In plants, metyl jasmonate (MeJA), which mediates various plant defense response, as well as abscisic acid (ABA) induces stomatal closure. It has been shown that there are various signaling factors in MeJA signaling in guard cells. RCN1, an Arabidopsis protein phosphatase 2A (PP2A) A subunit, is involved in auxin signaling in roots and ABA signaling in guard cells. Previously, our study suggested that RCN1 functions as a positive regulator in MeJA signaling in guard cells. RCN1 is required for MeJA regulation of reactive oxygen species production and inward-rectifying potassium ion channel currents in guard cells.
In order to gain further information about MeJA signaling in guard cells, we evaluated roles of RCN1 in MeJA regulation of nitric oxide production, calcium ion channel currents, and S-type anion channel currents in guard cells. Our results suggest a novel model of MeJA signaling in Arabidopsis guard cells.

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To avoid the generation of reactive oxygen species, makeup of PS II and PS I must be synchronized. We have found that a major sigma factor, SIG1, in RNA polymerase is phosphorylated in dim light to suppress the expression of photosynthesis genes, whereas SIG1 is dephosphorylated under irradiance to release the suppression.

We have employed a wheat germ cell-free protein production system in combination with AlphaScreen (PerkinElmer) to detect protein-protein interaction in vitro to search protein kinases responsible for phosphorylation of SIG1. SIG1 and approx. 800 protein kinases have been synthesized in vitro using RIKEN Arabidopsis Full-Length (RAFL) clones and subjected to the screening. We have obtained 49 candidate protein kinases. A phosphorylation assay has shown an activity to phosphorylate the Thr-170 of SIG1 in the wheat germ extracts.

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Positional information is crucial to determine cell fate, underlying basis of plant morphogenesis. Plasmodesmata constitute one pathway of such information, especially for transport of macromolecules. It is widely believed that plasmodesmata adjust their own size exclusion limit (SEL) to control intercellular movement of the signals, and accordingly play important roles for plant development.
To date, although spatio-temporal regulation of SEL has been proposed in several angiosperms, it remains largely unknown how the regulation of SEL influences developmental state of each cell.
We, therefore, decided to take advantage of a simple body plan of protonemata of the moss, Physcomitrella patens to approach this issue. Here we will report establishment of experimental systems: switching cell fate arbitrarily from proliferating to nonproliferating state and vice versa, and visualizing intercellular movement of macromolecles at a single cell level and at a certain cell cycle stage.

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The CLV3/ESR peptide family is a group of small dodecapeptides that play key roles in various cell-cell communications. Among them, CLE41, CLE42 and CLE44 has almost identical amino acid sequences, and the genes coding them falls in a single subgroup. These peptides were conventionally known as inhibitors of differentiation into tracheary elements. However, the analysis of GUS reporter gene under promoters of these genes indicated that, while CLE41 and CLE44 are expressed in vascular bundles, CLE42 is expressed in SAM and axillary buds. . Therefore, we decided to examine SAMs and axillary buds of the plants overexpressing these CLE genes.
Our results suggested that the overexperssed CLE41 and CLE42 or application of the CLE41 or CLE42 peptides can promote axillary bud formation and growth. Taken together with results from gene expression analysis, we will discuss through which pathway these CLE peptides affect the axillary buds.

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In Synechocystis sp. PCC 6803, a histidine kinase, SphS, induces expression of alkaline phosphates (AP) under phosphate deficiency. Since an amino terminal region of histidine kinase generally contributes to perceive the signal, we characterized function of a hydrophobic region and a PAS domain existing at the amino terminal region of SphS.
We expressed genetically modified SphSs in Synechocystis cells and abilities of the signal sensing were monitored by measuring the alkaline phosphatase (AP) activity. Deletion and point-mutation of PAS domain constitutively expressed AP activity, whilst deletion of the hydrophobic region lost expression of AP activity. Interestingly, substitution of the hydrophobic region with a membrane-bound sequence from another histidine kinase, NrsS, maintained the ability to respond to phosphate availability. These results suggested that the PAS domain of SphS is essential to regulate kinase activity. However, the hydrophobic region is not involved in the signal perception.

