Typically, metabolomics data show a few but significant correlations among metabolite levels when data sampling is repeated across individual plants grown under strictly controlled conditions. Although several researches have assessed topologies in metabolomic correlation networks, it remains unclear whether highly connected metabolites in these networks have specific functions in known tissue- and/or genotype-dependent biochemical pathways. In this study, we subjected root tissues to gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) and used published information on the aerial parts of 3 Arabidopsis genotypes, Col-0 wild-type, methionine overaccumulation 1 (mto1), and transparent testa4 (tt4) to compare systematically the metabolomic correlations in samples of roots and aerial parts. We then applied graph clustering to the constructed correlation networks to detect densely connected metabolites and evaluated the clusters by KEGG pathway enrichment analysis. This study demonstrated that the graph-clustering approach identifies tissue- and/or genotype-dependent metabolomic clusters related to the biochemical pathway.

View full abstract

MS/MS is a method for highly sensitive and selective detection of metabolites. The MS/MS fragment ion pattern of the metabolite is used for structural characterization. Thus, MS/MS database and data resource will accelerate the identification of diverse phytochemicals. In this study, we introduce a web-based database for MS/MS data of phytochemicals, Riken mass spectrometry (ReSpect, http://spectra.psc.riken.jp/). ReSpect currently contains 8459 records obtained by literature research and measurement of our standard compounds. The records were implemented in compatible format of MassBank (http://www.massbank.jp/) that can accept MS/MS data with its metadata. These data resources are freely available online. The spectrum search with ReSpect can automatically assign query (m/z value and relative intensity with text format) to identical/similar records with MS/MS similarity. The similarity scores between query and records are calculated using cosine similarity algorithm with user defined threshold. As the future work, we will obtain MS/MS-based large-scale annotations of un-targeted metabolomics that aid in the elucidation of phytochemical diversity.

View full abstract

Sphingolipids are composed of a ceramide backbone that consists of a C18 long-chain base (LCB) bound to a fatty acid via an amide linkage. Glucosylceramides (GlcCers) (also referred as 'cerebrosides' or 'glucocerebrosides' in plants) are a major class of complex sphingolipids that are formed by the addition of a glucose residue to a ceramide backbone. The LCBs in plant GlcCers are C-18 chain lengths and often include double bonds in C-4 or C-8 positions. Molecular species profiles of GlcCers that differentiate between Z and E stereoisomers of 8-unsaturated LCBs are not yet clear. In the present study, glucosylceramides molecular species were characterized by liquid chromatography tandem mass spectrometry in leaves of Poaceae. Liquid chromatography tandem mass spectrometry analysis was performed using a tandem quadrupole mass spectrometer (ACQUITY TQD, Waters). Molecular species including 2-hydroxy arachidic acids paired with 4-E, 8-Z-sphingadienine and 4-E, 8-E-sphingadienine were found. In addition, 4-hydroxy-8-Z-sphingenine and 4-hydroxy-8-E-sphingenine [t18:1(8Z) and t18:1(8E)] were exclusively attached to 2-hydroxy fatty acids (2-hFAs) with 22 to 26 carbon chain lengths.

View full abstract

Rumex obtusifolius L. (perennial weed; Polygonaceae) is one of the oxalate-rich plants. Nevertheless, the mechanism of oxalate accumulation in plants is still unclear. Recent metabolome analyses of R. obtusifolius revealed that oxalate contents were highly correlated to citrate and ascorbate (Miyagi et al, Metabolomics, 2010a, b). We reported that oxalate content in leaves was affected by carbon source (such as citrate) in stems. Using CE-MS, we analyzed the carbon effects on oxalate accumulation and other metabolite levels in leaves and stems of R. obtusifolius. The experiment using ¹³CO₂ showed that ¹³C-oxalate was highly accumulated in leaves and that ¹³C mobilization from stems to "new leaves" was observed. High CO₂ (1000 ppm) experiment showed that oxalate content was increased by high CO₂ with Hoagland's solution, although oxalate levels was not changed by CO₂ alone.

View full abstract

Gentians are mainly cultivated as ornamental flowers in Japan. Because they are allogamous plants, their heterozygosity have become a major problem. Recently, explants were cultured clonally in vitro to maintain genetic purity, but the culture conditions had not been studied. We therefore investigated the effects of deficiency of potassium (K) and phosphorus (P), in the culture medium. The results showed that the explants could grow under K or P deficiency and P deficiency caused formation of new buds similar to overwintering buds. To elucidate the metabolic mechanism behind the gentian response to mineral deficiency, we performed targeted metabolome analysis using CE/MS. Multivariate analysis using metabolite profiles clearly revealed that characteristic metabolite patterns arise in response to K or P deficiency. Under P deficiency, a severe decrease in energy metabolites was observed, in contrast to K deficiency and the control conditions. Therefore, exogenous P controlled low energy metabolism of explants, which may in turn trigger new bud formation and sprouting in vitro. These findings will contribute to understand an appropriate cultural condition of gentians.

View full abstract

Due to ever increasing environmental deterioration it is likely that influx of UV-B radiation will increase further due to the depletion of stratospheric ozone. Given this fact it becomes essential that we better understand both rapid and adaptive responses of plants to UV-B imposed stress. To understand the mechanism of production of these protective pigments in response to UV-B light, we compare the metabolic responses of wild type Arabidopsis to that of Arabidopsis mutants impaired in flavonoid (TRANSPARENT TESTA [tt]4 and tt5), or sinapoyl-malate (sinapoylglucose accumulator 1 [sng1]) biosynthesis, to a short, 24h or a longer 96h exposure to UV-B imposed stress. Our data reveals that short-term responses occur only at the level of primary metabolites suggesting that these effectively prime the cell in order to facilitate the later production of UV-absorbing secondary metabolites. Transcript profiling was conducted on samples to capture affected responses in transcript levels by irradiation of UV-B. The combined results of these studies are discussed in the context of current models concerning the metabolic response of plants to the stress imposed by excessive UV-B irradiation.

