Three protein kinase families, Aurora, Polo, and NIMA-related kinases (NEKs), regulate mitotic processes downstream from cyclin-dependent kinases. NEKs are highly conserved in eukaryotes and extended into multi-gene family. Previous studies in fungi and animals indicated that NEKs regulate mitotic progression, centrosomal separation, spindle formation, and cilia function, while functions of plant NEKs remain to be elucidated. Our recent work revealed that Arabidopsis NEK6 associates with cortical microtubules and modulates anisotropic cell growth and morphology interacting with armadillo-repeat kinesins (Sakai et al. 2008, Motose et al. 2008). Here, we describe functional analysis of NEKs in Arabidopsis with special emphasis on NEK6. NEK6 was highly accumulated in meristematic cells and associated with the cytokinetic phragmoplasts. NEK6 was concentrated in the dot-like structures exhibiting the dynamic movement along with the phragmoplasts and cortical microtubules. We also provide evidences for NEK-dependent microtubule regulation and functional relationship among NEKs.

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Plant cells have extraordinary pluripotency, but they are differentiated into a specific organ in a defined position and timing. Arabidopsis TCP3 regulates differentiation of cells in shoot lateral organs. We reported that expression of chimeric TCP3 repressor (TCP3SRDX), which was a dominant negative form of TCP3, induced wavy shaped organs and formation of shoots on cotyledons. Expression of TCP3SRDX induced ectopic expression of boundary-specific genes including CUC genes in association with reduction of accumulation of miR164, whose product cleaves CUC transcripts. To clarify the role of TCP3 in the negative regulation of CUC genes, we used a system for chemical induction of TCP3SRDX in transgenic plants and found that genes downstream of TCP3 are involved in transcription, auxin responses and regulation of chloroplast function. Transient gene expression assay indicated that TCP3SRDX suppressed the promoter activity of these genes. We discuss possible gene network regulated by TCP3.

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Arabidopsis leaf trichomes provide an excellent model system to study cell differentiation. Using a collection of Arabidopsis full-length cDNA over-expression (FOX) lines, we identified a transgenic FOX line that develops remarkably large leaf trichomes compared wild-type. Our sequencing analysis revealed that the corresponding gene, which we named WIND FARM, encodes a putative transcription factor that has not been previously characterised. Furthermore, the sequence alignment illustrated that the cDNA clone is strongly truncated, possibly leading to a dominant negative phenotype. In concert with this, three different T-DNA insertion lines display giant trichome phenotypes indistinguishable from FOX mutants, suggesting that WF acts as a negative regulator of trichome cell growth. Additional genetic studies show that WF acts downstream of trichome patterning and differentiation, confirming its exclusive role in cell expansion.

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The organogenesis of leaf blade and petiole as a whole has received less attention, although leaf is composed of these two parts. Based on the histological observation, we defined a meristematic region in a leaf as leaf meristem, which localizes at an expected leaf blade/petiole junctional region, and supplies cells to both directions toward the apex and base of a leaf primordium. Here, from the perspective of leaf meristem activities, we re-characterized the leaf development of a loss-of-function mutant of BLADE-ON-PETIOLE (BOP1) and BOP2, and an overexpression line of LEAFY PETIOLE. Histological analyses combined with marker genes suggested that phenotypes of these mutants could be caused by changes in leaf meristem activities. To reveal developmental functions of these genes on leaf meristem activities, genetic relationships between these mutants and several other leaf mutants are being investigated.

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Compensation is a phenomenon in which a decrease in cell number triggers an increase in mature cell size. This phenomenon is important to understand the coordination of cell proliferation and expansion during organogenesis. We have isolated and reported our analysis on five fugu mutants that exhibit compensation. Here, we report about the fugu2 mutant. Map-based cloning revealed that FUGU2 was FASCIATA1, which encodes for the chromatin assembly foctor-1 p150 subunit. To elucidate the molecular mechanisms of compensation, we performed micro-array analysis using mRNA extracted from leaf primordia of fugu2-1 and confirmed the results by RT-PCR. In fugu2 leaves, the expression of 46 genes was induced and that of 39 genes was repressed more than three fold. Particularly, 11 among the induced genes were involved either in DNA replication or repair. By using T-DNA insertional lines, we are now investigating relationships between compensation and these changes of gene expression.

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In leaves of Arabidopsis thaliana, excessive cell enlargement is often triggered by decreased cell proliferation activity. This has suggested that cell proliferation and cell expansion are fundamentally coordinated for proper organogenesis. Our recent analyses of the compensation exhibiting mutant fugu5 has revealed that compensation was totally cancelled when fugu5 was grown on MS medium. We found that sucrose was necessary and sufficient for fugu5 phenotype recovery. Furthermore, compensation in fugu5 occurred only in cotyledons and the first pair of rosette leaves, but not in rosette leaves of higher nodes. Map-based cloning of FUGU5 gene revealed that it is AVP1, which encodes for a vacuolar type H⁺-pyrophosphatase. Pyrophosphatase activity is totally lost in three fugu5 mutant alleles. Interestingly, the transition of protein bodies to central vacuole was significantly delayed in fugu5 cotyledons. Taken together, these results indicate that FUGU5 plays particularly an important role during early stages of seedling development.

