There are some papers reporting that the conductance for CO₂ diffusion from intercellular air space to the site of carboxylation (g_m) decreases when plants are exposed to drought. However, plants close their stomata and decrease stomatal conductance (g_s) when they are exposed to drought. Moreover, they tend to close in patchy. Because accurate estimation of CO₂ concentration in the intercellular air space (C_i) is prerequisite for the g_m measurement, the C_i and g_m values so far obtained under drought conditions are not reliable.
We have tested to use an ABA-deficient mutant, Nicotiana plumbaginifolia (aba1), to avoid inaccurate g_m estimation due to stomatal closure. As an index of drought stress, we set saturating soil water content as 100%. When it decreased to 20%, g_s decreased significantly in WT, whereas g_s value was still high in aba1. Therefore, we use this mutant for the experiment following change in g_m when the plants are exposed to drought and recovered from it. We are also measuring the amount of aquaporin, chloroplast surface area exposed to intercellular air space (S_c) and the activity of carbonic anhydrase to elucidate mechanisms underlying the changes in g_m.

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We studied how different color lights cause gradients of photoinhibition within a leaf, to help settle the controversy whether photon absorption by Chl or Mn is the primary cause of photoinhibition, suggested by the Excessenergy- or the Mn-hypothesis, respectively.
Lincomycin treated leaf discs were photoinhibited. A combination of a microfiber fluorometer, a fiber thinning technique and a micromanipulator enabled us to measure the Chl fluorescence signals within a leaf. Photoinhibition gradients were also compared with results of various conventional fluorometers.
The severity of photoinhibition was in the descending order of blue, red and green light near the adaxial surface, and in the descending order of blue, green and red light in the deeper tissue, which is correlated with the Chl and Mn absorption spectrum, respectively. These results cannot be explained by either hypothesis alone.
These data suggest that both the Excessenergy- and the Mn-mechanisms occur in photoinhibition, and fluorometers with red or blue measuring light give overestimated or underestimated Fv/Fm values of photoinhibited leaves, respectively; they measured deeper or shallower leaf tissue, respectively.

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The light condition in the forest understory in early spring was determined and possible acclimation of vernal species, Erythronium japonicum Dencne, inhabiting there was examined. Light intensities of the understory relative to the open were 38% and fluctuated between 113 and 913 μmol photons m^-2 s^-1. Fluctuations of the light intensities in the understory were predominated by 10 minutes cycle.
We compared photosynthetic characteristics of the plants acclimated to the understory with those to the open. The plants acclimated to the understory showed high maximum photosynthetic rate by 27.4% than those to the open. Their photosynthesis was induced rapidly under strong light soon after weak light for 10 minutes. Its efficiency decreased 7%. The efficiency under weak light soon after strong light decreased 10%. Soon after decreasing the light intensity, plants in the open showed temporary drop in photosynthetic rate. This result suggested development of photorespiration.

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Plants can fix carbons by photosynthesis, though they release about half of the fixed carbon by respiration. It was reported that photosynthesis rates per leaf area in many species increased in elevated CO₂. On the other hand, response of respiratory rates in plants grown in elevated CO₂ is poorly understood. In plants, respiratory rates are generally limited by amounts of substrates and ATP consumption rates. If there were any difference of respiratory rate in plants grown in elevated CO₂, it is expected that the difference is due to the change of substrate amounts and/or ATP consumption rates.
The study aim is to clarify the diurnal variation of respiratory rate and its limiting factor in plants grown in elevated CO₂. We grow Arabidopsis thaliana under 390 or 780 ppmv CO₂ and examined respiratory rates in plants at the beginning and end of the night at 20 days after germination. There were clear differences in respiratory responses between growth CO₂ conditions. Furthermore, to investigate factors leading to the difference, we determined amounts of primary metabolites at the beginning and end of the night and at noon using CE-MS.

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The photosynthesis mode of M. crystallinum is changed from C3 to crassulacean acid metabolism (CAM) within 10 days after onset of water stress when the plant age is 5 week old. Transcripts of many CAM-related genes increase during the CAM induction. To clarify the transcriptional regulation mechanism of a CAM-inducible gene, McGPT2 coding a plastidic glucose-6-phosphate/Pi translocator, we joined the -2.6 kb upstream region from the transcriptional start site of McGPT2 and its 5' deletion mutants to the firefly luciferase ORF. Transient expression studies with these constructs using M. crystallinum leaf tissues suggested that cis factors of the CAM induction and diurnal regulation were located within a region -0.24 kb upstream from the transcriptional start site and a region from -0.24 to -0.7 kb, respectively. The CAM induction of M. crystallinum takes more than 15 days after onset of the water stress if the plant age is 4 week old, which is much smaller than 5-week-old plants. We found that the increment of the McGPT2 promoter activity was also delayed much in the stressed 4-week-old plants suggesting that the CAM induction is affected developmentally at transcriptional level.

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Plants have mechanisms to acclimate to high-light conditions by extinction of the excess light energy. These mechanisms are important for plant to efficiently perform photosynthesis, and have to be controlled accurately. However, detailed mechanisms how plants diminish excess light energy are still unclear.Here we report a novel gene named LAP1(Light Acclimation Protein 1),that is involved in the high-light acclimation response in plants. Arabidopsis lap1 mutant displayed a pale green phenotype with higher NPQ values than those of wild type. We currently characterize lap1 mutant of synechocystis sp.PCC 6803.Together with these results, physiological function of LAP1 will be discussed in this presentation.

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The psaA gene, which encodes photosystem I reaction center subunit, is the critical target of regulation in environmental responses of at least cyanobacteria. We previously reported that the OmpR-type response regulator RpaA binds to the upstream region of psaA in Synechocystis sp. PCC 6803. RpaA was phosphorylated by histidine kinase SasA in a circadian manner in Synechococcus elongatus. Hik8 (SasA) controls expression of sugar catabolic genes in Synechocystis. In this study, we examined the role of RpaA in light- or glucose- induced expression of psaA. In wild type, the psaA transcription is inactive in the dark and induced by light illumination or glucose feeding. This induction was strongly reduced by rpaA or hik8 disruption. The induction was also interrupted by an electron transport inhibitor DBMIB. Thus, it is suggested that the Hik8-RpaA system mediates signal transduction from the redox state of the electron transport chain to the transcription of psaA. The rpaA disruption also affected the light- and glucose-induced expressions of cpcB and genes for glycolysis which are under control of Hik8, even though RpaA did not directly bind to their upstream regions.

