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Nitrogenase is an oxygen-vulnerable metalloenzyme that catalyzes nitrogen fixation. It largely remains unknown how nitrogenase coexists with oxygenic photosynthesis in nonheterocystous cyanobacteria, since there have been no appropriate model cyanobacteria so far. Here, we demonstrate in vivo transposon tagging in the nonheterocystous cyanobacterium Leptolyngbya boryana as a forward genetics approach. By conjugative transfer, a mini-Tn5-derived vector, pKUT-Tn5-Sm/Sp, was transferred from Escherichia coli to L. boryana cells. Of 1839 streptomycin-resistant colonies, we isolated three mutants showing aberrant diazotrophic growth. Genome resequencing identified the insertion sites of the transposon in the mutants. This in vivo transposon tagging mutagenesis of L. boryana provides a promising system to investigate molecular mechanisms to resolve the Oxygen Paradox between nitrogen fixation and oxygenic photosynthesis in cyanobacteria.

Chlorophyllase (CLH) is a common plant enzyme that catalyzes the hydrolysis of chlorophyll to form chlorophyllide, a more hydrophilic derivative. For more than a century, the biological role of CLH has been controversial, although this enzyme has been often considered to catalyze chlorophyll catabolism during stress-induced chlorophyll breakdown. In this study, we found that the absence of CLH does not affect chlorophyll breakdown in intact leaf tissue in the absence or the presence of methyl-jasmonate, which is known to enhance stress-induced chlorophyll breakdown. Fractionation of cellular membranes shows that Arabidopsis (Arabidopsis thaliana) CLH is located in the endoplasmic reticulum and the tonoplast of intact plant cells. These results indicate that CLH is not involved in endogenous chlorophyll catabolism. Instead, we found that CLH promotes chlorophyllide formation upon disruption of leaf cells, or when it is artificially mistargeted to the chloroplast. These results indicate that CLH is responsible for chlorophyllide formation after the collapse of cells, which led us to hypothesize that chlorophyllide formation might be a process of defense against chewing herbivores. We found that Arabidopsis leaves with genetically enhanced CLH activity exhibit toxicity when fed to Spodoptera litura larvae, an insect herbivore. In addition, purified chlorophyllide partially suppresses the growth of the larvae. Taken together, these results support the presence of a unique binary defense system against insect herbivores involving chlorophyll and CLH. Potential mechanisms of chlorophyllide action for defense are discussed.

The spectral absorbance of photoreceptor visual pigments and the opsin gene class of the visual pigments was investigated in Sardinops melanostictus. Microspectrophotometric (MSP) measurements showed that the rod photoreceptors had peak absorbance spectra (λmax) at 502 nm. The spectral sensitivity of single cones was centered at 393 nm. Double cones had a λmax of 493/522 nm, but a few displayed a red-shifted absorbance of the long-wave member at 542 nm. The mRNAs of six different opsins were isolated from the retina, retrotranscribed, cloned, and sequenced. Three genes encoded opsins in the green-sensitive class (RH2), and three genes encoded opsins in the red-sensitive class (LWS), the ultraviolet (UV)-sensitive (SWS1) class, and the rod class (RH1). A Southern blot analysis showed that the blue-sensitive (SWS2) opsin gene is absent from this species, hence it was concluded that the λmax of 393 nm was generated from the SWS1 opsin. Phylogenetic analyses of S. melanostictus RH1, LWS, and SWS1 sequences placed them with orthologs from other species (e.g., the cyprinids Danio rerio and Carrasius auratus) in Otomorpha. However, unexpectedly, the RH2 sequences were more similar to orthologs in members of the Euteleosteomorpha (e.g., Oryzias latipes and Takifugu rubripes) than to cyprinid RH2 opsins.

Hieracium praealtum forms seeds asexually by apomixis. During ovule development, sexual reproduction initiates with megaspore mother cell entry into meiosis and formation of a tetrad of haploid megaspores. The sexual pathway ceases when a diploid aposporous initial (AI) cell differentiates, enlarges, and undergoes mitosis, forming an aposporous embryo sac that displaces sexual structures. Embryo and endosperm development in aposporous embryo sacs is fertilization independent. Transcriptional data relating to apomixis initiation in Hieracium spp. ovules is scarce and the functional identity of the AI cell relative to other ovule cell types is unclear. Enlarging AI cells with undivided nuclei, early aposporous embryo sacs containing two to four nuclei, and random groups of sporophytic ovule cells not undergoing these events were collected by laser capture microdissection. Isolated amplified messenger RNA samples were sequenced using the 454 pyrosequencing platform and comparatively analyzed to establish indicative roles of the captured cell types. Transcriptome and protein motif analyses showed that approximately one-half of the assembled contigs identified homologous sequences in Arabidopsis (Arabidopsis thaliana), of which the vast majority were expressed during early Arabidopsis ovule development. The sporophytic ovule cells were enriched in signaling functions. Gene expression indicative of meiosis was notably absent in enlarging AI cells, consistent with subsequent aposporous embryo sac formation without meiosis. The AI cell transcriptome was most similar to the early aposporous embryo sac transcriptome when comparing known functional annotations and both shared expressed genes involved in gametophyte development, suggesting that the enlarging AI cell is already transitioning to an embryo sac program prior to mitotic division.

This paper describes an application of a metal hydride (MH) sheet, which consists of MH powder, carbon fiber, and aramid pulp, in a metal hydride heat pump (MHHP) system with a TiFe0.9Ni0.1/La0.6Y0.4Ni4.9Al0.1 working pair (MH1/MH2). In the experiments, the effect of the use of MH sheet on the system performances was investigated, in which the MH sheets were used to replace part of the MH powder to improve the heat exchange performance. The sheets and powder were packed alternately into the MH beds in layers with an aspect ratio less than one. The MH sheet significantly accelerated the heat exchange ratio of both MH packed beds. Using the MH sheet in both reactors, the specific cooling power increased by 1.2 times. The results also indicated that the role of heat exchange in an MH2 reactor as a cooling output side was more important in the enhancement of system performance than that in an MH1 reactor as a heat source side. In addition, the proposed MH sheet was effective not only for improving the system performance but also for decreasing the stress on the reactor vessel due to the expansion of MH during the hydrogen absorption/desorption.