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In leaf development, a defect in cell proliferation triggers enhanced cell enlargement, resulting in partial restoration of total leaf area. This phenomenon is termed compensation.
How differentiating cells in the tip region of developing leaf recognize the defect of cell proliferation in the basal region remain to be elucidated. Is there cell-to-cell communication with some mobile factors? These issues must be unraveled firstly. Thus, we started to analyze mosaic leaves expressing compensation-related factors induced by CRE/Lox system.
First, we carried out mosaic analysis of KRP2-overexpressor (ox). As a result, mosaic leaves contained two distinct cells, whose sizes were equivalent to wild-type and KRP2-ox cell, respectively. In these mosaics, cell size of wild-type clone was not affected whether KRP2-ox clone is directly adjacent or not, and vice versa. This fact suggested that the compensation in KRP2-ox is regulated in a cell-autonomous manner.

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The ROT4 (ROTUNDIFOLIA4) gene is widely conserved among land plants. In addition, constitutive overexpression of ROT4 in Arabidopsis exhibits pleiotropic phenotypes, such as shorter lateral organs, shorter statures, shorter roots, and protrusion at the base of pedicels and trichomes. Thus, it is suggested that ROT4 plays important roles in plant development, however, the molecular function is largely unknown.
To get insights about the functions of ROT4, we first generated a series of truncated version of the coding region and examined the phenotype of these overexpressors. As a result, we found that highly conserved region of 32 amino acid residues was necessary and sufficient for the ROT4 functions. We generated chimeric overexpressor of GFP:ROT4 using HSP::Cre/Lox system, and found that GFP:ROT4 functions in an autonomous fashion. We also found interesting phenotypes of chimeric transgenic plants. In this presentation, we will discuss the function of ROT4 on plant development.

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Chemo-attractants from the ovule have been thought to be key molecules in pollen tube guidance of flowering plants for more than 140 years. However, no molecule has been convincingly demonstrated to be the true attractant that actually controls pollen tube guidance. By using our in vitro Torenia system, we previously showed that the synergid cell emitted some diffusible attractant(s). The attractant molecule was species preferential even in closely relating species, implying that the molecule had rapidly evolved.
We have analyzed ESTs from isolated synergid cells of Torenia fournieri and identified small secreted proteins as candidate pollen tube attractants. To characterize their activity to attract pollen tubes, we tried to purify sufficient amount of recombinant proteins from E. coli and to refold them correctly. We also tried to develop novel in vitro assay to quantitatively analyze the attraction activity. We will demonstrate whether the candidates show the attraction activity or not.

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The synergid cell of flowering plants is necessary for pollen tube attraction and fertilization. We identified small secreted proteins as candidates of pollen tube attractants by EST analysis of the synergid cell of Torenia fournieri. To demonstrate that the candidates are true attractants, it is important to show that down-regulation of candidates impair pollen tube attraction. Thus we attempted to knock-down the candidate genes and to analyze the effects on pollen tube attraction. We developed a method and a device for Laser-assisted Thermal-expansion Micro-injection (LTM). Then morpholino antisense oligos (MOs) against these candidates were designed to inhibit both their translation and mRNA splicing. They were injected into the protruding embryo sac of Torenia by LTM. We will demonstrate our recent results to discuss whether the pollen tube attraction is impaired by injection of MOs or not.

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In the fertilization process of angiosperms, pollen tubes are not gathered around an ovule, but a single pollen tube is usually guided to each ovule. Such pollen tube guidance is considered to be involved in the mechanism that ensures blocking polyspermy.
We have recently identified small proteins as candidates of pollen tube attractants derived from the synergid cell of Torenia fournieri. The aim of this study is to investigate relation between the blocking polyspermy and attractants, by visualizing the candidates with immunological methods. We could establish methods for western blot analysis and immunostaining to show the candidate small proteins were secreted from mature ovules. We will show detailed time course analysis to discuss about the relation with blocking polyspermy.

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It is essential for fertilization of angiosperms that a pollen tube grows in a pistil and reaches an embryo sac. There must be various mechanisms that guide pollen tubes to embryo sacs in the fertilization process. However, most of molecules underlying the mechanisms, including pollen tube attractants, are unclear.
Our aim of this study is to identify pollen tube attractants of Arabidopsis thaliana that are likely to be derived from the ovule or, more precisely, the synergid cell. We have identified small proteins as candidates of pollen tube attractants derived from the synergid cell of Torenia fournieri. As previously suggested by physiological experiments using closely relating species, the candidate genes rapidly evolved, which made difficult to search homologs in Arabidopsis by conventional molecular phylogenetic analysis. Here we report our trial to identify candidates of attractants of Arabidopsis, including genetic analyses and semi in vitro assay for several candidates.

