The present investigation was performed to examine effects of changing the multimeric structure and composition of isoamylase (ISA) on starch synthesis in rice endosperm by reducing the expression of ISA1 or ISA2 gene or over-expression of ISA2 gene. Although suppression of ISA2 gene expression had no effects on the starch phenotypes, over-expression of the ISA2 gene showed a drastic decrease in the synthesis of starch with modified granules devoid of the thermal and crystalline properties and an accumulation of water-soluble highly branched malto-dextrins in the endosperm. In the over-expressed line all the ISA1 activity was accounted for by the form of ISA1-ISA2 whereas in the wild-type endosperm both the ISA1 and ISA1-ISA2 were present. The results indicate that the ISA1 is solely sufficient, but not the ISA1-ISA2, for the starch production in rice endosperm, sharply contrasting with the recent results that the ISA1-ISA2 is the only form for ISA activity playing a crucial role in the normal amylopectin biosynthesis in potato tuber and Arabidopsis leaf. Thus this study shows a functional diversity of ISA oligomeric structure between monocots and dicots.

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SSI and SSIIIa are the first and second major SS isozymes, respectively, in amylopectin biosynthesis of developing rice endosperm. We previously clarified the functions of these SS isozymes using the respective mutant lines (Fujita et al., (2006), Plant Physiol. 140: 1070-1084; Fujita etal., (2007), Plant Physiol. 144: 2009-2023). To isolate the double mutant of these major SS isozymes, the SSI mutant was crossed with the SSIIIa mutant. Two types of opaque seeds having ss1ss1/SS3ass3a and SS1ss1/ss3ass3a genotypes appeared in F₂ generation and SS activity of these lines was very low. On the other hand, we failed to detect developing endosperm that was deficient in both SSI and SSIIIa activity bands, indicating that the double recessive mutant was sterile. Two types of opaque seeds had unique amylopectin and high amylose content in the endosperm. The amount of granule-bound starch synthase I (GBSSI) protein that responsible for the biosynthesis of amylose and the activity of ADP-glucose pyrophosphorylase (AGPase) were higher than those of the wild type and their parent mutant lines.

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To reveal the function of SSIVb in starch biosynthesis of developing rice endosperm, we generated the ΔSSIIIa/ ΔSSIVb double mutant lines. The SSIVb single mutation did not affect seed morphology, grain weight, starch granule morphology, and amylopectin chain length distribution. In contrast, kernels of the double mutant lines had an opaque appearance. Their dehulled grain weight and starch content were lower (81%-86% and 71%, respectively) than those of the wild type. The endosperm starch granules of the double mutants formed entirely spherical shape and were loosely packed in the endosperm cells. The amylopectin B₂ to B₄ chains with degree of polymerization (DP) larger than 37 in the double mutant were much more reduced than these of the Δ SSIIIa . Amylose content was higher than WT and the Δ SSIIIa line. These results suggest that the function of SSIVb is overlapped with that of SSIIIa. We will also discuss the pleiotropic effects on the other enzymes involved in starch biosynthesis.

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Metabolic engineering of photosynthetic organisms is required for utilization of light energy and reducing carbon emission. We previously showed that a group 2 sigma factor SigE of Synechocystis sp. PCC 6803 globally activates transcription of sugar catabolic genes. In this study, we generated the strain overexpressing SigE and microarray analysis revealed that genes for the oxidative pentose phosphate pathway and glycogen catabolism increased in this strain. Immunoblotting revealed that protein levels of sugar catabolic enzymes such as glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glycogen phosphorylase, and isoamylase increased and the level of glycogen decreased in the SigE-overexpressing strain under light growth conditions. CE/MS analysis unraveled that metabolites of the TCA cycle and acetyl-CoA are altered by SigE overexpression. We also found that SigE-overexpressing strain exhibits defective growth under mixotrophic or dark conditions. We thus demonstrate that SigE overexpression activates sugar catabolism at transcript to phenotype levels, opening a sigma factor-based engineering for the modification of carbon metabolism in photosynthetic organisms.

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Most cyanobacteria have the single branching enzyme (BE) responsible for glycogen synthesis. In the BE mutants of Synechococcus elongatus PCC 7942, the amount of glycogen accumulated in the cells was decreased to 55% of that in the wild type, and the length of the most abundant α-1,4-chains in the polysaccharide was shifted from 8 to 4. However, the synthesis of the branched polysaccharides was still observed even in the absence of the prominent BE band in the zymogram, suggesting the presence of unidentified BE isoform(s) in cyanobacteria. Recently, it has been reported that a product of a GH57 family gene in the archaeon Thermococcus kodakaraensis exhibited BE activity. Mutants defective in the GH57 gene in S. elongatus PCC 7942 were therefore constructed and characterized. The amount of glycogen in the GH57 mutants was decreased only slightly. In contrast, when both BE and GH57 genes were disrupted, the amount of glucans accumulated in the cells was substantially decreased and the growth rates of the mutants were significantly reduced as compared to the wild type. The role of both of the gene products in the polysaccharide metabolism is discussed.