In this paper, we describe the combustion synthesis of a TiFe-based hydrogen storage alloy from Fe and TiO 2 using metallic calcium as the reducing agent and heat source. The effects of hydrogen on the combustion ignition temperature and the hydrogenation properties of the products were examined. In the experiments, Fe, TiO 2 , and Ca were mixed with a molar ratio of 1:1:4 and heated in a hydrogen atmosphere until the ignition due to the hydrogenation of calcium. For comparison, the same experiment was performed in an argon atmosphere. The ignition and maximum temperatures in the hydrogen atmosphere were drastically lower than those in the argon atmosphere. According to X-ray diffraction (XRD) analyses, all the peaks of the product combustion-synthesized in a hydrogen atmosphere were due to the TiFe phase, although some peaks of the product synthesized in argon indicated the existence of the TiO phase in addition to the TiFe phase. The product synthesized in hydrogen demonstrated a hydrogen storage capacity of 1.39 mass%, which is equal to that achieved using pure TiFe reagent. Moreover, a fine powdered product was obtained without any pulverization processes. This method creates an innovative production route for TiFe.

This paper describes the thermal and hydrogenation properties of a metal hydride (MH) sheet consisting of MH powder, aramid pulp, and carbon fiber. MH sheets were prepared by the wet paper method in which an agglutinated slurry of raw materials was dispersed onto a stainless steel mesh in water and then the sheet was dehydrated and dried. The cyclic characteristics and thermal conductivities of the MH sheets were experimentally investigated. The effects of changing the carbon fiber ratio and the measurement direction on the effective thermal conductivity were measured by the steady heat flow method. The thermal conductivity increased to 3.20 W/m·K with increasing carbon fiber ratio only in the planar direction. The decreases in mass due to removing MH powder and/or carbon fiber from sheet were less than 1 mass% after around 100 hydrogen absorption/desorption cycles. Moreover, the MH sheet was effective at decreasing the stress on the reactor vessel due to the expansion of MH during hydrogen absorption/desorption.

Gloeobacter violaceus PCC 7421 is considered, by molecular phylogenetic analyses, to be an early-branching cyanobacterium within the cyanobacterial clade. G. violaceus is the only known oxygenic photosynthetic organism that lacks thylakoid membranes. There is only one report on the development of a transformation system for G. violaceus [H. Guo, X. Xu, Prog. Nat. Sci. 14 (2004) 31–35] and further studies using the system have not been reported. In the present study, we succeeded in introducing an expression vector (pKUT1121) derived from a broad-host-range plasmid, RSF1010, into G. violaceus by conjugation. The frequency of transformation of our system is significantly higher than that described in the previous report. In addition, luciferase heterologously expressed in G. violaceus functioned as a reporter. The established system will promote the molecular genetic studies on G. violaceus., Highlights ▸ We developed a transformation system for Gloeobacter violaceus PCC 7421. ▸ We succeeded in producing G. violaceus harboring a broad-host-range plasmid. ▸ Luciferase was introduced into G. violaceus as a reporter gene. ▸ In the resultant transformant, luciferase was functionally expressed.

Among all photosynthetic and non-photosynthetic prokaryotes, only cyanobacterial species belonging to the genera Acaryochloris and Prochlorococcus have been reported to synthesize α-carotene. We reviewed the carotenoids, including their chirality, in unusual cyanobacteria containing diverse Chls. Predominantly Chl d-containing Acaryochloris (two strains) and divinyl-Chl a and divinyl-Chl b-containing Prochlorococcus (three strains) contained β-carotene and zeaxanthin as well as α-carotene, whereas Chl b-containing Prochlorothrix (one strain) and Prochloron (three isolates) contained only β-carotene and zeaxanthin but no α-carotene as in other cyanobacteria. Thus, the capability to synthesize α-carotene seemed to have been acquired only by Acaryochloris and Prochlorococcus. In addition, we unexpectedly found that α-carotene in both cyanobacteria had the opposite chirality at C-6': (6'S)-chirality in Acaryochloris and normal (6'R)-chirality in Prochlorococcus, as reported in some green algae and land plants. The results represent the first evidence for the natural occurrence and biosynthesis of (6'S)-α-carotene. All the zeaxanthins in these species were of the usual (3R,3'R)-chirality. Therefore, based on the identification of the carotenoids and genome sequence data, we propose a biosynthetic pathway for the carotenoids, particularly α-carotene, including the participating genes and enzymes.

Acaryochloris marina, a chlorophyll (Chl) d-dominated cyanobacterium, is a model organism for studying photosynthesis driven by far-red light using Chl d. Furthermore, studies on A. marina may provide insights into understanding how the oxygenic photosynthetic organisms adapt after the acquisition of new Chl. To solve the reaction mechanism of its unique photosynthesis, photosystem (PS) II complexes were isolated from A. marina and analyzed. However, the lack of a molecular genetic method for A. marina prevented us from conducting further studies. We recently developed a transformation system for A. marina and we introduced a chlorophyllide a oxygenase gene into A. marina. The resultant transformant accumulated [7-formyl]-Chl d, which has never been found in nature. In the current study, we isolated PS II complexes that contained [7-formyl]-Chl d. The pigment composition of the [7-formyl]-Chl d-containing PS II complexes was 1.96 ± 0.04 Chl a, 53.21 ± 1.00 Chl d, and 5.48 ± 0.33 [7-formyl]-Chl d per two pheophytin a molecules. In contrast, the composition of the control PS II complexes was 2.01 ± 0.06 Chl a and 62.96 ± 2.49 Chl d. The steady-state fluorescence and excitation spectra of the PS II complexes revealed that energy transfer occurred from [7-formyl]-Chl d to the major Chl d species; however, the electron transfer was not affected by the presence of [7-formyl]-Chl d. These findings demonstrate that artificially produced [7-formyl]-Chl d molecules that are incorporated into PS II replace part of the Chl d molecules and function as the antenna. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

