The uptake hydrogenase gene disrupted (ΔhupL) mutant of Nostoc sp. PCC 7422 has high hydrogen production activity, and accumulates hydrogen to about 30% (v/v) under a starting gas phase of Ar + 5% CO₂. The mutant cells cultured under 10% nitrogen accumulated hydrogen 2 and 10 times of those cultured under 20% and 50%, respectively. We studied the effects of nitrogen concentration (1%, 5% and 10%) of the first (heterocyst induction period) and the second (hydrogen accumulation period) gas phase on hydrogen accumulation. After 2 days (2nd phase), nitrogenase activity of 10% (2nd) nitrogen cells were lower than those of 1% and 5% cells. Irrespective of the above first gas conditions, 1% (2nd) nitrogen cells produced hydrogen about 1.5 times of 5% (2nd) cells. With 5% (1st) nitrogen cells, hydrogen production activity of 1% and 5% (2nd) cells were 9 and 5 times that of 10% cells, respectively.

View full abstract

Effects of light on expression of one of the glycolytic enzymes, fructose-1.6-bisphosphate aldolase (fbaA) were investigated in Synechocystis sp. PCC6803. The fbaA mRNA was induced by light pulse of 5 min in the presence of glucose, while glucose addition in the dark was not effective for induction of fbaA. Therefore, both light and glucose were required for the full induction of fbaA transcription. One of the response regulators, sll1330, was demonstrated to be involved in regulation of fbaA gene expression by light pulse and glucose. The transcript of fbaA appeared only slightly after light irradiation in the presence of glucose in the disruptant of this gene, while continuous light effectively induced transcription of fbaA. These results suggest that fbaA gene expression is controlled through two signal pathways: presumably photosynthetic processes and light pulses in the presence of glucose.

View full abstract

Transcriptomic analyses of the Synechocystis sp. PCC 6803 reveal that the expression of various genes for functionally uncharacterized proteins are up-regulated under environmental stress conditions. In this study, we focused on slr1674 gene, of which expression is drastically induced under the stresses, and its paraloge, slr1638. The expression of slr1674 gene was transiently induced under salt and oxidative stresses, whilst slr1638 gene was expressed constitutively. Each disruptant of slr1674 or slr1638 genes showed longer lag phase than wild-type strain, suggesting that the mutations might affect the ability of cells to protect fromexcess light stress at the initial growth phase after the dilution of culture. To confirm this hypothesis, we examined recovery rate of each disruptants from the photoinhibition. The recovery rates in the mutants were much slower than that in wild-type cells. These results suggested that these proteins might be involved in the recovery process from photoinhibition.

View full abstract

Protochlorophyllide (Pchlide) reduction is the penultimate step of chlorophyll (Chl) biosynthesis, which is catalyzed by two structurally unrelated enzymes; dark-operative Pchlide oxidoreductase (DPOR) and light-dependent Pchlide oxidoreductase (LPOR). Since LPOR is the sole Pchlide reductase in angiosperm, dark-grown seedlings become etiolated. The ternary complex Pchlide-NADPH-LPOR is highly accumulated in the etioplasts to form prolamellar body (PLB) characterized by a unique paracrystalline structure. Most oxygenic phototrophs except angiosperms produce Chl even in the dark and PLB is not formed as they have DPOR together with LPOR. In these organisms the expression level of LPOR is kept low in contrast to the high level of accumulation of angiosperm etioplasts. Here we show the impact of the overexpression of LPOR in the cyanobacterium Leptolyngbya boryana. We will present the results of phenotypic analysis including electron micrographic observation of a transformant overexpressing LPOR.

View full abstract

Diatoms contribute around 40% of the photosynthetic carbon fixation in the oceans, making them one of the most important photosynthetic groups on this planet. Recently, isolation of photosynthetic protein complexes from diatoms that are relatively easy to grow under laboratory conditions had been succeeded. However, one of the most cosmopolitan diatoms, Thalassiosira pseudonana, whose whole genome has been sequenced, has not been caracterized. Therefore, we initiated isolation, purification, and characterization of their photosynthetic complexes. In this study, T. pseudonana was grown on modified f/2 artificial sea water media to, while natural sea water has typically been used. The cultures were harvested and were then ruptured to isolate thylakoid membranes. The thylakoids were n-dodecyl-β-D-maltoside solubilized and then subjected to sucrose density gradient ultracentrifugation to separate the photosynthetic complexes (Fucoxanthin-chlorophyll proteins, Photosystem I, and Photosystem II). Here we report both pigments analyses by HPLC and protein identification using Mass spectrometry.

