transcribed by two types of RNA polymerases, eubacteria-type plastid-encoded RNA polymerase (PEP) and T7 phage-type nuclear-encoded RNA polymerase (NEP). In Arabidopsis thaliana, three T7 phage type RNA polymerase genes have been identified, and RpoTp and RpoTmp were suggested to localize in plastids. We have isolated a T-DNA inserted mutant in the rpoTp gene, identical to sca3-2. The mutant's seedlings showed almost albino, very poor growth and loss of glycolipids. We also found that the mutant cotyledons partially re-developed chloroplasts on MS-plates containing sucrose. Interestigly, the mesophyll cells of the mutant rosette leaves contained different type of aberrant plastids. This observation would suggest that RpoTp is necessary for synchronous proceeding of chloroplast development in a cell. We will discuss the functional difference between RpoTp and RpoTmp.

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Chloroplast gene expression in higher plants is thought to be regulated mainly at the post-transcriptional level. To study mRNA translation in the chloroplast, we used an improved in vitro translation system derived from tobacco chloroplasts. Using GFP reporter gene, we established non-radioactive detection system to measure rate of mRNA translation. We are currently analyzing cis-elements and trans-acting factors that are required for efficient translation initiation of chloroplast mRNAs in tobacco, and the results will be presented.

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In higher plants, RNA editing is a post-transcriptional process of altering a specific C to U in an RNA molecule in mitochondria and plastids. About 30 editing sites have been detected in the genomes of plastids and over 400 in mitochondria. We previously reported that a PPR protein CRR4 acts as a site-recognition factor in the ndhD-1 editing in chloroplasts.
Here, we discovered Arabidopsis crr21 mutants that are specifically impaired in the ndhD-2 editing that converts Ser128 of NdhD to Leu. The CRR21 gene encodes a member of the PPR protein family. Both CRR21 and CRR4 belong to the E+ class that is characterized by the presence of a conserved C-terminal region (the E/E+ domain). This E/E+ domain is highly conserved and exchangeable between CRR21 and CRR4. Our results suggest that the E/E+ domain has a common function in RNA editing rather than of recognizing specific RNA sequences.

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Pentatricopeptide repeat (PPR) protein has 35 conserved amino acids repeat. There are about 450 PPR proteins in both Arabidopsis thaliana and Oryza sativa. Since many of PPR proteins are predicted to localize in mitochondria or plastids, they are believed to play an important roles for organelle related functions. However, details of their function are unknown. We have found many PPR protein genes from draft sequence of the Physcomitrella patens genome. Most of A. thaliana PPR protein genes consist of a single exon, but in P. patens, large number of PPR protein genes contain more than two exons. Many P. patens PPR proteins are also predicted to localize in mitochondria or plastids. We will report the results of subcellular localization of moss PPR proteins used transient assay.

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RNA editing is a process converting specific C to U on chloroplast mRNA. crr4 mutants are defective in the RNA editing that creates an initiation codon of the chloroplast ndhD gene. CRR4 specifically recognizes and binds to the sequences surrounding this editing site. It is unlikely that CRR4 contains any domain responsible for RNA editing enzyme activity, such as cytidine deaminase, suggesting that CRR4 interacts with an unidentified RNA editing enzyme.
In this study, we have screened for suppressors of crr4-4 to elucidate the RNA editing machinery by determine the factor interacts with CRR4. crr4-4is a weak allele of crr4. We obtained 30 suppressor candidates from 25,000 M₂ plants treated with EMS. Although NDH protein level was restored in some lines, the editing phenotype was not drastically suppressed.