A new production system for rare-earth-based hydrogen storage alloys is proposed. We applied self-ignition combustion synthesis (SICS) utilizing hydrogenation heat of metallic calcium. The required primary energy and total exergy loss (EXL) for the production of 1 kg of LaNi5 alloy with the proposed and conventional systems were evaluated. The results revealed that the production of raw materials accounted for more than 90% of the total EXL in both systems. Specifically, the use of calcium had decisive effects on the total EXL of the system for producing LaNi5 alloy. The proposed system reduced the total EXL by 14.6 MJ/kg-LaNi5 as compared with the conventional system. The SICS was remarkably exergy-saving because the heating temperature was decreased by utilizing the hydrogenation heat of calcium and the product absorbed hydrogen without an activation treatment.

In this paper, we describe the self-ignition combustion synthesis (SICS) of LaNi 5 utilizing the hydrogenation heat of metallic calcium at different hydrogen pressures, and focus on the effect of hydrogen pressure on the ignition temperature and the initial activation of hydrogenation. In the experiments, La 2 O 3 , Ni, and Ca were dry-mixed, and then heated at 0.1, 0.5, and 1.0 MPa of hydrogen pressure until ignition due to the hydrogenation of calcium. The products were recovered after natural cooling for 2 h. The results showed that the ignition temperature lowered with hydrogen pressure. The products changed from bulk to powder with hydrogen pressure. This was probably caused by volume expansion due to hydrogenation at higher pressure. The product obtained at 1.0 MPa showed the highest hydrogen storage capacity under an initial hydrogen pressure of 0.95 MPa. The results of this research can be applied as an innovative production route for LaNi 5 without the conventional melting of La and Ni.

In a previous study, we measured the redox potential of the primary electron acceptor pheophytin (Phe) a of photosystem (PS) II in the chlorophyll d –dominated cyanobacterium Acaryochloris marina and a chlorophyll a –containing cyanobacterium, Synechocystis . We obtained the midpoint redox potential ( E m ) values of −478 mV for A. marina and −536 mV for Synechocystis . In this study, we measured the redox potentials of the primary electron acceptor quinone molecule (Q A ), i.e., E m (Q A /Q A − ), of PS II and the energy difference between [P680·Phe a − ·Q A ] and [P680·Phe a ·Q A − ], i.e., Δ G PhQ . The E m (Q A /Q A − ) of A. marina was determined to be +64 mV without the Mn cluster and was estimated to be −66 to −86 mV with a Mn-depletion shift (130–150 mV), as observed with other organisms. The E m (Phe a /Phe a − ) in Synechocystis was measured to be −525 mV with the Mn cluster, which is consistent with our previous report. The Mn-depleted downshift of the potential was measured to be approximately −77 mV in Synechocystis , and this value was applied to A. marina (−478 mV); the E m (Phe a /Phe a − ) was estimated to be approximately −401 mV. These values gave rise to a Δ G PhQ of −325 mV for A. marina and −383 mV for Synechocystis . In the two cyanobacteria, the energetics in PS II were conserved, even though the potentials of Q A − and Phe a − were relatively shifted depending on the special pair, indicating a common strategy for electron transfer in oxygenic photosynthetic organisms.

Asexual seed formation, or apomixis, in the Hieracium subgenus Pilosella is controlled by two dominant independent genetic loci, LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP). We examined apomixis mutants that had lost function in one or both loci to establish their developmental roles during seed formation. In apomicts, sexual reproduction is initiated first. Somatic aposporous initial (AI) cells differentiate near meiotic cells, and the sexual pathway is terminated as AI cells undergo mitotic embryo sac formation. Seed initiation is fertilization-independent. Using a partially penetrant cytotoxic reporter to inhibit meioisis, we showed that developmental events leading to the completion of meiotic tetrad formation are required for AI cell formation. Sexual initiation may therefore stimulate activity of the LOA locus, which was found to be required for AI cell formation and subsequent suppression of the sexual pathway. AI cells undergo nuclear division to form embryo sacs, in which LOP functions gametophytically to stimulate fertilization-independent embryo and endosperm formation. Loss of function in either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in total reversion to sexual reproduction. Therefore, in these apomicts, sexual reproduction is the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode genes essential for sexual reproduction, but may function to recruit the sexual machinery at specific time points to enable apomixis.

Plant male reproductive development is highly organized and sensitive to various environmental stressors, including high temperature. We have established an experimental procedure to evaluate high temperature injury in japonica rice plants. High temperature treatment (39 degrees C/30 degrees C) starting at the microspore stage repeatedly reduced spikelet fertility in our system. Morphological observations revealed that pollen viability in plants exposed to high temperatures was lower than that in control plants. Most pollen grains in high temperature-treated plants displayed a normal round shape and stained reddish purple with Alexander's reagent; however, the pollen grains were very poorly attached and displayed limited germination on the stigma. To investigate gene regulatory mechanisms in the anther in high temperature environments, DNA microarray analysis was performed by comparing non-treated samples with samples treated with 2-4 d of high heat. Genes responsive to high temperatures were identified from clustering of microarray data. Among these, at least 13 were designated as high temperature-repressed genes in the anther. Expression analyses revealed that these genes were expressed specifically in the immature anther mainly in the tapetum at the microspore stage and down-regulated after 1 d of high temperature. The expression levels of Osc6, OsRAFTIN and TDR, which are tapetum-specific genes, were unaffected by high temperatures. These results suggest that not all tapetal genes are inhibited by increased temperatures and the tapetum itself is not degraded in such an environment. However, high temperatures may disrupt some of the tapetum functions required for pollen adhesion and germination on the stigma.

