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The phytohormone gibberellins (GAs) are key regulators of growth, development and environmental responses in angiosperms. From an evolutionary perspective, all major steps of GA biosynthesis are conserved among vascular plants, while GA biosynthetic intermediates such asent-kaurenoic acid (KA) are also produced by bryophytes. Here we show that in the liverwortMarchantia polymorpha, KA and GA12are synthesized by evolutionarily conserved enzymes, which are required for developmental responses to far-red light (FR). Under FR-enriched conditions, mutants of various biosynthesis enzymes consistently altered thallus growth allometry, delayed the initiation of gametogenesis, and affected the morphology of gamete-bearing structures (gametangiophores). By chemical treatments and LC-MS/MS analyses, we confirmed these phenotypes were caused by deficiency of some GA-related compounds derived from KA, but not bioactive GAs from vascular plants. Transcriptome analysis showed that FR enrichment induced the up-regulation of genes related to stress responses and secondary metabolism inM. polymorpha, which was largely dependent on the biosynthesis of GA-related compounds. Due to the lack of the canonical GA receptors in bryophytes, we hypothesize that GA-related compounds are commonly synthesized in land plants but co-opted independently to regulate responses to light quality change in different lineages during the past 450 million years of evolution.

Strigolactones (SLs) are a plant hormone inhibiting shoot branching/tillering and a rhizospheric, chemical signal that triggers seed germination of the noxious root parasitic plant Striga and mediates symbiosis with beneficial arbuscular mycorrhizal fungi. Identifying specific roles of canonical and noncanonical SLs, the two SL subfamilies, is important for developing Striga -resistant cereals and for engineering plant architecture. Here, we report that rice mutants lacking canonical SLs do not show the shoot phenotypes known for SL-deficient plants, exhibiting only a delay in establishing arbuscular mycorrhizal symbiosis, but release exudates with a significantly decreased Striga seed–germinating activity. Blocking the biosynthesis of canonical SLs by TIS108, a specific enzyme inhibitor, significantly lowered Striga infestation without affecting rice growth. These results indicate that canonical SLs are not the determinant of shoot architecture and pave the way for increasing crop resistance by gene editing or chemical treatment.

Ectopic expression of the apple 2-oxoglutarate-dependent dioxygenase (DOX, 2ODD) gene, designated MdDOX-Co, is thought to cause the columnar shape of apple trees. However, the mechanism underlying the formation of such a unique tree shape remains unclear. To solve this problem, we demonstrated that Arabidopsis thaliana overexpressing MdDOX-Co contained reduced levels of biologically active gibberellin (GA) compared to wild type. In summary, (i) with biochemical approaches, the gene product MdDOX-Co was shown to metabolize active gibberellin A4 (GA4 ) to GA58 (12-OH-GA4 ) in vitro. MdDOX-Co also metabolized its precursors GA12 and GA9 to GA111 (12-OH-GA12 ) and GA70 (12-OH-GA9 ), respectively. (ii) Of the three 12-OH-GAs, GA58 was still active physiologically, but not GA70 or GA111 . (iii) Arabidopsis MdDOX-Co OE transformants converted exogenously applied deuterium-labeled (d2 )-GA12 to d2 -GA111 but not to d2 -GA58 , whereas transformants converted applied d2 -GA9 to d2 -GA58 . (iv) GA111 is converted poorly to GA70 by GA 20-oxidases in vitro when GA12 is efficiently metabolized to GA9 . (v) No GA58 was detected endogenously in MdDOX-Co OE transformants. Overall, we conclude that 12-hydroxylation of GA12 by MdDOX-Co prevents the biosynthesis of biologically active GAs in planta, resulting in columnar phenotypes.

Plants synthesize gibberellin (GA), a diterpenoid hormone, via ent-kaurenoic acid (KA) oxidation. GA has not been detected in the moss Physcomitrium patens despite its ability to synthesize KA. It was recently shown that a KA metabolite, 3OH-KA, was identified as an active regulator of protonema differentiation in P. patens. An inactive KA metabolite, 2OH-KA, was also identified in the moss, as was KA2ox, which is responsible for converting KA to 2OH-KA. In this review, we mainly discuss the GA biosynthetic gene homologs identified and characterized in bryophytes. We show the similarities and differences between the OH-KA control of moss and GA control of flowering plants. We also discuss using recent genomic studies; mosses do not contain KAO, even though other bryophytes do. This absence of KAO in mosses corresponds to the presence of KA2ox, which is absent in other vascular plants. Thus, given that 2OH-KA and 3OH-KA were isolated from ferns and flowering plants, respectively, vascular plants may have evolved from ancestral bryophytes that originally produced 3OH-KA and GA.