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Higher plants have two metabolic pathways for isoprenoid biosynthesis: the cytosolic mevalonate (MVA) pathway and the plastidal non-mevalonate (MEP) pathway. Little is known about the mechanisms that regulate these pathways and the metabolic crosstalk. To understand such regulatory mechanisms, we isolated and characterized the Arabidopsis mutant lovastatin insensitive 1 (loi1), resistant to lovastatin and clomazon, inhibitors of the MVA and MEP pathway respectively. The accumulation of the major products of these pathways, i.e., sterols and chlorophylls was less affected by lovastatin and clomazone, respectively. LOI1 encodes a pentatricopeptide repeat (PPR) protein. Most PPR proteins were predicted to take part in the post-transcriptional regulation of organelle gene expression. Our results demonstrate that LOI1 is predicted to localize in mitochondria and has the ability to bind single-stranded nucleic acids. Our investigation suggests that the post-transcriptional regulation of mitochondrial RNA may be involved in isoprenoid biosynthesis in both the MVA and MEP pathways.

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Since mitochondrial DNA contains many genes encoding the components of an oxidative phosphorylation system, transcription of mitochondrial genes and its regulation are very important when maintaining the metabolism of plant cells.
Many mitochondrial genes in higher plants are transcribed monocistronically but some genes are co-transcribed. These transcripts become mature and functional RNAs through the post-transcriptional processing reactions such as intron splicing, RNA editing and 5' and 3' processing. CRTAAGAGA (CNM) motif and CGTATATAA (TA) element were reported as a core promoter of a dicotyledonous plant. We found CNM motif in 5' upstream of some genes of tobacco mitochondria but the experimental proof has not been done. Then, analysis of the transcription is advanced by 5'-RACE and the circularized RT-PCR methods, and transcription start sites of some kinds of structural genes and rRNA genes were determined and CNM, CNM-like motif, and TA and TA-like element were found.

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Cyclin-dependent kinases (CDKs) are evolutionally conserved key regulators of the cell cycle. CDK activities are regulated by phosphorylation and binding of regulatory cyclin subunits. During the past decade, it has been shown that the posttranslational regulations of CDKs are well conserved among distantly related eukaryotes. On the contrary, little is known about transcriptional regulations of CDKs during post-embryonic development of plants.
Plants have six types of CDK, among which CDKA and CDKB play a key role in the cell cycle. The transcription of CDKB is under strict cell cycle control, and we have recently demonstrated that CDKB2 is regulated by proteolysis. The transcription of CDKA is not cell cycle phase-dependent, but it would be under developmental control to coordinate cell division and differentiation. In order to have insight into the transcriptional regulation of CDKA in planta, we have dissected the promoter region of Arabidopsis CDKA;1 and analyzed expression patterns.

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CDKF;1 is a plant-specific cyclin-dependent kinase (CDK) and functions as a CDK-activating kinase (CAK) in vitro. Such a function has been previously examined with transiently expressed CDKF;1 in root protoplasts. To reveal its in vivo functions, we isolated a T-DNA insertion mutant and investigated the phenotype. The cdkf;1 mutant exhibited a dwarf phenotype and delayed growth. Root growth stopped around 7 days after germination and the expression of CYCB1-GUS almost disappeared, suggesting that CDKF;1 is required for cell division in the root meristem. We also found that the leaf blade area, cell number and cell size were smaller than those of wild-type plants. Moreover, the cdkf;1 mutant showed a lower level of DNA ploidy. On the other hand, cell division pattern during embryogenesis has not been defected in the mutant. Based on these results, we concluded that CDKF;1 plays an essential role in cell divisions during post-embryonic development.

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srd2 is a temperature-sensitive mutant of Arabidopsis thaliana that exhibits peculiar defects in tissue culture responses including hypocotyl dedifferentiation and shoot regeneration. Its responsible gene, SRD2, was revealed to encode snRNA transcription activator. Our previous studies have shown involvement of SRD2-mediated increase of snRNAs in elevation of cell proliferation competence during dedifferentiation.
Here we report SRD2 expression analyzed using SRD2p::GUS transgenic lines. When hypocotyl segments were incubated on callus-inducing medium containing 2,4-D and kinetin, GUS was expressed strongly in the stele throughout the whole explant and also in non-stele tissues around the cut ends. A similar pattern was observed in explants incubated with 2,4-D alone. In culture with kinetin alone or no phytohormones, active GUS expression was limited to the near-cut-end region. In view of localization and phytohormone dependency of cell proliferation, the GUS expression in the stele induced by 2,4-D should reflect SRD2 expression required for dedifferentiation.