Chlorophyll (Chl) a in a cyanobacterium Synechocystis sp. PCC 6803 was replaced with di-vinyl (DV)-Chl a by knock-out of the specific gene (slr1923), responsible for the reduction of a 8-vinyl group, and optical and photochemical properties of purified photosystem (PS) II complexes (DV-PS II) were investigated. We observed differences in the peak wavelengths of absorption and fluorescence spectra; however, replacement of Chl a with DV-Chl a had limited effects. On the contrary, photochemical reactions were highly sensitive to high-light treatments in the mutant. Specifically, DV-Chl a was rapidly bleached under high-light conditions, and we detected significant dissociation of complexes and degradation of D1 proteins (PsbA). By comparing the SDS-PAGE patterns observed in this study to those observed in spinach chloroplasts, this degradation is assigned to the acceptor-side photoinhibition. The delayed fluorescence in the nanosecond time region at 77 K was suppressed in DV-PS II, possibly increasing triplet formation of Chl molecules. Our findings provide insight into the evolutionary processes of cyanobacteria. The effects of pigment replacement on the optimization of reactions are discussed.

We isolated highly-purified photochemically active photosystem (PS) II reaction center (RC) complexes from the cyanobacterium Synechocystis sp. PCC 6803 using a histidine-tag introduced to the 47 kDa chlorophyll protein, and characterized their spectroscopic properties. Purification was carried out in a one-step procedure after isolation of PS II core complex. The RC complexes consist of five polypeptides, the same as in spinach. The pigment contents per two molecules of pheophytin a were 5.8 ± 0.3 chlorophyll (Chl) a and 1.8 ± 0.1 β-carotene; one cytochrome b 559 was found per 6.0 Chl a molecules. Overall absorption and fluorescence properties were very similar to those of spinach PS II RCs; our preparation retains the best properties so far isolated from cyanobacteria. However, a clear band-shift of pheophytin a and β-carotene was observed. Reasons for these differences, and RC composition, are discussed on the basis of the three-dimensional structure of complexes.

The evolutionary route from anoxygenic photosynthetic bacteria to oxygenic cyanobacteria is discontinuous in terms of photochemical/photophysical reaction systems. It is difficult to describe this transition process simply because there are no recognized intermediary organisms between the two bacterial groups. Gloeobacter violaceus PCC 7421 might be a model organism that is suitable for analysis because it still possesses primordial characteristics such as the absence of thylakoid membranes. Whole genome analysis and biochemical and biophysical surveys of G. violaceus have favored the hypothesis that it is an intermediary organism. On the other hand, species differentiation is an evolutionary process that could be driven by changes in a small number of genes, and this process might give fair information more in details by monitoring of those genes. Comparative studies of genes, including those in Acaryochloris marina MBIC 11017, have provided information relevant to species differentiation; in particular, the acquisition of a new pigment, chlorophyll d, and changes in amino acid sequences have been informative. Here, based on experimental evidence from these two species, we discuss some of the evolutionary pathways for the appearance and differentiation of cyanobacteria.

Photochemically active photosystem (PS) I complexes were purified from the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina MBIC 11017, and several of their properties were characterized. PS I complexes consist of 11 subunits, including PsaK1 and PsaK2; a new small subunit was identified and named Psa27. The new subunit might replace the function of PsaI that is absent in A. marina. The amounts of pigments per one molecule of Chl d' were 97.0 +/- 11.0 Chl d, 1.9 +/- 0.5 Chl a, 25.2 +/- 2.4 alpha-carotene, and two phylloquinone molecules. The light-induced Fourier transform infrared difference spectroscopy and light-induced difference absorption spectra reconfirmed that the primary electron donor of PS I (P740) was the Chl d dimer. In addition to P740, the difference spectrum contained an additional band at 728 nm. The redox potentials of P740 were estimated to be 439 mV by spectroelectrochemistry; this value was comparable with the potential of P700 in other cyanobacteria and higher plants. This suggests that the overall energetics of the PS I reaction were adjusted to the electron acceptor side to utilize the lower light energy gained by P740. The distribution of charge in P740 was estimated by a density functional theory calculation, and a partial localization of charge was predicted to P1 Chl (special pair Chl on PsaA). Based on differences in the protein matrix and optical properties of P740, construction of the PS I core in A. marina was discussed.

The oxygen-evolving reactions of the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421 were compared with those of Synechocystis sp. PCC 6803. Four aspects were considered: sequence conservation in three extrinsic proteins for oxygen evolution, steady-state oxygen-evolving activity, charge recombination reactions, i.e., thermoluminescence and oscillation patterns of delayed luminescence on a second time scale and delayed fluorescence on the nanosecond time scale at − 196 °C. Even though there were significant differences between the amino acid sequences of extrinsic proteins in G. violaceus and Synechocystis sp. PCC 6803, the oxygen-evolving activities were similar. The delayed luminescence oscillation patterns and glow curves of thermoluminescence were essentially identical between the two species, and the nanosecond delayed fluorescence spectral profiles and lifetimes were also very similar. These results indicate clearly that even though the oxygen-evolving reactions are carried out in the periplasm by components with altered amino acid sequences, the essential reaction processes for water oxidation are highly conserved. In contrast, we observed significant changes on the reduction side of photosystem II. Based on these data, we discuss the oxygen-evolving activity of G. violaceus .