View full abstract

More than 30 unknown trans-membrane proteins putatively act as transporters of metabolites across the tonoplast membrane. However, actual functions of these trans-membrane proteins have not been clarified yet. We are currently investigating functions of several unknown tonoplast trans-membrane proteins, and mechanisms with which they control cellular and vacuolar metabolisms. In this study, we report changes observed in metabolite profiles of transgenic lines of Arabidopsis plants that overexpress a gene encoding a putative vacuolar transporter protein (At3g21690). In rosette leaves of the transgenic plants, significant increase was observed in the accumulation of glucosinolates and isothiocyanates. These glucosinolates were also detected in the vacuole isolated from wild-type Arabidopsis leaves. The results suggested that the At3g21690 protein transports glucosinolate to vacuole in Arabidopsis leaves. To test this hypothesis, we are currently producing T87 cultured cell line that overexpresses both At3g21690 gene and Myb28 gene that encodes a transcription factor regulating glucosinolate biosynthesis. This work was partly supported by CREST, JST.

View full abstract

LC-Orbitrap-MS provides accurate monoisotopic masses which enable the prediction of molecular formulas of individual compounds. It is well-known that some of the oxygen atoms found in natural products are incorporated from molecular oxygen by the action of monooxygenases such as cytochrome P450s. Incorporation of the oxygen atoms into metabolites is one of the driving forces leading to the diversification of natural products. To clarify the origin of the oxygen atoms in metabolites, we conducted an incorporation experiment with molecular ¹⁸O₂ using two phototrophs (Lotus japonicus and Physcomitrella patens).
L. japonicus was grown with molecular ¹⁸O₂ for a short term (one week) or a long term (four weeks) at 22ºC under a 16/8-h (day/night) photoperiod. After the long term cultivation, enrichment of ¹⁸O in the flavonoid aglycones has been confirmed by LC-Orbitrap-MS analysis. On the other hand, no detectable ¹⁸O has been found in all metabolites from P. patens grown with molecular ¹⁸O₂ for four weeks at 22ºC under continuous light condition. We will discuss the MS/MS analysis of the ¹⁸O labeled metabolites.

View full abstract

Phototropins (phot) are blue light receptors for phototropism, stomatal opening, and chloroplast relocation in angiosperms. To reveal the fundamental mechanisms of phot-mediated blue light responses, we used Marchantia polymorpha as a model plant. We analyzed the blue light responses of M. polymorpha and the structure of a PHOT gene in M. polymorpha. Firstly, as responses to blue light in M. polymorpha, we observed phototropism of thalli and protonemata, polarotropism of protonemata, and chloroplast relocation. By screening γ-irradiated spores, we obtained mutants about phototropism of protonemata. Secondly, we isolated a gene encoding phototropin (MpPHOT) in M. polymorpha. MpPHOT is present as a single copy, and grouped into the bryophyte PHOT clade. In addition, the transient expression of MpPHOT complemented the phenotype of chloroplast avoidance response in the phot2 mutant of the fern, Adiantum capillus-veneris. This suggested that Mpphot could function at least as a photoreceptor regulating the chloroplast avoidance response.

View full abstract

Protein phosphatase 1 (PP1) consists of a catalytic subunit (PP1c) and multiple regulatory subunits that determine the catalytic activity, the subcellular localization, and the substrate specificity. Previously, we have demonstrated that PP1 mediates blue light signaling between phototropins and the plasma membrane H⁺-ATPase in guard cells. However, the molecular mechanisms by which PP1 transmits the blue light signal and the regulatory subunit acts for this response remain unclear. Here we identified a plant specific PP1 regulatory subunit PRS2, which is expressed in guard cells. Arabidopsis genome contains nine PRS2-like proteins, and two of them are bound to PP1c through RVxF motif in PRSs, a PP1c binding sequence. Arabidopsis prs2 double mutants showed reduced blue light-dependent stomatal opening, H⁺ pumping, and phosphorylation of the H⁺-ATPase, but showed normal phosphorylation of phototropins. Wild type PRS2 complemented prs2 phenotype, but RVxF motif mutation did not. Tautomycin, an inhibitor of PP1c, mimicked the prs2 phenotype. These results suggest that PRS2 function as a regulatory subunit of PP1 and regulates blue light signaling between phototropins and H⁺-ATPase.

View full abstract

Blue light perceived by phototropins (phot1 and phot2) mediates stomatal opening through the activation of the plasma membrane H⁺-ATPase via unknown signaling. A recent study has suggested the possible involvement of RPT2 (ROOT PHOTOTROPISM2), a BTB/POZ family protein functioning for phototropism, in the stomatal opening in response to blue light. However, conclusive evidence for the requirement of RPT2 in the activation process of the H⁺-ATPase and the stomatal opening has not been obtained so far. In this study, We have generated double mutants of RPT2 and phototropins, and investigated the blue light-specific stomatal responses in the mutants. We first confirmed the impaired hypocotyl phototropic response in rpt2 mutants. However, blue light-dependent H⁺ pumping from guard cell protoplasts and binding of a 14-3-3 protein to the H⁺-ATPase were not affected both in rpt2 single mutant and phot1rpt2 and phot2rpt2 double mutants. In accord with these results, the stomata of all rpt2 mutants opened in response to blue light in epidermal peels and intact leaves, suggesting that RPT2 is neither required for the activation of H⁺-ATPase nor stomatal opening.

View full abstract

Phototropins (phot1 and phot2) are blue-light receptor kinases and mediate stomatal opening via activation of the plasma membrane H⁺-ATPase in guard cells. It has been demonstrated that blue light activates the guard-cell H⁺-ATPase through phosphorylation of a penultimate Thr and subsequent binding of the 14-3-3 protein to the phosphorylated C-terminus of H⁺-ATPase. So far, detection of the guard-cell H⁺-ATPase was performed by biochemical method using guard cell protoplasts (GCPs). However, the preparation of GCPs from Arabidopsis for this purpose needs over 10 g of rosette leaves and takes over 8 hrs. In the present study, we have established immunohistochemical method for detection of the guard-cell H⁺-ATPase in Arabidopsis using specific antibodies. The results showed that the H⁺-ATPase was mainly detected in guard cells and slightly in epidermal cells when we used the epidermal tissue from rosette leaves, and that the signals were found in peripheral of the cells. We are trying to establish whole-mount detection using rosette leaves at the present time and will report these results.