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Aquatic photosynthetic organisms, including unicellular green alga, Chlamydomonas reinhardtii, induce a carbon-concentrating mechanism (CCM) under limiting-CO₂ conditions in light. In Chlamydomonas, CCM is controlled by a gene Ccm1/Cia5, which encodes a zinc-containing soluble protein CCM1/CIA5. The ccm1 mutant cannot induce 51 genes under low-CO₂ (LC: aerated air containing 0.04% CO₂) condition. The cDNA array experiments revealed that the levels of 38 different transcripts were up-regulated under conditions that induces CCM but not under other conditions. In order to identify other CCM-related genes that had not been spotted on the cDNA array, transcriptome analyses were carried out by massive sequencing technique using Genome Analyzer IIx (Illumina). The mRNA samples extracted from wild type and mutant cells were reverse-transcribed and used for RNA-seq analysis by using GA IIx, in which 36-nucleotides (nt) of sequences were determined and they were analyzed using Kyoto Chlamydomonas Genome Database (KCGD http://chlamy.pmb.lif.kyoto-u.ac.jp/). Newly identified CCM-related genes will be discussed.

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Thioredoxin (Trx) is one of major mediators of light reducing power in the stroma of a number of photoautotrophs. On the other hand, the occurrences of chloroplastic Trxs are demonstrated but their target and physiological function are not solved in diatoms. Two pyrenoidal β-type carbonic anhydrases (CA) in the diatom Phaeodactylum tricornutum, PtCA1 and PtCA2, are supposed to be crucial factors for CCM in diatoms. Mature regions of PtCA1 and 2 were synthesized in Escherichia coli and purified. PtCAs were treated with DTT and Trxs from Arabidopsis or P. tricornutum and activity was measured. Oxidization of mPtCAs completely abolished the CA activity but DTT treatment resumed the activity. This DTT dependent activation of oxidized mPtCAs were synergistically stimulated by Trxs. mPtCA1 and 2 possessed two Cys residues besides Cys residues that form Zn ligands. Cys/Ser replaced mutation of mPtCA1 and 2 inhibited the inactivation of PtCAs under oxidation. These results indicate that PtCAs are inactivated in the stroma under the dark by forming intramolecular disulfide bond and activation accompanied by cleavage of this bond is governed by chloroplastic Trx.

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Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) from Bacillus subtilis catalyze mechanistically similar enolizations. Both enzymes require the activation to be catalytically competent. In the activation, the N^ε of K201 at the active site is carbamylated by non-substrate CO₂ to form carbamate, which is stabilized by the catalytically essential Mg²⁺. Interestingly, RLP shows higher activation level under low CO₂ condition (approximately 2μM) than RuBisCO. We focused on H294 completely conserved in RuBisCO and RLPs, because this residue is probably involved in stabilization of carbamate through a hydrogen bonding between N^ε of H294 and the carbamate oxygen (2.8A) in RLP. H294Q, H294N and H294A RLPs had 92-98% decreases in k_cat, 2.2- to 3.0-fold increases in K_m. All mutants required higher CO₂ concentration for maximal activities compared to wild-type. These results suggested that H294 stabilizes the carbamate in RLP. By contrast, H294 is far from the carbamate oxygen (3.4A) in plant RuBisCO, resulting in the destabilization of carbamate. Alternatively, this may enable to regulate the RuBisCO activity by activation in plants.

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The atmospheric CO₂ level has been increasing and is predicted to double by the end of this century. The elevation of atmospheric CO₂ level might lead to promotion of photosynthesis, which is directly associated with improvement of crop yields and better production of useful material including biofuel. However, the elevation of the atmospheric CO₂ level does not always promotes photosynthesis but represses both photosynthesis and nitrogen assimilation in some plant species. Previously, we reported that enhancement of nitrogen and sulfur assimilation in transgenic Arabidopsis expressing the Dof1 transcription factor facilitates activation of carbon fixation under the growth condition where ammonium ions and light energy are abundantly supplied. Here we investigate the effects of atmospheric CO₂ enrichment on the nitrogen and sulfur assimilation and carbon fixation in the Dof1 transgenic Arabidopsis plants by target metabolome analysis and discuss about the effects of genetic enhancement of nitrogen assimilation on plant response to elevation of atmospheric CO₂ level.

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Dark-adapted rice leaves were illuminated at PPFD = 1,500 μmol m^-2 s^-1 for five minutes, and NPQ size after the treatment was measured. Sasanishiki (Japonica cv.) showed NPQ size of around 2.0, whereas Habataki (Indica cv.) was around 1.5. We also measured around 50 cv.s of World Rice Core Collection, of which Japonica cv.s showed NPQ larger than 1.65 and Indica cv.s showed NPQ smaller than 1.65, except two cv.s. We then did QTL analysis between Sasanishiki and Habataki using backcross inbred lines. Tow QTLs qNPQ1-1 and qNPQ1-2, which regulate NPQ size, were identified on the chromosome 1. Of these two loci qNPQ1-2 had larger additive effect, and Sasanishiki allele of qNPQ1-2 positively regulates NPQ size. PsbS1, one of the two PsbS homologs, lies within qNPQ1-2 region, suggesting the possibility that PsbS is involved in the difference of NPQ size between Sasanishiki and Habataki. When we observed the relationship between PsbS1 expression levels and NPQ sizes in PsbS1 overexpressors, NPQ sizes were positively correlated with PsbS1 expression levels. Compared with the DNA sequence of Japonica cultivars, Indica cultivars had a 2.7-kb deletion at 0.4-kb upstream of PsbS1 gene.