Ruthenium catalysts were prepared in different alcohols by a chemical mixing technique, characterised by the preparation of a homogeneous solution containing catalyst components, and the uniform coagulation of the solution through hydrolysis. The technique has the potential for controlling the surface area of the catalysts and for making them porous. The ruthenium catalysts were much more effective for the partial hydrogenation of benzene to cyclohexene (maximum cyclohexene yield, 31.4%) in the absence of any poison such as alkali metal hydroxide or transitional metal sulphate in the reaction solution.

Sporophytic self-incompatibility (SSI) in the genus Ipomoea (Convolvulaceae) is controlled by a single polymorphic S locus. We have previously analyzed genomic sequences of an approximately 300 kb region spanning the S locus of the S1 haplotype and characterized the genomic structure around this locus. Here, we further define the physical size of the S locus region by mapping recombination breakpoints, based on sequence analysis of PCR fragments amplified from the genomic DNA of recombinants. From the recombination analysis, the S locus of the S1 haplotype was delimited to a 0.23 cM region of the linkage map, which corresponds to a maximum physical size of 212 kb. To analyze differences in genomic organization between S haplotypes, fosmid contigs spanning approximately 67 kb of the S10 haplotype were sequenced. Comparison with the S1 genomic sequence revealed that the S haplotype-specific divergent regions (SDRs) spanned 50.7 and 34.5 kb in the S1 and S10 haplotypes, respectively and that their flanking regions showed a high sequence similarity. In the sequenced region of the S10 haplotype, five of the 12 predicted open reading frames (ORFs) were found to be located in the divergent region and showed co-linear organization of genes between the two S haplotypes. Based on the size of the SDRs, the physical size of the S locus was estimated to fall within the range 34–50 kb in Ipomoea.

Sporophytic self-incompatibility (SSI) in Ipomoea trifida is controlled by a single, multi-allelic S locus. In previous map-based cloning studies of the SSI locus (S locus), we sequenced genomic regions covering the S loci of the S1 and S10 haplotypes. We identified a highly divergent region of about 50 kb in the S1 haplotype and suggested that the Ipomoea S locus genes are located within this genomic region. In the present study, we used the sequenced DNA fragments as probes for RNA blot analyses and found 14 genes that were expressed in tissues of S1 haplotype plants. Six of the genes were located within the S haplotype-specific divergent region (SDR), and were specifically expressed either in female or male tissues. RT-PCR analysis confirmed that three genes, named here SE1, SE2 and SEA, showed stigma-specific expression, and that one gene, named here AB2, exhibited anther-specific expression. Expression of these genes was developmentally regulated and was detected at high levels in young flower buds before anthesis. In situ hybridization also showed that AB2 was expressed in the tapetal cells of the anther. Analyses of cDNA clones derived from these stigma- or anther-specific genes revealed that they exhibited high levels of sequence polymorphism in three S haplotypes, although we also detected several transcripts produced by alternative splicing of the SE2, SEA, and AB2 gene products. The present study suggests that the highly polymorphic genes SE1, SE2 and SEA that are expressed in the stigma and AB2 that is expressed in the anther are candidates encoding pistil and pollen determinants, respectively, in the SSI system of Ipomoea.

A molecular method for detecting the epiphyte community on marine macroalgae was developed by using PCR-denaturing gradient gel electrophoresis. Selective amplification of 16S rRNA gene fragments from either cyanobacteria or algal plastids improved the detection of minor epiphytes. Two phylotypes of Acaryochloris , a chlorophyll d -containing cyanobacterium, were found not only on red macroalgae but also on green and brown macroalgae.

A reversible spectroscopic change in chlorophyll (Chl) d has been demonstrated for the first time, using reduction with sodium dithionite and oxidation with potassium ferricyanide in an aqueous solution of detergent micelles. Chl d treated with a low concentration of NaBH4 in methanol showed the same results. The chemical structure of the reduced Chl d was determined by mass spectrometry to be 3-desvinyl-3-hydroxymethyl Chl a. These findings lead to a new hypothesis for the biosynthetic pathway of Chl d, being that Chl d is synthesized by oxidation of 3-desvinyl-3-hydroxymethyl Chl a, which is first formed enzymatically from Chl a.

Fluorescence properties of the newly discovered chlorophyll (Chl) d -dominated cyanobacterium, Acaryochloris sp. strain Awaji were examined by a combination of steady state and the time-resolved fluorescence measurements. Acaryochloris sp. strain Awaji, in contrast to Acaryochloris marina MBIC11017, exhibited photosystem (PS) II fluorescence at 745 nm at room temperature and at 750 nm at −196 °C with a few Chl d components in the shorter wavelength region of the maxima. PS I fluorescence was not detected by steady state measurements but was by time-resolved spectroscopy as a transient component. The results clearly indicated diversity in the antenna systems in the genus Acaryochloris that contains Chl d as major pigments. Delayed fluorescence was observed only in the Chl a fluorescence region, consistent with A. marina . This strongly suggested that the primary electron donor for the PS II reaction center should be Chl a , which is identical to all other oxygenic photosynthetic organisms.