View full abstract

Green light is a major constituent of sunlight and is enriched in the leaf canopy. Compared with the stomatal responses to red light or blue light, virtually nothing is known for the stomatal responses to green light. Using intact sunflower leaves, we demonstrated that green light induces stomatal opening and suggested the existence of a green light receptor (Wang et al., 2008, PCE 31: 1307; PCP in press). This time, we present our recent data of the stomatal response to green light.
Stomatal opening in response to green light seems to be a phenomenon common to several herbaceous species, including sunflower, tobacco, and Arabidopsis thaliana. When the photosynthetic rate and stomatal conductance (Gs) of these leaves attained their steady states by a saturating red light, a weak green light pulse induced a further increase in Gs. It suggests that green light may employ a photosynthesis-independent pathway to induce stomatal opening. Moreover, a cyptochrome double mutant of A. thaliana showed significantly smaller green light response than the wild type. From these, we propose that cryptochromes to be a most likely candidate for green light receptor to mediate stomatal opening.

View full abstract

Phototropin (phot), a blue light receptor for phototropism, chloroplast movement, stomatal opening, etc. has two LOV domains and Ser/Thr kinase in its N- and C-terminal regions, respectively. In the dark, LOV2 acts as an inhibitor of the kinase. Blue light initiates photoreaction including a transient covalent bond formation between a Cys and a bound FMN in the LOV, which is supposed to cancel the inhibition via structural changes in the Jαhelix in the linker region with the kinase. Based on the predicted 3D model of Arabidopsis phot1 LOV2, we presumed two intradomain signaling pathways from FMN to Jαhelix (1)FMN (isoalloxazine)-N476-K475 and (2)FMN (phosphate)-R529-E537). In order to examine these two pathways, these amino acids were mutated to Ala and spectroscopic measurement, peptide mapping and kinase assay were performed. Amino acid mutations in the (1) little affected on the spectroscopic properties and peptide mapping patterns, but abolished the light-induced kinase activation. In contrast, those in (2) showed the light activation proposing the involvement of path (1) in the intradomain signaling responsible for the light-induced activation of the phot1 kinase.

View full abstract

We are investigating the Arabidopsis mutant showing abnormal photomorphogenesis isolated from gain-of-function mutant lines, which have glucocorticoid receptor (GR)-mediated induction system. The mutant isolated from these lines showed long hypocotyl and small cotyledons phenotypes by application of dexamethasone (DEX) under light condition. These phenotypes were observed under red and/or far-red light conditions, but not under blue and dark conditions.
The corresponding gene encoded C2H2 type zinc finger transcription factor, ZAT9. Interestingly, nuclear localization of ZAT9-GFP fusion protein was observed under blue, red and far-red light conditions, but not under dark condition. These results suggest light dependent transport of ZAT9 into nucleus. We also practiced microarray analysis to investigate genes regulated by ZAT9. As a result, the expression of several genes related with phytochrome dependent signal was affected in the mutant by application of DEX. We will discuss the functions of ZAT9 in light signal transductions.

View full abstract

CRES-T (Chimeric REpressor gene-Silencing Technology) is a novel and powerful gene silencing system. In this system, a chimeric repressor, produced by fusion of a transcription factor (TF) to a plant-specific repression domain, dominantly suppresses the expression of target genes over the activity of endogenous and functionally redundant TFs. This system makes it possible to produce transgenic lines with strong phenotype which cannot be seen in the single loss-of-function mutants due to the existence of functionally redundant paralogs.
In this study, we have screened for Arabidopsis CRES-T lines which show abnormal phenotype in dim light aiming for the identification of TFs which directly or indirectly participate in the regulation of light responses, and also finding lines which show visible phenotype only in dim light. So far, 250 lines with abnormal phenotype were isolated in dim light. Of these, 80 lines were selected and those causal TFs (or transcription related factors) were identified. We are now performing further analyses of the selected lines in the dim and regular light conditions.

View full abstract

Phytochrome B (phyB) is a major plant photoreceptor and its polypeptide is folded into two domains: the N-terminal photosensory domain and the C-terminal domain which has been widely believed to be a signaling domain of phyB. However, we have recently demonstrated that the N-terminal domain, but not the C-terminal domain, of Arabidopsis phyB transduces the signal to downstream components, which forces us to reconsider our view on phyB signal transduction. In order to efficiently identify downstream components of the N-terminal domain of phyB by a forward genetics approach, we conceived and established a novel mutant screening method in which several challenging attempts were made to exaggerate the mutant phenotype to a maximum extent and thus to overcome the problem of redundancy of gene function. Here we will describe the details of the strategy and will report on the progress of the mutant screening.

View full abstract

When phytochrome B deficient mutants (phyB mutants) in rice are grown under red light, they elongate their first leave and second leaf sheaths and became pale green seedlings. We have isolated several revertant mutants (phyB suppressor, PBS) from γ-ray mutagenized phyB-3 pool and then analyzed these mutants. Under ultrastructure of red light grown seedlings, chloroplasts in wild type (Norin-8-go) have well developed thylakoid membrane but those in phyB-3 mutant have small chloroplasts with little thylakoid membrane. The revertant mutants of PBS-19 and -25 have well developed chloroplasts and the chloroplasts were filled with a lot of thylakoid membranes. The elongation which were observed in phyB mutants were inhibited in the revertants. Interestingly, bend of second leaf blade was observed even under red light condition although it was not observed in either wild type or phyB mutants.
To identify these mutations, we are taking positional cloning using progeny crossed with phyB-1, which was Nipponbare genotype.