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To reveal the characteristics of three rice BE isozymes and their contribution to amylopectin biosynthesis, in vitro analysis was performed using purified recombinant BEI, BEIIa and BEIIb from rice. To our knowledge the present study shows for the first time the kinetic parameters of all the three BE isozymes towards both amylopectin and synthetic amylose as glucan substrate. Three BE isozymes had different chain-length preferences for substrate glucans. It is noted that BEIIb almost exclusively transferred chains of degree of polymerization (DP) 7 and 6 whereas BEIIa formed short chains of DP6-13 although a chain of DP6 was most effectively produced. On the other hand BEI preferentially formed chains of DP10-12. Although three BE could act on the chains of DP12 or slightly larger for amylopectin, BEI could only attack amylose of DP≥60, but BEIIs were unable to react to amylose of DP≤100. Based on these results, the functional distinction and interaction of BE isozymes during amylopectin biosynthesis in rice endosperm will be discussed.

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Root chilling increases the sugar concentration of tomato fruit. Without affecting the pattern in which starch accumulated in early expansion stage and disappeared before ripening while fructose and glucose increased with the fruit development, root chilling enhanced the accumulation level of starch. The increase in starch accumuation in early expansion stage was almost comparable to the following increase in fructose and glucose in a single fruit. However, the expression of invertase genes, LIN5 and TIV1, was decresed by root chilling. To help define this apparent conflict, we compared the related enzyme activities. The activity of soluble acidic invertase showed similar pattern as the expression of TIV1 gene. On the other hand, cell wall-bound neutral invertase activity was slightly increased by root-chilling, unlike the expression pattern of LIN5 gene. Sucrose phosphate synthase (SPS) and sucrose phospathase (SPP) was enhanced by root-chilling in early expansion stage. However sucrose synthase (SuSy) activity was not enhanced.

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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 fbaAgene expression by light pulse and glucose.
Expression of sll1330 gene were induced by glucose irrespective of light conditions, suggesting that the amount of sll1330 proteins increases in the presence of glucose and that sll1330 protein interacts with components involved in the signal transduction of light pulse.
We constructed mutants that expresses sll1330 even in the absence of glucose. The transcript of fbaA of this mutant appeared much more abundant than that of wild type. These results suggest that
fbaA is expressed by light pulse without glucose if sll1330 is expressed.

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Recycling of acyl group of glycerolipids remained obscure, because direct analysis of recycling of membrane lipids has been difficult. To analyze acyl recycling in glycerolipids, a computational methodology for the analysis of mass spectra of C-13 labeled molecules was developed. In the present study, some representative data will be presented to show the existence of acyl exchange without changing fatty acid composition of lipids. Based on the results, I will propose a new hypothesis of lipid flow within the membrane.

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Cyanobacteria are representative phototrophic organisms, so it should regulate metabolism flexibly. Although many studies for regulation of metabolism, such as photosynthesis, use these organisms, few studies have reported the amount of metabolites and its variation in these organisms. To investigate how cyanobacteria regulate metabolic reprogramming in poor nutrient conditions, we quantified amino acids pool that are center of carbon, nitrogen and sulfur metabolism. We collected cyanobacterial cells by centrifugation or filtration and froze in liquid nitrogen. The cell pellet was extracted with methanol. The extracts were dried up and dissolved in water. The dissolved extracts were applied to solid phase extraction, derivatized, and analyzed by gas chromatography mass spectrometry (GC-MS). We will report responses in amino acids pool to nitrogen or sulfur starvation.

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F₁-ATPase is the smallest mechanical motor enzyme, which works for ATP synthesis. In addition, this enzyme shows ATP hydrolysis activity when the electrochemical proton gradient is insufficient or not utilized. To maintain the efficient ATP synthesis activity, this motor protein seems to be highly regulated in vivo. The γ subunit of cyanobacterial ATP synthase contains the inserted sequence at the middle region of the molecule in contrast to the other bacterial and mitochondrial ATP synthase γ subunits. Though the deletion of this region emerged the higher ATP hydrolysis activity (Konno, et al. 2006), the reason of the observed change of the activity has not been clarified up to now. Based on the rotation analysis, we successfully clarified that the increase of the ATP hydrolysis activity by deletion of this inserted sequence was simply due to a low sensitivity to the intrinsic inhibition mechanism, ADP inhibition. In addition, we found that the unique property of this enzyme to prone to ADP inhibition, which is easily induced by the inserted sequence of γ, has the physiological significance to maintain the cellular ATP level.

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Molecular chaperone DnaK, cooperating with DnaJ and GrpE, has ATPase activity and functions in diverse cellular functions. In the cyanobacterium Synechococcus elongatus PCC 7942 (here after referred as S. 7942), three dnaK homologues (dnaK1, dnaK2, dnaK3) and four dnaJ homologues (dnaJ1, dnaJ2, dnaJ3, dnaJ4) have been identified. DnaK and DnaJ homologues in S. 7942 exhibit distinct dispensability, expression profiles, and localizations, suggesting that each DnaK and DnaJ proteins plays distinct roles in the S. 7942 cells.
In order to reveal the partnership between three DnaK and four DnaJ proteins in vitro, each recombinant DnaK, DnaJ and GrpE protein was purified and the DnaK ATPase activity was compared among the combinations with DnaJ and GrpE by malachite green assay. Among three DnaK proteins, DnaK2 showed the highest ATPase activity. In addition, the ATPase activity in each DnaK was enhanced by the addition of DnaJ and/or GrpE. In case of DnaK1 and DnaK2, the activity was most enhanced by DnaJ2, whereas the activity of DnaK3 was most enhanced by DnaJ3.