Acaryochloris marina is a unique photosynthetic prokaryote containing chlorophyll(Chl)-d as a major photoactive pigment (over 95%). The molecular structure of Chl-d is proposed as the 3-formyl analog of Chl-a. However, the stereochemistry of Chl-d at the 13(2)-, 17- and 18-positions has not yet been established unambiguously. In the first part of this paper, we describe the determination of their stereochemistries to be 13(2)-(R)-, 17-(S)- and 18-(S)-configurations by using 1H-1H NOE correlations in 1H-NMR and circular dichroism spectra as well as chemical modification of Chl-a to produce stereochemically defined Chl derivatives. In the second part of the paper, we report a facile synthesis of a self-aggregative Chl by modifying isolated Chl-d. Since Chl-d was characterized by its reactive 3-formyl group, the formyl group was reduced with t-BuNH2BH3 to afford the desirable Chl, 3-deformyl-3-hydroxymethyl-pyrochlorophyll-d (3(1)-OH-pyroChl-d). The synthetic 3(1)-OH-pyroChl-d molecules spontaneously self-organized to form well-ordered aggregates in a non-polar organic solvent. The self-aggregates are a good model of major light-harvesting antenna systems of green photosynthetic bacteria, chlorosomes, in terms of the following three findings. (1) Both the red-shifted electronic absorption band above 750 nm and its induced reverse S-shape CD signal around 750 nm were observed in 0.5% (v/v) THF-cyclohexane. (2) The stretching mode of the 13-carbonyl group was downshifted by about 35 cm(-1) from the wavenumber of its free carbonyl. (3) The self-aggregates were quite stable on titration of pyridine to the suspension, in comparison with those of natural chlorosomal bacteriochlorophyll-d possessing the 3-(1-hydroxyethyl) group.

An extra copy of a rice pistil chitinase gene, termed chip, which is expressed predominantly in floral organs just before anthesis, was introduced into an economically important cultivar of rice (Oryza sativa L. cv. Koshihikari) together with the hygromycin-resistance gene (hph) as a marker gene by co-transformation. Over 100 regenerated rice plants (R0) and their self-pollinated first and second generations (R1 and R2) were genotypically characterized by PCR-based genomic DNA analysis of the two transgenes to obtain stable transgenic lines, in which chip was inserted into a single locus separately from hph. We obtained homozygous chip-transgenic R1 lines free of hph that stably expressed the chip mRNA and protein in rice leaf tissue. Chitin-hydrolytic activity in the leaves of these transgenic lines was higher than in those of non-transgenic controls. The resistance of the transgenic lines to rice blast disease fungus (Magnaporthe grisea) was examined by smearing water agar mixed with overcrowded conidia (race 007.0) on mechanically injured leaves, showing tendency to blast-disease resistance. Thus, the genotype-based selection was demonstrated to be effective at obtaining genetically stable transgenic plant lines without marker genes.

application/pdf, The secondary electron acceptor of photosystem (PS) I in the cyanobacterium Gloeobacter violaceus PCC 7421 was identified as menaquinone-4 (MQ-4) by comparing high performance liquid chromatograms and absorption spectra with an authentic compound. The MQ-4 content was estimated to be two molecules per one molecule of chlorophyll (Chl) a′, a constituent of P700. Comparative genomic analyses showed that six of eight men genes, encoding phylloquinone/MQ biosynthetic enzymes, are missing from the G. violaceus genome. Since G. violaceus clearly synthesizes MQ-4, the combined results indicate that this cyanobacterium must have a novel pathway for the synthesis of 1,4-dihydroxy-2-naphthoic acid.

A 'metal-free' chlorophyll (Chl) a, pheophytin (Phe) a, functions as the primary electron acceptor in PS II. On the basis of Phe a/PS II = 2, Phe a content is postulated as an index for estimation of the stoichiometry of pigments and photosystems. We found Phe a in a Chl d-dominant cyanobacterium Acaryochloris marina, whereas Phe d was absent. The minimum Chl a:Phe a ratio was 2:2, indicating that the primary electron donor is Chl a, accessory is Chl d, and the primary electron acceptor is Phe a in PS II of A. marina. Chl d was artificially formed by the treatment of Chl a with papain in aqueous organic solvents. Further, we will raise a key question on the mechanisms of water oxidation in PS II.

During the processes of pollen recognition, pollen-tube guidance, fertilization, and early embryogenesis, a complicated gene network is highly regulated in plants. To visualize the profile of gene expression during these processes, we performed cDNA microarray analysis with RNA isolated from rice pistils at several stages. We identified 32 cDNA clones, in 21 non-redundant groups, with pistil-specific expression patterns. These genes could be clustered into five groups based on expression patterns, and included pathogenesis-related genes, transcription factors, and genes related to pollen germination, pollen tube elongation, and starch degradation. The microarray analysis identified several previously undescribed genes that are expressed in a pistil-specific manner. These results provide new clues to the molecular mechanisms that underlie plant reproduction.

Diploid Ipomoea trifida is an ancestral wild species of the cultivated hexaploid sweet potato, and displays a sporophytic self-incompatibility (SI) that is controlled by a single multiallelic S-locus. To characterize the genomic region of the S-locus using a map-based cloning method, a BAC library consisting of approximately 40,000 clones was constructed from genomic DNA of S_1-homozygote, and screened using S-linked DNA markers which were mapped in our previous study. We constructed a contig covering the S-locus region with additional screening of fosmid and λ phase libraries. RFLP analysis of recombinant plants using terminal end sequences of the BAC clones as probes indicated that the S-locus region was delimited within a map distance of 0.57 cM, spanning approximately 300 kb in physical distance. Remarkable suppression of genetic recombination was detected in the S-locus region. From sequence analysis of the 313-kp region, 43 ORFs, many repetitive sequences and 5 transposable elements were predicted. None of the ORFs, however, showed a high homology with the SI genes reported to date at the S-locus of other plant families, suggesting that a unique molecular mechanism is involved in the SI system of the Convolvulaceae family.

The complicated genetic pathway regulates the developmental programs of male reproductive organ, anther tissues. To understand these molecular mechanisms, we performed cDNA microarray analyses and in situ hybridization to monitor gene expression patterns during anther development in rice. Microarray analysis of 4,304 cDNA clones revealed that the hybridization signal of 396 cDNA clones (271 non-redundant groups) increased more than six-fold in every stage of the anthers compared with that of leaves. Cluster analysis with the expression data showed that 259 cDNA clones (156 non redundant groups) were specifically or predominantly expressed in anther tissues and were regulated by developmental stage-specific manners in the anther tissues. These co-regulated genes would be important for development of functional anther tissues. Furthermore, we selected several clones for RNA in situ hybridization analysis. From these analyses, we found several novel genes that show temporal and spatial expression patterns during anther development in addition to anther-specific genes reported so far. These results indicate that the genes identified in this experiment are controlled by different programs and are specialized in their developmental and cell types.