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BiP is an Hsp70 in the endoplasmic reticulum (ER), and Arabidopsis thaliana has three BiP genes, BiP1, BiP2 and BiP3. We found that simultaneous deletion of the BiP1 and BiP2 genes resulted in defect in the polar nuclei fusion during female gametophyte formation. Transmission electron microscopy revealed that the defect was at the nuclear membrane fusion step. Since BiP has shown to function in the nuclear membrane fusion during mating in budding yeast, our results suggests that the BiP-mediated nuclear fusion is conserved from yeast to plant. In yeast, two J domain-containing co-chaperones (J proteins) in the ER, Sec63p and Jem1p, function in nuclear membrane fusion during mating as partners for BiP. While Arabidopsis has two orthologs of the SEC63 and one ortholog of JEM1, single mutants of these J protein genes did not show female gametophyte formation defect. We are now analyzing double mutants of these genes.

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Abnormal endosperm development is observed in inter-specific or inter-ploidy crosses of many plant species. Several reports suggested that the endosperm failure is caused either by promotion or suppression of the endosperm development and this can be due to the functional difference between parental genomes. One of the possible molecular mechanisms underlying this phenomenon can be genomic imprinting, an epigenetic mechanism with parent-of-origin dependent mono-allelic gene expression.
We conducted inter-specific crosses between several cultivated and wild rice species and found abnormality in hybrid endosperm. In these crosses, the rate of nuclear division of the endosperm was not affected but the timing of cellularization was found to be delayed or advanced. We also carried out the microarray analysis using hybrid endosperm of O. sativa and O. longistaminata, and identified the candidate genes related to the endosperm failure. Possible involvement of these genes and their effects on the endosperm development will be discussed.

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Plants have higher regeneration capacity than animals. It is hence an enticing task to delineate the mechanism underneath the remarkable regeneration capacity of plants. Plants maintain the stem cell activity at roots,shoots and flowers. The homeodoman protein WUSCHEL is necessary to maintain the stem cell activity. In flower development, WUS activity is abolished after proper number of floral organ primordia is created. We show that the nuclear matrix protein GIANT KILLER is involved in negative regulation of WUS through epigenetic regulation. We also show that after the stem cell activity is abolished by ectopic GIK induction, new meristems are regenerated. I will discuss on the regulation of WUS by GIK and associated meristem regeneration.

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Active development of rachis branches leads to the production of more spikelets, and finally to generation of larger panicle. We identified a dominant mutant, APO1-D3, containing increased number of rachis branches and spikelets. APO1-D3 was caused by an insertion of an active transposon nDart1-0 in promoter region of APO1. The expression of APO1 was increased in APO1-D3, suggesting that APO1 regulates the transition timing from rachis branch meristem to spikelet meristem. The effect of APO1 on rachis branch development was confirmed by the APO1 over-expression (APO1ox) analysis. Approximately half of the APO1ox plants showed a hyper-branching phenotype with the severity in proportion to the APO1 expression level. In addition, the size of inflorescence meristem of APO1-D3 was enlarged compared with that of wild type. These results indicate that the expression level of APO1 regulates the panicle size through controlling cell proliferation in inflorescence meristem.

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For the comprehensive understanding of the evolution of plant flowers, it is important to study the development of monocot flowers as well as eudicot flowers. In grasses, inflorescence comprise of unique structural units, spikelet and floret. The rice spikelet has one floret, which is subtended by two tiny glume-like organs, called sterile lemma. The sterile lemma is thought to be a rudimentary organ of two lateral florets, which have been degenerated during rice evolution. We have focused on a rice mutant, sterile lemma of which are longer than those of wild type, to elucidate the developmental mechanism of the sterile lemma and their evolutionary origin. Here, we will report the results of molecular genetic analyses and discuss evolution of rice spikelet.

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Floral organs are considered to be modified leaves that have the adaxial-abaxial polarity. However, the morphologies of stamens and carpels that differentiate into reproductive organs are different from leaves. It is interesting to address how the polarity is regulated in thsese organs.
We are focusing on a flower mutant, named rod-like lemma (rol) in rice. Flowers of rol show pleiotropic phenotype probably because of defects in the adaxial-abaxial polarity. To examine the relationship between anther development and the adaxial-abaxial polarity, we investigated expression pattern of the marker genes for this polarity. The results lead a hypothesis that conversion of the adaxial-abaxial polarity may take palce during anther development and subsequent anther development may be dependent on the new polarity. Our observations of anther development in rol support this hypothesis. The results also suggest that one theca is a developmental unit that has the adaxial-abaxial polarity.

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MOSAIC FLORAL ORGANS 1 (MFO1) / OsMADS6 is one of the rice class-G MADS-box genes. We have analyzed mfo1 mutants and RNAi knocked-down lines, and unveiled that MFO1 is involved in determination of floral organ identity and meristem determinacy.
We generated double knocked-down mutants of MFO1 and other classes of MADS-box genes, SPW1 (class B), OsMADS3 (class C), and LHS1 (class E), respectively. SPW1 was expressed strongly in lodicules like MFO1. OsMADS3 was expressed in carpel like MFO1, and increase of lodicules in similar arrangement and floral meristem indeterminacy were observed in both mfo1 and the osmads3 mutant. In the phylogentic analysis, LHS1 showed high homology with MFO1, and both LHS1 and MFO1 were expressed in palea and carpel and possess common function associated with meristem determinacy. From the analyses of theses double mutants, we will discus the relationship between MFO1 and the other classes of MADS-box genes.