View full abstract

Rice has three phytochromes, PHYA, PHYB and PHYC. Expression analysis of PHYA protein has been difficult because the amount of PHYA is low in light-grown plants. We improved method of protein extraction and were able to detect PHYA protein by immunoblot in green leaf in rice. We examined the expression of PHYA in plants with fully expanded fifth leaf. Amount of PHYA in leaf sheath (LS) was higher than that in leaf blade (LB). The fourth and fifth LB contained the same level of PHYA, but a higher level was detected in the sixth LB, which were enclosed by the fifth LS. In the fifth LB and LS, PHYA decreased during the light-phase and increased during the dark-phase, and the change of PHYA transcript showed correlation with the change of PHYA protein in LB. But in LS, the change of PHYA transcript showed only a weak correlation with the change of PHYA protein. It has been reported that PHYA is suppressed by PHYB in pea. We examined this phenomenon using phyB, phyC and phyBphyC mutants, and found that the expression of PHYA was suppressed mainly by PHYB and partly by PHYC. These results indicate that further studies are needed to elucidate the function of PHYA in green leaf.

View full abstract

Phytochromes are one of the photoreceptors sensing red- and far-red-light and encoded by small gene families in higher plants. In the case of rice, three genes (PHYA, PHYB, PHYC) encode phytochrome apoproteins. Each member of rice phytochromes has distinct as well as cooperative roles in light perception. In this study, we made each promoter-GUS transgenic rice plants to analyze the expression patterns of each phytochromes. PHYA showed vascular bundle specific expression patterns at almost all tissues and developmental stages while PHYB and PHYC showed lower expression and tissue specificity than PHYA. The results of extensive analysis indicate that the vascular bundle specific patterns of PHYA are affected by light circumstance. In order to discuss biological meanings of the expression patterns, we tested complementation of each phytochrome mutations with own or swapped promoter-phytochrome cDNA transgene. Photomorphogenesis of T1 seedlings indicated that PHYA promoter could regulate enough for PHYB function but PHYB promoter could not for PHYA. However, the results of flowering time complementation showed a partial effect of PHYB promoter on PHYA function.

View full abstract

Iron (Fe) is an essential nutrient for all organisms including plants. Graminaceous plants release mugineic acid family phytosiderophores (MAs), which bind and solubilize insoluble Fe in the rhizosphere. The resulting Fe(III)-MAs complexes are reabsorbed by the root cells. Maize belongs to C(4) grasses, and have high photosynthetic efficiency. However, under Fe deficient condition such as on calcareous soil, maize shows easily Fe deficient symptoms and its growth decreases dramatically. Yellow stripe 1 (ys1) and ys3 were both the mendelian recessive mutants which showed intercostal chrolosis in their leaves, typical symptoms of Fe deficiency. ys1 has a mutation in MAs-phytosiderophores transporter YS1 genes, and lacks to absorb Fe(III)-MAs complexes. ys3 has been shown to be defect in MAs release, however the causative gene was not identified. Using microarray analysis, we surveyed the genes whose expression levels were changed by Fe deficiency, and analyzed their function by comparing with rice information. In addition, we also analyzed the genes expression profile in ys1 or ys3 mutants.

View full abstract

Plants induce iron uptake/utilization system in response to iron deficiency. We have previously identified and characterized cis-acting elements and trans-acting factors involved in iron deficiency response in graminaceous plants, whereas identity of signals that trigger this response remains unknown. The rice transcription factor IDEF1 (Iron Deficiency-responsive Element-binding Factor 1), which specifically binds the IDE1 sequence, confers iron deficiency response and tolerance in rice. IDEF1 expression is constitutively observed in various organs including roots, leaves, flowers and seeds throughout rice life, and is not affected by iron nutritional status. During iron sufficiency and the early stages of iron deficiency, the majority of known iron uptake/utilization-related genes are positively regulated by IDEF1. In subsequent stages of iron deficency, however, IDEF1 partially changes the species of its downstream genes. These observations raise the possibility that IDEF1 may possess the ability to sense iron nutritional status. Identification of putative functional domains involved in this iron sensing will be reported.

View full abstract

Plants have a mechanism for solubilization and uptake of iron to root cells. It is expected that the response of iron deficiency in roots are regulated by systemic long-distance signals and intracellular local signals. However, the long-distance signal itself is unknown, and it is not clear how these two signals affect iron uptake in roots. Therefore, we carried out the expression analysis in Arabidopsis using microarray.
The iron deficiency responsive genes were categorized by the strength of the influence of the long-distance signals or the local signals. The responsiveness to the long-distance signals or the local signals was correlated with categorization based on the Gene Ontology, indicating that there was qualitative difference between the two signals. Furthermore, we screened the genes responding to iron deficiency in both shoots and roots in order to find out the producer of the long-distance signals. Unexpectedly, only eight genes were induced in both shoots and roots, which included four bHLH type transcription factors and nicotianamine synthase. Our results suggest that nicotianamine possibly play a role in the long-distance signaling for iron uptake.

View full abstract

In Arabidopsis, primary root elongation is known to be inhibited by excess iron, while promoted by iron deficiency. Since iron is generally one of the limiting factors for plant growth, it is important for plants to control root development in response to iron status. However, molecular mechanism underlying the control of root development in relation to iron is still unclear. Here we report the (i) establishment of a novel bioassay system that detects responses of root elongation in response to iron status, and the (ii) isolation of mutants that exhibit defects in such responses. We first found that primary root elongation speed of Arabidopsis seedlings was approximately 10 mm/day on 1/2 MS medium when iron and phosphorous are omitted. However, the root elongation speed was decreased when excess iron was supplied during the assay. Using this bioassay system, we obtained at least 4 mutant lines with altered responses to exogenous iron supply through the screening of 8,000 M2 seeds of fast neutrons mutants (65 Gy), and confirmed that the traits are inheritable in their progeny. These mutants might be useful genetic tools to understand the iron sensing mechanism of plants.

View full abstract

The role of rice monogalactosyldiacylglycerol synthase gene (OsMGD) was examined under salinity stress by overexpressing it in tobacco (Nicotiana tabacum L. SR1). The transgenic tobacco showed better growth and maintained higher photosystem II photon yield under salinity stress than wild type. The transgenic tobacco showed 2.0 to 4.5 folds higher MGD activity than wild type and singnificantly higher contents of monogalactosyldiacylglycerol and digalactosyldiacylglycerol than wild type. Subcellular localization confirmed the presence of OsMGD protein in inner envelope of chloroplast. Ultrastructure observation of chloroplast showed slightly more dense grana structure in transgenic plant. Interestingly, the transgenic plant showed better growth in phosphorus deficient medium.