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The calcium ion (Ca²⁺) plays a major role in stress signal transduction in plant cells. However, little is known about stress- and pathogen-induced Ca²⁺ oscillation in chloroplasts. Here we provide the first direct evidence that stromal Ca²⁺ levels are elevated by various biotic and abiotic stresses, including pathogen-derived elicitors, H₂O₂, high salinity and high osmolarity. For example, bacterial elicitor (flg22) induced a rapid elevation of cytoplasmic Ca²⁺ concentration within a min and subsequent slower and long lasting stromal Ca²⁺ elevation. We recently found that chloroplast protein CAS is involved in the generation of cytosolic Ca²⁺ signals and subsequent stomatal closure (Nomura et al., 2008 Plant J). Flg22-induced stromal Ca²⁺ elevation was significantly suppressed in the CAS KO mutants. These results suggest that CAS plays a crucial role in the generation of stress- and pathogen-induced stromal Ca²⁺ signals.

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We tried to perform the chemical genetics screening by Brz (brassinazole) that was synthesized as the first specific inhibitor of brassinosteroid biosynthesis. Target of Brz220 is the cytochrome P450 enzyme encoded by DWF4. In order to analyze in detail the mechanisms of brassinosteroid signal transduction, we screened for mutants that showed longer hypocotyls than wild type when grown with Brz220 in the dark, and designated bil mutants (Brz-insensitive-long hypocotyl). We identified a semidominant mutant, bil5, from fast neutron-treated lines. Hypocotyl elongation of these plants on Brz medium was at least twice that of the wild type. Adult bil5 plants showed pale green and thin leaves, and thin and shortened inflorescences. The palisade cell and spongy cell of bil5 mesophyll tissues were smaller and disturbed. Number and size of secondary xylem cell of bil5 inflorescences were decreased. The root elongation, flower size and silique size of bil5 was also inhibited.

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Brassinosteroids (BRs) are steroid hormones of plants that regulate plant growth and development. BRs are perceived by a cell surface receptor kinase BRI1, then various components are predicted to be involved to transduce the BR signal in the cell. To identify additional components involved in BR signal, we had analyzed various dwarf and semidwarf mutants from Arabidopsis mutant, which is insensitive to the BR synthetic inhibitor Brz. Brz causes deetiolation and dwarf phenotype similar to those of BR-deficient mutants. We had tried to screen mutants that showed to etiolate on medium containing Brz. Here we identify a bil4 (Brz-insensitive-long hypocotyl 4) mutant from activation tagging line. bil4 mutant showed slender dwarf phenotype which small and twisted leaves, and short and thin inflorescences. From genetic and functional analyses to bil4 mutant, we would like to clarify in detail the mechanism of brassinosteroid signaling transduction.

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We tried to screen new brassinosteroid signaling mutants from Arabidopsis activation tag lines and isolated two mutants.
In the light, Brz treated plant showed dark green leaves as compared to non-treated leaves. bpg2 was screened as pale green phenotype with Brz in the light. A T-DNA insertion site of bpg2 mutant was identified in an ORF of novel GTPase homologous gene that were conserved in many organisms. Expression of BPG2 gene was induced by light and Brz, and was highest in greening organs. BPG2-GFP fusion protein analysis revealed that BPG2 located in chloroplasts. In bpg2 mutant, abnormal chloroplast rRNA processing and protein accumulation were observed. These results might suggest that BPG2 plays an important role of posttranscriptional and/or translational regulation for chloroplast genome on brassinosteroid signaling.

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We had identified brassinosteroid biosynthesis inhibitor Brz insensitive mutant bil1 from EMS-mutation lines, and bil5 from fast neutron-mutation lines. To identify additional components of the brassinosteroid signaling, trial to screen new mutants as Brz-insesitive-long hypocotyls in the dark was applied to Arabidopsis activation-tag lines. We screened Arabidopsis activation-tag lines and isolated bil2 that showed long hypocotyls on the medium containing Brz in the dark. Light-grown bil2 mutants had long petiole phenotype similar to wild-type plants treated with brassinosteroid or BRI1-ox mutants. These phenotypes suggested that bil2 mutants enhanced the brassinosteroid signaling. Identification of overexpressing the bil2 candidate gene is progress.