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We have isolated bisexual (R025) and asexual (K034) mutants of Silene latifolia and analyzed them using in situ hybridization of SlSTM (SHOOT MERISTEMLESS) 1 and 2, SlCUC (CUP-SHAPED COTYLEDON), and SlCLV1 (CLAVATA1). They would control the gynoecium suppression pathway in the early-stage of sexual differentiation in R025 and K034. Particularity, K034 produces asexual and imperfect female (female-like) flowers on one individual. In the in situ hybridization, two kinds of expression patterns were observed prior to any morphological differentiation as male or female. In K034, 14.3% of flowers were female type and the rest were male, corresponding to the frequency of female-like flowers. Male- and female- type expression patterns of SlSTM, SlCUC, and SlCLV1 corresponded to carpel suppression patterns, suggesting that the mutation(s) responsible for gynoecium suppression acts upstream of the pathway involved in SlSTM, SlCUC, and SlCLV1, and switches carpel suppression patterns at a constant rate.

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White-rot fungi, produce extracellular enzymes such as lignin-peroxidase and laccase for degrading wood materials. The laccase catalyzes reduction of oxygen and the one-electron oxidation of a variety of phenolic substrates. In previous study, we produced transgenic tobacco plants transformed with a fungal laccase gene under CaMV35s promoter. Some of the transgenic plants exhibited higher laccase activity comparing to the wild-type plants. As expected, these plants can remove some phenolic compounds dissolved in hydroponic medium. In contrast, some unexpected phenotypes were observed in the transgenic plants. For example, anther and filament of stamen transformed to petal-like tissues. In some transgenic plants, color of anther turned to brown and the brownish anther failed to dehiscence at maturing stage. Furthermore, unidentified compound was deposited in epidermis and endothecium of the transgenic anther tissue. These results indicate that ectopic expression of laccase gene has biological effect on plant morphogenesis.

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The tapetum is believed to play important roles for maturation of pollen grains, but many of its functions have not been uncovered yet. To reveal such functions by reverse genetics, tapetum-specific promoters are good tools. However, few such promoters have been reported. We analyzed a promoter of GRP17, which encodes a protein accumulating specifically in tapetal cells. A 150 bp fragment upstream from initiation codon was sufficient for tapetum-specific expression. This promoter activity was abolished by base substitutions in a motif conserved among GRP gene promoters, suggesting that this motif might be a target site of tapetum-specific transcription factors. Some lipid transfer proteins (LTP), which are involved in lipid metabolism and transfer, act only in the tapetum. Their promoters worked specifically in tapetal cells at different stages of pollen development.

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Double fertilization is a unique reproductive system specific to angiosperm. During double fertilization, one of the two sperm cells fertilizes with egg cell and the other fertilizes with the central cell of the female gametophyte. Because the female gametophyte is deeply embedded in the ovule, it is difficult to observe the process of double fertilization in a live condition. We have developed novel systems to observe the entire process of Arabidopsis double fertilization in vitro. Video-rate imaging revealed that sperm nuclei were rapidly released into the embryo sac and delivered to the space between the egg and central cell after pollen tube discharge. We also found that the fusion of male and female gametophyte cells finished around 10 minutes after pollen tube discharge. In addition to this, we will introduce a trial to distinguish two sperm cells using fluorescent proteins.

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Female gametophyte plays a critical role in essentially every step of the reproductive process. One of the most important functions of the female gametophyte is the pollen tube guidance. Within the female gametophyte, synergid cells are required for pollen tube guidance (Higashiyama et al. 2002). Although the cellular functions of the synergid cells have been understood, little is known for the molecular-level function of the synergid cells. However, Kasahara et al. identified a key gene, MYB98. MYB98 is the most important gene to understand the synergid cells at a molecular level. MYB98 is expressed exclusively in the synergid cells and myb98 mutant phenotype showed pollen tube guidance defect. To investigate genes essential for the development and function of the synergid cell, we started forward genetics approach by using MYB98::GFP visible screening. In this meeting, we discuss about the newly isolated mutants and the future research plans for this project.

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In the female gametophyte of most angiosperms, the egg, central and synergid cells have distinct functions for fertilization. However, neither physiological mechanisms nor gene expression patterns responsible for the establishment of these cell features are elucidated. We are now trying two approaches; one is the in vitro analysis using Torenia, a unique plant having a protruding embryo sac, and the other is microgenomics using isolated gametophytic cells of Arabidopsis. We have developed a method to cultivate immature ovules of Torenia with the 4-nucleate female gametophyte stage to be fully matured. Live cell imaging and laser ablation experiments are now in progress. On the other hand, we have developed a new method to isolate synergid cells using novel micromanipulation system and cell-specific fluorescent marker lines in Arabidopsis. We will report our results of gene expression analysis by using the isolated cells.