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Photosynthetic organisms, such as cyanobacteria, must be able to respond to oxidative stress. We have reported several transcriptional regulators such as an oxidant-inducible PerR-like transcriptional regulator Slr1738 and ROS-inducible transcriptional regulator SufR. In the course of microarray screening of Synechocystis sp. PCC 6803, we found that genes from sll1158 to sll1161 were strongly induced by hydrogen peroxide stress under the high light conditions. On the Synechocystis genome, a LysR-type transcriptional regulator, Slr1245, is located immediately upstream of the sll1158 gene in the divergent direction. The Δslr1245 mutant did not induce the genes from sll1158 to sll1161 upon the hydrogen peroxide stress. Gel shift assay confirmed that the Slr1245 protein bound to an upstream promoter region of sll1158. These results suggest that Slr1245 is the hydrogen peroxide-responsive transcriptional activator to induce expression of the genes from sll1158 to sll1161. Further studies are being done to know the physiological role of these genes and the hydrogen-peroxide-sensing mechanism of Slr1245.

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An alkaliphilic halophilic cyanobacterium, Arthrospira (Spirulina), is an important organism for industrial applications and as a food supply. Almost the complete genome of A. platensis NIES-39 was determined in this study. The genome structure of A. platensis is estimated to be a single, circular chromosome of 6.8 Mbp, based on optical mapping. Annotation of this 6.7 Mbp sequence yielded 6630 protein-coding genes as well as 2 sets of rRNA genes and 40 tRNA genes. Of the protein-coding genes, 78% are similar to those of other organisms; the remaining 22% are currently unknown. A total 612 kbp of the genome comprises group II introns, phage-like sequences, insertion sequences and some repetitive elements. Genome rearrangement mediated by the phage-like sequences was detected. As A. platensis is the first genome for filamentous non-N₂-fixing cyanobacteria, filament-specific genes were highlighted by comparative genomic analysis. Further, we will report the unique characteristics of A. platensis genome especially about group I introns, restriction-modification systems, cAMP signal transduction systems and chemotaxis proteins.

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Fresh water cyanobacteria, such as Synechococcus elongatus PCC 7942 (hereafter refferd as S. 7942) and Synechocystis sp. PCC 6803, have multi-genome per cell. However, replication mechanism in fresh water cyanobacteria is largely unknown. In S. 7942, it has been predicted that replicational origin exists on the upstream region of dnaN gene (Liu and Tsinoremas, 1995, Gene, 172, 105-109), although experimental validation has not been carried out.
In order to investigate the site of the replicational origin in S. 7942, we constructed the assay strain S. 7942^TK containing thymidine kinase gene (TK) in S. 7942 genome, since TK is essential for bromodeoxyuridine (BrdU) incorporation. The DNA replication in S. 7942^TK was completely blocked by dark treatment. After addition of BrdU, DNA synthesis and BrdU labeling was initiated by light irradiation. DNA labeled with BrdU was isolated by immunoprecipitation, and analyzed for the newly replicated region using next-generation sequencer. The results indicated that the replicational origin exists on the upstream region of dnaN gene, consistent with previous report.

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It is empirically known that cyanobacteria easily give rise to spontaneous mutations on their genome sequences. These differences of genotype and phenotype between the laboratory strain and the strain whose genome had been sequenced sometimes cause severe problems on post genome analyses. To solve this problem, it is necessary to resequence the genome of laboratory strains. Here, we carried out resequence analysis of the laboratory strain of a cyanobacterium, Synechococcus elongatus PCC 7942 using next generation sequencer, Genome Analyzer II. Mapping analysis of 700 Mb data of short-read sequences to the reference genome enables us to identify 7 SNPs and a large deletion of continuous 48.7 kb region in the laboratory strain. These results suggest that the importance of resequence analysis on the bacterial genome research.

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Protein kinases are major signaling molecules that are involved in a variety of cellular processes. However, molecular mechanisms of how protein kinases discriminate specific substrates are still largely unknown. Ca²⁺-dependent protein kinases (CDPKs) play central roles in Ca²⁺ signaling in plants. Previously, we found that a tobacco (Nicotiana tabacum) CDPK negatively regulates transcription factor REPRESSION OF SHOOT GROWTH (RSG) that is involved in gibberellin (GA) feedback regulation. Here, we found that the variable N-terminal domain of NtCDPK1 is necessary for the recognition of RSG. A mutation (R10A) in the variable N-terminal domain of NtCDPK1 reduced both RSG-binding and RSG phosphorylation while leaving kinase activity intact. Furthermore, R10A mutation suppressed in vivo function of NtCDPK1. The substitution of variable N-terminal domain of an Arabidopsis CDPK AtCPK9 with that of NtCDPK1 conferred RSG kinase activities. Our results open the possibility of engineering the substrate specificity of CDPK by manipulation of the variable N-terminal domain, enabling a rational rewiring of cellular signaling pathways.