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Abscisic acid (ABA) is a plant hormone involved in the regulation of seed dormancy and germination. We have shown that AtNCED6 and AtNCED9 play a key role in ABA biosynthesis in seeds. Promoter-reporter analysis, in combination with in situ hybridization and RT-PCR, indicated that AtNCED6 was expressed in endosperm during early to mid seed development, whereas AtNCED9 expression was observed in the seed coat in the middle of seed development and in the embryo during late stages of seed development. These data indicate that ABA biosynthesis is regulated in tissue specific manners depending on developmental stages. However, factors controlling tissue-specific ABA biosynthesis as well as physiological roles of ABA synthesized in different tissues are not well understood. We are conducting promoter analysis of AtNCED6 and AtNCED9. In addition, identification of the genes whose expression is regulated by ABA in tissue specific manners is under progress.

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Abscisic acid (ABA) regulates physiologically important stress and developmental responses. ABI1 encodes a protein phosphatase 2C that functions in early in ABA signaling. To address the mechanism of ABI1-mediated ABA signaling, we generated tagged ABI1 Arabidopsis expression lines in an abi1 knockout mutant and performed affinity column purification of ABI1-associated proteins. Transgenic tagged ABI1 plants show a strong ABA insensitive phenotype at the seed germination stage. After silver staining, visible bands overlapped with controls, and specific bands associated with purified tagged ABI1 samples were consistently observed. Mass-spectrometrical analyses allowed identification of proteins associated with ABI1. These included some known ABA signaling components. These results suggested that our strategy has the potential of identifying ABA signaling components. We found that a sub-group of gene family members interacted with ABI1 in an ABA dependent manner. The functional relationship between ABI1 and this protein family is being characterized.

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Abscisic acid is a ubiquitous hormone that plays a central role in land plants'responses to environmental stresses. The molecular mechanisms of ABA responses have been revealed in higher plants. In contrast, the mechanisms of ABA responses in lower land plants are not well understood. In order to understand molecular mechanisms underlying ABA responses in bryophytes, we analyzed Marchantia polymorpha MpABI1, a homologue of ABSCISIC ACID INSENSITIVE 1 (ABI1) in Arabidopsis. MpABI1 encoded a protein phosphatase 2C (PP2C) with an N-terminal regulatory domain. Transient expression of MpABI1 in Physcomitrella patens resulted in suppression of ABA-induced gene expression. Furthermore, ABA-induced freezing tolerance was impaired in transgenic mosses expressing the PP2C domain of MpABI1. These results suggest that MpABI1 is a negative regulator of ABA responses and that the functions of PP2Cs in ABA signaling are conserved in land plants.

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Plant regulates transpiration by controlling the stomatal conductance. In addition to such rapid responses, stomatal development is also regulated by environmental factors. The phytohormone abscisic acid (ABA) is known to regulate stomatal closure in response to the changing water status. We previously reported that the density of stomata was increased in both ABA-insensitive abi1-1 and ABA-deficient aba2-2 mutants and suggested that ABA regulates stomatal differentiation. In the present study, we investigated the effect of exogenous ABA application on the defect in stomatal development of the aba2-2 mutant. The suppressed stomatal density of aba2-2 was restored by ABA application. This is possibly due to both enlargement of the pavement cell size and decreased formation of stomatal meristemoids. Moreover, we found the up-regulation of the genes for stomatal development in the aba2-2 and abi1-1 mutants. Taken together, we conclude that ABA regulates developmental programs of stomatal formation.

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Levels of endogenous ABA are changed dynamically in response to environmental conditions. We examined physiological roles of Arabidopsis CYP707As in the plant's response to the change in humidity. When wild-type plants were transferred to high humidity condition, CYP707A1 and CYP707A3 transcript levels increased, followed by reduction of ABA levels. cyp707a3 mutant exhibited high ABA levels even after transferring to high humidity conditions, whereas cyp707a1 mutant exhibited low ABA levels comparable to wild type. High humidity-induced expression of CYP707A1 and CYP707A3 occurred in guard cells and vascular tissues, respectively. Stomatal closure of cyp707a1 mutant, but not cyp707a3 mutant, was ABA-hypersensitive when epidermal peel was treated with exogenous ABA. These results implicate that CYP707A3 reduces the amount of mobile ABA in vascular tissues in response to high humidity, whereas CYP707A1 inactivates local ABA pools inside the guard cells.