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In skunk cabbage, developmental changes in reproductive organ, spadix, are closely related to the transition of thermogenic stages. Spadix can keep its internal temperature at around 20 degrees Celsius in female stage, but cannot keep it in male stage. Although growing reproductive organs in non-thermogenic plants exhibit extensive morphological changes and active cellular dynamics, those features in thermogenic plants have remained unclear. Therefore, we investigated the internal structure of spadices during the transition of thermogenic stages. Cross-sectional analyses of anthers clearly show extensive anther development, such as disruption of tapetam structure and pollen development. Ultrastructural analyses show that mitochondrial contents in female tissue, such as petal, pistil, and stamen, are higher than those in male. Although the level of uncoupling proteins is higher in female mitochondria than in male mitochondria, their respiratory levels are similar. Based on these results, we will discuss the thermogenic mechanisms in skunk cabbage.

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Postembryonic development in higher plants relies on the activity of the shoot apical meristem (SAM). In Arabidopsis, PASTICCINO2 (PAS2) encodes a major very-long-chain fatty acid (VLCFA) elongase, and its mutant shows ectopic cell division in aerial organ. However, it remains unknown how VLCFA regulates cell division and organ formation at the tissue level. Detailed phenotypic analyses of the pas2 mutants showed that they have enlarged meristematic region in the SAM that accompanied with CLAVATA3 expression. Despite a wide variety of developmental defects in aerial tissues, PAS2 was predominantly expressed in the shoot epidermis. We shall discuss a functional link between VLCFA synthesis and organ formation in terms of cell division control in Arabidopsis.

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Cyclin-dependent kinases (CDKs) regulate cell cycle progression, being activated by phosphorylation of the conserved threonine residue within T-loop. This phosphorylation is catalyzed by CDK-activating kinase (CAK). Arabidopsis has four genes encoding CAKs, namely CDKD;1, CDKD;2, CDKD;3 and CDKF;1. We previously reported that the cdkf;1 mutants showed severe defects in post-embryonic development but not in embryonic development, while the cdkd;1 cdkd;3 double mutants were embryonic-lethal. These results suggest diverged functions of CAKs during plant development. However, the spatio-temporal expression patterns of CAKs, which may cause differential roles in developmental processes, remain unknown. Hence, we are conducting detailed expression analyses of CAKs, especially during embryogenesis. We shall discuss why the CAK functions are so diverged in terms of expression patterns during embryogenesis.

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Genome integrity is maintained by coupling DNA damage repair to the cell cycle arrest during the repair. The fact that plants do not possess orthologs of some of the key factors that are important for the cell-cycle arrest in animal cells, implies that they probably have different mechanisms to cope with DNA damage.
In plant cells, A- and B-type cyclin-dependent kinases (CDKA and CDKB) are assumed to mainly control cell cycle progression. CDKAs are expressed throughout the cell cycle, while CDKBs are plant-specific CDKs and expressed specifically from the late S- to the M-phase. Recently, we found that the expression of CDKB2 is specifically suppressed in response to DNA damage. Together with the results of cultured cells, we hypothesize that this quantitative regulation of CDKB2 is required for inhibiting progression into mitosis in response to DNA damage.

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Cells undergoing endoreduplication replicate chromosomal DNA without intervening mitosis. The resulting larger, higher-ploidy nucleus is often associated with an increase in cell size. In Arabidopsis, inhibition of G2/M transition enhances endoreduplication. A linkage between DNA damage response and endocycle control is also suggested. On the other hand, increased ploidy level in rice is only reported in endosperm, DNA damage treatments do not enhance polyploidy in rice. Cell cycle regulation mechanism is not well understood in rice. B-type CDKs are plant-specific CDKs and CDKB2 expression is restricted from the late S-to the M-phase. These results suggest CDKB2 plays an important role in G2/M transition in plants. To investigate the role of rice CDKB2, we created OsCDKB2 knockdown rice via RNAi and found polyploidy cell in calli of knock-down plants. Increased number of chromosomes detected in these polyploid cells indicates endomitosis was occurred by the suppression of CDKB2 function.

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The proper segregation of chromosomes during mitosis is required for accurate distribution of genetic information by two daughter cells. Aurora kinases are serine/threonine protein kinases with essential roles in cell division through eukaryotes. Although functions of animal and yeast Aurora kinases have been analyzed in detail, those of plant Aurora kinases are unknown. Our live cell imaging analyses indicated that the plant Aurora kinase has dual roles; correction of aberrant kinetochore-microtubule attachment and dissociation of cohesin during chromosome alignment and segregation. We also analyzed transgenic plants of Arabidopsis thaliana to express AtAUR3 with tdTomato. AtAUR3 localized around kinetochores of mitotic chromosomes in A. thaliana.