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Carbon and nitrogen availability is one of the most important factors to regulate plant development. To utilize limited resource of carbon and nitrogen efficiently, plants enable to sense and respond to balance of carbon (C) and nitrogen (N) metabolites, called C/N response.
We previously showed a novel ubiquitin ligase ATL31 has essential role to regulate C/N response in Arabidopsis thaliana. In this study, we tried to isolate target protein ubiquitinated by ATL31. As a result of immunoprecipitation and MS analysis used epitope-tagged ATL31, we found several candidates of target protein for the ubiquitination. Details of the ubiquitinated proteins and biochemical function of ATL31 will be discussed.

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Plant respiratory burst oxidase homolog (rboh) proteins have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Ten rboh genes (AtrbohA-J) have been identified in the genome of Arabidopsis. By applying a heterologous expression system using HEK293T cells, we recently showed that AtrbohC and D possess ROS-producing activity synergistically activated by binding of Ca²⁺ to the EF-hand region of the cytosolic domain and phosphorylation. (Ogasawara et al., JBC, 2008; Takeda et al., Science, 2008). Rboh proteins have recently been suggested to be regulated by cytosolic accessory factors including small GTPases (Wong et al., Plant Cell 2007; Oda et al., JBC 2009). We here established a heterologous co-expression system for functional characterization of possible regulators of Atrbohs. We will report effects of co-expression of AtRac/ROPs on the ROS-producing activity of Atrbohs and discuss their possible functions in the regulation of ROS production.

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Production of reactive oxygen species (ROS) by NADPH oxidases has been shown to play crucial roles in the regulation of biotic and abiotic stress responses, programmed cell death and root hair development. In contrast to the NADPH oxidase complex in mammalian phagocytes that consists of multiple regulatory subunits, only the homologs of the catalytic subunit NOX2, rboh (respiratory burst oxidase homolog), and the cytosolic small G proteins, Rac, have been found in plants. By applying a heterologous expression system using HEK293T cells, we recently showed that AtrbohC and AtrbohD possess ROS-producing enzyme activity synergistically activated by binding of Ca2+ and phosphorylation (Ogasawara et al., JBC, 2008). We here screened for proteins that interact with the N-terminal cytosolic region of AtrbohD and AtrbohF using yeast two-hybrid assay, and so far identified 19 candidates. We further heterologously coexpressed the putative interactors with Atrbohs in HEK293T cells and characterized their effects on the ROS-producing activity. Possible involvement of the putative interactors in the regulation of Atrbohs will be discussed.

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Phosphatidylinositol-4-phosphate-5-kinase genes (PIP5Ks) play important roles in root hair formation. We investigated functions of PIP5K2, PIP5K3, and PIP5K4 genes in the regulation of root hair elongation during phosphate starvation. pip5k2 and pip5k4 mutants showed short root hair phenotypes similar to that of pip5k3. In pip5k2pip5k3 and pip5k3pip5k4 double mutants, those phenotypes were additively enhanced. These results indicate a redundant function in root hair elongation between these genes. pip5k3 and pip5k4, but not pip5k2, affected the enhancement of root hair elongation by phosphate starvation, indicating that PIP5K3 and PIP5K4 are involved in the phosphate starvation response. This is supported by the fact that promoters of PIP5K3 and PIP5K4 have recognition sites of the transcription factor, PHR1, which mediates phosphate-starvation responses. We constructed partial complementation plants by introducing PIP5K3 and PIP5K4 transgenes lacking PHR1-recognition sites into pip5k3 and pip5k4, respectively. These partial complementation plants are being used to investigate the biological significance of root hair elongation during phosphate starvation.

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Recently, we found novel calcium-binding proteins in Arabidopsis thaliana. One of these proteins, AtPCaP2 consists of 168 amino acid residues and contains N-myristoylation signal. To estimate its physiological role we characterized molecular properties of AtPCaP2. When PCaP2::GFP protein was expressed in Arabidopsis cells, green fluorescence was clearly observed in the plasma membrane. But fluorescence of PCaP2^G2A::GFP whose Gly-2 was replaced with Ala was observed in the cytosol. The results indicate that PCaP2 binds to the plasma membrane via myristoylation at Gly-2. Promoter-GUS analysis showed the expression in root epidermal cells, root hairs and pollens. And we found PCaP2 has the binding capacity to Ca²⁺ and specific phosphatidylinositol phosphates (PtdInsPs). Furthermore, calmodulin was associated with PCaP2 in a Ca²⁺-dependent manner, and its association weakened the interaction of PCaP2 with PtdInsPs. Expression of PCaP2 was significantly enhanced by NaCl and KCl. We propose that PCaP2 may be a molecular switch or break of PtdInsPs-mediated intracellular signaling in root hairs and/or pollen tubes.