View full abstract

Proline transporters (ProTs) originally isolated as a highly selective transporter for proline, have been also shown to transport glycinebetaine (betaine). In this study, we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus and Atriplex), and non-accumulating (Arabidopsis) plants. Results revealed that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH dependent and inhibited by a proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). These transporters exhibited a higher affinity for choline rather than betaine in the choline uptake-deficient mutant. Uptake of choline was independent of proton gradient, weakly inhibited by CCCP, and not induced by (Na⁺). In situ hybridization experiments showed that sugar beet BvBet/ProT1 is a salt-induced transporter mainly localized in phloem and xylem parenchyma cells. Physiological and functional properties of BvBet/ProT1 will be discussed.

View full abstract

The Mrp (multiple resistance and pH) systems are multi-subunit monovalent cation/proton antiporters. Here, we show that a halotorerant cyanobacterium Aphanothece halophytica has a mrp homologue gene cluster mrpCD1D2EFGAB (Ap-mrp). In proximity to mrpC, NapA type Na⁺/H⁺ antiporter (Ap-napA1-2) and bicarbonate transporter (Ap-bicA) genes were found. The Ap-mrpC like cluster was amplified and expressed in the antiporter-deficient Escherichia coli TO114. Cells of TO114 expressing the cluster exhibited resistance to Na⁺ and Li⁺. The everted membrane vesicles of TO114 cells expressing the cluster exhibited Na⁺/H⁺ and Li⁺/H⁺ exchange activities. Physiological role of mrp was analyzed using a mrpA-disrupted mutant of fresh water cyanobacterium Synechococcus elongatus PCC 7942.

View full abstract

Alkaline phosphatases (APases) are important enzymes in organophosphate utilization. A recent study showed that PhoD is more abundant in the marine bacteria than PhoA or PhoX. However, functional characterization of cyanobacterial PhoD remains to be clarified. Here, we cloned the phoD gene (ApphoD) from a halotolerant cyanobacterium Aphanothece halophytica. The deduced protein ApPhoD contains the Tat consensus motifs and peptidase cleavage site in N-terminal tail. The enzyme was activated by Ca²⁺ and exhibited the alkaline phosphatase (APase) and phosphodiesterase (APDase) activities. Subcellular localization experiments showed the secretion and processing of ApPhoD in transformed cyanobacterium. Expression of ApphoD gene in A. halophytica cells was upregulated not only by phosphorus starvation but also under salt stress conditions. These facts will be discussed.

View full abstract

Recently, it has been reported that various environmental stresses induce galactinol synthase genes (GolS) and accumulation of raffinose-related oligosaccharides leading to aquired tolerance in Arabidopsis and tomato (Yuasa et al., this meeting, 2009). Our previous studies indicated that reactive oxygen species (ROS) treatment appeared to improve drought tolerance of soybean. So, we investigated expression profiles of GmGolS in soybean under drought and phytophrmonal signals involving GmGolS. Semi-quantitative RT-PCR indicated that GmGolS and ethylene signaling genes, GmACS and GmERF, were induced in response to drought treatment. GmGolS, GmACS and GmERF were induced by hydroperoxide. Furthermore, immunoblot with anti-Ein3 specific antibody revealed that drought and hydroperoxid treatments on soybean up-regulated a protein level of Ein3 homolog, which is known to be involved in ethylene-dependent induction of ERF. These results suggested that induction of GmGolS in soybean under drought is involved in ROS and ethylene signal.

View full abstract

Divinyl chlorophyllide reductase (DVR) is one of the enzymes in the chlorophyll biosynthetic pathway. DVR reduces the vinyl group on the pyrrole ring B to the ethyl group. The DVR gene of Arabidopsis (AtDVR) was identified by the genetics approach. AtDVR homologs were not found in cyanobacteria including Synechocystis sp. PCC6803. Slr1923 was identified as the DVR gene (SyDVR) candidate of Synechocystis sp. PCC6803 by the bioinformatics approach and SyDVR disruptant accumulated divinyl chlorophyll instead of monovinyl chlorophyll. We found that both AtDVR and SyDVR recombinant proteins reduce the vinyl group to the ethyl group, but the reductant of AtDVR and SyDVR were different.
Some cyanobacteria possess AtDVR homolog instead of SyDVR homolog. We examined whether AtDVR homolog in cyanobacteria reduces the vinyl group. Marine Synechococcus WH8102 has AtDVR homolog and the recombinant protein reduced the vinyl group to the ethyl group.
These results showed the presence of two types of DVR in cyanobacteria.

View full abstract

Chlorophyll(Chl)-c is widely distributed in oxygenic photosynthetic organisms and is supposed to be a light-harvesting pigment in photosynthesis. Comparing to other chlorophyllous pigments, the pigment is quite unique in its structure. Chl-c is characterized by a fully conjugated porphyrin skeleton as well as an acrylic residue at the 17-position. However, the details of distribution of the stereoisomers at the chiral 13²-position (prime (13²S) and non-prime (13²R) types of Chls) and photosynthetic functions have not yet been clarified. We established the method for determination of the stereochemistry by chiral HPLC and CD spectra. To generalize natural selection of (13²R)- and/or (13²S)-enantiomers of Chl-c, we analyzed various classes of photosynthetic organisms containing Chl-c based on the method. The (13²R)-enantiomer could be exclusively observed for all organisms investigated here. The results strongly indicate that Chl-c functions as a light-absorbing pigment in photosynthesis together with the other pigments, Chl-a and carotenoids. This natural selection was consistent with that of Chl-b found as an accessory pigment in plant.