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For molecular genetics of plant organogenesis in vitro, we have isolated and characterized many temperature-sensitive mutants of Arabidopsis that are impaired in adventitious root formation. Here we report analysis of the process of cell proliferation reactivation with one of these mutant, rid2, whose responsible gene encodes a putative methyltransferase with a nuclear localization signal. Callus formation from hypocotyl explants of rid2 is highly temperature-sensitive. In hypocotyl explants, high temperature inhibited the re-initiation of cell cycle, and induced structural abnormality of nuclei accompanied with nucleolar enlargement and uneven cell expansion. By contrast, root explants of rid2 are partially temperature-sensitive for callus formation, and they show discontinuous callusing phenotype at high temperature. Closer observation revealed that in the non-callusing region, not cell cycle re-initiation but cell proliferation maintenance is interfered with. These results suggest that RID2 functions at two different points in the process of reactivation of cell proliferation.

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In plants most organs are composed of cells of various ploidy levels and endoreduplication is a key process in the plant's developmental plan.
We have isolated ilp1-1D as a dominant mutant in which polyploidy level is increased in the seedlings. ilp1-1D showed correlation between increased cell volume with polyploidy. ILP1 gene encodes for a novel nuclear protein and this protein functions as a transcriptional repressor in vivo.
The expression of all the member of CYCA2 family was reduced in an ILP1 over-expressing line. This indicates that ILP1 is a repressor that control CYCA2 expression. T-DNA insertion mutants of CYCA2;1 has increased ploidy level. Here we demonstrate that ILP1 regulates endoreduplication through control of CYCA2 expression in Arabidopsis.
ILP1 has homologs not only in plants but also in insects and mammals, and the mouse ortholog of the ILP1 also repressed cyclin A expression in mouse NIH3T3 cells.

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The moss Physcomitrella patens development starts from a germinating spore that gives rise to a filamentous structure called protonema. Bud that is arisen form protonema develops to give rise to a leafy shoot called gametohpore. After dissection of a leaf from a gametophore, leaf cells facing to the dissected cells change to protonemal apical stem cells. However, the molecular mechanisms of this process have been unknown. To understand the mechanisms, we focused on the cell cycle regulation during the transdifferentiation after leaf dissection. In this study, we searched the DNA database for P. patens genes encoding core cell-cycle regulators. We next investigated the accumulation pattern of transcripts of the cell-cycle regulators in dissected leaves with real-time RT-PCR analysis. The result revealed that transcripts encoding some cell-cycle regulators increased in dissected leaves. Further experiments are directed to the functional analysis of the cell cycle-related genes during the transdifferentiation.

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Regulation of plant cell cycle shares highly similarity with the system controlling in mammals, in which the signal pathway mediated through Rb (retinoblastoma) plays important roles in regulation of the G1/S transition. A previous study reported that AtRBR1, encoding the only Arabidopsis Rb, is related to the stem cell maintenance of the root. However, it remains to be elucidated that AtRBR1 function concerns the phosphorylation status of AtRBR1 protein in plants.
In this study, we analyzed this issue by using Arabidopsis suspension cultured MM2d cells. When MM2d cells are subjected to re-entry into cell cycle, hyper-phospholylated AtRBR1 protein increases during G1/S phase. Additionally, pull-down assay revealed that hypo-phospholylated AtRBR1 binds to E2F, but hyper-phospholylated AtRBR1 is unable to bind. These results suggest that, like mammalian Rb, phosphorylation probably results in inactivation of AtRBR1 and the subsequent release of active E2F/DP transcription factors, thereby progressing the cell cycle to S phase.