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We previously isolated the Arabidopsis sog1-1 (suppressor of gamma response) mutant as a second-site suppressor of the Ionizing Radiation (IR) sensitive phenotype of the repair defective xpf mutant. sog1-1 mutant appeared to be defective in a DNA damage-induced checkpoint, but the SOG1 gene was not identified. Here, we report that SOG1 encodes a putative transcription factor. This gene is a member of the NAC family (a family of proteins unique to land plants) and plays a central role in response to IR. Hundreds of genes are normally upregulated by IR in wild-type Arabidopsis. Surprisingly, almost every one of these genes is not induced in the sog1-1 mutant, suggesting that SOG1 is the master regulator of the transcriptional response to IR. These studies suggest that, in plants, signals related to genomic stress are processed through a single, central transcription factor- SOG1.

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ANQ/MAPKK6 of Arabidopsis thaliana regulates cytokinesis. anq mutant plants exhibit cytokinesis defect. ANQ was expressed preferentially in proliferating tissues. ANQ localized at the equatorial plane of phragmoplast. We showed that ANQ acts as a MAPKK in yeast when it was co-expressed with HINKEL/AtNACK1 kinesin-like protein and ANP1 (and ANP3) MAP kinase kinase kinase. We identified MAPKs of group B as downstream factor of ANQ with in vitro phopholylation and activation experiment and with yeast two-hybrid analysis. These results proposed that HINKEL-ANP1 (and ANP3)-ANQ-group B MAPK constitute a MAPK cascade. We will discuss the role of this cascade in Arabidopsis cytokinesis.

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Cyclin-dependent protein kinases (CDKs) are main regulators during cell cycle. Here we show that CDKs might regulate the activation of NACK-PQR MAPK pathway that is known as a key-regulator of plant cytokinesis. The NACK-PQR pathway is activated by the direct interaction between NACK1 kinesin-like protein and NPK1 MAPKKK at anaphase. Previously we showed that both NACK1 and NPK1 are highly phosphorylated before the activation in vivo, and that they are phosphorylated by CDKs in vitvo. In this study, we show that NACK1 and NPK1 are phosphorylated at CDK-phosphorylation sites during early M phase when NPK1 are inactive. The levels of phosphorylation of NACK1 and NPK1 depended on the levels of CDK activities. In addition, the phosphorylation in vitro of NACK1 and NPK1 by CDKs inhibited the interaction between NACK1 and NPK1. These results suggest that CDKs function in suppression of the activation of NACK-PQR pathway, namely the entry of cytokinesis.

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The NACK-PQR pathway is MAPK cascade that consists of NPK1 MAPKKK, NQK1 MAPKK, NRK1 MAPK and NACK1 kinesin like protein in tobacco and is activated during late M phase. During cytokinesis, these factors are found at the phragmoplast equator. These results have proposed that this cascade is involved in cytokinesis. Homologs of these factors are identified in Arabidopsis thaliana. Here we searched target proteins of AtMPK4 that is a homolog of NRK1 to identify downstream factors of this cascade. We found proteins phosphorylated by NRK1 in microtubule-associated proteins fraction purified from tobacco BY-2 cells. Atkinesin13B was identified from these proteins by in silico assays with database of Arabidopsis thaliana. When we performed kinase assays using Atkinesin13B, Atkinesin13B was phosphorylated by AtMPK4. Furthermore Atkinesin13B localized at the phragmoplast equator during cytokinesis. These results suggested the possibility that Atkinesin13B is a downstream factor of AtMPK4.

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Plant cytokinesis is accomplished with the formation of phragmoplast, which is formed between two daughter chromatins during anaphase. The development of phragmoplast is regulated by the MAP kinase (MAPK) cascade. Although the MAP65, one of microtubule binding proteins, has shown to be phosphorylated by MAPK, most of targets are unidentified. We have tried to identify the new targets of MAPK and found PATL2 of Arabidopsis. PATL2 contained SEC14 domain which thought to be involved in the phospholipid metabolism and vesicle transport. The recombinant PATL2 protein was phosphorylated by MAPK and showed the binding activity to phospholipids. The binding activity of the phosphorylated PALT2 was different from the native form. The GFP-fused PATL2 expressed in plant cells was localized to plasma membrane and phragmoplast. These findings suggest that PATL2 is phosphorylated by MAPK at phragmoplast during cytokinesis and that changes the composition of phospholipids on phragmoplast through the activity of PALT2.

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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 from protonema develops to give rise to a leafy shoot called gametohpore. After excision of a leaf from a gametophore, leaf cells facing to the dissected cells change to protonemal cells. To explore molecular framework governing the cell cycle reactivation, we investigated temporal and spatial expression patterns of cell cycle regulators in the excised leaves. PpCDKA transcript and protein were constantly detected in P. patens leaf cells and the PpCDKA activity increased in 24 hours after excision. At the same time, PpCYCD gene expression, which is responsible for the PpCDKA activation, was observed. Taken together, our results indicate that the constant expression of PpCDKA is likely related to the immediate reentry of a cell cycle with response to the excision signal, through the PpCYCD expression.

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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 59 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 less in differentiated protonemal cells. We will also report results of functional analyses on the two candidate genes.

