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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.

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.

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.

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.

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 .

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.

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.

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.

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.

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.

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.

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.

In oxygenic photosynthetic organisms, the properties of photosynthetic reaction systems primarily depend on the Chl species used. Acquisition of new Chl species with unique optical properties may have enabled photosynthetic organisms to adapt to various light environments. The artificial production of a new Chl species in an existing photosynthetic organism by metabolic engineering provides a model system to investigate how an organism responds to a newly acquired pigment. In the current study, we established a transformation system for a Chl d-dominated cyanobacterium, Acaryochloris marina, for the first time. The expression vector (constructed from a broad-host-range plasmid) was introduced into A. marina by conjugal gene transfer. The introduction of a gene for chlorophyllide a oxygenase, which is responsible for Chl b biosynthesis, into A. marina resulted in a transformant that synthesized a novel Chl species instead of Chl b. The content of the novel Chl in the transformant was approximately 10% of the total Chl, but the level of Chl a, another Chl in A. marina, did not change. The chemical structure of the novel Chl was determined to be [7-formyl]-Chl d(P) by mass spectrometry and nuclear magnetic resonance spectroscopy. [7-Formyl]-Chl d(P) is hypothesized to be produced by the combined action of chlorophyllide a oxygenase and enzyme(s) involved in Chl d biosynthesis. These results demonstrate the flexibility of the Chl biosynthetic pathway for the production of novel Chl species, indicating that a new organism with a novel Chl might be discovered in the future.

Summary A complement of cone visual pigments was identified in the Japanese anchovy Engraulis japonicus, one of the engraulid fish species that has a retina specialized for polarization and color vision. The nature of the chromophore bound to opsin proteins was investigated using high performance liquid chromatography (HPLC). The opsin genes were then cloned and sequenced, and the absorption spectra of different types of cones were obtained by microspectrophotometry (MSP). Two green (EJ-RH2-1, EJ-RH2-2) and one red (EJ-LWS) cone opsin genes were identified and are presumably related to the Vitamin A1-based visual pigments (i.e., rhodopsins) with λmax values of 492, 474 and 512 nm for EJ-RH2-1, EJ-RH2-2, and EJ-LWS, respectively. The long and short cones from the ventro-temporal retinal zone consisted of a pure population of RH2 class gene-based pigments (λmax value of 492 nm). The long and short cones from other retinal areas and the lateral components of the triple cones possessed a mixture of RH2 and LWS class gene-based pigments that exhibited a λmax value approximately 502 nm. The central component of the triple cones contained only RH2 class gene-based pigments (λmax value of 474 nm). Thus, E. japonicus possesses a middle-wave range of spectral sensitivity and acquires different color vision systems in distinct visual fields. .

ビリジンの存在下,トルエン溶媒中でフェニルイソシアナートを高圧下に反応させると主成分として二量体である1,3-ジフェニル-1,3-ジアゼチジン-2,4-ジオンが高収率で得られた.この反応は大きな圧力効果を示し,30℃ におけるみかけの活性化体積は-23ml/molと計算された.3-メチルピリジンおよび4-メチルピリジソはピリジンよりもさらに高い触媒活性を示したが,2-メチルピリジン,2,6-ジメチルピリジンはほとんど触媒活性を示さず,ビリジン環の窒素のまわりのかさ高さが触媒能に大きく影響した.圧力を上げたり,ピリジン触媒の量を増やすと二量体の選択性が向上したが,トリエチルアミソのような脂肪族アミン触媒を用いたり,アセトニトリルなどの極性溶媒中の反応では三量体が主成分となった.二量体の生成機構として四員環遷移状態を経由する機構を推定した.高圧下の反応により,衡またはか位に匿換基を有するフェニルイソシアナートでは好収率で二量体が得られるが,ゲクロロフェニル,1-ナフチル,及びアルキルイソシアナート類では二量化が進行しなかった.

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.

S-チオカルバモイル,S-アロイル,および8-(N-フニニルベンズイミドイル)チオヒドロキシルアミン類におけるアミノ基の反応性に注目し,安息香酸無水物によるベンゾイル化とベンズアルデヒド類によるイミン化合物の生成について検討した。これらのチオヒドロキシルアミン類はいずれも,熱および酸に対してやや不安定であるにもかかわらず,比較的に穏和な条件で対応するN-ベンゾイル誘導体および一連の薪規なイミン誘導体が,かなりよい収率で得られた。このことから,これらの化合物のアミノ基はアミン類ほどの活性はないものの,アミド類のものよりも高い活性を有しているものと考えられた。3-チオカルバモイルチオヒドロキシルアミンとアルデヒドとから誘導されるイミン化合物は銀イオンに対して特異的な親和性を示し,銀塩と1:1の付加体を生成することも見いだした。