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During plant development, regulation of cell proliferation is considered to be mediated through cell cycle regulators in each cell. However, the mechanisms of such regulation are still poorly understood. Transcription of a group of G2/M phase-specific genes is regulated by a common cis-acting element and the R1R2R3-Myb transcription factors that bind to the element. Arabidopsis contains 5 genes encoding R1R2R3-Myb proteins, which can be divided into 3 groups (A-type, B-type and C-type) based on their primary structures. When two C-type Myb genes are doubly inactivated by T-DNA insertions, mRNA levels of various G2/M phase-specific genes are elevated. This up-regulation was more pronounced in developmentally old leaves with low cell division activities than in young leaves with high cell division activities. Our results suggest that C-type Myb proteins may down-regulate G2/M phase-specific genes and thereby inactivate cell proliferation during development of plant organs.

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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. 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).
We examined the isolation and characterization of the mutants which are defective in RPT subunit in Arabidopsis. The function of RPTs will be discussed in terms of cell size regulation.

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Proper regulations of cell number and shape are essential for normal development in multicellular organisms. Spatial and temporal controls of microtubules are prominent molecular mechanisms managing these cellular processes. Sister Type II ubiquitin-like proteins, PUBL1 and PUBL2 are expressed in meristematic cells in the moss Physcomitrella patens and are essential for proper cell division and elongation via organization of microtubule arrays. To understand the molecular mechanism of microtubule organization, we have analyzed molecular function of PUBLs.
Double disruption of PUBL1 and PUBL2 retarded the collapse of phragmoplasts. In the wild type, the phragmoplast is composed of antiparallel microtubules that overlap and closslink at its equatorial plane. In the phragmoplasts of the disruptants, the overlapping microtubules were expanded. The PUBLs-GFP fusion proteins were localized to the phragmoplast equator. Thus we propose that PUBLs regulate the stability of phragmoplast microtubules by acting on antiparallel microtubules at the phragmoplast equator.

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There are a lot of genes encoding membrane proteins in plants. However, it is difficult to analyze their biological functions, because membrane proteins are active only as integrated form in membranes and are insoluble after separation from membranes. Hence, functions of almost all of those proteins remain to be clarified. In this research, we tried to improve proteoliposome reconstituting system for analyzing plant membrane proteins by using wheat germ cell-free protein synthesis system. At first, we started this study by testing phosphoenolpyruvate/phosphate translocator OsPPT1 as a model protein. OsPPT1 was easily synthesized by the cell-free system, but the synthesized proteins were insoluble. In order to improve the solubility of the call-free synthesized OsPPT1, we tested several detergents, and found that some detergents increased the solubility of the products in the cell-free reaction. We eventually succeeded in detection of the phosphate-transporting activity of OsPPT1 on liposome prepared from phosphatidylcholine.

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Aluminum (Al) tolerance in wheat is primarily controlled by the expression of the ALMT1 gene encoding Al-activated malate transporter at root apex. We investigated genomic sequences of the ALMT1-upstream region in several wheat lines varied in Al tolerance. The first 1,000-bp immediately upstream of the ALMT1 was variable and exhibited six different patterns with some repeats (duplication or triplication) in lines of non-Japanese origin. The number of repeats positively correlated with the ALMT1-expression level and Al tolerance. Thus it is possible that the upstream sequence of the ALMT1 is involved in the control of the gene expression level and degree of Al tolerance in wheat. In lines of Japanese origin, however, Al tolerance was correlated with level of Al-activated malate efflux but not with ALMT1-expression level, suggesting that some other gene(s) are involved in the post-transcriptional process to control the functional expression of ALMT1 protein and Al tolerance.