View full abstract

There are two structurally unrelated protochlorophyllide (Pchlide) reductases in photosynthetic organisms; one is dark-operative reductase (DPOR) and the other is light-dependent reductase (LPOR). Since angiosperms employ LPOR as the sole Pchlide reductase, seedlings in the dark accumulate Pchlide that binds to LPOR to form prolamellar body (PLB) characterized by a unique paracrystalline structure in etiolplasts. However, it is unclear if LPOR has an intrinsic property to form PLB and how LPOR have evolved to form PLB during evolution. Here we show aberrant structure formation by overexpression of a cyanobacterial LPOR in a mutant lacking DPOR of the cyanobacterium Leptolyngbya boryana. We isolated a transformant overexpressing the L. boryana LPOR. Aberrant structures apparently different from thylakoid were observed in the dark grown cells by electron microscopy. LPOR localization in the aberrant structures is supported by immunocytochemical microscopy with anti-LPOR antiserum. Further cellular and biochemical analysis suggested that the cyanobacterial LPOR forms the aberrant structures but fails to form a photoactive complex.

View full abstract

Comprehension of plant carbon kinetics is important for understanding of plant growth and development. Here we speak about an improved method regarding the positron-emitting tracer imaging system (PETIS) and carbon-11-labeled carbon dioxide (¹¹CO₂) for analyzing the carbon kinetics in soybean plants. The method enables quantitative imaging of plant carbon during the translocation of leaf photoassimilate carbon into sink organs. The modification of the method kept the total amount of carbon during imaging to enable absolutely quantitative analysis of sink-source carbon kinetics. Based on feasibility study, we will discuss how the modified method is useful for carbon kinetics in soybean plants.

View full abstract

We examined the effect of lack of Psb28 on photosynthetic machinery using a psb28 disruptant of Synechocystis sp. PCC 6803 under high temperature stress conditions. The mutant cells showed normal growth under low-light and high-light conditions at 30^oC. However, the growth of mutant cells was retarded compared to wild-type cells under high-light conditions when the growth temperature was changed to higher temperatures above 38^oC. Results obtained by experiments on photoinhibition of photosynthesis showed that photosynthetic machinery in the psb28 disruptant is more susceptible to photoinhibition at 40^oC than that in the wild-type cells. These results suggest that Psb28 is involved in the repair cycle of photosynthetic machinery at higher temperatures.

View full abstract

There are three genes encoding the D1 protein (PsbA) of photosystem II (PSII) in a thermophilic cyanobacterium T. elongatus genome. The psbA1 gene mainly expresses under normal cultivation conditions, whereas the expression of psbA3 is induced by high light. It has been shown that natures of the PsbA1- and PsbA3-PSII significantly differ, which would be due to different 21 amino acid residues among 344 ones constituting of PsbA. Especially, the oxygen-evolving activity of PsbA3-PSII is ca. 1.7 times higher than that of the other. By investigating the redox potential E_m of the primary electron acceptor pheophytin (Ph), we concluded that the difference in the activity is due to the difference that E_m(Ph) of the PsbA3-PSII is more positive by 17 mV than that of the other, which would be induced by substitution of the 130th residue. Thermoluminescence measurements on the PSIIs, however, implied that the different activity cannot be explained simply by the difference in the E_m(Ph) values. In this work, by measuring E_m of the secondary acceptor Q_A, we will discuss, based on the free energy change from Ph to Q_A, on the relationship between the energetics and activity of the PSIIs.

View full abstract

We are interested in stress responses during hydrogen production in Chlamydomonas reinhardtii at the genomic levels. At the first step, in order to understand genetic information including transcription and gene structures on the genome, we have developed genome database, Kyoto Chlamydomonas Genome Database (KCGD) using mating type-minus (mt-) strain C-9. In order to map the transcribed regions on the Chlamydomonas genome, full-length cDNAs derived from mRNA isolated from the cells that suffered to sulfate-limiting stress were fragmented and subjected to Illumina genome analyzer, in which 76-nucleotide sequences were determined. Of 46,920,630 total reads, 12,268,213 (26.1.%) were uniquely mapped to the Chlamydomonas genome. We have mapped short reads of 36 nucleotides on the genome, sulfate deficient-related genes and genes related to hydrogen production on the genome database KCGD. (*http://chlamy.pmb.lif.kyoto-u.ac.jp/) RNAseq data will also reveal the coding regions which may contribute to the hydrogen production or adaptation for S-deficient stress conditions.

View full abstract

The photosystem 1 (PS1) complex of the green alga Chlamydomonas reinhardtii contains fourteen subunits (PsaA-L, PsaN-O) and nine light-harvesting chlorophyll a/b complex (LHCI). In contrast to plant PSI complexes, Chlamydomonas PS1 complex looses some peripheral subunits during solubilization and/or purification. In order to purify PS1 complexes with more intact structure, it is necessary to develop a mild purification procedure. Here we report our recent development of a rapid and efficient purification method that uses glycerol gradient ultracentrifugation. This method separates photosystems 1 and 2 more perfectly than sucrose density gradient ultracentrifugation and need shorter time for centrifugation. The resulting preparation contained 12 photosystem 1 subunits and nine LHCI subunits but lost PsaO and PsaN. The subsequent purification by ion exchange chromatography removed PsaH as well as small contaminations, indicating the association of PsaH with photosystem 1 core complex is not stable. The compositions of lipids in the purified preparation will also be reported.

View full abstract

Sll1252 was identified as a novel protein in photosystem II (PS II) complexes from Synechocystis sp. PCC 6803. To address the function of Sll1252, the corresponding gene, sll1252, was deleted in Synechocystis 6803. The growth rate and cell morphology of the mutant were not affected in normal growth medium. The growth and oxygen-evolving activity of the mutant cells was highly suppressed compared with those of wild-type cells in the Cl^- and/or Ca²⁺-depleted medium. PS II recovery from photoinhibition was suppressed in the mutant. Despite the defects in PS II, in the light, the acceptor side of PS II was more reduced and the donor side of PS I was more oxidized compared with wild-type cells, suggesting that functional impairments also occurred in cytochrome b₆/f complexes. Furthermore, the amount of IsiA protein increased in the mutant, especially in the Cl^--depleted medium, indicating that the mutant cells perceive environmental stress to be greater than it is. Overall, the Sll1252 protein appears to be closely related to redox-sensing of the plastoquinone pool to balance the photosynthetic electron flow and the ability to cope with global environmental stresses.