A marine cyanobacterium, Prochlorococcus, is a unique oxygenic photosynthetic organism, which accumulates divinyl chlorophylls instead of the monovinyl chlorophylls. To investigate the molecular environment of pigments after pigment replacement but before optimization of the protein moiety in photosynthetic organisms, we compared the fluorescence properties of the divinyl Chl a-containing cyanobacteria, Prochlorococcus marinus (CCMP 1986, CCMP 2773 and CCMP 1375), by a Synechocystis sp. PCC 6803 (Synechocystis) mutant in which monovinyl Chl a was replaced with divinyl Chl a. P. marinus showed a single fluorescence band for photosystem (PS) II at 687 nm at 77 K; this was accompanied with change in pigment, because the Synechocystis mutant showed the identical shift. No fluorescence bands corresponding to the PS II 696-nm component and PS I longer-wavelength component were detected in P. marinus, although the presence of the former was suggested using time-resolved fluorescence spectra. Delayed fluorescence (DF) was detected at approximately 688 nm with a lifetime of approximately 29 ns. In striking contrast, the Synechocystis mutant showed three fluorescence bands at 687, 696, and 727 nm, but suppressed DF. These differences in fluorescence behaviors might not only reflect differences in the molecular structure of pigments but also differences in molecular environments of pigments, including pigment–pigment and/or pigment–protein interactions, in the antenna and electron transfer systems.

Endoglucanase Cel5A from Clostridium josui contains a family 17 carbohydrate-binding module (CBM) (CjCBM17) and a family 28 CBM (CjCBM28) in tandem. These two CBMs bound to non-crystalline cellulose and beta-1,3-1,4-glucan. Our results indicate that the CBMs recognized different components on the cell wall of a sweet potato root. The root was cut into longitudinal sections. We used CjCBM17 and CjCBM28 fused to two different fluorescent proteins to visualize differential recognition of the plant cell wall. When they were microscopically observed, CjCBM28-fused cyan fluorescent protein (CFP) differentially bound to the root cap, but CjCBM17-fused blue fluorescent protein (BFP) did not. CjCBM17-BFP bound to the central part or the root apical meristem. These results suggest that CjCBM17 and CjCBM28 recognize different sites of the cell wall and that the cell-wall components and the polysaccharides configuration in the cell wall differ between tissues.

トリエチルアミンの存在下,ベンゼン溶媒を用いてイソチオシアン酸メチルを800MPa,100℃ で20時間反応させると三量体である1,3,5-トリメチル-1 ,3,5-トリアジソ-2,4,6(1H,3H,5H)-トリチオンが高収率で得られた。同じ反応を常圧で行っても三量体はまったく生成せず,この反応が高圧力下で大きく促進されることが明らかになった。第三級アルキルアミンはみな高い触媒活性を示したが,フェニル基のついたN,N-ジメチルアニリンは活性を示さなかった。ピリジンは高い触媒活性を示したが,2-位にメチル基のついた2-メチルピリジン,2,6-ジメチルピリジンは活性がなく,窒素のまわりのかさ高さが触媒能に大きく影響した。イソチオシアソ酸エチルはメチルエステルと同様な条件で三量体を高収率で与えたが,より錬長の長いアルキル基をもつイソチオシアン酸プロピルおよびブチルでは,そのような条件では反応性が低く,三量体は低収率であった。しかしDBU触媒を用い,130℃ に温度を上げることによりこれらのイソチオシアソ酸エステルにおいても好収率で三量体が得られた。分子内に二重結合を持つイソチオシアン酸アリルでは,DBU触媒を用いたり高温では重合が優先したが,トリエチルアミン触媒を用い,圧力を1200MPaまで上げることにより娃収率で三量体が得られた。

Summary In angiosperms, chlorophyll biosynthesis is light dependent. A key factor in this process is protochlorophyllide oxidoreductase (POR), which requires light to catalyze the reduction of protochlorophyllide to chlorophyllide. It is believed that this protein originated from an ancient cyanobacterial enzyme that was introduced into proto-plant cells during the primary symbiosis. Here we report that PORs from the cyanobacteria Gloeobacter violaceus PCC7421 and Synechocystis sp. PCC6803 function in plastids. First, we found that the G. violaceus POR shows a higher affinity to its substrate protochlorophyllide than the Synechocystis POR but a similar affinity to plant PORs. Secondly, the reduced size of prolamellar bodies caused by a knockdown mutation of one of the POR genes, PORA, in Arabidopsis could be complemented by heterologous expression of the cyanobacterial PORs. Photoactive protochlorophyllide in the etioplasts of the complementing lines, however, was retained at a low level as in the parent PORA knockdown mutant, indicating that the observed formation of prolamellar bodies was irrelevant to the assembly of photoactive protochlorophyllide. This work reveals a new view on the formation of prolamellar bodies and provides new clues about the function of POR in the etioplast–chloroplast transition.