The plant hormones strigolactones (SLs) regulate shoot branching and mediate the communication with symbiotic mycorrhizal fungi, but also with noxious root parasitic weeds, such as Striga spp. SLs derive from carlactone (CL) and are divided structurally into canonical and non-canonical SLs. However, the questions about particular biological functions of the two groups and the identification of the SL that inhibits shoot branching are still unanswered, hampering targeted modification of SL pattern towards improving plant architecture and resistance against Striga. Here, we reported that 4-deoxyorobanchol (4DO) and orobanchol, the two canonical SLs in rice, do not have major role in determining rice shoot architecture. CRISPR/Cas9 mediated Osmax1-900 mutants, lacking these two SLs, do not show the high tillering and dwarf phenotype typical for SL-deficient plants. However, the absence of 4DO and orobanchol in root exudates significantly decreased their capability in inducing Striga seed germination, while caused only a delay in root colonization by mycorrhizal fungi. To confirm the genetic evidence, we used the SL-biosynthesis inhibitor TIS108. Our results showed that TIS108 is a MAX1-specific inhibitor that lowers 4DO and orobanchol synthesis, conferring a resistance to Striga without a severe impact on rice architecture. Hence, our work uncovers the specific function of canonical SLs as rhizospheric signals and paves the way for establishing chemical and genetic based approaches for combating the root parasitic weeds, by targeted depletion of their release.

The orange leafhopper, Cicadulina bipunctata (Melichar) (Hemiptera: Cicadellidae), is an insect pest of various Poaceae plants. Feeding by this leafhopper species induces growth suppression and gall formation, which is characterized by severe swelling of leaf veins, named wallaby ear symptoms. In this study, we performed histological analysis of the cell differentiation in the galled leaves of rice seedlings and devised a new method to quantify the degree of gall formation. In the histological study, abnormal vascular cell patterning and differentiation were observed in the galled leaves. Morphological changes during gall formation were quantitatively evaluated through image analysis.

Orange leafhopper Cicadulina bipunctata feeding induces wallaby ear symptoms, namely growth suppression and gall formation characterized by severe swelling of leaf veins, on various Poaceae, thereby leading to low crop yields. Here, we investigated the development of wallaby ear symptoms on rice seedlings due to C. bipunctata feeding. After confirming that C. bipunctata feeding induces growth suppression and gall formation on rice seedlings, we further demonstrated that gall formation score decreased with decreasing levels of nitrogen in the medium and that C. bipunctata feeding induces the expression levels of nitrogen transporter genes. These gene expression changes may participate in the nutrient accumulation observed in galled tissues and in gall formation. In addition, these expression changes should induce growth promotion but the inhibition of gibberellin signaling by C. bipunctata feeding might be the reason why growth is suppressed. Treatment with plant growth regulators did not affect gall formation, suggesting the existence of a complex gall formation mechanism by C. bipunctata feeding.

Momilactones are bioactive diterpenoids that contribute to plant defense against pathogens and allelopathic interactions between plants. Both cultivated and wild grass species of Oryza and Echinochloa crus-galli (barnyard grass) produce momilactones using a biosynthetic gene cluster (BGC) in their genomes. The bryophyte Calohypnum plumiforme (formerly Hypnum plumaeforme ) also produces momilactones, and the bifunctional diterpene cyclase gene CpDTC1/HpDTC1, which is responsible for the production of the diterpene framework, has been characterized. To understand the molecular architecture of the momilactone biosynthetic genes in the moss genome and their evolutionary relationships with other momilactone-producing plants, we sequenced and annotated the C. plumiforme genome. The data revealed a 150-kb genomic region that contains two cytochrome P450 genes, the CpDTC1 / HpDTC1 gene and the “dehydrogenase momilactone A synthase” gene tandemly arranged and inductively transcribed following stress exposure. The predicted enzymatic functions in yeast and recombinant assay and the successful pathway reconstitution in Nicotiana benthamiana suggest that it is a functional BGC responsible for momilactone production. Furthermore, in a survey of genomic sequences of a broad range of plant species, we found that momilactone BGC is limited to the two grasses ( Oryza and Echinochloa ) and C. plumiforme , with no synteny among these genomes. These results indicate that while the gene cluster in C. plumiforme is functionally similar to that in rice and barnyard grass, it is likely a product of convergent evolution. To the best of our knowledge, this report of a BGC for a specialized plant defense metabolite in bryophytes is unique.