View full abstract

In summer season, huge amount of fresh water is supplied from Mackenzie River in the south-eastern area in Beaufort Sea, Canada. This seasonal event drastically fluctuates the oceanic condition for the growth of microalgae such as irradiance, salinity, temperature and nutrient. In this study, we investigated the photosynthetic properties of phytoplankton collected from surface water (less than 5 m) during summer season (from 13 May to 04 Aug, 2004) in Beaufort Sea by using a pulse amplitude modulation fluorometer (Phyto-PAM).
The properties of photosynthesis in phytoplankton assessed in this work were influenced by irradiance rather than salinity, nutrients, or temperature. The values of the maximum yield of photosystem II (Fv/Fm), the maximal electron transport rate (rETRmax) and the maximum of NPQ (NPQmax) decreased according to the increase of irradiance. In summer season, the photosynthetic activity in phytoplankton was suppressed by irradiance in this area.

View full abstract

Plants adjust the light harvesting capacity of two photosystems (PSI and PSII) by redistributing light-harvesting complex II (LHCII). Recently, we revealed the phosphorylation of major LHCIIs, minor LHCIIs, and PSII core proteins triggered the detachment of all LHCIIs. Also, using the RNAi mutant, we showed that the lack of minor LHCII (CP29) caused a severe effect in state transitions. To investigate the importance of major LHCIIs during state transitions, we studied Chlamydomonas reinhardtii mutant, npq5. After fusing a His-tag to CP47, we isolated PSII-LHCII supercomplexes from npq5 by nickel affinity chromatography. Gel filtration showed that three different PSII complexes were existed, as in wild type. However, no significant difference in their abundance during state transitions was observed, indicating that LHCIIs were not detached from PSII. Thus this result suggests that LHCII type IV is involved in the LHCII detachment. We will report the detailed mechanism more related to major LHCIIs.

View full abstract

Photosystem II (PSII) is known to suffer from oxidative damage even under moderate light intensity and be actively repaired to sustain its activity. This mechanism is called PSII repair cycle, and factors involved in the cycle and how it proceeds are studied by genetic and biochemical approaches. However, it is not clear how PSII extrinsic subunits in thylakoid lumen called oxygen evolving proteins (OEC proteins) are involved in this process. In previous report, we showed that suppression of PsbP, the OEC protein specifically found in land plants and some green algae, leads to the decrease of accumulation of PSII-LHCII super-complex in tobacco leaves. In this study, we further analyzed super-complex organization on thylakoid membranes by Blue-Native PAGE and sucrose density gradient centrifugation, and examined the effect of PsbP knock-down to clarify the function of PsbP in PSII repair cycle.

View full abstract

PsbQ, an extrinsic subunit of photosystem II, is a nuclear-encoded protein that optimaizes the water-splitting reaction. In addition to PsbQ, Arabidopsis has three PsbQ paralogs, the PsbQ-Like (PQL) proteins. Phylogenetic analysis suggested that PQL proteins specifically present in higher plants.
To estimate the function of PQL proteins, we investigated the expression profiles of Arabidpsis PQL genes using the microarray data from AtGenExpress using ATTED-II program.
Our in silico analysis showed that PQL genes were coexpressed with genes encoding subunits of the chloroplast NAD(P)H dehydrogenase(NDH) complex; NdhL, NdhN, NdhO, Ndf4, and PPL2. Consistent with co-expression profiles, the activity and protein level of the NDH complex in thylakoids were severely decreased in each of pql mutants. These results suggest that PQL proteins would be novel thylakoid lumenal proteins required for accumulation of the NDH complex in Arabidopsis.

View full abstract

To avoid photoinhibition, an efficient degradation of D1 protein in the repair cycle of photosystem II is important. Mounting evidence indicates that FtsH, an ATP-dependent metalloprotease in thylakoid membranes, plays a key role in this process. On the other hand, light-induced phosphorylation of D1 is suggested to regulate D1 degradation, provided that phosphorylated D1 may be a poor substrate of proteases. During light irradiation, phosphorylated-D1 level was assayed in mature leaves of Arabidopsis var2 (lacking FtsH2) using immuno-blot against anti-phosphothreonine antibodies. These assays showed that the phosphorylated D1 was readily accumulated in var2 compared with wild-type, suggesting the connection between D1 degradation and phosphorylation. In this study, we further attempt to in vivo assess the role of phosphorylation, mediated by a novel STN8 kinase in D1 degradation. To do this, a double mutant var2 and stn8 was generated. Photosynthetic properties of the double mutant will be presented.