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Aluminum (Al) tolerance in wheat appears to be controlled by Al activated malate transporter (ALMT1) localized on the plasma membrane. Hydrophobicity profile of ALMT1 protein predicts 5 to 8 transmembrane regions. To elucidate the transmembrane topology of this transporter, we examined the orientations of N and C termini and intra-membrane loop domains of ALMT1 protein. The ALMT1 protein tagged with GFP or His epitope was transiently expressed in animal cells. To determine the localization of epitops in the cells, the anti-ALMT1 antiserums raised against the peptides of ALMT1 N or C terminus were used, without or with a detergent (Triton X) to penetrate the antibodies into cells. The observations of GFP as well as His tag, N- and C- terminal epitopes under fluorescence microscope suggest that the N and C termini of ALMT1 protein face toward the outside of the membrane.

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Arabidopsis thaliana BOR1 is an efflux-type borate transporter required for xylem loading of boron(B) under low B condition. BOR1 accumulates at plasma membrane under B limitation, and is degraded under high B condition.
In the present study, we report the generation of the transgenic Arabidopsis tolerant to excess B by overexpression of BOR4, one of the six BOR1 paralogs present in the Arabidopsis genome.
Transgenic A.thaliana lines expressing BOR4 under the control of CaMV 35S RNA promoter were generated. Immunoblot analysis showed that BOR4-GFP accumulation was not greatly changed upon high B supply. When they were grown in solid medium containing toxic levels of B, six independent transgenic lines showed improvement of growth compared to the wild type plants.
These results indicate that BOR4 has a distinct character from BOR1 in terms of protein accumulation and is capable of conferring extremely high B tolerance to Arabidopsis.

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Zinc is an essential nutrient for cells playing a vital role in controlling cellular processes such as growth, development and differentiation. OsZIP4 is a zinc transporter which localizes to apical cells. We have produced transgenic rice over expressing OsZIP4 under the control of CaMV 35S promoter. Zinc concentration in 35S-OsZIP4 transgenic plants was higher than that of control in roots, and was lower in shoots and seeds. Northern blot analysis revealed that transcripts of OsZIP4 expression driven by CaMV 35S promoter were detected in roots and shoots of 35S-OsZIP4 transgenic plants, but endogenous OsZIP4 transcripts were decreased in roots, and increased in shoots as compared with control. Microarray analysis revealed that the genes expressed in 35S-OsZIP4 shoots coincided with those induced of zinc deficient shoots. These results indicated that OsZIP4 changed the zinc distribution inside rice, and that OsZIP4 is a critical zinc transporter that should be strictly regulated.

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SULTR1;1 and SULTR1;2 are two high-affinity sulfate transporters co-localizing at epidermis and cortex of Arabidopsis roots for the uptake of sulfate from the environment during sulfur limitation (-S). Previously, we have reported that -S induction of SULTR1;1 and SULTR1;2 transcripts are dependent on their promoters activities. To analyze post-transcriptional regulation of SULTR1;1 and SULTR1;2, we generated transgenic plants that rescue the sulfate uptake capacity of sultr1;1 sultr1;2 double knockout mutant by constitutively over-expressing SULTR1;1 or SULTR1;2 under the control of 35S RNA promoter. In transgenic plants, SULTR1;1 mRNA was expressed only in roots, and SULTR1;1 protein content was increased by -S. By contrast, SULTR1;2 mRNA was expressed in both roots and leaves, however SULTR1;2 protein was accumulated exclusively in roots during -S. The present study suggested existence of post-transcriptional mechanisms for the control of SULTR1;1 and SULTR1;2, playing important roles in optimizing the uptake of sulfate during -S.

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In higher plants, Vacuole, which occupies most of a plant cell, plays important roles in a variety of plant growth and development. However, regulatory mechanism of vacuolar development has remained largely unknown.
We analyzed vacuolar aquaporins which transport water into the vacuoles and increase their volume.First, we analyzed the gene expression of a tobacco BY-2 vacuolar aquaporin,NtγTIP,using miniprotoplasts. The miniprotoplasts which are removed the vacuoles by centrifugation,start to regenerate the vacuoles as network-like structures.Subsequently,the network-like structures swelled to tube-like structures and finally the large vacuoles developed.In this process,NtγTIP was induced at the transition of network to tube-like structures and the large vacuolar development.Next,we prepared transgenic tobacco BY-2 cells that overexpress NtγTIP.In the NtγTIP-overexpressing cells,development of the tube-like vacuolar structures and large vacuoles were accelerated compared to the control ones. Furthermore, overproduction of NtγTIP promoted the vacuolar development and cell elongation. These results suggest that NtγTIP is involved in the vacuolar development.