We previously found a chemical, designated as NJ15, which inhibited both auxin and brassinosteroid responses in dark-grown Arabidopsis. To study its mode of action, we performed a phenotypic screening of NJ15-low-sensitive lines among mutant pools of Arabidopsis. One line (f127) showed clear NJ15-low-sensitivity in terms of hypocotyl elongation and shoot gravitropism. After further testing, it was determined that DCR, an enzyme involved in cutin polymerization, had lost its function in the mutant, which caused its low sensitivity to NJ15. Fatty acids are the base materials for polymers such as cutin and cuticular wax. We confirmed that NJ15 affects fatty acid biosynthesis, and that it does differently from cafenstrole, a known inhibitor of cuticular wax formation. Based on these results, we propose that the target of NJ15 is likely located within the cutin polymer formation pathway. Abbreviations: Caf: cafenstrole; DEG: differentially expressed gene; FDR: false discovery rate; FOX: full length cDNA-overexpressor; VLCFA: very-long-chain fatty acid NJ15 inhibits auxin and brassinosteroid responses in dark-grown Arabidopsis. Its target was supposed to be within the cutin polymer formation pathway.

Helminthosporol, a natural growth regulator isolated from a fungus, stimulates hypocotyl growth and seed germination, similar to gibberellin (GA). We recently reported that helminthosporic acid (H-acid), a synthetic analog of helminthosporol, acts as an agonist of GA receptor. In this study, we showed that a H-acid analog, in which the hydroxymethyl group at the C-8 position of H-acid was converted to a keto group, acts as a selective GA receptor agonist. 1) This analog shows higher hypocotyl elongation activity in Arabidopsis than H-acid does, and induces the degradation of DELLA protein and 2) leads to the formation of the GID1-DELLA complex and 3) regulates the expression of GA-related genes. In addition, 4) its hypocotyl elongation activity was not observed in a atgid1a single mutant, and 5) this analog could promote only the interaction between specific GA receptors and DELLA proteins in vitro. Taken together, our results strongly suggest that the selectivity of the reported H-acid analog depends on the specificity of its GA receptor binding activity.

Helminthosporol was isolated from a fungus, Helminthosporium sativum, as a natural plant growth regulator in 1963. It showed gibberellin-like bioactivity that stimulated the growth of the second leaf sheath of rice. After studying the structure–activity relationship between the compound and some synthesized analogs, it was found that helminthosporic acid (H-acid) has higher gibberellin-like activity and chemical stability than helminthosporol. In this study, we showed that (1) H-acid displays gibberellin-like activities not only in rice but also in Arabidopsis, (2) it regulates the expression of gibberellin-related genes, (3) it induces DELLA degradation through binding with a gibberellin receptor (GID1), and (4) it forms the GID1-(H-acid)-DELLA complex to transduce the gibberellin signal in the same manner as gibberellin. This work shows that the H-acid mode of action acts as an agonist for gibberellin receptor.

CYP701B1 of the moss, Physcomitrella patents, might be a unique cytochrome P450 having the ent-kaurene oxidase (KO) activity occurring in nonvascular plant. Phylogenetic analysis suggested that the gene encoding CYP701B1 was diverged from a common ancestral gene encoding KO of vascular plants. CYP701B1 expressed in Phichia yeast microsomes was purified and characterized. The purified CYP701B1 catalyzed the oxidation of ent-kaurene to ent-kaurenoic acid through three successive monooxygenations, and the rate-limiting step of this oxidation might be the initial step that forms ent-kaurenol. CYP701B1 was a typical ferric low-spin cytochrome P450 and was completely moved to high-spin state upon binding with ent-kaurene, and apparent Kd of ent-kaurene estimated by the spectral change caused by this spin-state shift was 2.5 μM. The potent KO inhibitor uniconazole, an azole compound with molecular size similar to ent-kaurene, bound CYP701B1 with high affinity. However, ketoconazole, an azole compound whose molecular size is larger than ent-kaurene could not bind to CYP701B, though it binds strongly with CYP51, lanosterol 14-demethylase. The results indicated that the active site of CYP701B1 is fitted for the molecular size of ent-kaurene. The P450 monooxygenase adapted for ent-kaurene oxidation might appear in land plants before evolutionary divergence into vascular and nonvascular plants.