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Photosystem II(PSII) performs light-induced electron transfer and water-splitting reactions, leading to the formation of molecular oxygen. After initial light excitation and charge separation at the reaction center of PSII, an electron is transferred from Q_A to Q_B. Q_B leaves the complex to cyt b₆f as PQH₂ after uptake of two protons and two electrons.
Our final goal is to realize an artificial system of photosynthesis using PSII with functional molecules newly designed and synthesized. The electron acceptor parts of the molecules, connecting electron transfer and reduction parts in series, may bind to the Q_B site. In order to obtain structural information for binding mode at the Q_B site, we introduced four herbicides, DCMU, Bromacil, Bromoxynil, and Terbutryne, into PSII crystals by soaking, well known to bind into the Q_B site and to inhibit electron transfer beyond Q_A. X-ray diffraction data were collected at BL44XU, SPring-8. Crystal structures were solved with molecular replacement technique and refined around 1.9- 2.3A resolutions. Based on the structures of herbicides found at the Q_B site, we will discuss inhibitor binding modes and electron transfer mechanism from Q_A to Q_B.

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Generally, photosystem (PS) I complex functions as a trimer in cyanobacteria and as a monomer associated with light-harvesting chlorophyll complex in higher plants. Recently, we demonstrated a filamentous N2-fixing cyanobacterium Anabaena sp. PCC 7120 has tetramer and dimer of PSI but no trimer at all (Watanabe et al. 2010 PCP in press). Here, we improved the protocol and successfully isolated from Anabaena a supercomplex consisting of the PSI tetramer and phycocyanin. Normally, phycobiliproteins are assembled via linker polypeptides into rod and core complexes of phycobilisome (PBS), which preferentially transfers light energy to PSII. On the other hand, the Anabaena supercomplex lacked the core subcomplex of PBS based on the spectroscopic and polypeptide analyses. However, a rod-core linker polypeptide CpcG3 was identified in the supercomplex. The Anabaena genome encodes 4 copies of cpcG, while only three except CpcG3 have been detected in the conversional PBS. Since CpcG3 has a hydrophobic C-terminus, it may directly connect the rod subcomplexes with the PSI. We will also discuss the possible universal role of the antenna-PSI supercomplex in cyanobacterial photosynthesis.

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Photosynthetic water oxidation takes place in the water oxidizing center (WOC) of photosystem II (PSII) through the S-state cycle of five intermediates (S₀-S₄). Determining the efficiencies of the S-state transitions is essential for clarifying the mechanism of water oxidation. In this study, we have estimated the efficiencies of individual S-state transitions using Fourier transform infrared (FTIR) difference spectroscopy. Flash-induced FTIR difference spectra were measured using PSII core complexes of T. elongatus in the presence of ferricyanide as an electron acceptor. Flash-induced electron transfer from WOC to ferricyanide was detected by the CN stretching bands of ferricyanide/ferrocyanide. The flash-number dependence of the intensities of these signals showed a typical period-four oscillation pattern. Simulation of this pattern provided the efficiencies of 96, 97, 95, and 92% for the S₀-to-S₁, S₁-to-S₂, S₂-to-S₃, and S₃-to-S₀ transitions, respectively. The tendency of the lowest efficiency in the S₃-to-S₀ transition was also observed in spinach PSII membranes, indicating that this is a general feature in the water oxidation reaction.

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Photosynthetic oxygen evolution takes place by water oxidation at the Mn cluster in photosystem II (PSII). Ammonia has been known to be an effective inhibitor of O₂ evolution as a water analogue. However, the detailed mechanism of ammonia inhibition is yet to be understood. In this study, we investigated the molecular mechanism of ammonia inhibition by examining the pH dependence of the inhibition rate and the changes in Fourier transform infrared (FTIR) difference spectra. The treatment of 100 mM NH₄Cl on either intact or PsbP, PsbQ-depleted PSII membranes of spinach showed decreases in the O₂ evolution activity by ~40% at any pH in the pH5.0-8.0 range. S₂/S₁ FTIR difference spectra exhibited clear changes by NH₄Cl treatment in the asymmetric and symmetric COO^- stretching regions. The amplitude of the spectral changes as a function of the NH₄Cl concentration was correlated with the concentration dependence of the inhibition rate. These results indicate that NH₄⁺ rather than NH₃ functions as a major inhibitor and that the interaction of NH₄⁺ with carboxylate ligands to the Mn-cluster or those forming a hydrogen bond network causes inhibition of O₂ evolution.

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PSII complexes in Synechocystis sp. PCC 6803 co-exist with several uncharacterized proteins, including Sll1252, which has a N-terminal extension and an S4-like domain and is conserved in all the known photosynthetic organisms. We inserted a kanamycin-resistant gene into an upstream site of S4 domain of the sll1252 gene. Although cultures of the mutant were sustainable at 5 μmole photons m^-2 s^-1, they were severely photodamaged at 70 μmole photons m^-2 s^-1. Activity of PSII and rate of respiration in the mutant were similar to those in wild-type cells. However, activity of net photosynthesis, from H₂O to MV, was drastically dropped in the mutant. Profiles of transcriptome in the mutant cells were resembled those in cells treated with DBMIB and PQ-pool in the mutant was more reduced than that in wild-type cells. These results indicate decrease in the rate of electron transfer from PQH₂to Cyt_b6/f.
Each suppressor mutant had an transposon insertion between kanamycine-resistant gene and S4 domain, suggested that imbalance of the expression level of the S4 domain and the N-terminal portion of Sll1252 might cause the severe phenotypes.

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Cyanidium caldarium is a unicellular primitive red alga. Photosystem II complex (PS II) isolated from C. caldarium contained four extrinsic proteins, PsbO, PsbV, PsbU and PsbQ'. PsbQ' subunit was also found in other alga, such as diatom and other species of red algae, however, the localization of PsbQ' subunit is not clear at present. To reveal the localization of PsbQ' subunit, we crosslinked C. caldarium PSII with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. We obtained several crosslinked products, and similar results were obtained with PSII from Cyanidioschyzon merolae. Additionally, we introduced a spin-label at the cysteine residue of the PsbQ' subunit, and measured the pulse ESR spectra. Electron electron double resonance for the radical pair of spin-labeled PsbQ' subunit and Y_D were observed after illumination of the sample. The results revealed that the average distance between the two radicals was 34-35 angstrom. From these results, we discuss the localization of the PsbQ' subunit in the red algal PSII.