View full abstract

FtsH is an ATP-dependent metalloprotease and present as a hetero-complex in thylakoid membranes. FtsH2, encoded in Arabidopsis VAR2 locus, is a major isoform. Mutants lacking FtsH2 (var2) result in a typical leaf-variegated phenotype. In this study, we assessed the importance of the catalytic center (zinc-binding domain) in FtsH2. We generated transgenic var2 and var1 var2 plants expressing a proteolytically-inactive version of FtsH2 (H488L). Expression of FtsH2 (H488L) in var2 rescued leaf variegation. On the other hand, expression of FtsH2 (H488L) in var1 var2 did not fully rescue the variegation phenotype. In mature leaves, H488L seemed to slow the rate of D1 protein degradation but did not lead to photoinhibition in vivo. Thus, the protease activity of FtsH2, though slightly affecting D1 degradation, is dispensable. Our results suggest that all the catalytic domains are not necessary when provided by at least several FtsH isomers.

View full abstract

Photosystem I (PSI) cyclic electron transport is essential for photosynthesis and photoprotection in Arabidopsis. Based on its physiological significance, function of PSI cyclic electron transport is likely to be conserved in rice. In fact, the related genes are conserved between Arabidopsis and rice.
PSI cyclic electron transport consists of two pathways: the NDH-dependent and PGR5-dependent pathways. The Arabidopsis gene CRR6 (CHLORORESPIRATORY REDUCTION 6) is essential for the NDH pathway and the crr6 mutant cannot accumulate the NDH complex. CRR6 is also conserved in rice (OsCRR6) and we obtained its knockout line. Consistent with Arabidopsis crr6, rice crr6 was deficient in the accumulation of the NDH complex. From these results, we conclude that the function of CRR6 and the NDH complex is conserved in rice. We also constructed RNAi lines of rice homologs of PGR5 (OsPGR5).

View full abstract

In response to environmental cues, plants fine-tune activity of photosynthesis. Physiological functions of CEF-PSI were proposed to be the production of additional ATP for CO₂ fixation and induction of NPQ via the formation of trans-thylakoid ΔpH. Thus the defect in CEF-PSI drastically affects the cellular metabolisms. In this study, we analyzed the effect of the defect in CEF-PSI on gene expression in response to CO₂ concentration by microarray analyses in Arabidopsis wild type and pgr5 lacking the main pathway of CEF-PSI.
The transcriptome in both wild-type and pgr5 plants grown at elevated CO₂, i.e., 2,000 ppm CO₂, or ambient CO₂ for 10 days were analyzed. The gene expression did not respond to the increase in CO₂ concentration in pgr5. This result suggests that CEF-PSI is involved in the gene regulation in the acclimation process to elevated CO₂.

View full abstract

Chloroplast ATP synthase is the well-studied thiol enzyme, which is reduced and activated by the chloroplast thioredoxin. Under the light, thioredoxin is reduced by the electrons from the photosynthetic electron transfer system via ferredoxin-thioredoxin reductase, and the reduced form thioredoxin attacks the disulfide bond on the γ subunit of the ATP synthase thus giving the active enzyme. However, not much is known on the redox state of this γ subunit in the intact chloroplasts nor the green leaves so far. There is only the report on the change of the redox state of the γ subunit, which was estimated from the fluorescence change of the chloroplasts itself.
In this study, we intended to directly determine the redox state of the γ subunit in vivo. To this end, we applied the combined methods using thiol specific modification and SDS-PAGE. The physiological significance of redox regulation of ATP synthase is discussed.

View full abstract

Chloroplastic 2-oxoglutarate/malate transporter (OMT) functions in mediating between carbon and nitrogen metabolic pathways. Additionally, we estimated that OMT also functions as an oxaloacetate transporter. Now we examined the uptake activity of oxaloacetic acid (OAA) in chloroplasts. It showed that chloroplasts of omt1-T2 had lower affinity for OAA than that of wild type. The addition of OAA rapidly lowered the high NADP-MDH activation state due to drive of malate valve. However the extent of the decrease was lower in omt1-T2 chloroplasts. These data indicate that the reduction of OAA uptake activity in omt1-T2 chloroplasts leads to deteriorate the malate valve function. Furthermore, omt1-T2 plants accumulated less Ser and Gly, and more glyoxylate than wild type. Therefore, the photorespiration rate was thought to be declined in omt1-T2 plants. The present results indicate a crucial role of OMT in avoiding photoinhibition by regulating malate valve and photorespiration.

View full abstract

The biomass production of rice grown under red light supplemented with blue light was higher than that grown red light alone, which was caused by an increase in photosynthetic rate associated with a higher leaf N content. However, it is unclear whether the increase in photosynthetic rate by increased stomatal conductance is related to the promotion of growth by supplemental blue light. We investigated the photosynthetic characteristics under short-term red, blue and mixture of red and blue light irradiation in rice and spinach.
The photosynthetic rate decreased and stomatal conductance did not change with increasing the ratio of blue light PFD in the measurement light PFD. Blue light did not promote photosynthesis more than red light. To analyze the mechanisms underlying an increase leaf N content by supplemental blue light, some measurements including nitrate reductase activity and nitrate content in xylem sap are being analyzed.