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In animals, NO-mediated production of cGMP is a crucial signaling pathway that controls numerous different cellular functions. In plants, conflicting results are obtained. This signaling mechanism remains a mystery.
Sawa et al. (2007) reported that a novel NO derivative of cGMP, 8-NO2-cGMP (NcGMP), acts as a second messenger in animals. We intend to study the role of this compound in plant cell signaling. Stomatal closure is induced by NcGMP, but not by 8-BrcGMP, whereas stomatal opening in the dark is induced by 8-Br-cGMP but not NcGMP, indicating that cGMP and its nitrated derivative act as signaling molecules in different signaling pathways. Furthermore, inhibitors of cADP-ribose synthesis and EGTA reversed NcGMP-induced stomatal closure, indicating that NcGMP is upstream of cADPR and Ca. The inhibitor of phospholipase D reversed NO-induced stomatal closure but not NcGMP-induced stomatal closure; this pathway is NO signaling from NcGMP.

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Glutamate is well established as an important signaling molecule in the mammalian central nervous system. However, it has long been believed that this signaling pathway does not function in plants, which have no nervous system. During the past decade, evidence has unexpectedly emerged that glutamate signaling occurs in higher plants. Completion of the Arabidopsis genome project revealed that the 20 AtGLR genes are homologous to mammalian ionotropic glutamate receptor genes. Glutamate receptors were found in roots; they mediated Ca influx through the cell membrane. Glutamate triggered an increase in cytoplasmic Ca concentration in Arabidopsis seedlings and affected root growth and branching.
At this meeting, we report the effect of glutamate on stomatal movement. Glutamate induced stomatal closure, which might occur as a result of increased cytoplasmic Ca concentration.

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Plants perceive moisture gradients by their roots and elongate them toward water sources to effectively obtain a limited amount of water in soil (hydrotropism). Little is known about the genes involved in hydrotropism of roots. We previously reported the reduced hydrotropic response of an Arabidopsis mutant defected in AKT2, which encodes a voltage-gated K⁺ channel. To gain a insight into the functional role of AKT2 in hydrotropism, we examined the spatial expression pattern of AKT2 during hydrotropic response using pAKT2::GUS transgenic plants. In the absence of a moisture gradient, AKT2 expression was uniformly observed in the phloem of roots. Under a moisture gradient, however, AKT2 expression was preferentially detected in the phloem of the outer (dry) side of bending roots rather than that of the inner (moistened) side. The results suggest that asymmetric accumulation of AKT2 in phloem plays a role in the hydrotropic response of roots.

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Cell differentiation involves temporally and spatially distinct stages, in which cell fate is established by an appropriate genetic program and then maintained by epigenetic regulation through the subsequent mitotic events. We identified a novel mutant, named bouquet-1 (boq-1), exhibiting frequent stem fasciation caused by the postembryonic development of extra shoot apical meristems in a SHOOT MERISTEMLESS (STM)-dependent manner. The boq-1 mutation enhanced adventitious meristematic activity observed in the stm mutant, and partially suppressed the floral defects in the stm and wuschel mutants. Molecular cloning revealed that the BOQ gene encodes a WD40 repeat protein. Overproduction of the boq-1 allele in the wild-type background mimicked the boq-1 phenotypes in a dose-dependent manner, suggesting that the mutant BOQ-1 protein acts in a dominant negative manner. Taking these results together, we propose that the BOQ gene is involved in the epigenetic regulation of cell differentiation directly or indirectly in a novel fashion.

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The phytohormone auxin transport and signaling are the key determinants of embryo patterning. To clarify the mechanisms underlying this process, we have analyzed the macchi-bou2 (mab2) mutant identified by a pinoid (pid) enhancer mutant screen. While pid single mutant seedlings showed only a mild phenotype in cotyledon number and/or positioning, the mab2 pid double mutant completely lacked cotyledons. The mab2 single mutant showed abnormal cell division during embryogenesis in addition to the abnormal cotyledon patterning. Using map-based cloning we identified the point mutation in the gene encoded a putative component of the Mediator complex, a general transcriptional regulator for RNA polymerase II, in mab2. MAB2 protein interacted with another mediator component in yeast, suggesting that MAB2 functions as the Mediator complex in Arabidopsis to regulate the transcription during embryogenesis.