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Many plants monitor seasonal changes in day length in order to regulate flowering time for successful reproduction. We focused on the liverwort, M.polymorpha, to understand the origin and evolution of growth-phase transitions regulated by the photoperiod. M.polymorpha belongs to a group of basal land plants, and was reported to be a long-day plant. Firstly, we searched for genes associated with the photoperiod pathway in M.polymorpha genome database, and found homologues of TOC1/PRR family gene, GI and ADO/FKF/LKP/ZTL family gene. These genes were named as MpTOC1, MpPRR2, MpGI and MpFKF1, respectively. Phylogenetic analysis supported the orthologous relationship of these genes to their homologues in vascular plants. Furthermore, northern blot analyses showed rhythmic expression pattern of MpGI, MpTOC1, and MpPRR2 under LD conditions. The phase peaks of these genes were similar to those in vascular plant. Interestingly, expression level of MpPRR2 was lower in MpPHY RNAi-line than in WT, suggesting positive regulation of MpPRR2 by Mpphy. Taken together, these results suggest that M.polymorpha would have a basic and simple regulatory mechanism of its circadian clock.

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The plant circadian clock generates daily rhythms in the activity of many biological processes and serves their photoperiodic responses. In A. thaliana, the clock regulates the long-day-specific promotion of flowering time and short-day-specific elongation of hypocotyl length. For the photoperiodic control of flowering, it was known B-box containing transcriptional factor CO is activated at the end of day time specifically under long day conditions to induce the FT gene expression. On the other hand, it is speculated bHLH transcriptional factors PIF4/5 are activated at the end of nigh time to enhance hypocotyl elongation. To make sure the assumption, here, we screened PIF4/5-dependent genes whose expressions are induced at the end of night specifically in short days. We identified the ATHB2 gene encoding a HD-Zip protein as a candidate. Examination of ATHB2 transcriptional profiles under various photoperiodic conditions in photoreceptor and clock mutant backgrouds makes it possible to suggest that both the circadian clock and photoreceptor phyB coordinately control the PIF4-ATHB2 output pathway responsible for the short-day-specific photoperiodic control of hypocotyl elongation.

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The biological clock plays essential roles for sleep and hormonal regulation in animals, and for flowering and leaf movement regulation in plants. In animals, it is well known that a strong master clock in the brain tunes peripheral clocks in peripheral organs. In contrast, it is unknown whether a master clock and peripheral clocks exist in plants. However, there are evidences to suggest that plants may have master and peripheral clocks similar to animals- (1) animal and plant clocks have similar underlying molecular mechanisms, (2) plants integrate individual cellular circadian rhythms into a single individual rhythm to achieve flowering and leaf movement. This suggests the existence of tissue-specific clock in plants similar to animals. Since plants don't have an organ equivalent to the brain, and it is difficult to excise specific tissues from plants, tissue -specific clock function has remained elusive. Here we show two novel methods for monitoring clock gene expression in the tissue-specific manner. One is direct tissues isolation with high time resolution and another is non-invasive imaging for clock gene dynamics.

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Legume plants open their leaves early in the morning and close them in the evening. This phenomenon is called nyctinasty, which is controlled by circadian clock regulating the balance of concentrations between two endogenous bioactive substances, leaf-opening factor (LOF) and leaf-closing factor (LCF). Recent studies of Prof. M. Ueda et. al., (Tohoku University) identified a candidate for LOF binding protein in Cassia plant. Identified fragment sequences of the protein showed similarity to methionine synthase (MS). Thus, to examine whether MS is able to bind to LOF, how this binding affects MS activity, and whether MS plays the same role in other plants, we isolated cDNA clone of MS from Cassia obtusifolia. An anti-MS antibody generated against recombinant MS was used to examine the tissue distribution of MS. As a result, bacterially expressed recombinant proteins exhibited MS activity and were bound to LOF, though the activity was not changed by their binding. These data showed that MS appears to be a LOF receptor in vivo, though the role was still uncertain.

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Carrot (Daucus carota) somatic embryogenesis has been extensively used as an experimental system to investigate the development of zygotic embryogenesis. In zygotic and somatic embryos, abscisic acid (ABA) is involved in acquisition of desiccation tolerance and dormancy, and the VP1/ABI3 factor functions as a transcriptional factor on seed specific ABA signal transduction. We isolated two carrot homologs of the VP1/ABI3 factor, C-ABI3-1 and C-ABI3-2. Each gene exhibited similar expression profile during somatic embryogenesis and seed development. In the transgenic carrot cells showing ectopic expression of C-ABI3-1 or C-ABI3-2, the expression of some seed specific ABA-inducible genes was successfully induced by ABA-treatment. On a trans-activation assay using Arabidopsis abi3-6 or carrot non-embryogenic cells, both C-ABI3-1 and C-ABI3-2 decreased the expression of C-ABI3-1. These suggest that C-ABI3-1 and C-ABI3-2 may function collaboratively on ABA signal transduction in carrot embryos.

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To clarify relationship between cell cycle regulation and embryo morphogenesis, we focus on the role of cell cycle inhibitor(s) during somatic embryogenesis in carrot.
For isolation of KRP-homologue-genes, we design degenerated primers based on amino acid sequences in conserved CDK-cyclin binding domain. Conclusively, two cDNA fragments were amplified by RT-PCR. After 5-, 3-RACE, full length-cDNAs had been isolated for both genes (DcICK1 and DcICK2). DcICK1 and DcICK2 consist of 229 and 231 amino acids, respectively. They are highly homologous with AtKRP3 from Arabidopsis, and, motif compositions are also similar to the KRP. These genes highly express around shoot and root meristems. However, no significant change of these expressions is observed during somatic embryogenesis.When localization of DcICK1-mRNA was detected by in situ hybridization method, it is detected in whole site of globular shaped embryos or primodia of cotyledon in torpedo shaped embryo.