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rid2 is a temperature-sensitive mutant of Arabidopsis that is defective in the initial process of root and callus formation, and therefore is expected to serve as a useful tool for studying dedifferentiation and reactivation of cell division. rid2 is also characterized by enlargement of the nucleolar cavity at the restrictive temperature, which directed our attention to the relationship between RID2 and nucleoli. We analyzed processing of pre-rRNAs, the main function of nucleoli, and showed that it is significantly impeded in the rid2 mutant. Analysis with RID2::GFP indicated that the RID2 protein is abundant in nucleoli.
For a genetic approach to understanding the action mechanism of RID2, we isolated a suppressor mutant of rid2, sriw1. Chromosomal mapping and genome sequencing detected a base substitution in At5g09330, which encodes a putative NAC transcription factor, of the sriw1 genome possibly responsible for suppression of rid2.

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5-Bromodeoxyuridine (BrdU), a thymidine analog, exerts promotive and inhibitory effects on shoot regeneration in hypocotyl tissue culture of Arabidopsis when administered during dedifferentiation at low and high concentrations, respectively. bro1 and bro2 are mutants resistant to BrdU with respect to these effects. bro2 is also resistant to fluorodeoxyuridine (FdU), another thymidine analog, while bro1 is not. bro1 carries a missense mutation in a gene encoding an RNA-binding protein, and bro2 carries a missense mutation in a gene encoding a thymidine kinase.
Here we report new BrdU-resistant mutants, bro3 and bro4. bro4 was shown to be resistant to FdU like bro2, while bro3 was not resistant to FdU like bro1. Chromosome mapping located these mutations on different regions of chromosome 4. Four nucleoside transporter genes, including FUR1, exist in the region where bro4 was mapped. Sequencing analysis of the bro4 genome is underway with a special note on these genes.

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Plant body size is thought to be tightly regulated by nuclear ploidy levels and cell numbers.
sd5 mutant was isolated as a dwarf and seedling lethal mutant from RIKEN Ds insertional mutant lines. Cell numbers rather than ploidy levels were decreased in sd5 cotyledons and CYCB1;1 expression was also down-regulated, indicating that cell proliferation was strongly affected in sd5.
Corresponding gene of sd5 encodes a homologue of yeast DIM1, a component of U5 spliceosome. Loss of DIM1 caused lethality as a result of defect of cell cycle in S. pombe. Interestingly, SD5 did not complement to the pombe dim1-35 mutation when SD5 was expressed under limited temperature. On the other hand Arabidopsis another homologue of DIM1 could complement to this mutation. These results suggested SD5 plays different functions of authentic DIM1 protein.
Here we will demonstrate the detail of sd5 phenotypes and physiological functions of SD5.

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Chloroplasts evolved from a cyanobacterial endosymbiont and chloroplast division requires the formation of an FtsZ division ring, which is descended from the cytokinetic machinery of cyanobacteria. As in bacteria, the positioning of the chloroplast FtsZ ring is regulated by MinD and MinE proteins. However, chloroplast division also involves mechanisms invented by the eukaryotic host cell. Here we show that a plant-specific protein MULTIPLE CHLOROPLAST DIVISION SITE 1 (MCD1) regulates FtsZ ring positioning in Arabidopsis thaliana chloroplasts. MCD1 is required for chloroplast division, localizing at the division sites and punctate structures dispersed on the inner envelope. Further analyses revealed that MCD1 and MinD affect each other through direct interaction to position the FtsZ ring. These results suggest that the plant cell has a new component to modulate the bacteria-derived Min system in the initiation of the chloroplast division.

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Consistent with their endosymbiotic origin, chloroplasts only proliferate by division. To establish the permanent endosymbiotic relationship, the host cell has been regulated proliferation of chloroplasts during proliferation of the cell. Furthermore, in higher plants, size and number of chloroplasts changes according to cell differentiation. But the regulatory mechanism of chloroplast division is poorly understood. Recently, we found that the overexpression of PDV2, which was identified as a component of chloroplast division machinery, caused the increase of number of chloroplasts per cell and the decrease the size of chloroplasts. The expression of PDV2 was high in shoot apical meristem and decreased according to leaf development. We also found that cytokinin increased PDV2 expression. These results indicate that PDV2 regulates chloroplast division during leaf development under cytokinin signaling pathway. We also analyze a PDV2 ortholog in Physcomitrella patens, and the functions of PDV2 in land plants will be discussed.

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Chloroplast division is driven by both prokaryotic component and eukaryotic component. Our previous study, we isolated an YLMG cDNA overexpressing Arabidopsis with altered chloroplast number and size from the FOX hunting transgenic lines. In this study, we investigated the localization of YLMG protein and analyzed in detail the YLMG-overexpressing plant and -deficient plant. YLMG-deficient plant did not have effect of chloroplast division, and exhibited young leaves of pale-green. Moreover, chloroplast nucleoids of YLMG-deficient plants were larger than those of the wild type. On the other hand, chloroplast nucleoids of YLMG1-overexpressing plants became filamentous and formed a network instead of multiple small nucleoids dispersed throughout the stroma. We discuss the role of AtYLMG1 in the chloroplast division and the morphology of chloroplast nucleoids.