Acaryochloris marina is a unique cyanobacterium that is able to produce chlorophyll d as its primary photosynthetic pigment and thus efficiently use far-red light for photosynthesis. Acaryochloris species have been isolated from marine environments in association with other oxygenic phototrophs, which may have driven the niche-filling introduction of chlorophyll d . To investigate these unique adaptations, we have sequenced the complete genome of A. marina . The DNA content of A. marina is composed of 8.3 million base pairs, which is among the largest bacterial genomes sequenced thus far. This large array of genomic data is distributed into nine single-copy plasmids that code for >25% of the putative ORFs. Heavy duplication of genes related to DNA repair and recombination (primarily recA ) and transposable elements could account for genetic mobility and genome expansion. We discuss points of interest for the biosynthesis of the unusual pigments chlorophyll d and α-carotene and genes responsible for previously studied phycobilin aggregates. Our analysis also reveals that A. marina carries a unique complement of genes for these phycobiliproteins in relation to those coding for antenna proteins related to those in Prochlorococcus species. The global replacement of major photosynthetic pigments appears to have incurred only minimal specializations in reaction center proteins to accommodate these alternate pigments. These features clearly show that the genus Acaryochloris is a fitting candidate for understanding genome expansion, gene acquisition, ecological adaptation, and photosystem modification in the cyanobacteria.

The trimerization of phenyl isocyanate in the presence of triethylamine was accelerated under high pressure to give triphenyl isocyanurate almost quantitatively. The reaction in benzene was remarkably accelerated by compression. The effects of pressure, temperature, catalysts, and solvents were examined on the trimerization of phenyl isocyanate. Aryl and normal alkyl isocyanates trimerized under high pressure to give the corresponding isocyanurates in good yields, whereas isocyanates having bulky alkyl groups such as t-butyl and cyclohexyl did not trimerize even under 800 MPa.

S-Thioaroylsulfenamides (ArCSSNH2), formed by reaction of ArCSS−Na+ with hydroxylamine-O-sulfonic acid, were aroylated or acylated to give N-aroyl- or N-acyl-S-thioaroylsulfenamides, respectively, which were then cyclized with dehydration to afford 3-substituted 5-aryl-1,4,2-dithiazolium salts. The behavior of 3,5-diphenyl-1,4,2-dithiazolium perchlorate toward nucleophiles such as active methylene and amino compounds was studied systematically. The reaction can be classified into two cases depending on fission modes of initial adducts, i.e., ring opening–ring closure reaction (Path B) and fragmentation of dithiazole ring (Path C), and in some cases the initial adducts were isolated.

The composition of photosystem II (PSII) in the chlorophyll (Chl) d -dominated cyanobacterium Acaryochloris marina MBIC 11017 was investigated to enhance the general understanding of the energetics of the PSII reaction center. We first purified photochemically active complexes consisting of a 47-kDa Chl protein (CP47), CP43′ (PcbC), D1, D2, cytochrome b 559 , PsbI, and a small polypeptide. The pigment composition per two pheophytin (Phe) a molecules was 55 ± 7 Chl d , 3.0 ± 0.4 Chl a , 17 ± 3 α-carotene, and 1.4 ± 0.2 plastoquinone-9. The special pair was detected by a reversible absorption change at 713 nm (P713) together with a cation radical band at 842 nm. FTIR difference spectra of the specific bands of a 3-formyl group allowed assignment of the special pair. The combined results indicate that the special pair comprises a Chl d homodimer. The primary electron acceptor was shown by photoaccumulation to be Phe a , and its potential was shifted to a higher value than that in the Chl a /Phe a system. The overall energetics of PSII in the Chl d system are adjusted to changes in the redox potentials, with P713 as the special pair using a lower light energy at 713 nm. Taking into account the reported downward shift in the potential of the special pair of photosystem I (P740) in A. marina , our findings lend support to the idea that changes in photosynthetic pigments combine with a modification of the redox potentials of electron transfer components to give rise to an energetic adjustment of the total reaction system.

6, 7-Aryl-2-oxa-7-azabicyclo[3. 2. 0]heptane compounds were obtained from arylimines with 2, 3-dihydrofuran under high pressure. When benzylidene aniline used as imine, 6, 7-diphenyl-2-oxa-7-azabicyclo[3. 2. 0]heptane was obtained 66% yield with 87% selectivity. This reaction was promoted by pressure and the yield and the selectivity of the reaction were increased by temperature. The mechanism of the reaction was considered zwittenonic route was favored.