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Organ regeneration in tissue culture involves de novo formation of apical meristems, which should provide a useful system to study how meristems arise. We isolated a number of temperature-sensitive mutants of Arabidopsis with defects in organ regeneration. Of these, rid3 is characterized by the inability to form adventitious roots and shoots at the restrictive temperature. Our previous analysis revealed that the RID3 gene encodes a novel WD-40 repeat protein and that, in tissue culture, its expression increases during callus development and decreases locally before the formation of the shoot apical meristem. Here we report some recent results on the function of RID3 in embryogenesis. We found that the rid3 mutation interferes with embryonic pattern formation and with expression of meristem-related genes and that RID3 expression, which is uniformly high in early-stage embryos, is gradually excluded from pre-meristematic regions as embryogenesis proceeds. Our results indicate that RID3 is involved in the formation of apical meristems during embryogenesis as well as during organ regeneration.

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While molecular mechanisms underlying axis formation and organ initiation have started to emerge, our knowledge on the mechanism controlling early embryogenesis is still limited. By using a root-specific activation tagging system of Arabidopsis, we have revealed that members of the RKD family of plant-specific putative transcription factors possess an ability to promote cell division and de-differentiation. Reporter analyses indicated that all five members of the Arabidopsis RKD family are transcribed at small domains in early embryos, though, only mutants of RKD4 showed abnormal cell division in early embryos. GFP-tagged RKD4 proteins localized to the nuclei and the amino-terminal region of RKD4 activated gene transcription in yeasts, supporting the notion that RKDs function as transcription factors. Induced ectopic expression of RKD4 in the seedlings activated expression of early embryo-specific genes, and some of them were down-regulated in rkd4 mutants. Taken together, these results suggest that RKD proteins regulate gene expression in early embryos, and whereby promote their correct development.

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Recent environmental issues have prompted an increase in the demand for wood, so it is important to understand the process of wood formation. We focused on 16 poplar (Populus trichocarpa) NAC domain protein genes, PtVNS (VND, NST/SND and SMB related proteins) genes, which are homologous to Arabidopsis transcription factors regulating the differentiation of xylem vessels and fiber cells. 12 PtVNS genes including both VND and NST groups were expressed in developing xylem tissue and phloem fiber, whereas in primary xylem vessels, only the VND group genes were expressed. By using the post-translational induction system of AtVND7, a master regulator of xylem vessel differentiation, many poplar genes functioning in xylem vessel differentiation were identified. Transient expression assays showed the variation in PtVNS transactivation activity toward downstream genes. Moreover, overexpression of PtVNS genes induced ectopic secondary wall thickening in poplar leaves as well as in Arabidopsis seedlings with different levels of induction efficiency according to the gene. These results suggest that wood formation in poplar is regulated by cooperative functions of the NAC domain proteins.

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We established the in vitro system for vessel element transdifferentiation in Arabidopsis Col-0 suspension cells. Through a microarray analysis of the system we identified a number of genes with drastic changes in expression during the transdifferentiation. Moreover, we revealed that VASCULAR-RELATED NAC-DOMIN7 (VND7) encoding a NAC-domain transcription factor functions as a master regulator for vessel formation. However, the regulatory mechanism of VND7 expression is still largely unknown. Hence, in this study we aimed to identify transcription factors that regulate VND7 expression. So we first selected transcription factors up-regulated at the phase that most of the cells were transdifferentiated into vessel elements. Then we performed the particle bombardment-based transient assays to evaluate whether each candidates can induce VND7 expression using VND7pro:Luciferase construct as a reporter. As a result, we found that several genes could up-regulate VND7 expression including all VND family members. So now we are further analyzing these transcription factors to reveal its function during vessel formation and try to clarify the transcriptional relationships among these factors.

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To understand the epigenetic regulation of gene expression during transdifferentiation of plant cells, using a xylogenic Zinnia cell culture system, in which isolated mesophyll cells transdifferentiate into tracheary elements, we analyzed the dynamics of histone modification during differentiation process. We first found a dynamic change in the level of acetylated histone H3. And then we revealed that TSA, an inhibitor of histone deacetylase, inhibited tracheary element differentiation. The inhibition rate depended on the concentration of TSA and the time when it was supplied to culture. Zinnia gene chip analyses showed that approximately 60 genes for transcription factors were expressed >2-fold more in TSA-treated cells than control 6 hours after the treatment. Based on these results we will discuss about the role of histone acetylation / deacetylation in gene regulation during tracheary element transdifferentiation.

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Phospholipase D (PLD) is the enzyme that hydrolyzes glycerophospholipids, a major component of biological membranes, to produce phosphatidic acid, a signaling molecule on membranes. Plants encode two classes of PLD; one exists generally in eukaryotes and contains PX and PH domains, and the other exists specifically in plants and contains a C2 domain. PLDζ1 is one of the two PX-PH-type PLDs in Arabidopsis thaliana. Its gene has been reported to be directly regulated by the transcription factor GL2 and act as a positive factor for root hair formation during root hair patterning. Examined T-DNA insertion mutants of the gene, however, have shown no significant phenotypes in root hair patterning. We found that another T-DNA insertion line, which is likely to be a loss-of-function mutant of the gene, exhibited unstable, but significant abnormality in growth. This low-penetrance phenotype could be complemented with a transgene encoding PLDζ1-YFP, confirming that the phenotype is due to a defect in the PLDζ1 gene function. Using this transgenic line, we investigated intracellular localization of the functional PLDζ1 fusion protein.

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In Arabidopsis roots, vascular bundles are arranged with a bilateral symmetry. Recent analyses of the mutant of LONESOME HIGHWAY (LHW) have suggested that LHW is a key factor for establishing the bilateral symmetry. In the lhw mutant, only single xylem and phloem poles are formed, and the number of cells in the stele of the root reduces. LHW encodes a transcription factor, which contains bHLH-domain. There are three paralogs of LHW in the Arabidopsis genome. However, all of them are still unknown about function and expression. To get a hint of functions of these genes, we first investigated expression patterns of them. For this purpose, we made transgenic plants harboring promoter-GUS and promoter-YFP-NLS of each paralog gene. Next we observed the phenotype of a knockout line of a paralog gene. Based on these analyses, we will discuss function of the LHW paralog genes.