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Amyloplasts are a differentiated form of plastids that serve for synthesis and storage of starch. Amyloplasts have double envelope membranes and divide by binary fission, as the leaf chloroplasts do. So far, research into the molecular mechanisms of plastid division has been conducted mainly using chloroplasts from leaf or algal cells. It remains unknown whether proliferation of non-photosynthetic plastids follows the 'chloroplast division model' in higher plants. In this study, we focus on the mechanisms of amyloplast proliferation in seed integuments of Arabidopsis. Using four chloroplast division mutants (arc5, arc6, minD and minE) and transgenic lines expressing stroma-targeted fluorescent proteins, we visualised and investigated amyloplast populations in outer ovule integument cells of the mutants. It was indicated that the control of amyloplast division is considerably different from that of chloroplasts.

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In cyanobacterium, peptidoglycan synthesis is essential for its cell division. It is widely agreed that plastid is originated from a single cyanobacterial ancestor, though no wall-like structure has been observed in plastids of green plants. We found all homologs to bacterial Mur genes for peptidoglycan synthesis from Physcomitrella patens. Pbps catalyze the final step of peptidoglycan synthesis. Disruption of PpPbp gene in P. patens causes appearance of macrochloroplasts. Main subfamily of Pbp possesses two functional domains, transglycosidase (TG) and transpeptidase (TP). To examine each function of putative domain in PpPbp, two mutations were introduced for each domain. When the plasmid containing PpPbp gene without mutations was transferred into the PpPbp KO line, the macrochloroplast phenotype was recovered. On the other hands, each PpPbp with two mutations for TG or TP domain could not recover the macrochloroplast phenotype. This result suggests that both activities are essential in P. patens chloroplast division.

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The number of chloroplasts per cell is maintained virtually constant by chloroplast division and chloroplast partition at the time of cell division. Protonema of Physcomitrella patens grows by tip growth or branching. It is also noted that ampicillin decreases number of chloroplasts. But how single large chloroplasts are formed remains unclear. When protonemata of Physcomitrella patens were treated with ampicillin, the number of chloroplasts decreased earlier than expected, assuming equal partitioning. We analyzed chloroplast partitioning at the time of cell division, and found that unequal partitioning occurs preferentially when the number of chloroplasts is small.

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Arabidopsis thaliana crumpled leaf(crl) mutants have yellowish green and crumpled leaves, and contain fewer and giant chloroplasts (Asano et al., 2004).In this study, we analysed the structure of CRL gene homologs in the moss Physcomitrella patens and characterized their function. A. thaliana has a single AtCRL gene whereas P. patens has three CRL genes(PpCRL1,PpCRL2,PpCRL3). We generated gene disruptants by homologous recombination. Single gene disruptants exhibited similar phenptypes of the wild-type mosses. In contrast, the PpCRL1/2 double disruptants showed severe retardation of growth, aberrant morphogenesis and abnormal plastid division in the protonemata. However, PpCRL1/3 double disruptants resulted in a similar phenotype of the wild type. This observation suggests that PpCRL1 and 2 gene plays a critical role in protonemal growth of P. patens. To prove this we have generated PpCRL2/3 double and PpCRL1/2/3 triple disruptants.

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Among 2,090 chloroplast proteins identified by computational prediction in Arabidopsis, we collected 3,244 tag-lines disrupted by transposon or T-DNA that encode 1,369 chloroplast proteins. From this collection, we have not only collected 1,234 homo-lines without visible phenotypes, but also isolated 100 mutants showing abnormal seedling (apg mutants) and 141 mutants of which homozygous lines were not obtained. We are constructing the RIKEN Arabidopsis Chloroplast Encyclopedia (RACE) database that presents genetic and phenotypic information on our resources, as well as essential, non-redundant genes that show an abnormal phenotype at seedlings and developing seeds stages when disrupted by mutation. The RACE database also provides tools for searching by AGI code, mutant line number and phenotype, and rapid access to detailed information on the resources. Moreover, our homozygous collections can be expected to yield powerful tools to accelerate and enhance the functional genomics of nuclear-encoded chloroplast proteins in Arabidopsis.

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Plastids are descended from a cyanobacterial symbiosis and have bacterial type RNA polymerase (PEP). However, recent plastids proteome analyses revealed that higher plant plastids contain not only prokaryotic proteins derived from the ancestor, but also several eukaryotic proteins acquired from host cells during plant evolution. We focused on a component of chloroplast transcriptional active chromosome: pTAC3, which has a eukaryotic-type DNA binding domain (sap domain). The pTAC3 knock-out mutants showed albino phenotype and expression of PEP-dependent genes was significantly suppressed in the mutants. To determine the binding sites for pTAC3 in Arabidopsis chloroplast genome, we performed a chloroplast ChIP assay. pTAC3 binds mainly to transcription units that are transcribed by PEP such as psbA. Gel shift analysis revealed that a GST-tagged sap domain fragment has DNA binding activity, while the sequence specificity is weak. These results suggest that pTAC3 might function as a regulatory factor of PEP.

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