The non-seed land plant Physcomitrella patens is a model species for developmental, cellular, and molecular biology studies in mosses and also for performing genetic analyses. Previously, it was shown that wild-type P. patens displays a unique photomorphogenetic behavior, in which chloronemal filaments grow in the opposite direction to a blue-light source. Here, we describe bioassay systems that can be used to study light avoidance responses as well as other aspects of photomorphogenetic regulation in P. patens grown under red- and blue-light sources.

Insect gall structures have many characteristic forms and colors, which are distinguishable from host plants. In this study, we identified an anthocyanin from red color insect galls and revealed that the anthocyanin biosynthesis of plants was induced by the gall extracts. The galling insects presumably regulate the anthocyanin biosynthesis of host plants to protect their larvae from environmental stresses.

The non-seed land plant Physcomitrella patens is a model species for developmental, cellular, and molecular biology studies in mosses and also for performing genetic analyses. Previously, it was shown that wild-type P. patens displays a unique photomorphogenetic behavior, in which chloronemal filaments grow in the opposite direction to a blue-light source. Here, we describe bioassay systems that can be used to study light avoidance responses as well as other aspects of photomorphogenetic regulation in P. patens grown under red- and blue-light sources.

Salicylic acid (SA) plays important roles in the induction of systemic acquired resistance (SAR) in plants. Determining the mechanism of SAR will extend our understanding of plant defenses against pathogens. We recently reported that PAMD is an inhibitor of SA signaling, which suppresses the expression of the pathogenesis-related PR genes and is expected to facilitate the understanding of SA signaling. However, PAMD strongly inhibits plant growth. To minimize the side effects of PAMD, we synthesized a number of PAMD derivatives, and identified compound 4 that strongly suppresses the expression of the PR genes with fewer adverse effects on plant growth than PAMD. We further showed that the adverse effects on plant growth were partially caused the stabilization of DELLA, which is also related to the pathogen responses. These results indicate that compound 4 would facilitate our understanding of SA signaling and its cross talk with other plant hormones.

ent-Kaurenoic acid (KA) is a key intermediate connected to a phytohormone gibberellin. To date, the general procedure for quantifying KA is by using traditional gas chromatography–mass spectrometry (GC–MS). In contrast, gibberellins, which are more hydrophilic than KA, can be easily quantified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). In this study, we have established a new method to quantify KA by LC–MS/MS by taking advantage of a key feature of KA, namely the lack of fragmentation that occurs in MS/MS when electrospray ionization (ESI) is in the negative mode. Q1 and Q3 were adopted as identical channels for the multiple reaction monitoring of KA. The method was validated by comparing with the results obtained by selected ion monitoring in GC–MS. This new method could be applicable for the quantification of other hydrophobic compounds., Highlights • ent-Kaurenoic acid (KA) can be quantified by LC–MS/MS. • Detection limit of KA acid was 0.59 ng ml−1. • We set Q1 and Q3 to the same MRM channel. • Endogenous KA in a moss was detected and quantified.