The excited-state dynamics of delayed fluorescence in photosystem (PS) II at 77 K were studied by time-resolved fluorescence spectroscopy and decay analysis on three samples with different antenna sizes: PS II particles and the PS II reaction center from spinach, and the PS II core complexes from Synechocystis sp. PCC 6803. Delayed fluorescence in the nanosecond time region originated from the 683-nm component in all three samples, even though a slight variation in lifetimes was detected from 15 to 25 ns. The relative amplitude of the delayed fluorescence was higher when the antenna size was smaller. Energy transfer from the 683-nm pigment responsible for delayed fluorescence to antenna pigment(s) at a lower energy level was not observed in any of the samples examined. This indicated that the excited state generated by charge recombination was not shared with antenna pigments under the low-temperature condition, and that delayed fluorescence originates directly from the PS II reaction center, either from chlorophyll a D1 or P680. Supplemental data on delayed fluorescence from spinach PS I complexes are included.

Ultrafast excitation relaxation dynamics and energy-transfer processes in the light-harvesting complex II (LHC II) of Arabidopsis thaliana were examined at physiological temperature using femtosecond time-resolved fluorescence spectroscopy. Energy transfer from lutein to Chl a proceeded with a rate constant of k(ET) = 1.8-1.9 x 10(13) s(-1) and a yield of approximately Phi(ET) = 0.70, whereas that from neoxanthin to Chl a had a rate constant of k(ET) = 6.5 x 10(11) s(-1) and a yield at the most of Phi(ET) = 0.09. Fluorescence anisotropic decay of lutein in LHC II showed a value larger than 0.4 at the initial state and decayed to approximately 0.1 in 0.3 ps, indicating that two lutein molecules interact with each other in LHC II. In solution, anisotropy of lutein remained constant (0.38) independent of time, and thus a new excited state inferred between the S(2) (1B(u)) state and the S(1) (2A(g)) state was not applicable for lutein in solution. Energy migration processes among Chl a or Chl b molecules were clearly resolved by kinetic analysis. On the basis of these results, relaxation processes and energy-transfer kinetics in LHC II of A. thaliana are discussed.

Carotenoid composition and its biosynthetic pathway in the cyanobacterium Gloeobacter violaceus PCC 7421 were investigated. β-Carotene and (2S,2′S)-oscillol 2,2′-di(α-l-fucoside), and echinenone were major and minor carotenoids, respectively. We identified two unique genes for carotenoid biosynthesis using in vivo functional complementation experiments. In Gloeobacter, a bacterial-type phytoene desaturase (CrtI), rather than plant-type desaturases (CrtP and CrtQ), produced lycopene. This is the first demonstration of an oxygenic photosynthetic organism utilizing bacterial-type phytoene desaturase. We also revealed that echinenone synthesis is catalyzed by CrtW rather than CrtO. These findings indicated that Gloeobacter retains ancestral properties of carotenoid biosynthesis.

Constitution of the photosystem I complex isolated from the cyanobacterium Gloeobacter violaceus PCC 7421 was investigated by tricine–urea–SDS–PAGE, followed by peptide mass fingerprinting or N-terminal sequencing. Eight subunits (PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaL and PsaM) were identified as predicted from the genome sequence. A novel subunit (PsaZ) was discovered, but PsaI, PsaJ, PsaK and PsaX were absent. PsaB has a C-terminal extension with 155 amino acids in addition to the conserved region and this domain is similar to the peptidoglycan-binding domain. These results suggest that PS I complexes of G. violaceus have unique structural properties.

The nucleotide sequence of the entire genome of a cyanobacterium Gloeobacter violaceus PCC 7421 was determined. The genome of G. violaceus was a single circular chromosome 4,659,019 bp long with an average GC content of 62%. No plasmid was detected. The chromosome comprises 4430 potential protein-encoding genes, one set of rRNA genes, 45 tRNA genes representing 44 tRNA species and genes for tmRNA, B subunit of RNase P, SRP RNA and 6Sa RNA. Forty-one percent of the potential protein-encoding genes showed sequence similarity to genes of known function, 37% to hypothetical genes, and the remaining 22% had no apparent similarity to reported genes. Comparison of the assigned gene components with those of other cyanobacteria has unveiled distinctive features of the G. violaceus genome. Genes for PsaI, PsaJ, PsaK, and PsaX for Photosystem I and PsbY, PsbZ and Psb27 for Photosystem II were missing, and those for PsaF, PsbO, PsbU, and PsbV were poorly conserved. cpcG for a rod core linker peptide for phycobilisomes and nblA related to the degradation of phycobilisomes were also missing. Potential signal peptides of the presumptive products of petJ and petE for soluble electron transfer catalysts were less conserved than the remaining portions. These observations may be related to the fact that photosynthesis in G. violaceus takes place not in thylakoid membranes but in the cytoplasmic membrane. A large number of genes for sigma factors and transcription factors in the LuxR, LysR, PadR, TetR, and MarR families could be identified, while those for major elements for circadian clock, kaiABC were not found. These differences may reflect the phylogenetic distance between G. violaceus and other cyanobacteria.