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ABA hypersensitive germination2-1 shows pleiotropic phenotypes including an ABA hypersensitivity, an SA hypersensitivity, and a dwarf phenotype. The ahg2-1 mutation seems to reduce the expression of the polyA specific ribonuclease. To address the molecular basis of the AHG2/PARN function and the effect of the ahg2-1 mutation, we conducted a genetic screen for suppressor mutants of ahg2-1 using root elongation as a physiological marker. We isolated a dozen of candidates with various suppressor strengths. Interestingly, most of the suppressor mutants suppressed all the ahg2-1 phenotypes; ABA and SA hypersensitivities, and dwarf phenotype, suggesting that the functions of these suppressors and AHG2/PARN are closely related. We identified the corresponding gene for a suppressor mutant. We will discuss the function of the suppressor and the mechanisms by which ahg2-1 causes such the diverse and complicated effects.
Nishimura et al., Plant J., 44, 972-, 2005.
Nishimura et at., Plant Cell Physiol., in press.

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The two-component regulatory systems involving histidine-kinases (HK), which serve as signal receptors, are widespread signal transduction strategies in higher plants. In fact, HKs act as cytokinin receptors, which play crucial roles in hormone responses in Arabidopsis. It should also be emphasized that the plant two-component systems had evolved form the ones in prokaryotic bacteria. In fact, it was demonstrated that the Arabidopsis HKs (AHK2/3/4) are able to function as plant hormone cytokinin sensors in Esherichia coli. Hence, it is of interest to characterize the plant two-component systems from the evolutional viewpoint. In this respect, Physcomitrella patens is particularly interesting, because this species of moss is a basal lineage of land plants, having diverged before the acquisition of well developed vasculature. Here, we conducted genome-wide bio-informatics with special reference to the two-component systems-associated genes, including clock-associated ones, by adopting the genome databases of P. patens. Furthermore, we will provide experimental evidence for that this moss had a set of AHK2/3/4 homolog, which has an ability to act as cytokinin sensors in E. coli.

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The two-component regulatory systems involving histidine-kinases (HK), which serve as signal receptors, are widespread and general signal transduction strategies in plants. In fact, HKs act as cytokinin receptors, which play crucial roles in hormone responses in Arabidopsis. Furthermore, the two-component systems-related factors (PRRs) are known as important components of the circadian oscillator. Hence, it is timely and important to generalize the concept of two-component systems to understand the greatly diverse physiology of a wide variety of plant species other than Arabidopsis. For instance, a cytokinin receptor HK is crucially implicated in the root nodule formation in legume species, which yearly provide us with 1/3 of grain production in the whole world.
From this viewpoint, here we conducted an extensive and genome-wide bio-informatics with special reference to the two-component systems- and/or circadian clock-associated genes by adopting the genome databases of Lotus japonicus, which is a model legume species. We will present comprehensive information from such analyses, together with some critical experimental results with the plant.

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We have reported that Arabidopsis transcription factors DREB1s regulate expression of abiotic stress-responsive genes. Rice has 10 DREB1 family genes, OsDREB1s. Overexpression of OsDREB1A in rice activated expression of target stress-responsive genes such as lip9 and improved stress tolerance to drought, high-salinity and cold. Recently, we reported that OsDREB1G had the highest transactivation activity among 10 OsDREB1s and OsDREB1F activated the GUS reporter gene driven by an rd29A promoter, but did not activate the gene driven by the lip9 promoter.
In this study, we analyzed DNA binding specificities of OsDREB1s by gelshift assays. 9 OsDREB1 proteins bound a DRE in the rd29A promoter. However, binding of OsDREB1F to a DRE in the lip9 promoter was very weak. We analyzed downstream genes of OsDREB1G using transgenic rice harboring OsDREB1G under control of a promoter responsive to dexamethasone (DEX-OsDREB1G plants). More than 200 genes were up-regulated in the transgenic rice. Northern analysis confirmed that many downstream genes were induced by DEX in both DEX-OsDREB1G and DEX-OsDREB1A plants commonly and at least two genes were induced specifically in the DEX-OsDREB1G plants.

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Deciduous trees such as poplar show annual periodicity in shoot growth and development. However such periodicity of growth and function of root is not well known. In order to clarify seasonal fluctuation of function of root annually, xylem sap was collected from poplar cut branch and content of sap and gene expression in root were investigated. Many xylem sap components showed seasonal fluctuation and peaks are different by components. PIP and CYCB were strongly expressed in root around April when xylem sap quantity increases and around June when growth of root becomes active, respectively. In addition, the gene for25 kDa protein (XSP25) which most abundantly exists in xylem sap was identified by MS analysis and XSP25 was expressed in root in winter and highly up-regulated by ABA application to autumn root, but not by cold treatment. On the other hand, NCED encoding the enzyme for ABA synthesis was up-regulated in shoot, but not in root of cold-treated plants. This study suggests that ABA synthesized in shoot in response to low temperature in early winter may possibly move to root and promote the synthesis of XSP25 in root to protect shoot tissues against cold and/or dry in winter.