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The Arabidopsis thaliana NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), acts as a master regulator of the differentiation of xylem vessels. To understand the mechanism by which VND7 regulates xylem vessel differentiation, we screened for proteins that interact with VND7 with a yeast two-hybrid system, and identified a cDNA encoding a NAC domain protein, VND-INTERACTING2 (VNI2). The expression of VNI2 overlaps with that of VND7 in elongating vessel precursors in roots, whereas the initiation and termination of VNI2 expression always precedes those of VND7 expression. Transient reporter assays showed that VNI2 is a transcriptional repressor and can repress the expression of vessel-specific genes regulated by VND7. The expression of the VNI2 under the control of the VND7 promoter resulted in the inhibition of the normal development of xylem vessels. These data suggest that VNI2 regulates xylem cell specification as a transcriptional repressor that interacts with VND7.

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In a vascular tissue of Arabidopsis roots, specific cell types are arranged in diametrically opposed longitudinal files that maximize the distance between them and create a bilaterally symmetric root. Mutations in the LONESOME HIGHWAY (LHW) gene eliminate bilateral symmetry and reduce the number of cells in the stele of the root, resulting in roots with only single protoxylem and protophloem poles. LHW does not appear to be required for the creation of any specific cell type, but coordinately controls the number of all vascular cell types by regulating the size of the pool of cells. LHW encodes a protein with weak sequence similarity to basic helix-loop helix (bHLH)-domain proteins. Now efforts are underway to reveal how LHW regulates vascular development in roots.

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We have developed a novel activation tagging system for Arabidopsis in which expression of tagged genes is specifically activated in postembryonic roots. Using this system, several mutants with altered room meristem patterns have been isolated. Two of the mutants, designated urp4 and urp5, were characterized by enhanced cell divisions in roots and their causal genes were found to encode homologous proteins with transcription factor motifs. While loss-of-function mutants of these genes did not show obvious abnormality, mutants of another member in the same family lost functional meristem in primary roots. Observation of their embryogenesis and marker analysis revealed that the mutants are defective in cell proliferation and formation of organ primordia for both roots and cotyledons. In yeast amino-terminal region of the protein was shown to activate gene transcription. Taken together these results suggest that this novel transcription factor-like protein acts in embryonic pattern formation and axis formation.

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Axis formation is one of the fundamental events in early development. In the Arabidopsis embryo, the main body axis is evident by the asymmetric division of the zygote forming a small embryonic apical cell and a large basal cell with mostly extra-embryonic fate. The homeobox gene WOX8 (WUSCHEL RELATED HOMEOBOX8) is expressed in the zygote and its basal derivatives and is crucial for axis development. Nevertheless, how polar WOX8 expression is achieved was totally unknown. We identified the transcription factors binding to WOX8 cis-elements. The mutants of these factors affected not only WOX8 expression, but also the zygotic polarity and the embryonic axis, indicating that these genes are the novel regulators of axis initiation. Based on these results, the mechanism regulating plant axis development will be presented.

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Cytokinins (CKs) are required to maintain the shoot apical meristem(SAM). LONELY GUY (LOG) gene of rice, encoding a CKs activating enzyme, controls meristem activity. The SAM of log-6 is smaller than that of WT from early embryogenesis and terminates prematurely. The reduction of SAM size is caused by the decrease in the meristem cell number, suggesting that LOG promotes cell proliferation in the SAM.
To investigate how meristem cell number is regulated by CKs, we examined the phenotype of LOG over-expression (LOGox)ansgenic plants. LOGox plants seem to be normal except that LOGox plants produce multiple embryo seeds frequently. These results indicate that CKs play an important role for the formation of the SAM in embryogenesis. We will discuss the function of CKs in early embryogenesis of rice.

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Carrot inhibitory factor of somatic embryogenesis, 4-hydroxybenzyl alcohol (4HBA), is released from carrot embryogenic cells (EC), and inhibit somatic embryogenesis in liquid culture. We reported that 4HBA and its related compound, vanillyl alcohol (VA) had not inhibited somatic embryogenesis on solidified medium from carrot hypocotyls, which were induced embryogenic competence (the 48th Annual Meeting of JSPP in 2007). 4HBA and VA also have weak affect Arabidopsis somatic embryogenesis (the 49th annual meeting of JSPP in 2008).
In this study, we reported that the effect of 4HBA and VA on somatic embryogenesis in various Arabidopsis accessions. We found several accessions, such as Ti-1 and Tsu-0 shows relatively higher sensitivity to VA and 4HBA, respectively, than Col-0. We are also analyzing the effect of 4HBA and VA on the expression patterns of various embryogenesis-specific genes. The function of these phenolic compounds for plant embryogenesis will be discussed.