View full abstract

The leaf temperature of terrestrial plants is influenced by such factors as not only the air temperature but the radiant heat from the sun and the vaporization heat by the transpiration. The leaf temperature of crassulascean acid metabolism (CAM) plants which make very little transpiration in daytime can be high. We measured leaf temperatures of CAM plant, Kalanchoe daigremontiana, and C₃, Hybiscus cannabinus, in summer daytime. The leaf temperature of C₃ plant was almost equal to the air; however that of CAM was 14.9^oC higher than air. This results suggest that the leaf temperature of CAM plants rise to over 45^oC at summer day. For discussing the influence on photosynthesis we measured photosynthetic rate in the phytotron. At 36^oC, the induction time of photosynthesis was longer. At 40^oC, the photosynthesis remarkably declined.

View full abstract

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

View full abstract

In Synchococcus elongatus, nitrate reductase (NR) is inhibited upon addition of NH₄⁺ to the medium. The inhibition involves the PII protein, but little is known about the detail of the regulatory mechanism. Since NR was recently found to interact with the DNA-binding domain of NblR, the response regulater that responds to high light and nitrogen/sulfur deficiency, we characterized an nblR mutant in this study to determine whether or not NblR regulates NR. Since NH₄⁺ inhibits the nitrate-nitrite transpoter (NRT) as well as NR in the wild-type cells, we constructed the nblR mutant from NC2, a mutant having NH₄⁺-resistant NRT activity, and examined the effects of NH₄⁺ thereon. The mutant showed NH₄⁺-responsive complete inhibition of NO₃^- uptake, indicating that NR is tightly regulated. Thus, NblR was shown to be clearly not essential for the regulation of NR.

View full abstract

In the yellow and colorless mutant of C. kesseleri, grown with nitrate as the sole nitrogen source, the addition of glucose or the irradiation of blue light inhibited the uptakes of the nitrate and amino acids in growing cells, while these enhanced the uptake of nitrate and amino acids in starved cells. The addition of methionine sulphoximine (MSX), a potent inhibitor of glutamine synthetase, to growing cells resulted in intracellular ammonium-accumulation and inhibition of uptakes nitrate and amino acids. For the colorless mutant, blue light was shown to activate purified nitrate reductase. These results indicate that in the mutant cells of Chlorella examined, uptake of nitrogenous compounds seems to be influenced by the release of ammonia occurred in the presence of glucose or under blue light.

View full abstract

Plants acquire C in the form of CO2 from the air, and obtain N in the form of nitrate or ammonium and S in the form of sulfate from the soil. These elements are converted into materials necessary for the growth. In the past meetings, we reported co-activation of nutrient assimilation pathways in the transgenic Arabidopsis plants expressing maize Dof1, a transcriptional activator for expression of the genes encoding enzymes involved in carbon skeleton production. To investigate molecular mechanisms for co-activated assimilation pathways in the Dof1 transformants, we are currently performing identification of the direct target genes of Dof1 in the transformants and detailed analyses of mechanisms underlying enhanced N assimilation-dependent activation of S assimilation and C fixation. We will report the results of these analyses.

View full abstract

AnPixJ is a novel cyanobacteriochrome of a cyanobacterium Anabaena(Nostoc)sp. PCC 7120. Upon the irradiation of red- light and green-light, it shows a reversible photoconversion between a green-absorbing (λ_max=543 nm) and red-absorbing (λ_max=648 nm) forms. His-tagged chromophore-binding GAF domain of AnPixJ protein (AnPixJ-GAF) with a molecular weight of about 20 kDa was purified by using Ni affinity column. We introduced this water-soluble protein into SiO2 mesoporous materials and tested its photoconversion. AnPixJ introduced into a silica mesoporous material showed photoconversion activity. We studied the effects of pore diameter on the extent of the adsorption and photoconversion activity.

View full abstract

We have studied effect of high temperature stress on reproductive development in barley plants. Exposed to high temperature (30°C day/25°C night) for five days at the panicle differentiation stage, the following pollen development was completely aborted. In this study, we investigated prediction of coexpression by pearson's correlation coefficient among 22k gene probes, using 664 microarray experiments published on ArrayExpress at the European Bioinformatics Institute. We found expression of mitochondria-related genes were highly correlative to expression of DNA replication-related genes. In contrast, strong negative correlation was observed between expression of chloroplast-related genes and expression of DNA replication-related genes. Actually cell-proliferation arrest, abnormal mitochondria and mature chloroplast were observed in the exposed anther cells. Now we are attempting to investigate correlation between transcripts of the genes associated with each plant hormone.