In response to environmental stressors such as blast fungal infections, rice produces phytoalexins, an antimicrobial diterpenoid compound. Together with momilactones, phytocassanes are among the major diterpenoid phytoalexins. The biosynthetic genes of diterpenoid phytoalexin are organized on the chromosome in functional gene clusters, comprising diterpene cyclase, dehydrogenase, and cytochrome P450 monooxygenase genes. Their functions have been studied extensively using in vitro enzyme assay systems. Specifically, P450 genes (CYP71Z6, Z7; CYP76M5, M6, M7, M8) on rice chromosome 2 have multifunctional activities associated with ent-copalyl diphosphate-related diterpene hydrocarbons, but the in planta contribution of these genes to diterpenoid phytoalexin production remains unknown. Here, we characterized cyp71z7 T-DNA mutant and CYP76M7/M8 RNAi lines to find that potential phytoalexin intermediates accumulated in these P450-suppressed rice plants. The results suggested that in planta, CYP71Z7 is responsible for C2-hydroxylation of phytocassanes and that CYP76M7/M8 is involved in C11α-hydroxylation of 3-hydroxy-cassadiene. Based on these results, we proposed potential routes of phytocassane biosynthesis in planta.

We report a novel physiological response to blue light in the moss Physcomitrella patens . Blue light regulates ent -kaurene biosynthesis and avoidance response to protonemal growth. Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurenoic acid via ent-kaurene. While the moss Physcomitrella patens has part of the GA biosynthetic pathway, from geranylgeranyl diphosphate to ent-kaurenoic acid, no GA is found in this species. Caulonemal differentiation in a P. patens mutant with a disrupted bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase (PpCPS/KS) gene is suppressed under red light, and is recovered by application of ent-kaurene and ent-kaurenoic acid. This indicates that derivatives of ent-kaurenoic acid, not GAs, might act as endogenous developmental regulators. Here, we found unique responses in the protonemal growth of P. patens under unilateral blue light, and these regulators were involved in the responses. When protonemata of the wild type were incubated under blue light, the chloronemal filaments grew in the opposite direction to the light source. Although this avoidance was not observed in the ent-kaurene deficient mutant, chloronemal growth toward a blue-light source in the mutant was suppressed by application of ent-kaurenoic acid, and the growth was rescued to that in the wild type. Expression analysis of the PpCPS/KS gene showed that the mRNA level under blue light was rapidly increased and was five times higher than under red light. These results suggest that regulators derived from ent-kaurenoic acid are strongly involved not only in the growth regulation of caulonemal differentiation under red light, but also in the light avoidance response of chloronemal growth under blue light. In particular, growth under blue light is regulated via the PpCPS/KS gene.

Momilactones, which are diterpenoid phytoalexins with antimicrobial and allelopathic functions, have been found only in rice and the moss Hypnum plumaeforme. Although these two evolutionarily distinct plant species are thought to produce momilactones as a chemical defence, the momilactone biosynthetic pathway in H. plumaeforme has been unclear. Here, we identified a gene encoding syn-pimara-7,15-diene synthase (HpDTC1) responsible for the first step of momilactone biosynthesis in the moss. HpDTC1 is a bifunctional diterpene cyclase that catalyses a two-step cyclization reaction of geranylgeranyl diphosphate to syn-pimara-7,15-diene. HpDTC1 transcription was up-regulated in response to abiotic and biotic stress treatments. HpDTC1 promoter-GUS analysis in transgenic Physcomitrella patens showed similar transcriptional responses as H. plumaeforme to the stresses, suggesting that a common response system to stress exists in mosses. Jasmonic acid (JA), a potent signalling molecule for inducing plant defences, could not activate HpDTC1 expression. In contrast, 12-oxo-phytodienoic acid, an oxylipin precursor of JA in vascular plants, enhanced HpDTC1 expression and momilactone accumulation, implying that as-yet-unknown oxylipins could regulate momilactone biosynthesis in H. plumaeforme. These results demonstrate the existence of an evolutionarily conserved chemical defence system utilizing momilactones and suggest the molecular basis of the regulation for inductive production of momilactones in H. plumaeforme.

Natural terpenoids have elaborate structures and various bioactivities, making difficult their synthesis and labeling with isotopes. We report here the enzymatic total synthesis of plant hormone gibberellins (GAs) with recombinant biosynthetic enzymes from stable isotope-labeled acetate. Mevalonate (MVA) is a key intermediate for the terpenoid biosynthetic pathway. 13C-MVA was synthesized from 13C-acetate via acetyl-CoA, using four enzymes or fermentation with a MVA-secreted yeast. The diterpene hydrocarbon, ent-kaurene, was synthesized from 13C-acetate and 13C-MVA with ten and six recombinant enzymes in one test tube, respectively. Four recombinant enzymes, P450 monooxygenases and soluble dioxygenases involved in the GA4 biosynthesis from ent-kaurene via GA12 were prepared in yeast and Escherichia coli. All intermediates and the final product GA4 were uniformly labeled with 13C without dilution by natural abundance when [U-13C2] acetate was used. The 13C-NMR and MS data for [U-13C20] ent-kaurene confirme...