View full abstract

Haptophyte algae acquired their plastid through the secondary endosymbiosis. Therefore they have many distinct characteristics compared to well-studied primary symbiotic algae and plants. We previously reported that the Haptophyte alga Emiliania huxleyi actively produces C₄ compound via anaplerotic β-carboxylation reaction during photosynthesis. We also isolated a cDNA encoding pyruvate carboxylase (EhPYC) which is known as major β-carboxylation enzyme of non-photosynthetic organisms. In the present study, we confirmed the expression of EhPYC at protein level by western blot analysis. The recombinant EhPYC produced in a bacterium exhibits activity of PYC. The deduced EhPYC is predicted to have a bipartite plastid targeting signal that functions to deliver proteins into the four-membrane plastid of Haptophyte algae. Results of immunofluorescence labeling also supported the plastid localization of EhPYC. Genomes of green algae, diatoms, and brown alga possess homologs of PYC genes, but not in any species of cyanobacteria and higher plants. The wide distribution of PYC genes among eukaryotic algae suggests that PYC play crucial roles in the algal primary carbon metabolism.

View full abstract

We have previously demonstrated that transgenic tobacco plants enhancing cytosolic fructose-1,6-bisphosphatase (FBPase) had increased lateral shoots and leaves at elevated CO₂ levels. These results suggest that alteration of the sucrose partitioning affects development of shoot branching. To clarify the effect of alteration of sugar partitioning on shoot branching, we generated and evaluated transgenic Arabidopsis expressing cyanobacterial FBPase-II in cytosol (AcF). AcF plants showed the phenotypes in growth, photosynthetic activity, and fresh weight similar to the wild-type plants at ambient CO₂ levels. In contrast, at elevated CO₂ levels (1000 ppm), the lateral shoot, leaf number and fresh weight of AcF were increased compared with the wild-type plants. Next we found that transcript levels of various genes involved in the biosynthesis of plant hormones significantly changed in the AcF plants.

View full abstract

Lowering of the CO₂ concentration in the environment induces development of a pyrenoidal starch sheath, as well as that of pyrenoid and CO₂-concentrating mechanisms, in many microalgae. In the green alga Chlamydomonas, activity of granule-bound starch synthase (GBSS) concomitantly increases under these conditions. In this study, effects of the GBSS-defective mutation (sta2) on the development of pyrenoid starch were researched in Chlamydomonas. Stroma starch- and pyrenoid starch-enriched samples were obtained from log-phase cells grown with air containing 5% CO₂ (high-CO₂ conditions) and from those transferred to low-CO₂ conditions (air level, 0.04% CO₂) for 6 h, respectively. The morphology of the pyrenoid starch was thinner and more fragile than the wild type, suggesting that GBSS does affect the morphology of pyrenoid starch. Surprisingly normal GBSS activity is shown to be required to obtain the high A-type crystallinity levels that we now report for pyrenoid starch. A model is presented explaining how GBSS-induced starch granule fusion may facilitate the formation of the pyrenoidal starch sheath. (Izumo et al., Plant Science, in press)

View full abstract

The promoter activity of chloroplastic carbonic anhydrase gene (ptca1) in the marine diatom Phaeodactylum tricornutum is repressed mediated by cAMP under high CO₂ condition. The ptca1 promoter includes CO₂/cAMP responsive elements (CCRE1-3), but the details of CO₂ signal transduction and regulatory mechanisms of ptca1 promoter is unclear. In this study, we retrieved 8 candidates which may bind to CCREs (PtbZIP1-8) according to the structure of putative DNA binding domain from P. tricornutum genome database. PtbZIP1-8 were cloned and expressed in E. coli, and solubilized PtbZIPs were purified by introduced His-Tag using Ni sepharose. Gel-shift assay was carried out using DNA probes labeled by Cy5; i.e., CCREs pentamers and the native promoter sequence including CCRE1 or CCRE2. The results clearly indicated that PtbZIP7 binds to CCREs specifically, while PtbZIP1 and PtbZIP2 showed non-specific bindings to CCREs. Expression profiles of PtbZIP7 were also analyzed using qPCR, and the localization of PtbZIP7 was observed in P. tricornutum. As a result, transcription levels of PtbZIP7 were constitutive and PtbZIP7 was localized in the nucleus, independently of CO₂ concentration.

View full abstract

The transcription of chloroplastic β-carbonic anhydrase gene (ptca1) in the marine diatom Phaeodactylum tricornutum is known to respond to environmental CO₂ concentration mediated by a second messenger cAMP. Our recent studies revealed that the promoter of the ptca1 has three tandemly-aligned cis-elements, CO₂/cAMP responsive elements (CCRE1-3), and alternative combinations of CCRE1/3 or CCRE2 are essential to repress the ptca1 in response to CO₂ increase. Our preliminary results also suggested the existence of cAMP independent pathways for CO₂ signal transduction. In this study, we screened putative CO₂-responsive genes in P. tricornutum by semi qRT-PCR, and obtained three candidate genes. We obtained for these candidate genes putative promoter regions. In order to evaluate promoter activities, those promoter regions were fused with GUS reporter gene, uidA and introduced into P. tricornutum cells. Results of GUS reporter assay demonstrated that the promoter of one of the candidate genes, ptsbt4 showed cAMP dependent CO₂ response. However deletion assays of ptsbt4 promoter indicated that regions required of CO₂ or cAMP response were independent.

View full abstract

Chloroplast interior contains thylakoid membranes, the stacked and unstacked regions of which are called the grana and stroma lamella, respectively. Photosystem I (PSI) predominantly exists in the stroma lamella, and photosystem II and its light-harvesting antenna proteins (LHCII) usually exist in the grana. Recent biochemical studies show that LHCII optimizes the rate of photochemistry by physically shuttling between the two photosystems. However, it is still unknown how LHCII migrates along thylakoid membranes due to the lack of direct observation. In this study, we directly observed the photosynthetic membrane proteins in the isolated thylakoid membranes by using atomic force microscopy. The results indicated that most part of the thylakoid membranes were crowded by membrane proteins. This will draw attention to reconsider how LHCII migrates in the crowded membranes to counterbalance the energy level of each photosystem during photoacclimation in a short period of time.