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Anabaena sp. PCC 7120 develops specialized cells called heterocysts for nitrogen fixation. Heterocysts are terminally differentiated cells, which do not divide. In previous studies, it was considered that the cell division is necessary for the process of heterocyst development and that the decision of heterocyst is random. In the present study, we followed the process of heterocyst differentiation in single filaments of Anabaena sp. PCC 7120. We analyzed the time-dependent change of heterocyst pattern, the relationship between heterocyst differentiation and phycobilisome fluorescence and the positioning of the heterocysts at initial stage of heterocyst induction. These results suggested that a set of 4 cells at initiation of heterocyst induction was important for the decision of the cell that became heterocyst early during the induction. We propose a model of initial heterocyst pattern formation at early stage.

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Pentatricopeptide repeat (PPR) proteins are thought to possess important physiological roles in plant organelle biogenesis, organ development, cell differentiation, and so on. Although several PPR proteins was reported to be involved in RNA processing of RNA precursors in plastids and mitochondria, the function of most PPR proteins remains to be elucidated. To investigate the function of PPR proteins, we have been performing loss-of-function analyses of Physcomitrella patens PPR protein genes. In the present study, we report the phenotypes of PpPPR_63 gene disruptant. PpPPR_63 consists of two PPR motifs and the C-terminal NYN domain. The disruptant showed the impaired growth of protonemal colony and abnormal cell shape of branched protomemata. To investigate the subcellular localization of PpPPR_63, stable transformants with green fluorescent protein (GFP) knocked-in the PpPPR_63 gene was produced. The GFP fluorescence was observed in the nucleus of the protonemata, rhizoids and buds.

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Transdifferentiation from a differentiated cell to a pluripotent stem cell is frequently observed in plants, in contrast to the firmness of differentiated states in animals. A leaf cell of Physcomitrella patens begins apical growth and cell division after leaf excision and exposure to light within 24 hours and ultimately grows into a complete plant body. To identify the factors that promote transdifferentiation, we collected a time-course profile of gene expression by using custom-microarrays, and identified 29 transcription factors (TFs) that were significantly up-regulated within 12 hours in excised leaves. Next, we investigated whether these TFs are involved in transdifferentiation by dominant repression by conditionally overexpressing chimeric genes in which TFs are fused with a modified EAR-motif repression domain (SRDX). Overexpression of a bZIP TF SRDX fusion protein severely inhibited the transdifferentiation, indicating the bZIP TF is a potential promoting factor of transdifferentiation.

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Next generation sequencing technologies, or deep sequencing, is revolutionizing the genomics. Our group is exploiting the 454 Sequencer by Roche, Switzerland, installed in Functional Genomics Center Zurich (FGCZ). A run of The 454 Sequencer yields >1,000,000 fragments with read length of 400 bp, which is the longest in next-generation sequencers. The 454 Sequencer has been exploited for the identification of microbial community and metagenomics, because it can avoid the significant bias of culture or cloning process during Sanger sequencing. We have surveyed bacterial community living within carnivorous plants. We also exploit the 454 Sequencer for the molecular analysis of so-called "non-model" plants, in which prior genomic information is limited.

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Expansins are proteins that function in cell enlargement, fruit tissue softening, abscission, pollen tube elongation and others and the α-expansin subfamily is composed of 26 genes in Arabidopsis. Based on the DNA microarray analysis we examined four α-expansins (AtEXPA1, AtEXPA4, AtEXPA6, AtEXPA15) which highly express in the elongating tissues such as hypocotyls and stems. The observation of T-DNA inserted mutants showed that the hypocotyls of atexpa1/atexpa15 double mutant significantly shorten compared with wild type. As a result of promoter GUS analysis, expression of AtEXPA15 was observed in the hypocotyls just after germination. Taken together it is suggested that AtEXPA15 involves in the elongation growth of the hypocotyls. We now analyze the expression pattern of AtEXPA1.