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Stomatal movement that originates from changes in shape and volume of the guard cells is discussed in combination with the vacuolar structure. By using transgenic Arabidopsis thaliana line expressing GFP-AtVam3p, we reported last year that during stomatal closure, many intra-vacuolar membrane structures appeared and the surface area of stomatal vacuole increased. In this study, we investigated the origin of the increased vacuolar membrane (VM). Inhibition of the activity of phosphatidylinositol- 3-kinase that is involved in vesicle transport inhibited both the stomatal closure and the internalization of the VM. Measurement of fluorescence intensities of a vital staining-dye, FM4-64 that firstly incorporated into plasma membrane (PM) of Vicia faba guard cells, showed that its movement from PM to VM was accelerated in proportion to ABA-induced stomatal closure. These results suggest a possibility that excess PM produced during stomatal closure was stored as VM.

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Potassium ion (K⁺) is one of the fundamental mineral nutrients for plant growth and is involved in cell elongation and expansion through osmotic regulations in plant cells. In addition, K⁺ uptake through K⁺ channels is necessary for proper cell cycle progression. Therefore, in this study, we examined the role of K⁺ in cell division and cell cycle progression of tobacco BY-2 cells. When we measured the cell osmotic pressure with plasmolysis method and a pressure probe, it was high in the elongating cells and low in the dividing ones. Subsequently, when we examined the effect of cytoplasmic alkalizing reagents, they restored the decreased cell division rate and the delay in cell cycle progression observed in the condition of K⁺ depletion. Therefore, K⁺ will be involved in cytoplasmic alkalization in cell division and cell cycle progression rather than in the regulation of cell osmotic pressure.

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Blue light induces stomatal opening through the activation of the plasma membrane H⁺-ATPase. Recent investigations have demonstrated that blue light activates the H⁺-ATPase via phosphorylation on threonine residue in the C-terminus with subsequent binding of 14-3-3 protein to the phosphorylated C-terminus in guard cells. However, protein kinase and protein phosphatase, which catalyze phosphorylation and dephosphorylation of the H⁺-ATPase, are still unknown. In this study, we analyzed the H⁺-ATPase complex by gel filtration and blue native-PAGE in Vicia guard cells. The H⁺-ATPase complex was detected 300-500 kDa. Illumination of blue light had no effect on size of the H⁺-ATPase complex in guard cells. Interestingly, treatment of guard cells with a fungal toxin fusicoccin, an activator of the H⁺-ATPase, increased size of the H⁺-ATPase complex to 750 kDa. We will report analyzes of in vitro phosphorylation and dephosphorylation of the H⁺-ATPase in the purified complex.

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Arabidopsis thaliana have CACTA transposons in their genomes. We previously reported that CACTAs are mobilized by ddm1 mutant. Moreover, we described that CACTA activated by the ddm1 mutation remains mobile in the DDM1 wild-type background. These findings indicated that DNA methylation works as a heritable epigenetic mark to silence CACTA transposon. In this study, we examined the developmental timing of the CACTA mobilization. We estimated CACTA1 transposition frequency by PCR. PCR analysis showed that the first ddm1 homozygous generation plants contained less genome copies that had the excised original CACTA1 locus than the next generation did. Currently, we are analyzing transgenic plant with 35S-dCACTA-GUS construct for histological evidence of CACTA excision. We will discuss the mechanism of the activation of the CACTA transposition during the developmental process.