View full abstract

High-temperature stress causes abortive male reproductive development in many plant species.
We have showed that high-temperature stress induces premature cessation of cell division on anther wall cells and pollen mother cells , premature degradation of tapetal cells and alters of particular genes expression . In the process, we noticed increased expression of polyamine oxidase (PAO) that is important enzyme to cause programed cell death on hypersensitive response in tobacco plants and notable alteration of expression on various phytohormone related genes.
To address the function of PAO and phytohormones during high-temperature male sterility, we dosed Inhibitor of PAO and phytohormones in barley plant under high-temperature condition. As a result, we found that the treatment of exogenous phytohormones conferred high-temperature tolerance on male reproductive organs in barley plants.
These results indicate the reduction of quantity and/or sensitivity to the phytohormone causes high-temperature male sterility in barley plant.

View full abstract

Apple flowering genes AFL, MdFT and MdTFL1, which are orthologues of LEAFY, FT and TFL1 from Arabidopsis respectively, has been detected the relation between the expression traits and the flowering. At the former meeting (49th; Sapporo), the MdFT gene was not induced at the flowering initiation and expressed at a meristem at anytime. The transgenic apple overexpressed with MdFT showed early flowering within several months after transformation. The expression level of MdFT was relative to the flower setting. The transgenic apples keep setting one to two flowers per month. This suggested the MdFT involved in the flower induction. The induced flowers often had increased petals and short of carpels. The MdFT transgenic shoots grafted on rootstock had same flowers. Some ornamental apple cultivar had same flowers as the transgenic apple. Six cultivars expressed much higher MdFT than normal apple. This strongly suggested the MdFT effected the flower formation of apple.

View full abstract

Although Pharbitis nil is known to flower by short-days or stresses, we found that var. Kidachi flowers without these cues. In tap water (stress condition) under long-days, Kidachi formed floral buds at around the 5-6 nodes and higher nodes above around the 9th node. Even with nutrient solution, it formed floral buds at the higher nodes. The flowering at the higher nodes was not inhibited by phenylalanine ammonia-lyase inhibitor which inhibits stress-induced flowering and by night break which inhibits photoperiodic flowering. If young Kidachi seedlings were grafted onto aged Kidachi plants, the former formed floral buds at the nodes lower than the 9th nodes. The older rootstocks were used, the scions formed floral buds at the lower nodes. These suggest that the flowering at the higher nodes in Kidachi was non-photoperiodic and non-stress-induced flowering, and induced by transmissible flowering stimulus which was generated with increasing the plant age.

View full abstract

The transition from vegetative to reproductive phase is a crucial moment in the life of higher plants in order to ensure a better reproductive success. Flowering time is controled by both external and internal factors and is precisely regulated by a complex genes network. In 1991, Koorneef divided the flowering-related genes into four pathways : photoperiodic, autonomous, GA-dependant and vernalization-dependant pathways.
My work aimed to identify new genes involved in the regulation of Arabidopsis thaliana flowering. Three late-flowering mutants have been identified and named fla1 to fla3. First results have shown that fla1 (At5g06850) belongs to the photoperiodic and fla2 (At1g61940) to the autonomous pathway. RT-PCR analysis have shown that photoperiodic genes downstream of CO and some of the autonomous pathway genes are down-regulated in fla1 and fla2. FLC expression in fla2 and FLM expression in fla1 and fla2 are up-regulated.

View full abstract

Arabidopsis thaliana PROTODERMAL FACTOR 2 (PDF2) encodes the HD-ZIP type IV transcription factor and is expressed in the L1 layer. pdf2 atml1 double mutant has a severe defect in the differentiation of epidermal cells. PDF2 and ATML1 are indicated to redundantly play a critical role in maintaining the identity of the L1 cell.
The PDF2 overexpressing plant exhibited normal shoot development. However, it showed late flowering phenotype. In the PDF2 overexpressing plant, the expression of a floral integrator gene FT was down-regulated. Further genetic analyses indicated that the late-flowering phenotype was caused by alteration of CO expression in the photoperiod pathway.
We isolated four EMS mutants that suppress the late-flowering phenotype of the PDF2 overexpressing plant. The FT expression in these mutants was recovered as in wild-type. We expect that genetic analyses of these mutants will provide us the molecular basis of novel genes that function up-stream of CO.

View full abstract

Phytochrome is one of the major photoreceptor families in plants and regulates flowering mechanism. It is known that phytochrome translocates from the cytoplasm into the nucleus in a light-dependent manner and transmits a signal. However, the detail of the phytochrome-mediated signaling mechanism in the late-vegetative phase remains unknown. The VOZ (Vascular plant One-Zinc finger) protein, first reported as a transcription factor, was isolated as a phytochrome-interacting factor by yeast-two hybrid screening, using a cDNA library derived from bolting Arabidopsis plants. Arabidopsis possesses two VOZ genes, AtVOZ1 and AtVOZ2, and voz1 voz2 double mutant showed late-flowering phenotype under long-day condition. Previous analysis revealed that VOZ functions downstream of phyB and that VOZ upregulates the FT mRNA level. Here we report the intracellular localization of VOZ under different light quality conditions, using lines expressing GFP-VOZ2 fusion protein.