In recent years, development of environment measure cars is an important theme of the earth environment protection and energy saving. Various kinds of hybrid electric vehicles increase the production rapidly. However, the vehicle price is one of the big themes for the hybrid electric vehicle’s growth. High voltage harnesses necessary to connect between hybrid units (inverter to motor, battery) that must be fitted in a limited vehicle space. Especially, high voltage and high current wire harnesses for hybrid electrical vehicle need thicker cross section and higher connection reliability in comparison with wires for 12V.In this paper, we set our goal of the terminal which is proof against vehicle vibration and is possible to apply high and continuous current. We successfully selected the structure and the material and developed the terminal at low cost, which is proof against vehicle vibration and is able to pass over 100A current. The developed terminals are used in Lexus GS450h.

The structure of poly(N-isopropylacrylamide)−clay nanocomposite gels (NC gel) is investigated. The sophisticated contrast variation small-angle neutron scattering technique leads to obtain the partial scattering functions of each component quantitatively. The extracted partial scattering functions of clay, polymer, and the polymer−clay cross-term are analyzed in detail, and the selective interaction between clay and polymer is revealed. On the basis of these structural observations, it is confirmed that the clay nanoparticles serve as 2-dimensional cross-linkers.

Summary: The polymer-clay nanocomposite gels (NC gels), developed by Haraguchi et al. (Adv. Mater., 2001), exhibit extraordinarily high mechanical properties, such as high elongations and high ultimate strengths. In order to understand the origin of these properties, contrast-matched small-angle neutron scattering (SANS) studies were carried out for stretched NC gels. It was found that (1) the scattering function for NC gels is simply given by a Lorentz function, a function for semidilute polymer solutions, without a characteristic cross-link inhomogeneity term for polymer gels, and (2) the unique mechanical properties are ascribed to its network structure, i.e., long polymer chains connected by “plane” cross-links.

The gelation mechanism of poly(N-isopropylacrylamide)−clay nanocomposite gels (NC gel) was investigated by dynamic light scattering (DLS) and contrast variation small-angle neutron scattering (SANS). It was found that the gelation mechanism of NC gels is similar to that of conventional gels made with organic cross-linker (OR gels). Namely, time-resolved DLS measurements captured all of the characteristic features of gelation at the threshold. This indicates that the gelation of NC gels is also classified to an ergode−nonergode transition. However, the size of the clusters at the gelation threshold is much larger than that of OR gels. This results in a significant depression of optical transmittance exclusively at the gelation threshold for NC gels. Partial scattering functions, i.e., two self-terms SPP(q) and SCC(q) and the corresponding cross-term SCP(q), were obtained by contrast-variation SANS, where P and C denote polymer and clay, respectively, and q is the magnitude of the scattering vector. The det...

The microscopic structure of poly(N-isopropylacrylamide)−clay nanocomposite gels (NC gels) having various clay concentrations (Cclay) was investigated by mechanical measurement and small-angle neutron scattering (SANS) as a function of stretching ratio λ. The mechanical measurements indicated that the number of poly(N-isopropylacrylamide) (PNIPA) chains bridging clay platelets, Nchain, per unit volume increased with Cclay4/3. Two-dimensional SANS intensity patterns exhibited the so-called “abnormal-butterfly” pattern. However, both sector-averaged scattering intensities parallel and perpendicular to the stretching direction were well represented by Ornstein−Zernike (OZ) scattering functions, suggesting that the NC gels have very little cross-linking inhomogeneities regardless of Cclay and of λ (λ ≤ 2.0). The correlation length, ξ, obtained by the OZ functions was a decreasing function of Cclay, i.e., ξ ∼ Cclay-1/3, and was in proportion to the inter-platelet distance (∼Cclay-1/3). This suggests that the c...