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The vascular meristem comprising procambial/cambial cells localizes between phloem and xylem tissues and is essential for the indeterminate radial secondary growth of plant body. Previously, we showed that a CLE-family secretory peptide TDIF acts as an intercellular signal for stem-cell maintenance in this meristem. TDIF, which is secreted from phloem cells, is perceived by procambial cells via a leucine-rich repeat receptor kinase TDR/PXY. This signal suppresses xylem differentiation of procambial cells and maintains the vascular meristem organization. However, how TDIF acts during the indeterminate secondary growth of vascular tissues remains unclear.
In this study, we aimed to analyze the function of TDIF signaling in detail during secondary growth. We generated promoter–reporter constructs for vascular marker genes including CLE41 (encoding TDIF) and TDR and introduced them into vascular mutants. Based on these results, we will discuss the function of TDIF signaling genes in secondary growth.

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The WUSCHEL-related HOMEOBOX (WOX) family of homeodomain transcription factors plays a crucial role in plant growth and development. Although genetic studies have uncovered the function of WOX genes, its molecular mechanism regulating their functions in cellular level remains elucive.
Previously, we have revealed that the WOX4 gene is expressed in procambial cells of vascular bundles and promotes their proliferation in Arabidopsis. In this study, we aimed to analyze the transcriptional regulation by WOX4. The WOX4 protein contains a transcriptional repressor domain called WUS box (TLXLFP) near its C-terminus (Leibfried et al., 2005; Ikeda et al., 2009). We generated a modified version of WOX4 gene, in which the repressor domain was removed and an activator domain was added. Using this construct, we established an Arabidopsis cell culture in which the expression of the modified WOX4 gene can be induced by estrogen. We will report the results from gene expression analysis with this line.

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Previous studies demonstrated that the CLE41/44 (TDIF) peptide plays a critical role in vascular formation: TDIF is secreted from phloem cells and received by TDR-expressing procambial cells, in which TDIF-TDR signaling enhances proliferation of procambial cells and prevents xylem differentiation from procambial cells. CLE42 and CLE46 also possess a CLE motif similar to that of CLE41/44. Although CLE42 has the TDIF activity, CLE46 does not. Thus function of CLE46 is yet poorly understood.
To understand the function of CLE46, we first analyzed promoter activity of CLE46 in pCLE46:GUS transgenic plants. Next, we generated CLE46-overexpressed and RNAi-CLE46-suppressed transgenic plants. Based on these results, we discuss the function of CLE46 peptide.

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Cell fate separation between the meristem and lateral organs is a central process in plant morphogenesis. CUP-SHAPED COTYLEDON1 (CUC1), a NAC transcription factor, is a key regulator for boundary specification in Arabidopsis thaliana. To investigate the molecular mechanism of boundary establishment, genes acting downstream from CUC1 were screened by transcriptome analyses. Among these genes, LIGHT-DEPENDENT SHORT HYPOCOTYLS 4 (LSH4) encodes a nuclear-localized protein of the ALOG family. LSH4 is expressed in shoot organ boundaries, and its transcription is directly regulated by CUC1. Constitutive expression of LSH4 at the shoot apex resulted in enlargement of the meristem and suppression of leaf primordium growth. In these plants, some sepals have split at the upper part, and ectopic flowers or floral organs are formed within a flower. These results indicate that LSH4 is expressed in the shoot organ boundaries under the direct transcriptional regulation of CUC1, and modulates the cell proliferation and differentiation.

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Lateral root (LR) formation in Arabidopsis is regulated by auxin signaling that depends on transcriptional activator Auxin Response Factors such as ARF7 and ARF19 and repressor auxin/indole-3-acetic acid (Aux/IAA) including IAA14/SOLITARY ROOT (SLR). In gain-of-function slr-1 mutant, stabilized IAA14 inactivates ARF7/19 functions and causes the block of LR initiation like arf7 arf19 double mutant. However, the mechanism of auxin-responsive transcription during LR formation is still unknown. Here, we show that MACCHI-BOU 2 (MAB2), which encodes MED13 subunit in a regulatory module of the Mediator complex, is required for the regulation of auxin-responsive transcription during LR formation. Genetic interaction between mab2 and slr-1 suggests that MAB2 negatively regulates LR formation. Furthermore, the mab2 mutation also restored expression of LBD16 and LBD29, which are direct targets of ARF7/19 in LR formation, in the slr-1 mutant backgrounds. These results indicate that MAB2 acts as a transcriptional repressor in coordination with IAA14 to inactivate ARF7/19 functions during LR formation.

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Most of the higher plants produce leaf-like structures, called bracts, at the base of flowers, while Arabidopsis thaliana does not. However, previous studies showed that cryptic bracts are formed at the base of Arabidopsis flower primordia, suggesting the existence of a mechanism of bract suppression. Recently, BLADE ON PETIOLE 1 (BOP1)/BOP2 and PUCHI have been shown to coordinately function in Arabidopsis bract suppression.
Here, we report MACCHI-BOU 2 (MAB2), AtMed13, as a novel factor involved in bract suppression. Detailed phenotype analysis of mab2 mutants showed that bract primordia developed coincidentally with floral meristem in the inflorescence meristem. To elucidate the relationship between MAB2 and other factors involved in bract suppression, we constructed multiple mutants. Results from the genetic study indicate that MAB2 functions in the same pathway as BOP1/BOP2 and differently from PUCHI in bract suppression. In addition, using a yeast two-hybrid assay, we found that BOP2 could interact with a subunit of CDK8 complex including MAB2/AtMed13. Based on these results, we will present the molecular mechanism of bract suppression in Arabidopsis .