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A unique peroxidase isoenzyme, cationic cell-wall-peroxidase (CWPO-C), from poplar oxidizes sinapyl alcohol, ferrocytochrome c and synthetic lignin polymers. It was suggested that Tyr-177 and Tyr-74 on the protein surface are considered to be important for the oxidation activities of CWPO-C with a wide range of substrates, including lignin.
In this study, we focused on CWPO-C homolog genes of Arabidopsis thaliana, and these T-DNA mutant lines were analyzed. Within the 7 mutant lines, lignins in mutant line 6 and 7 were reduced by 12.6% and by 15.9% compared to wild type, respectively. These mutant lines are defective in the genes encoding peroxidase carries Tyr-177 and Tyr-74. Moreover, yield of DFRC monomers were increased in these mutant lines, compared to other mutants. These results suggest that peroxidase carries Tyr-177 and/or Tyr-74 has an impact on the amount of lignin and lignin assembly in the cell walls.

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Arabinogalactan-proteins (AGPs) are a family of proteoglycans found in plasma membrane and cell walls of higher plants. As backbones of the carbohydrate moieties, AGPs commonly have beta-3,6-galactans, to which other auxiliary sugars such as L-arabinose (L-Ara), glucuronic acid (GlcA), and 4-O-methyl-GlcA (4-Me-GlcA) are attached. Although a variety of proteins are expected to undergo the arabinogalactan (AG) modification in vivo, the physiological functions of AG moieties remain obscure. In the present study, the structure of carbohydrate moieties of AGPs was altered with fungal alpha-L-arabinofuranosidase (Arafase) and beta-glucuronidase (GlcAase). Recombinant Arafase (rArafase) released L-Ara from AGP from radish roots in vitro. Recombinant GlcAase acted on GlcA and 4-Me-GlcA residues of the AGP in the collaborative manner with rArafase while it failed to act alone. Transgenic Arabidopsis plants expressing the Arafase and GlcAase exhibited higher activities of Arafase and GlcAase than wild-type plants.

Konishi et al., (2008) Carbohydr. Res. 343, 1191-1201

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UDP-arabinopyranose mutase (UAM), which catalyzes to interconvert UDP-arabinopyranose and UDP-arabinofuranose, is essential for production of arabinofuranose-containing polysaccharides and glycoproteins. UAM is identical to plant polypeptides previously known as reversibly glycosylated polypeptides (RGPs) which reversibly bind UDP-Glc to give glucosylated polypeptides. Little is known about the catalytic mechanism and there is controversy that RGPs have other functions besides mutase. We now report identification of glycosylated residues.
LC/MS analysis revealed that glucosyl residue from UDP-Glc linked to Arg158. We generated the recombinant proteins from which some arginines were substituted for alanine. Mutase activity was not detected in an R158A mutant protein. The mutase activity of an R151A mutant was the same as wild type, but of R165A decreased to 6% of wild one. From the results, it was concluded that Arg158 of UAM was the sugar-binding amino acid residue, and the RGP and mutase activities occurred on same Arg158.

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brittle culms (bcs) are mutants of grasses such as rice, barley and maize whose plant bodies are easily broken and expected to have defects in the cell wall formation. The brittle phenotype of rice bc5 mutant exclusively appears in the developed nodes. In the present study, cell walls of bc5 nodes were analyzed. Decreased accumulation of lignin in the sclerenchyma tissues was observed in bc5 nodes stained with Safranin O and phloroglucinol-HCl. Amounts of cellulose and hemicellulose were reduced 47% and 35%, respectively, compared with those in the wild-type plants. The sugar composition and linkage analyses of the hemicellulose indicated that the accumulation of glucuronosylarabinoxylan is perturbed in bc5 nodes. These results indicate that BC5 regulates the accumulation of cellulose, glucuronosylarabinoxylan and lignin thereby affecting the deposition of secondary cell wall in sclerenchyma tissue of nodes.

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Secondary walls are synthesized only in cells of limited tissues such as stem, hypocotyl, anther and silique. In the course of systematic functional analysis of NAC transcription factors (TF), we found that NST1, NST2 and NST3 (=SND1) are key regulators for secondary wall formation except vascular vessel. We are now trying to identify TFs functioning downstream of NSTs by microarray data, complementation analysis and some other experiments. As a result, some MYB TFs including MYB46 and NAC TFs including SND2, which belongs to different subfamily from that containing NSTs, are found to play important roles by regulating each other. We will present entire framework of gene regulatory network during secondary wall formation.