The primary electron donor of photosystem (PS) II in the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina was confirmed by delayed fluorescence (DF) and further proved by pigment contents of cells grown under several light intensities. The DF was found only in the Chl a region, identical to Synechocystis sp. PCC 6803, and disappeared following heat treatment. Pigment analyses indicated that at least two Chl a molecules were present per each two pheophytin a molecules, and these Chl a molecules are assigned to PD1 and PD2. These findings clearly indicate that Chl a is required for water oxidation in PS II.

Ferrochelatase catalyzes the insertion of Fe2+ into protoporphyrin IX to generate protoheme. In higher plants, there is evidence for two isoforms of this enzyme that fulfill different roles. Here, we describe the isolation of a second ferrochelatase cDNA from cucumber (CsFeC2) that was less similar to a previously isolated isoform (CsFeC1) than it was to some ferrochelatases from other higher plants. Inin vitro import experiments, the two cucumber isoforms showed characteristics similar to their respective ferrochelatase counterparts of Arabidopsis thaliana. The C-terminal region of CsFeC2 but not CsFeC1 contained a conserved motif found in light-harvesting chlorophyll proteins, and CsFeC2 belonged to a phylogenetic group of plant ferrochelatases containing this conserved motif. We demonstrate that CsFeC2 was localized predominantly in thylakoid membranes as an intrinsic protein, and forming complexes probably with the C-terminal conserved motif, but a minor portion was also detected in envelope membranes. CsFeC2 mRNA was detected in all tissues and was light-responsive in cotyledons, whereasCsFeC1 mRNA was detected in nonphotosynthetic tissues and was not light-responsive. Interestingly, tissue-, light-, and cycloheximide-dependent expressions of the two isoforms of ferrochelatase were similar to those of two glutamyl-tRNA reductase isoforms involved in the early step of tetrapyrrole biosynthesis, suggesting the existence of distinctly controlled tetrapyrrole biosynthetic pathways in photosynthetic and nonphotosynthetic tissues.

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.

Acaryochloris marina MBIC 11017 possesses chlorophyll (Chl) d as a major Chl, which enables this organism to utilize far-red light for photosynthesis. Thus, the adaptation mechanism of far-red light utilization, including Chl d biosynthesis, has received much attention, though a limited number of reports on this subject have been published. To identify genes responsible for Chl d biosynthesis and adaptation to far-red light, molecular genetic analysis of A. marina was required. We developed a transformation system for A. marina and introduced expression vectors into A. marina. In this study, the high-frequency in vivo transposon mutagenesis system recently established by us was applied to A. marina. As a result, we obtained mutants with the transposon in their genomic DNA at various positions. By screening transposon-tagged mutants, we isolated a mutant (Y1 mutant) that formed a yellow colony on agar medium. In the Y1 mutant, the transposon was inserted into the gene encoding molybdenum cofactor biosynthesis protein A (MoaA). The Y1 mutant was functionally complemented by introducing the moaA gene or increasing the ammonium ion in the medium. These results indicate that the mutation of the moaA gene reduced nitrate reductase activity, which requires molybdenum cofactor, in the Y1 mutant. This is the first successful forward genetic analysis of A. marina, which will lead to the identification of genes responsible for adaptation to far-red light.

Synechocystis sp. PCC 6803 (Synechocystis) is the first sequenced photosynthetic organism and has two advantages: natural transformation and light-activated heterotrophic growth. Such characteristics have mainly promoted reverse genetic analysis in this organism, however, to date approximately 50% of genes are still annotated as 'unknown protein' or 'hypothetical protein'. Therefore, forward genetic analysis is required for the identification of significant genes responsible for photosynthesis and other physiological phenomena among the genes of unknown function. The in vivo transposon mutagenesis system is one of the major methods for random mutagenesis. However, present in vivo transposon mutagenesis systems for cyanobacteria face problems such as relatively low frequency of transposition and repeated transposition in the host cells. In this study, we constructed vectors based on a mini-Tn5-derived vector that was designed to prevent repeated transposition. Our vectors carry a hyperactive transposase and optimized recognition sequence of transposase, which were reported to enhance frequency of transposition. Using the vector, we succeeded in highly frequent transposition (9×10(-3) per recipient cell) in Synechocystis. Transposon insertion sites of 10 randomly selected mutants indicated that the insertion sites spread throughout the genome with low sequence dependency. Furthermore, one of the 10 mutants exhibited the slow-growing phenotype, and the mutant was functionally complemented by using our expression vector. Our system also worked with another model cyanobacterium, Synechococcus elongatus PCC 7942, with high frequency. These results indicate that the developed system can be applied to the forward genetic analysis of a broad range of cyanobacteria.

Unicellular cyanobacterium Gloeobacter violaceus is an only known oxygenic photosynthetic organism that lacks thylakoid membrane. Molecular phylogenetic analyses indicate that G. violaceus is an early-branching cyanobacterium within cyanobacterial clade. Therefore, the photosynthetic system of G. violaceus is considered to be partly similar to that of the ancestral cyanobacteria that would lack thylakoid membrane. G. violaceus possesses chlorophyll (Chl) a as the only chlorophyll species like most cyanobacteria. It was proposed that the ancestral oxygenic photosynthetic organism had not only Chl a and phycobilins but also Chl b. However, no organism which contains both Chl a and Chl b and lacks thylakoid membrane has been found in nature. Therefore, we introduced the chlorophyllide a oxygenase gene responsible for Chl b biosynthesis into G. violaceus. In the resultant transformant, Chl b accumulated at approximately 11% of total Chl independent of growth phase. Photosystem I complexes isolated from the transformant contained Chl b at 9.9% of total Chl. The presence of Chl b in the photosystem I complexes did not inhibit trimer formation. Furthermore, time-resolved fluorescence spectrum demonstrated that Chl b transferred energy to Chl a in the photosystem I complexes and did not disturb the energy transfer among the Chl a molecules. These results show that G. violaceus is tolerant to artificially produced Chl b and suggest the flexibility of photosystem for Chl composition in the ancestral oxygenic photosynthetic organism.