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The auxin distribution is established by polar auxin transport that is dependent on polar-localized auxin efflux carriers, PIN-FORMED (PIN). Recently, a NONPHOTOTROPHIC HYPOCOTYL 3 (NPH3)-like protein, MACCHI-BOU 4 (MAB4), has been reported to regulate polar auxin transport through the control of subcelluar localization of PIN1 proteins in cotyledon development. MAB4 has four homologs, named MAB4/ENP LIKE 1 (MEL1), MEL2, MEL3 and MEL4. In root tips of mel1 mel2 mel3 mel4 quadruple mutants, PIN2 abundance was severely reduced and PIN2 polarization was weakened. To investigate the mechanism by which the MAB4 subfamily genes regulate PIN2 localization, we used pharmacological approaches in PIN2-GFP expressing mutants. When treated with BFA or wortmannin, PIN2 protein accumulated in induced intracellular compartments of mel1 mel2 mel3 mel4 roots as well as wild-type roots. Meanwhile, when treated with thyrphostin A23 that is a blocker for clathrin-dependent endocytosis, PIN2 internalization was not blocked in mel1 mel2 mel3 mel4 roots. These results suggest that the MAB4 subfamily genes regulate PIN endocytosis in the plasma membrane.

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PIN-FORMED1 (PIN1)-dependent polar auxin transport is essential to organ formation. Previous studies on PIN1 localization uncovered a two-step process in organ formation. (1st) PIN1 polarity converges toward organ initiation sites and auxin accumulates there. (2nd) PIN1 polarizes basally in the center and auxin flow sinks. We have identified a NPH3-like protein MAB4 and its homologs MEL1/MEL2, involved in polar auxin transport in organogenesis. To elucidate the function of MAB4 family genes in PIN1 polarization, we analyzed PIN1 localization in pin-like inflorescences of mab4 mel1 mel2. In the mutant, PIN1 polarity converged normally, but followed basal shift of PIN1 polarity did not occurred, suggesting that MAB4 family genes establish an auxin sink through the control of PIN1 polarity in the meristem. In addition, it is possible that auxin accumulation and its signaling induce the MAB4-dependent basal shit of PIN1 polarity, which is the transition from the 1st step to the 2nd step. To confirm the possibility, we examined the relationship between auxin signaling and MAB4 family genes. Our results indicate that MAB4 family genes function downstream of auxin signaling.

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Many steps of lateral root development were positively regulated by auxin, including initiation step. However, it was not clear how to control the lateral root initiation, spatially and temporary. We analyzed auxin accumulation spots in primary root continuously. About a half of them disappeared without cell division in wild type. Although the primary root elongated constantly, lateral root primodia were synthesized periodically from the auxin accumulation spots presented about 2 mm away from root tip. These suggested that the lateral root initiation was regulated by a periodic signal. The number of lateral root was slightly increased in puchi mutant and some of them were close. It took a long time when auxin accumulation spots were disappeared without cell division in puchi. Additionally, auxin accumulation spots could be observed adjacent to the newly synthesized lateral root primodia, which never detected in wild type. The expression of PUCHI was induced by auxin. About 85% of G₂/M marker expressed sites were developed to LRP in both wild type and puchi. These suggested that PUCHI negatively regulated the lateral root initiation before cell division.

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In vascular plants, the maintenance of the activity in the root apical meristem is important for the development of the root architecture. To investigate the mechanisms of root meristem maintenance (RMM), we isolated the recessive mutant LR11-4 that showed the aberrant root growth from Arabidopsis. In the LR11-4 mutant, the length of the primary root is remarkably shorter than that of the wild type, and the frequency of lateral root (LR) formation is not affected but the LR growth is severely inhibited. Fine mapping and sequence analysis revealed that LR11-4 mutant genome has a non-sense mutation in the FBA1 gene, encoding a fructose-1,6-bisphosphate aldolase1 that functions in Calvin cycle. The complementation test showed that the LR11-4 phenotype is caused by the mutation in the FBA1. FBA1-GFP fusion protein expressed under the control of FBA1 genomic region was localized in the plastids of the root cap columella and leaves, strongly suggesting that the function of FBA1 in the plastids is necessary for RMM in Arabidopsis. We are now investigating which organs should express FBA1 for RMM.

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In most dicot plants, lateral root (LR) formation, which is important for the construction of root system, is initiated from asymmetric cell divisions (ACDs) of the xylem pole pericycle cells of the parent roots such as the primary root and the existing lateral roots. In Arabidopsis, two Auxin Response Factors, ARF7 and ARF19, positively regulate LR formation through activation of the plant-specific transcriptional regulators, LATERAL ORGAN BOUNDARIES-DOMAIN16/ASYMMETRIC LEAVES2-LIKE 18 (LBD16/ASL18) and LBD29/ASL16. Detailed expression analysis using GFP-tagged LBD16 reporter protein indicated that LBD16-GFP was mainly expressed at the LR initiation sites in three cell files of the xylem pole pericycle of the primary roots. Furthermore, expression of the artificial dominant repressor of LBD16 (LBD16-SRDX) under the control of its own regulatory region completely blocked both the polar nuclear migration and the first ACDs of the xylem pole pericycle cells for LR initiation. Taken together, these results indicate that the localized activity of auxin-inducible LBD/ASL proteins links to the regulation of ACDs necessary for LR initiation in Arabidopsis.

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CLE (CLAVATA3/ESR) peptides precisely regulate cell proliferation and differentiation by acting as signaling molecules in cell-cell communication. In Arabidopsis, there are 32 CLE genes including CLV3 and CLE41/44, which encode TDIF (Tracheary element differentiation inhibitory factor). However, the roles of most CLE peptides have not been revealed yet. To know novel function of CLE peptides, we comprehensively examined the effects of chemically synthesized CLE peptides on root xylem development. This physiological analysis revealed that CLE peptides inhibited protoxylem vessel formation, independently of root growth arrest. This function was also proved by phenotype analyses of CLE receptor mutant and CLE-overexpressing transgenic plants. Next we performed transcriptome analysis when treated with or without CLE peptides. Interestingly, CLE peptides application reduced the expression levels of type-A ARRs, which negatively regulate cytokinin signal transduction. These results together with data from genetic analysis using cytokinin-related mutants clarified cross-talk between CLE peptides and cytokinin signaling pathways in protoxylem vessel formation.