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Heterotrimeric G-proteins have been implicated in having a role in many plant signalling pathways. To understand further the role of G-proteins, a preliminary experiment was performed to assess the impact of the Gα subunit loss-of-function mutation gpa1-1 on the Arabidopsis transcriptome. The analysis indicated that the Gα subunit may play a role in response to jasmonic acid (JA). Consistent with this, Gα mutants showed a reduced response to JA in inhibition of chlorophyll accumulation and root growth, whilst Gα gain-of-function plants overexpressing Gα showed the opposite phenotype. Gene expression studies indicated that the Gα subunit regulates a subset of JA-regulated genes defining a branch point in this signalling pathway in Arabidopsis. Further analysis of the impact of Gα loss of function upon the JA-regulated transcriptome using Arabidopsis full genome arrays indicated that up to 29% of genes that are >2-fold regulated by JA in the wild type are misregulated in the Gα mutant. This supports the observation that a significant proportion of, but not all, JA-regulated gene expression is mediated by Gα.

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Signaling through heterotrimeric G-proteins is highly conserved among divergent eukaryotes. In plants, heterotrimeric G-proteins play an essential role in auxin, abscisic acid (ABA), and sugar response. If G-protein-coupled receptors (GPCR) bind their ligands, subunits of heterotrimeric G-proteins dissociate from each other and regulate activities of other proteins, thus mediating signaling from GPCR to downstream pathways. In plants, however, there have been few reports on factors regulated by heterotrimeric G-proteins. In this study, we performed yeast two-hybrid screening and identified a bZIP transcription factor VIP1 as a novel interacting partner of Arabidopsis heterotrimeric G protein beta (AGB1). AGB1-VIP1 interaction in planta was studied by the BiFC assay, and it was suggested that the interaction occurred in the nucleus. When VIP1 was expressed constitutively in Arabidopsis under the control of the CaMV35S promoter, seed germination was severely inhibited in the presence of NaCl, ABA, or glucose. AGB1 was known to be a negative regulator of ABA signaling. Taken together, our results propose possibility that VIP1 participates in the ABA signaling pathway mediated by AGB1.

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Deepwater rice is cultivated in Southeast Asia where serious flood occurs periodically. To avoid flood condition, deepwater rice elongates its internode along with the rising water. QTL analysis revealed one major QTL for internodal elongation on the long arm of chromosome 12. By positional cloning, we identified two genes, named Snorkel1 (SK1) and Snorkel2 (SK2) regulated deepwater esponse. However, Snorkels are insufficient to elongate under the deep-water, and it is necessary for other QTL to interact. Inthis study, to elucidate the mechanism of internode elongation in rice, we performed the genetic analysis to identify the other QTL, using NIL-lines and pyramiding-lines.

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The most remarkable characteristic of deepwater rice is rapid internode elongation when subjected to deep water. Deepwater rice does not show significant internode elongation in shallow water, whereas deep water induces dramatic internode elongation. Most ordinary rice cultivars planted in irrigated fields do not have this characteristic. Deepwater rice is mainly cultivated in lowland areas of South and Southeast Asia where the severe flood is occurred during the rainy season. Rapid internode elongation in response to deep water in deepwater rice is necessary to avoid anoxia. Deepwater rice elongates internodes to keep the top leaves above the water surface to allow gas exchange.
Recently, we isolated the SNORKEL1 (SK1) and SNORKEL2 (SK2) which are responsible for deepwater response. The functional analyses of SK1 and SK2 indicated that these genes were ERF type transcription factors which encoded AP2/ ERF domain and involved in signaling of gaseous phytohormone, ethylene. Our previous results suggested that the SK genes are involved in the ethylene signal transduction. Here we show the analyses of the relationship between SK genes and downstream genes.

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CO₂ is an environmental signal that regulates stomatal movements. High CO₂ concentrations reduce stomatal aperture, whereas low concentrations lead to stomatal opening. However, molecular mechanisms underlying stomatal CO₂ sensing and the downstream signaling pathway are largely unknown. To identify genes that function in CO₂ responses in guard cells, we previously isolated Arabidopsis CO₂ insensitive mutants that were roughly categorized into two-types according to their phenotypes; (1) impaired in stomatal opening under low CO₂ concentrations (2) impaired in stomatal closing under high CO₂ concentrations. Among these, we especially focused on a novel type mutant cdi6 (carbon dioxide insensitive 6) that impaired in both CO₂ responses. The CDI6 is a plant transcription factor, and is localized specifically to the nucleus of guard cells. We examined transcriptional profiles in guard cell protoplasts using Arabidopsis DNA microarray. From the results, the expression of several genes that were involved in stomatal function or development was changed in cdi6 mutant. These results suggest that CDI6 has an important role in regulation of guard cell gene expression.

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Stomata in the epidermis regulate gas exchange between plants and atmosphere. Stomatal opening, which is mediated by blue-light receptor phototropins, is driven by activation of the plasma membrane H⁺-ATPase. Under drought stress, a phytohormone abscisic acid (ABA) induces stomatal closure to prevent water loss from plants. However, the signaling pathways for blue light-induced stomatal opening and ABA-induced stomatal closure have yet to be determined. Here, we performed the mutant screening focused on stomatal aperture-dependent transpiration in detached leaves from EMS-treated Arabidopsis thaliana and isolated ftd (fast transpiration in detached leaves) mutants. Of these, ftd2 showed dwarf and pale green phenotypes in addition to open-stomata phenotype with ABA-sensitivity in stomatal guard cells. Rough mapping analysis revealed that FTD2 locus locates on the 5^th chromosome. We will report the results of fine mapping.