View full abstract

Penetrating light into terrestrial plant leaf is refracted and absorbed by epidermis, palisade and spongy tissue, and then its qualities and quantities change. In this study microenvironment of light in broad bean leaf was determined by the spectrophotometer with a microprobe of fiber optics. The broad bean leaf was illuminated from the upper side with a halogen lamp simulating a scattered light and the microprobe was inserted into the leaf from the lower side. Blue (λ=480nm) and red (λ=680nm) light were well absorbed at palisade tissue and light intensity at the bottom of the tissue decreased to 21% of light on the upper surface. Green light (λ=550nm) penetrating to the lower side of the leaf was then absorbed by the spongy cells and only 5.8% of the light reached to the lower side. Scattered light determined at different angles was similarly absorbed by the leaf. A laser light (λ=680nm) simulating a direct sunlight was also used as a light source. The light was refracted by epidermis and palisade tissue and then got to be scattering light in the leaf.

View full abstract

AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient roots. Since ammonium derived from urea degradation might cause toxicity, the process of urea transport should be regulated. Although the physiological function of AtDUR3 is well characterized, its biochemical property needs to be elucidated. Plasmalemma phosphoproteome analysis and prediction analysis with topology showed that AtDUR3 may have 11 potential phosphorylated amino acid residues, suggesting a post-translational regulation of AtDUR3 activity by phosphorylation.
We substituted Asp and Ala for Tyr, Ser and Thr in AtDUR3 by site directed mutagenesis to mimic phosphorylation and dephosphorylation. Comparing the growth of transformed yeast strain on the medium containing urea as sole nitrogen, some of yeast strain harboring mutated AtDUR3 grew faster than a strain harboring wild-type while some grew slower. Furthermore we screened 11 candidate proteins that may interact with AtDUR3 by mating based split-ubiquitin system. The effect of amino acid substitution and interacting protein co-expression on isotope labeled urea uptake in yeast will be discussed.

View full abstract

Nitrogen is an essential nutrient for plant cells. We are using a unicellular red alga, Cyanidioschyzon merolae, as the model system to analyze the nitrogen regulation in photosynthetic eukaryotes. In this study, we examined in detail the physiological changes of C. merolae in case of nitrogen depletion. In addition, we identified and characterized a transcription factor that is responsible for expression of nitrogen assimilation genes in C. merolae. DNA microarray and Northern blot analyses revealed that transcript of the gene encoding CmMYB1, an R2R3-type MYB transcription factor, increased 1 hour after nitrogen depletion. The CmMYB1 protein started to accumulate after 2 hours and reached a peak after 4 hours after the nitrogen depletion, correlating with the expression of key nitrogen assimilation genes. The nitrogen depletion-induced gene expression disappeared in a CmMYB1 null mutant. Chromatin immunoprecipitation analysis and electrophoretic mobility shift assays using crude cell extract demonstrated that CmMYB1 specifically occupied these nitrogen-responsive promoter regions only under nitrogen-depleted conditions.
Ref) Imamura et al. (2009) PNAS 106, 12548-1255

View full abstract

Osppc4, a rice chloroplastic PEPC, is involved in a novel pathway of organic acid synthesis, which acts as a major route of carbon skeleton supply for the nitrogen assimilation in leaves. To understand functioning mechanisms of Osppc4 in the nitrogen assimilation, effects of ammonium addition to the culture solution on expression of genes for PEPCs and enzymes involved in ammonium assimilation were examined in leaves and roots.
In leaves, expression of Osppc4 and gln2 encoding GS2 was largely induced after the ammonium addition. Expression of Osppc2a and Osppc2b for cytosolic PEPCs was also enhanced, albeit to a lesser extent, while other genes did not show enhanced expression. In roots, by contrast, almost all genes examined showed enhanced expression. Among them, Osppc4 and Osppc1 for a major cytosolic PEPC most rapidly responded to the ammonium addition. The Osppc4 protein level was also increased concomitantly with induction of gene expression. These results suggest that Osppc4 acts to regulate ammonium assimilation by rapidly responding to ammonium status around roots.

View full abstract

On the reducing side of photosystem I (PSI), electrons are distributed into several pathways; reduction of NADP+ and PQ as liner and cyclic electron transports, respectively, and transfer of reducing power to amino acid metabolic pathway. To characterize electron transfer reaction around PSI, we screened Arabidopsis mutants that show a slow or no decay kinetics of fluorescence yield from the peak Fp and isolated several mutants from an EMS-treated M2 population. Of these mutants, two lines 32-33 and 7-2-33 showed a slower decrease in fluorescence yield, and were subjected to conventional map-based cloning. Both lines were found to have a mutation causing amino acid substitution in the GUL1 gene, coding for ferredoxin-dependent glutamate synthase (Fd-GOGAT). Overexpression of Fd-GOGAT rescued the defective fluorescence induction kinetics as well as the pale-leaf phenotype of 32-33, confirming that GUL1 is responsible for these phenotypes in 32-33. Immunoblot analyses revealed that 32-33 accumulates Fd-GOGAT at 25% of WT level, with the slightly retarded electrophoretic mobility. Studies on the kinetics of the redox rate of P700 are underway in 32-33.

View full abstract

Self-incompatibility (SI) is widespread in flowering plants as a genetic system to prevent self-fertilization. We now know that SI is controlled by a single multi-allelic locus (named S-locus). In the Brassicaceae, SI is determined by the female determinant SRK (S-locus receptor kinase) expressing on stigmatic papilla cells and its ligand and the male determinant SP11 (S-locus protein 11) on the surface of pollen grains. When self-pollen attaches on the papilla cell surface, interaction between SRK and SP11 activates an SI signal to reject self-pollen. It is also known that SI can be overcome by supplying 4-5% CO₂ gas, and this method has been effectively used on the large-scale production of parental inbred lines in F₁-hybrid breeding. However, nearly nothing is known about the molecular mechanism of the SI breakdown system by the CO₂ treatment.
In this study two inbred lines of Brassica rapa with different reaction level of SI to CO₂ and their F₁ and F₂ progeny were used, looking for new insights into the SI overcome induced by the CO₂ treatment. Our results indicate that the CO₂ sensitivity is S-haplotype independent and further investigation is in progess.

View full abstract