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Paraserianthes falcataria is known to be one of the fastest-growing tropical trees on the earth. The tree grows symbiotically with nitrogen-fixing Rhizobium and phosphorus-promoting mycorrhizal fungi. We produced the transgenic trees overexpressing poplar cellulase. They did not decrease the level of cellulose but wall-bound xyloglucan in the walls. Such a cell wall modification not only accelerated growth by increasing plastic extensibility but also brought changes in opening and closing movements of their leaves, which were caused by expansion and contraction of motor cells. Nevertheless, the levels of enzymatic cellulose degradation were higher in the transgenic trees than that in the wild-type tree. We suggest that the enhancement of saccharification could be caused by a decrease in xyloglucan cross-linkings between cellulose microfibrils.

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Pectins are commonly found in the primary wall and the middle lamella region of the wall where two adjacent cells abut. Homogalacturonans are the major components of pectic polysaccharides and play an important role in plant development and morphogenesis. We have specifically degraded homogalacturonans in poplar by expressing apple polygalacturonase. Cell walls of transgenic poplars showed a 15% reduction in galacturonic acid content as compared with the wild type. In comparison to the wild type, transgenic plants showed a reduced growth rate with stem height and diameter. Anatomical analysis of stem transverse sections revealed that the cells from transgenic poplars were smaller than those from the wild type. Moreover, length of xylem fibers from transgenic poplars were also shorter than those from the wild type. These observations indicate that the homogalacturonan is a critical factor in plant cell growth.

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Pectin methylesterases (PMEs) are a class of enzymes capable of catalyzing the demethylesterification of cell wall pectins. Although PMEs are implicated in regulating mechanical properties of the cell wall, functions of individual members are not clear. Our comprehensive analyses on cell-wall related genes in Arabidopsis thaliana have identified a PME, which is expressed specifically in certain cell files within cortical cells in basal part of stems. A null mutant for this gene, pme61, carries thinner and more fragile stems compared to wild type plants. The mutant exhibits aberrant cell-wall morphology in the specific cell files. We also have revealed that degree of metylesterification is higher and that the PME activity is lower in pme61 than in that of wild type. Thus, we conclude that AtPME61 works as a primary PME in the basal part of stem, and plays an essential role for formation of supporting tissues.

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Xyloglucans are considered to be function as cross-links among cellulose microfibrils in most dicotyledonous cell walls. Xyloglucan endotransglucosylase/hydrolase (XTH) catalyzes cutting and tethering of xyloglucans, thereby mediating construction and reorganization of cell walls. In certain monocotyledonous cell walls, on the other hand, xyloglucans are not considered important in cross-linking cellulose microfibrils. As a matter of fact, however, rice contains 29 XTH genes, which show tissue specific and growth stage dependent expression patterns, suggesting their importance in the rice cell walls. To gain insight into functions of XTHs in rice, we focused on OsXTH19, which was specifically expressed in the dividing/elongating zone of leaf blade and internode. We will report substrate specificity of OsXTH19 using a recombinant protein, and phenotypic traits of transgenic rice lines in which expression levels of OsXTH19 are modified.

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The xyloglucan endotransglucosylase/hydrolases (XTHs) are a family of enzymes that catalyze xyloglucan endotransglucosylase and/or xyloglucan endohydrolase activities and, thereby, play a principal role in the construction and restructuring of xyloglucan cross-links in the cellulose/xyloglucan framework. In angiosperm, XTHs constitute large multigene families, which probably reflect the diverse roles of XTHs for the diversity of cell types. Although the bryophyte has fewer cell types than angiosperm, the Physcomitrella patens XTH (PpXTH) gene family consists of 32 members, which is comparable in size with those previously defined in two fully sequenced angiosperm genomes. Extensive comparative analyses of XTH families in P. patens and angiosperms suggest that numerous bryophyte-specific XTH genes have evolved to meet the morphological and physiological needs of the bryophytes. These surprising findings raise interesting questions about the biological importance of XTHs in basal land plants.

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