The nanocomposite (NC) gels made of poly(N-isopropylacrylamide) (PNIPA) and synthetic clay show extraordinarily high mechanical properties, e.g., high extensibility and high strength at break. The structure and deformation mechanism of the NC gels have been investigated by small-angle neutron scattering (SANS). Two-dimensional SANS intensity patterns were obtained for deformed NC4 gels with the stretching ratio up to 6 times, where 4 denotes the clay concentration being 0.04 mol/L (≈3.05 wt %). A so-called abnormal-butterfly pattern was observed in the low q region, i.e., q < 0.02 A-1, while a normal-butterfly pattern was obtained in the larger q (≥0.02 A-1). Here q is the magnitude of the scattering vector. However, contrast-matched SANS experiments disclosed that the abnormal-butterfly pattern is not originated from cross-link inhomogeneities observed in conventional gels but from oriented-clay platelets by deformation. These results indicate that clay platelets, 300 A in diameter and 10 A thick embedde...

Gibberellins (GAs) are diterpenoid hormones that regulate growth and development in flowering plants. The moss Physcomitrella patens has part of the GA biosynthetic pathway from geranylgeranyl diphosphate to ent-kaurenoic acid via ent-kaurene, but it does not produce GA. Disruption of the ent-kaurene synthase gene in P. patens suppressed caulonemal differentiation. Application of ent-kaurene or ent-kaurenoic acid restored differentiation, suggesting that derivative(s) of ent-kaurenoic acid, but not GAs, are endogenous regulator(s) of caulonemal cell differentiation. The protonemal growth of P. patens shows an avoidance response under unilateral blue light. Physiological studies using gene mutants involved in ent-kaurene biosynthesis confirmed that diterpenoid(s) regulate the blue-light response. Here, we discuss the implications of these findings, and provide data for the ent-kaurene oxidase gene-disrupted mutant.

The moss Physcomitrella patens produces both ent-kaurene and ent-kaurenoic acid, which are intermediates of gibberellin biosynthesis in flowering plants. The CYP701 superfamily of cytochrome P450s functions as ent-kaurene oxidases in the biosynthesis of ent-kaurenoic acid. A candidate gene encoding ent-kaurene oxidase in P. patens, CYP701B1, was cloned and heterologously expressed in yeast to examine enzyme activities in vitro. The recombinant CYP701B1 protein catalyzed the oxidation reaction from ent-kaurene to ent-kaurenoic acid. CYP701B1 activity was highly resistant to the ent-kaurene oxidase inhibitor uniconazole-P (IC50 64μM), even though the activity of Arabidopsis ent-kaurene oxidase (CYP701A3) was sensitive (IC50 0.26μM).

Natural terpenoids have elaborate structures and various bioactivities, making difficult their synthesis and labeling with isotopes. We report here the enzymatic total synthesis of plant hormone gibberellins (GAs) with recombinant biosynthetic enzymes from stable isotope-labeled acetate. Mevalonate (MVA) is a key intermediate for the terpenoid biosynthetic pathway. ¹³C-MVA was synthesized from ¹³C-acetate via acetyl-CoA, using four enzymes or fermentation with a MVA-secreted yeast. The diterpene hydrocarbon, ent-kaurene, was synthesized from ¹³C-acetate and ¹³C-MVA with ten and six recombinant enzymes in one test tube, respectively. Four recombinant enzymes, P450 monooxygenases and soluble dioxygenases involved in the GA₄ biosynthesis from ent-kaurene via GA₁₂ were prepared in yeast and Escherichia coli. All intermediates and the final product GA₄ were uniformly labeled with ¹³C without dilution by natural abundance when [U-¹³C₂] acetate was used. The ¹³C-NMR and MS data for [U-¹³C₂₀] ent-kaurene confirmed ¹³C-¹³C coupling, and no dilution with the ¹²C atom was observed.

Rice ent-kaurene oxidase 2 (OsKO2) perhaps functions in the early steps of gibberellin biosynthesis. We found that microsomes from the methylotropic yeast Pichia pastoris expressing both OsKO2 and a fungal cytochrome P450 monooxygenase (P450) reductase converted ent-kaurene to ent-kaurenoic acid. This is direct evidence that OsKO2 is involved in the sequential oxidation of ent-kaurene to ent-kaurenoic acid in gibberellin biosynthesis in rice.