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The salt stress is one of the representative abiotic stresses that decreases growth and productivity of crops plants. The root water permeability of three barley (Hordeum Vulgare) cultivars that show a difference in the salt sensitivity was measured under salinity stress using a pressure chamber. Water permeability of salt sensitive variety did not show any difference in response to salt stress, in contrast to significant reductions of water permeability found in more salt tolerant varieties. Root water permeability of tolerant cultivars exposed to 100 mM NaCl was found to be extremely down-regulated after 1h. Interestingly, however, water permeability was partially recovered after 4 h, which was followed by a re-down-regulation afterwards. Further measurements using various inhibitors indicated that at least protein phosphorylation and recycling plays an essential role in the regulation mechanism.

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Aquaporins facilitate water flux across biomembranes, and are involved in various physiological phenomena in several plant tissues. We analyzed the water-flux activity of plant aquaporins using lily (Lilium longiflorum) pollen protoplasts. Large protoplasts with a uniform diameter of approximately 95 μm were isolated from lily nearly mature pollen grains. The genes for plasma membrane aquaporins (PIPs) of Arabidopsis and carrot were put under the control of the strong pollen-specific promoter, maize Zm13 promoter, and then they were introduced into the protoplasts by electroporation. The pollen protoplasts expressed AtPIP2;1 or DcPIP2;1 increased significantly their volumes in hypotonic solution (350 mM mannitol) compared with the vector control. By contrast, the volume changes of the protoplasts expressed AtPIP1;1 or DcPIP1;1 were similar to that of the vector control. These results suggest that PIP2s show higher water-flux activity in plant cells, whereas PIP1s do not. Thus, we propose the lily pollen protoplasts as a simple and useful experimental system to analyze the function of plant aquaporins.

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In Arabidopsis genome, about 1,400 transporter-related genes were fond in silico, of which function is almostly unknown. To overcome round robin screening of novel transporter genes from candidates, we prepared transorter-specific gene library Amethyst (Arabidopsis membrane transporter and hydrophobic protein cDNA clones stack).
In this study, we prepared over-expression library of Amethyst in mammalian culture cell. 961 genes were subcloned to CMV promoter driven mammalian expression vector. Then, as a test case, we screened molybdenum, one of a trace elements, related transporter. pCMV-Amethyst and molybdenum-sensing MolyProbe sensor protein were co-expressed in 293T culture cell, and molybdenum depending FRET pattern were analysed.

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Blue light induces stomatal opening through the activation of the plasma membrane H⁺-ATPase. Recent investigations have demonstrated that blue light activates the H⁺-ATPase via phosphorylation on a penultimate threonine residue in the C-terminus and subsequent binding of 14-3-3 protein to the phosphorylated C-terminus in guard cells. However, protein kinase and protein phosphatase, which regulate the phosphorylation level of the H⁺-ATPase, are largely unknown. In this study, we analyzed biochemical properties of the H⁺-ATPase phosphorylation. in vitro phosphorylation on threonine residue in the C-terminus of the H⁺-ATPase was detected in the microsome from Vicia guard cell protoplasts and also detected in the plasma membrane from etiolated seedlings of Arabidopsis. Moreover, we found that a nonionic surfactant TritonX-100 has an inhibitory effect on in vitro phosphorylation. These results suggested that the protein kinase localized in the plasma membrane is common to plant cells and that structure of the plasma membrane may be requisite for the protein kinase activity.

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Plant plasma membrane H⁺-ATPase creates an electrochemical gradient of H⁺ across the plasma membrane. Recent studies have demonstrated that the H⁺-ATPase is activated via phosphorylation on a penultimate threonine residue in the C-terminus and subsequent binding of 14-3-3 protein to the phosphorylated C-terminus. However, biochemical properties of protein kinase and protein phosphatase, which regulate phosphorylation level of the H⁺-ATPase, are largely unknown. In this study, we investigated in vitro dephosphorylation of the H⁺-ATPase. Dephosphorylation of the H⁺-ATPase was detected in the plasma membrane from etiolated seedlings of Arabidopsis. Furthermore, the dephosphorylation was inhibited by EDTA, a chelating agent for divalent cations, but not by calyculin A, an inhibitor of type 1 and type 2A protein phosphatases. These results suggest that a divalent cation-dependent protein phosphatase localized in the plasma membrane is involved in dephosphorylation of the H⁺-ATPase. We will report analysis of the H⁺-ATPase complex.