View full abstract

Cyanobacteria are the simplest organisms known to have a circadian clock. A circadian clock gene cluster kaiABC was cloned from the cyanobacterium Synechococcus elongatue PCC 7942 and a transcription/translation-based autoregulatory loop of kaiBC gene expression has been proposed to drive circadian rhythms. KaiA and KaiC were proposed as positive and negative regulators of kaiBC expression, respectively. In addition, KaiA-mediated activation of kaiBC expression was KaiC dependent, suggesting that KaiC also functions in a positive feedback process in the molecular oscillatory mechanism. However, we showed that self-sustainable oscillation of KaiC phosphorylation was reconstituted in vitro. Therefore, KaiC phosphorylation cycle is assumed to be a basic timing process of the circadian clock. In this study, we investigated cyanobacterial circadian oscillator in vitro and in vivo.

View full abstract

Cyanobacterial circadian clock can be reconstituted in vitro by mixing KaiA, KaiB, KaiC and ATP. KaiC forms hexamer in the presence of ATP. KaiC has weak ATPase activity, which defines the circadian period. As ATPase activity of KaiC is temperature-independent, ATP hydrolysis should be involved in the process of temperature compensation of the period. KaiC has a duplicated structure; N-terminal CI and C-terminal CII domains have a set of Walker motifs A and B, and a pair of catalytic Glu residues. We obtained full length and CI domain proteins of KaiC containing E77Q;E78Q mutations. These mutations lowered the ATPase activities. Moreover, ATPase activity was highly temperature-dependent in CI mutant. We also examined the association state of this protein, and revealed that the hexamer gradually dissociated into monomer even in the presence of ATP. These results suggest the relationship between hexamer formation and ATP hydrolysis.

View full abstract

In a cyanobacterium, Synechococcus elongatus PCC 7942, a circadian input protein Pex accumulates in dark transiently, and then down-regulates the transcription of the clock gene kaiA. Herein, we analyzed molecular size of a protein complex of Pex in vivo. The extract of the cyanobacterial cells were fractionated. Then, Pex in the obtained fractions were examined by western blotting method. A protein band in 17 kDa was detected as Pex protein in 34 kDa complex, in addition to previously reported 13 kDa protein in 17 kDa complex. The 17 kDa protein signal was absent in pex deficient mutant cell extract, therefore we determined the 17 kDa protein was Pex. The size of 17 kDa is consistent to the one deduced from the open reading frame of pex. We also present significance of hydrophobic amino acids important for the dimer formation of Pex.

View full abstract

In plants, most shoot organs are derived from the shoot apical meristem (SAM), which is formed between cotyledon primordia during embryogenesis. The Arabidopsis genes CUP-SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3 encoding NAC transcriptional activators regulate SAM formation and morphogenesis of shoot organ boundaries. We have identified a number of candidate downstream genes whose expression is positively controlled by CUC genes during embryogenesis. To investigate their functions, we expressed each candidate under the CUC2 promoter in cuc1 cuc2 embryos, which develop fused cotyledons and fail to form a SAM. Two of them were able to rescue the phenotype, resulting in formation of partially separated cotyledons and a functional SAM. Moreover, the two genes also affected formation of sepal boundaries, another location that required CUC gene activity for proper morphogenesis. These results indicate that the two genes act downstream of CUC1 and CUC2 and promote development of SAM and shoot organ boundaries.

View full abstract

Land plants have haploid gametophyte and diploid sporophyte generations. Molecular mechanism causing the difference between two generations is not clearly known. In this meeting, we show Physcomitrella patens gene orthologous to Arabidopsis thaliana Polycomb gene CURLY LEAF (PpCLF) regulates an alteration of the program of two generations.
In ppclf deletion mutant, sporophyte-like tissues with morphology and gene expression were formed from protonemata in haploid gametophyte generation. Together with the PpCLF expression pattern analyzed by using a modified YFP gene Citrine knocked-in lines, PpCLF represses a program managing diploid generation in haploid generation.
The ppclf deletion mutant sporophyte-like tissue has never developed into a sporangium. To examine PpCLF function in the development into sporangium, exogenous PpCLF cDNA was transiently expressed in ppclf deletion background. Sporangium-like tissues with outer and partially inner morphology were formed from sporophyte-like tissues by PpCLF induction. We also discuss this finding and PpCLF function in diploid generation.