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We introduced the inverted repeat (IR) silencer DNA construct to trigger RNAi in a unicellular green alga Chlamydomonas reinhardtii, which is targeted aadA mRNA Robust gene silencing was observed at some transformants immediately after transformation, however when the efficiency was reexamined after long period of continuous culture and subsequent single colony isolation, RNAi transformants showed heterogeneously decreased efficiency of RNAi. Prominent negative correlation between the amounts of accumulated aadA hairpin RNA and the RNAi efficiency was demonstrated by Northern blot analysis. Methylated cytosines were heavily accumulated in the IR region of silencer DNA and the modified cytosine ratio negatively correlated with accumulated hairpin RNA. Moreover, treatment of histone deacetylase inhibitor, immediately increased the accumulated hairpin RNA. Taking together, we propose that IR DNA structure of silencer construct induced CG-methylation at the region, then histone modification change followed to repress the transcription of the hairpin RNA.

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The unicellular green alga chlamydomonas reinhadtii has no enzyme to convert a single strand RNA into a double strand. This leads to that continuous supply of large amount of double strand RNA is required to achieve stable RNAi. However, we have been observed that inverted repeat DNA construct drove by the rbcS2 promoter, which is one of the most effective polII promoter, can induce a transient RNAi. We noticed the report that silence DNA construct drove by tRNA promoter is very successful in human cell. So far, we made six kinds silencer DNA constructs bearing modification at the acceptor stem of C.reinhardtii Asp tRNA, which is targeted the aadA mRNA conferring spectinomycin resistance. Preliminary experiment showed that some of them have much bigger efficiency than the polIII promoter drove type silence construct.

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DDM1 (Decreased in DNA Methylation) encodes a chromatin-remodeling factor is thought to be necessary for maintenance of cytosine methylation in genomic DNA. The ddm1 mutants in Arabidopsis confer hypomethylation in repetitive sequences and induce epi-alleles stochastically. While Arabidopsis bears the single DDM1 gene, two redundant DDM1 genes, OsDDM1a and OsDDM1b, were annotated in rice. We have developed an efficient and generally applicable gene targeting (GT) procedure in rice, which is based on a strong positive-negative selection in a large-scale Agrobacterium-mediated transformation, followed by PCR screening. To elucidate the function of rice DDM1 genes, we are attempting to isolate transgenic rice plants having these OsDDM1 genes modified by GT. We have obtained several independent transgenic lines with either OsDDM1a or OsDDM1b modified in the heterozygous condition and their segregants with one of the mutated alleles in the homozygous condition. We will present our results of these mutants.

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The MET1(Methyltransferase1) gene copies the pattern of cytosine methylation at CpG sites to the newly synthesized strand, which plays a critical role in epigenetic inheritance. While Arabidopsis carries the single MET1 gene, it is known that rice bears two redundant MET1 genes, OsMET1a and OsMET1b. We have developed an efficient and generally applicable gene targeting (GT) procedure in rice, which is based on a strong positive-negative selection in a large-scale Agrobacterium-mediated transformation, followed by PCR screening. To elucidate the function of rice MET1 genes, we are attempting to isolate transgenic rice plants having the GUS reporter gene fused with the endogenous promoters of these OsMET1 genes by GT. We have obtained 15 and 3 independent transformed lines with OsMET1a and OsMET1b modified in the heterozygous condition. We will discuss the results obtained from the OsMET1 knock-in mutants and technological aspects of our knock-in targeting.

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Gene targeting by homologous recombination (HR) is a powerful tool for reverse genetics. We have developed an efficient and generally applicable gene targeting (GT) in rice based on a large-scale transformation with strong positive-negative selection followed by PCR screening. To investigate the function of DNA methylation in rice, we generated transgenic rice plants having OsDRM1a disrupted by GT and obtained six independent T0 lines containing the mutated OsDRM1a gene in the heterozygous condition. Homozygous T1 plants showed characteristic phenotypes including dwarfism, reduced tillers, late heading, abnormal flowers, and activation of certain retrotransposons, indicating that OsDRM1a plays important roles in rice normal development and requires for the transcriptional suppression for some retrotransposons in the genome.

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