We have shown that a metal target covered with water, Water Cannon target (WC), provides higher Cm than a pure metal target. We improved repetitive water supply system and succeeded in driving a miniature car of 70g weight by only 1.4J YAG laser. Then we invented new target, Metal Free Water Cannon target (MFWC) which can be driven into any direction. In addition, we improved it and invented Water Film Cannon (WFC). Cm of 3680[N.s/MJ] was achieved with WFC. By using this system, we propose a piston engine and vertical launch system. We also examined a similar propulsion system with CO2 laser and found some enhancement with water cannon.

We propose an automobile driven by piston motion, which is driven by water-laser coupling. The automobile can load a solar-pumped fiber laser or can be driven by ground-based lasers. The vehicle is much useful for the use in other planet in which usual combustion engine cannot be used. The piston is in a closed system and then the water will not be exhausted into vacuum. In the preliminary experiment, we succeeded to drive the cylindrical piston of 0.2g (6mm in diameter) on top of water placed inside the acrylic pipe of 8 mm in inner diameter and the laser is incident from the bottom and focused onto the upper part of water by the lens (f=8mm) attached to the bottom edge.

We propose a new concept controlling a satellite by a fiber laser loaded in it and demonstrated the acceleration of pendulum with 7kW/2n and 2kHz fiber laser, and measured the Cm of 16Ns/MJ corresponding to the scaling of YAG laser. This laser can be easily bundled to generate much larger power. For more efficient acceleration, we propose “metal‐free water cannon target”, the new concept of propulsion using only water. The momentum coupling coefficient of 2500[Ns/MJ] was achieved with vacuum pump oil instead of water, and we succeeded in controlling the driving direction by the system based on the new concept. This can be used for thrusting a satellite and controlling its posture in combination with fiber lasers.

We found that water overlay on a metal layer is more effective than solid overlay. By using this target we demonstrated the successful flight of paper‐airplane of 5 cm‐size over a distance of 1–2m. In this paper, repetitive water supply system and levitation system are proposed for practical application, and examined by experiments. We succeeded in driving an object continuously using repetitive water supply and air slider. We also succeeded in driving 300g object by 0.5J laser using these equipments. In this paper, we try to find out a new possibility of water‐powered laser propulsion.

The microscopic structure of shear-induced gels for a mixed solution of 2-hydroxyethyl cellulose and nanometer-size spherical droplets has been investigated by in situ small-angle neutron scattering (SANS) with a Couette geometry as a function of shear rate gamma. With increasing gamma, the viscosity increased rapidly at gamma approximately 4.0 s(-1), followed by a shear thinning. After cessation of shear, the system exhibited an extraordinarily large steady viscosity. This phenomenon was observed as a shear-induced sol-gel transition. Real-time SANS measurements showed an increase in the scattering intensity exclusively at low scattering angle region. However, neither orientation of polymer chains nor droplet deformation was detected and the SANS patterns remained isotropic irrespective of gamma. It took about a few days for the gel to recover its original sol state. A possible mechanism of gelation is proposed from the viewpoint of shear-induced percolation transition.

The pressure-induced structural changes of a block copolymer, poly(2-ethoxyethoxyethyl vinyl ether)-block-poly(2-hydroxyethyl vinyl ether) (pEOEOVE-b-pHOVE) in aqueous solutions, were studied by means of small-angle neutron scattering (SANS) and dynamic light scattering (DLS) from atmospheric pressure up to 400 MPa. pEOEOVE-b-pHOVE formed a spherical micellar structure above 40 degrees C due to poor solubility of pEOEOVE. Micellization phase diagram was determined by DLS, and a covex-upward pressure-temperature (P-T) phase diagram was obtained having a peak around (P,T)=(150 MPa,48 degrees C). The SANS curves at 50 degrees C were analyzed as a function of P. The micellar core size decreased by pressurizing at low P's (Por=150 MPa) and then increased by further pressurizing (150 MPaP400 MPa). It was confirmed that the water volume fraction in the micellar core was larger at high P's than that at low P's even when the core sizes are the same at both pressure regions. This means that the temperature dependence of hydration is more selective at low pressures than at high pressures, in good accordance with our previous results on concentrated aqueous solutions of block copolymers [Phys. Rev. Lett. 96, 048303 (2006)].