View full abstract

Cell fate change from matured cells to stem cells is a well-known feat of plants, but the molecular mechanisms are mostly unexplored. Using the moss Physcomitrella patens, we have previously reported that light signals mediated by phytochromes and cryptochromes are required for cell fate change from a leaf cell to a stem cell. To elucidate the downstream mechanism of light signaling, we analyzed the function of SBP-box genes that were suggested to be negative effectors of cryptochrome signaling. Cell fate change was suppressed in plants that were conditionally expressed the PpSBP4, but promoted in plants that were conditionally expressed the dominant repression fusion protein of PpSBP4 (PpSBP4-SRDX). Furthermore, the transcript levels of PpSBP4 were reduced during the process of cell fate change. These results suggest that PpSBP4 act as negative regulator of cell fate change from leaf cells to protonemal stem cells.

View full abstract

Stomata play important roles such as gas exchange, through which plants absorb CO₂ and release O₂ to their environment. MC79 was isolated as a responsible gene for the Arabidopsis thaliana mutant, which showed the morphologically abnormal stomata. The MC79 encodes a leucine rich repeat receptor like kinase. To examine the expression and the intracellular localization of MC79, we produced the P_MC79:MC79-GFP transgenic plant. MC79-GFP was detected in the meristemoid and guard mother cell (GMC), and localized at surrounding plasma membrane. Since the expression of MC79 disappeared in guard cells (GC), we considered morphogenesis of GC is determined by transient expression of MC79. We hypothesized that MC79 is involved in the signal transduction pathway determine the morphogenesis of GC during GMC stage.

View full abstract

It is not easy to inspect structural changes inside seeds, because seed coats intercept optical conditions, and fixation and cutting seed coats are necessary to examine at microscope level. Here we introduce X-ray computed tomography (CT) to test whether structural changes during seed germination can be traced. The present work used the Beamline BL20B2 of SPring-8. A seed of Lotus japonicus was placed on a rotating stage and the successive images with 2.3μm or 4.9μm pixel size were obtained using an X-ray beam at 10keV. Tomograms were constructed and analyzed using SP-μCT and IMOD software. Using this method, not only vascular bundles in cotyledons, but also borders between seed coats and the endosperm, outlines of the embryo, and void spaces in the seed were clearly seen. Scanning electron microscope observation supported our observation. Three-dimensional structure of Arabidopsis and onion seeds using X-rayCT were also reported.

View full abstract

Temperature is an environmental signal that defines the season of the seed germination. Inhibition of germination by high temperature in summer enables the winter annual seeds to germinate and to establish seedling growth in autumn. To understand the mechanism of germination regulation by temperature, we screened T-DNA insertion lines of Arabidopsis to select mutant seeds which don't germinate at upper limit permissive temperature (28^oC). The seeds of the mutant, nekojita1 (nkj1), showed enhanced dormancy and enhanced sensitivity to abscisic acid (ABA). Germination of nkj1 seeds at 28^oC was recovered by ABA biosynthesis inhibitor, fluridone. Germination of the seeds of known ABA hyper-sensitive mutants, era1-2 and era3-1, was also inhibited at 28^oC. These results suggest that ABA hyper-sensitivity is the main cause of germination inhibition at the permissive temperature. nkj1 was recessive and mapped to the bottom arm of chromosome 4 where no known ABA related genes have been located.

View full abstract

Suppression of seed germination by high temperature (thermoinhibition) during summer is crucial for winter annuals to establish vegetative growth in autumn. Abscisic acid (ABA) has been shown to have a decisive role in thermoinhibition of lettuce and many winter annual seeds including Arabidopsis. Germination of red-light pulse irradiated Arabidopsis seeds were almost completely inhibited at 30^oC in the dark. In contrast to continuous light condition, the seeds of ABA deficient aba2-2 showed little thermoinhibition tolerance in the dark after the red-light treatment. The seeds of knockout mutant of PIL5, a light-labile germination suppressor, were thermoinhibition sensitive under the continuous light but were thermoinhibition tolerant in the dark after the red-light pulse. Preliminary results indicate high temperature represses the decrease of PIL5 protein induced by red-light. These results suggest that PIL5 works as a main suppressor of GA action in thermoinhibition of Arabidopsis seeds in the dark after the red-light pulse.

View full abstract

ABA plays vital roles in the maintenance of seed dormancy. In Arabidopsis, fus3 mutant seeds fail to undergo dormancy. Several studies have shown that FUS3 positively regulates ABA biosynthesis. However, little is known about the molecular mechanisms underlying this process. To this end, we mutagenized the seeds of a transgenic line in which ectopic expression of FUS3 could be artificially induced by estrogen and screened for novel mutants defective in FUS3-imposed growth arrest and resistance to a GA biosynthesis inhibitor at seedling stages. Interestingly, several identified mutants showed organ-specific defects in FUS3-imposed growth arrest. To further investigate the regulation of ABA biosynthesis downstream of FUS3, map-based cloning of isolated mutants is being carried out. Based on these results, we will discuss possible mechanism underlying the FUS3 regulation of ABA biosynthesis during seed development.