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Cytokinin plays crucial roles in diverse aspects of plant growth and development. Most active cytokinin is trans-zeatin (tZ). In the biosynthesis pathway, the nucleotide form is initially produced as cytokinin precursor. It is expected that two pathways be involved in production of tZ from the nucleotide: two-step activation pathway and direct activation pathway. In the two-step pathway, the nucleotides are converted to the nucleosides by nucleotidase and then to tZ by nucleosidase. On the other hand, in the direct pathway, the nucleotides are converted to tZ in one step. However, the biological roles of each pathway remain unclear.
Although the genes related in two-step pathway are not indentified, LONELY GUY (LOG) was previously identified as a cytokinin-activating enzyme that works in the direct pathway.
To study the biological roles of the direct pathway, we analyzed multiple knock out mutant of Arabidopsis LOGs (AtLOG1-5, 7-8). In addition, we monitored cytokinin metabolic fluxes by using stable isotope.

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Plant hormones play an important role as signaling molecules in the regulation of almost all phases of plant development, from embryogenesis to senescence. As well as metabolome and transcriptome, introduction of omics approach into hormone research will give us the clues for deeply understanding the regulation of hormone action and metabolism. We have developed a highly sensitive and high-throughput method for the simultaneous analysis of 45 molecular species of cytokinins, auxins, ABA, gibberellins (GAs), Jasmonic acid, and salicylic acid using an automatic liquid handling system for solid phase extraction, UPLC-ESI-qMS/MS, and chemical derivatization with bromocholine. Our current method needs less than 100 mg (fresh weight) of plant tissues to determine phytohormone profiles and enables us to analyze simultaneously more than 180 plant samples. Application of this method to plant hormone profiling enabled us to draw organ-distribution maps of hormone species in rice and Arabidopsis.

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E.globulus, one of the major afforestation species is known to be difficult for vegetative-propagation. So far, it was necessary to develop novel method for promotion of adventitious root (AR) formation. First, we measured endogenous levels of several hormones. As a result, the IAA level of easy-rooting line was two times higher than that of the poor-rooting line. Next, we forcused on the cytochrome P450s. Several triazole-containing chemical compounds have previously been shown act as efficient inhibitors of cytochrome P450. A chemical library of triazole derivatives to find chemicals which have the effect of promoting AR formation was screened. Consequently, five compounds effectively promoted AR formation.
Finally, we investigated how these chemicals affected the growth of Arabidopsis thaliana. Arabidopsis seedlings were grown on agar medium containing the chemicals. One of the selected chemicals, MA65 increased the numbers of root in Arabidopsis seedlings. The amounts of endogenous IAA in Arabidopsis grown in the presence of MA65 were analyzed. The IAA content was increased by two-fold. Taken together, MA65 may increase endogenous IAA level and promotes an AR formation.

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Auxin plays a key role in emergence of complexity in morphogenesis. Auxin induces interaction of the receptor TIR1 and AUX/IAA proteins, which causes poly-ubiquitination and subsequent degradation of the AUX/IAA proteins. Degradation of AUX/IAA proteins induces auxin responses. Five TIR1 homologues AFB1-5 are also implicated in auxin perception, and they may have different auxin-specificities. Also, there may be different interaction-preferences between these TIR1/AFBs and AUX/IAAs. These diversities may be responsible for complex auxin responses. Here we aimed to test interaction-specificities between every receptor and AUX/IAA using a heterologous yeast system, because yeasts expressing TIR1 have been reported to degrade IAA17 in an auxin-dependent manner. We here constructed yeast strains expressing a pair of a receptor and a luciferase-fused AUX/IAA, for all combinations, and measured luciferase activity after addition of different auxin molecular species. The results suggested that TIR1 and AFB2 have partially different target specificities.

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In several angiosperm species, excised plant tissues give rise to the entire organism through transdifferentiation from differentiated cells to stem cells when cultured with phytohormones including auxin. However, the action mechanism of auxin on this process has remained elusive. In our current study with Physcomitrella, which has a high-capacity for transdifferentiation, we show that the anti-auxin probe BH-IAA suppresses transdifferentiation from leaf cells to stem cells. Because BH-IAA blocks auxin response through the suppression of the SCF^TIR1-Aux/IAA pathway and the transcriptional regulation by auxin response factors (ARFs), the transcriptional network of ARFs is likely to be important in transdifferentiation of leaf cells. As a first step to elucidate this network, we isolated 13 ARF genes from Physcomitrella, and found 2 to 3 splice variants of 4 genes. In addition, we will show recent studies on the expression of ARFs during transdifferentiation and the phenotypes of transgenic lines expressing high levels of ARFs.

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We reported that L-aminooxy-phenylpropionic acid (AOPP) inhibits indole-3-acetic acid (IAA) biosynthesis. Since AOPP is unstable in MS medium and has side effects, e.g., inhibiting phenylalanine ammonia-lyase (PAL), more stable inhibitor having higher specificity was developed. We have designed and synthesized various compounds structurally related to AOPP. Among these compounds, KOK1169 inhibited a recombinant enzyme of IAA biosynthesis enzyme, TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1, stronger than AOPP. Furthermore, KOK1169 inhibited recombinant enzyme, AtPAL2 weaker than AOPP. In Arabidopsis seedlings, treatment with KOK1169 at 30 μM for 3 h was as effective as that with AOPP to reduce endogenous IAA. However KOK1169 inhibited the seedling growth for 8 days, while AOPP did not. The seedling growth was recovered to the control level with exogenous IAA treatment. These results suggested that KOK1169 has higher specificity as an auxin-biosynthesis inhibitor than AOPP. This work was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN).

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Luciferase reporter system was used for the measurement of AUX/IAA19 expression in Arabidopsis. Application of 100 nM NAA induced a transient increase of the luciferase signal in both wt and the dominant mutant, msg2-1. The basal expression level and maximum auxin response were low in msg2-1, which was consistent with previous report (Tatematsu et al., 2004). The supposed accumulation of msg2-1 protein with a long half-life may attribute to the defects. Kinetic analysis suggested that the inhibitory effect of msg2-1 protein was only observed 1 h after NAA application. This may explain why msg2-1 showed wild-type response in root gravitropism. In fact, when we applied longer presentation time of gravi-stimulation for root tips, msg2-1 roots showed a reduced gravitropism, ie, plagiotropism. These observations suggest that a combination of multiple AUX/IAAs with different half-life may determine gravity setpoint angle (GSA) of root and may be one of the critical factors to exert a plagiotropism in the root system.

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