Epitaxial InN layers were deposited on MgAl 2 O 4 (1 1 1) substrates by microwave-excited metalorganic vapor-phase epitaxy. The crystallographic orientation relationships between the InN layer and MgAl 2 O 4 (1 1 1) were InN (0 0·1)∥MgAl 2 O 4 (1 1 1) and InN [1 1·0]∥MgAl 2 O 4 [1 0 0]. The full-width at half-maximum of the X-ray rocking curve of 97 arcsec was obtained on MgAl 2 O 4 (1 1 1).

The initial growth stage of InN on both α-Al 2 O 3 (0 0·1) and MgAl 2 O 4 (1 1 1) substrates has been investigated. Both the reflection high-energy electron diffraction (RHEED) and the atomic force microscope (AFM) measurements indicated that the growth of InN on α-Al 2 O 3 (0 0·1) was three-dimensional, but was two-dimensional on MgAl 2 O 4 (1 1 1).

The mechanism responsible for the degreening of plants and the degradation of chlorophyll was unclear for many years. However, recent studies have identified the colorless intermediates and helped to construct a basic pathway for degradation. After the successive removal of phytol and Mg21 from the chlorophyll molecule by chlorophyllase and 'Mg dechelatase', pheophorbide a is cleaved and reduced to yield a colorless, open tetrapyrrole intermediate. After further modifications, this is finally transported to the vacuole. Cloning the genes for chlorophyllase isozymes and the reductase should help to elucidate the physiological roles of each enzyme at a molecular level.

The genus Ipomoea (Convolvulaceae) has a sporophytic self-incompatibility system that is under the genetic control of a single multiallelic S -locus. Self-incompatibility reactions occur on the stigma surface at an early stage after pollination, and result in the complete arrest of pollen germination such that no seed is set. In a genetic analysis of 224 plants of diploid I. trifida , collected from six native populations in Central America, we identified 49 different S -alleles that showed a linear dominance-recessive hierarchy. We also obtained a spontaneous self-compatible mutant and showed that this self-compatibility trait is due to mutation at the S -locus. To investigate the molecular basis of sporophytic self-incompatibility in Ipomoea , we analysed stigma proteins and mRNAs extracted from several different S -genotypes, using two-dimensional gel electrophoresis (2D-PAGE) and AFLP-based mRNA fingerprinting (AMF), respectively. From the 2D-PAGE analyses, S -locus-linked stigma proteins (SSPs) were identified. The amino acid sequences of these proteins have a high homology to non-metallo short-chain alcohol dehydrogenases reported in several plant species. The SSP gene was mapped at a distance of 1.2 cM from the S -locus. In the AMF analysis, we obtained a clone homologous to the Brassica SRK , which showed no genetic linkage to the S -locus of Ipomoea . This suggests that the sporophytic self-incompatibility system of Ipomoea is mediated through a different molecular mechanism to that of Brassica .

We have established an efficient Agrobacterium-mediated transformation method in some leading varieties of Japonica rice (Oryza sativa, L.), including Koshihikari. Scutellum calli were induced from mature seeds on our revised medium, KA1, with high frequencies (50 to 70%) and were used for co-culture with Agrobacterium tumefaciens EHA101 which carries binary vector harboring either β-glucuronidase (GUS) gene or synthetic green fluorescent protein (sGFP (S65T)) gene driven by CaMV 35S promoter. Scutellum calli at 3 weeks old were highly efficient for the regeneration of transformants. The transformation efficiencies ranged from 15 to 34 % in seven leading varieties of nonglutinous rice. The presence of the foreign genes in the genome was confirmed by southern blot analysis, and the expression of sGFP (S65T) gene was detected in several tissues of transformants with bright fluorescent signals under a fluorescent microscopy. The present study demonstrates the usefullness of sGFP (S65T) gene as a reporter in transformed rice plants.

The [2 + 2]cycloaddition of phenyl isocyanate to 2,3-dihydrofuran was appreciably accelerated by compression to produce a β-lactam ring; its apparent activation volume was estimated to be −28 ml mol−1. Similar β-lactams were obtained under high pressure in the reaction of phenyl isocyanate with various vinyl ethers. Although the [2 + 2]cycloaddition of alkyl isocyanates to 2,3-dihydrofuran was also accelerated under high pressure to give β-lactams with good yields; the reactions of alkyl isocyanates with ethyl vinyl ether were slow, even at high pressure.

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) catalyzes the first step in the synthesis of phenylpropanoids. An antisense or sense PAL cDNA of sweet potato under the control of a tapetum-specific promoter of rice was introduced into tobacco. A reduction in pollen fertility was observed in two out of seventeen antisense PAL transformants and in six out of nineteen sense PAL transformants. The pollen fertility of these plants ranged from 8% to 60%. The distorted pollen grains that did not germinate lacked starch and flavonols. PAL activity in anthers at the microspore stage was also reduced to some extent and the level of PAL activity was positively correlated with the number of fertile pollen grains at the flowering stage. Although it was unclear how the antisense or sense transgene affected the PAL activity in anthers, our results clearly demonstrate that the PAL activity in the anther tapetum has a significant effect on the development of microspores.