View full abstract

HSI2 and HSL1 are B3 domain transcriptional repressors of Arabidopsis thaliana. Seeds of double knockouts of hsi2 and hsl1 (KK mutant) obtained by a cross express seed maturation genes about 4 days after germination, accumulate seed storage proteins and oil in swollen hypocotyls, and stop growth 7 days after germination. To understand the mechanisms by which HSI2 and HSL1 repress the seed maturation program after germination, we introduced dexamethasone (DEX)-inducible RNAi for HSL1 in hsi2 mutant (HSL1-RNAi) and DEX-inducible RNAi for HSI2 in hsl1 mutant (HSI2-RNAi). Although germination of seeds of both transformants in the presence of DEX caused swelling of hypocotyls and severe growth retardation, DEX-induced gene expression pattern of HSL1-RNAi was more similar to that observed in KK mutants. We are also examining the possibility that B3 domain of HSI2 recognize RY motif in the promoters of many seed maturation genes.

View full abstract

To investigate the flow of the nutrients iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) during rice seed germination, we performed microarray analysis to examine the expression of genes involved in metal transport. Many kinds of metal transporter genes were strongly expressed and their expression levels changed during rice seed germination. Furthermore, imaging of the distribution of elements (Fe, Mn, Zn, and Cu) was carried out using Synchrotron-based X-ray microfluorescence at the Super Photon ring-8 GeV (SPring-8) facility. The change in the distribution of each element in the seeds following germination was observed by in vivo monitoring. Iron, Mn, Zn, and Cu accumulated in the endosperm and embryos of rice seeds, and their distribution changed during rice seed germination. The change in the patterns of mineral localization during germination was different among the elements observed.

View full abstract

rid3 and rgd3 are mutants of Arabidopsis that were characterized by being highly temperature-sensitive for organogenesis in vitro while being much less sensitive for dedifferentiation and callus growth. The RGD3 and RID3 genes encode BTAF1 and WD40-repeat protein, respectively.
Here we report the effects of the rid3 and rgd3 mutations on spatial patterns of cell proliferation and expression of the CUC-STM pathway genes during shoot regeneration. After callus was induced to regenerate shoots, cell division ceased and then was reactivated locally in the surface region, resulting in the formation of pre-SAM cell mounds. The rgd3 mutation inhibited cell division reactivation, and the rid3 mutation caused excess unorganized division. These defects were related to the expression level of CUC1. RID3 was expressed outside the cell mounds while RGD3 and CUC1 were expressed at the cell mounds. Based on the results, a fundamental mechanism of SAM neoformation will be discussed.

View full abstract

ANGUSTIFOLIA (AN; 636 aa), a plant homolog of CtBP/BARS, controls the polar elongation of leaf cell and trichome-branching pattern in Arabidopsis thaliana. AN has a plant-specific long C-terminal region of about 200 aa. Gene encoding AN without this long C-terminus was not found in A. thaliana genome. To decide functions of AN homologs in moss, we determined the full-length cDNA sequences of four Physcomitrella patens (Pp)AN genes (1-1, 1-2, 2-1, 2-2). Homologs of PpAN1-1 and 1-2 have the long C-terminus, while PpAN2-1 and 2-2 do not contain. PpANs show high similarity to AN: 44.1%, 45.5%, 35.3% and 35.0% for PpAN1-1, 1-2, 2-1 and 2-2, respectively.Single KO lines with insertion of drug-resistant gene were produced for each PpAN genes by a gene-targeting technique. At present, we can not observe any changes in P. patens morphology.Production of double KO lines with loss of a pair of genes is in progress

View full abstract

In recessive bop1/bop2 double mutant of arabidopsis, leaf growth continues much longer period than in wild type. Thus, BOP1 and BOP2 genes (BOPs) appear to be required for determinate leaf development in arabidopsis. Similar indeterminacy is observed in leaves of genus Monophyllaea (Gesneriaceae). Therefore, we are examining if indeterminate leaf growth of Monophyllaea glabra could be attributable to deficiency of the BOP gene. First, we isolated two homologs (MgBOPs) and revealed that both MgBOPs could functionally complement the bop1/bop2 mutations. Then, RT-PCR analysis revealed that expression patterns of MgBOPs resembled that of the BOPs in arabidopsis. Now, we are conducting analysis to reveal the spatio-temporal expression patterns of MgBOPs. We are also examining structure of leaf blade, SAM and boundary region of those tissues of M. glabra by using some molecular markers. Based on those results, we will discuss mechanisms responsible for indeterminate leaf growth of M. glabra.

View full abstract