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The transport and gating activities of K⁺ channels are thought to be regulated by multiple posttranslational modifications of the C-terminal region, such as phosphorylation and binding of cyclic nucleotides. These phenomena indicate that multiple signaling pathways likely exist for channel regulation in response to various environmental stresses. We previously identified the phosphorylation target sites of the Arabidopis Shaker-type K⁺ channel, KAT1, and found that the modification of one of these sites strongly modulates K⁺ channel activities of KAT1 expressed in Xenopus oocytes and yeast (Sato et. al. Biochem. J. 2009). The phopshorylation site is highly conserved between all Arabidopis K⁺ channels. Thus, we examined the phosphorylation of the other Arabidopsis K⁺ channels, and the effect of modification of the conserved site on the K⁺ channel activities. We will also discuss the regulatory mechanism involving the modification of C-terminal region.

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We have identified MCA1 and MCA2 genes encoding Ca²⁺-permeable mechanosensitive channel candidates from Arabidopsis thaliana. MCA1 and MCA2 are highly homologous in amino acid sequence and involved in Ca²⁺ uptake. Although there are homologs of MCA1 and MCA2 in various plants, their functions are unknown. We reported previously that MCA1 and MCA2 are localized in the yeast plasma membrane. Here, we performed further molecular characterization of MCA1 and MCA2 using yeast. To investigate relationships between the structure and function in Ca²⁺ uptake, we made a series of truncation mutants lacking the N-terminal or C-terminal region of MCA1 and MCA2 and a D/E to A substitution mutant of an EF hand-like motif and introduced these mutants to yeast cells. Ca²⁺ uptake assays indicated that functional mechanism of MCA1 or MCA2 is different. We also demonstrate that MCA2 is localized in the Arabidopsis plasma membrane and that it is an integral plasma membrane protein in yeast cells.

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Vacuoles have diverse functions within all plant cells. Vacuoles get rid of wastes, accumulate nutrients, and regulate pressure within a cell. They are also known to store sugars. However, only a small number of vacuolar sugar transporters have been identified and characterized.
We first identified a vacuolar sucrose transporter from tobacco BY-2 cells (NtSUT4) and established transgenic tobacco BY-2 cells that overexpress NtSUT4-GFP (BY-SUTG cells). Using model system for synchronous cell elongation in miniprotoplasts (evacuolated cells) prepared from tobacco BY-2 cells, we found that overexpression of NtSUT4-GFP inhibit cell growth towards cell major axis. Moreover, in the same condition, we also found that, in BY-SUTG cells, more amount of cellulose accumulated than in wild type BY-2 cells. These results suggest that NtSUT4 affects cell wall formation and involved in cell shape via sucrose homeostasis in plant cells.

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Graminaceous plants have developed a chelation strategy to efficiently acquire insoluble iron, which includes the synthesis and secretion of the iron-chelator phytosiderophore (PS) such as mugineic acids (MAs) and the transportation of Fe(III) complexed with PS. We have identified a specific Fe(III)-PS transporter HvYS1 in barley. In this reports, we have introduced the HvYS1 gene to petunia, which have reduction mechanism, but not chelation strategy to acquire iron upon iron deficiency. When these transgenic petunia were grown hydroponically in medium containing Fe(III)-2'-deoxymugineic acid (DMA) complex, the Fe-DMA complex were detected in the root by ESI-FT-ICRMS. Consequently, transgenic petunia exhibited an enhanced tolerance to low iron concentration in alkaline hydroponic culture in the presence of Fe(III)-DMA complex, which led to 2 times mass weight and 1.5 times iron concentration as compared with nontransformed plants. These results show that plants, which possess reduction mechanism for iron acquisition, confer resistance to Fe deficiency in alkaline condition by acquiring the chelation strategy.

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High salinity restricts the growth of most higher plants. Vacuolar compartmentation constitutes a major cellular strategy for salt tolerance. During salt accumulation, we have previously observed a rapid increase of vacuolar volume without an increase in cell size in barley and Arabidopsis root cells and mangrove suspension cultured cells.
In the present study, we analyzed the behavior of membrane vesicles and vacuoles in Arabidopsis under high salt condition. Upon treatment with NaCl, a rapid increase in the vacuolar volume in Arabidopsis root cells and suspension cultured cells was observed. Experiments involving GFP-AtVam3 suspension cultured cells revealed an active movement of small vesicles around the main vacuole due to salt treatment. Furthermore, using the fluorescence Na+ indicator (Sodium Green), Na+ accumulation in the vesicles and vacuole was apparent. The immunostaining of suspension cultured cells with antibodies against AtNHX1 showed the vesicle-like fluorescence in the cytoplasm under salt treatment. Data support the involvement of vesicle transport in vacuolar Na+ accumulation.

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Aluminum ion (Al³⁺) inhibit the root growth in acidic soils. Nevertheless, a variety of plants have a capability to grow in acidic soils by excluding and/or detoxifying the Al³⁺. By the exposure to Al³⁺, a sort of organic acids such as malate or citrate are secreted from the root tips, and the organic acids chelate and detoxify Al³⁺.
Al-resistant cultivars of wheat release malate, which is controlled by the Al-activated malate transporter encoded by the ALMT1 gene. Heterologous expression of the ALMT1 in Xenopus oocytes conferred Al³⁺-activated malate efflux. To clarify the underlying processes for Al³⁺-activated malate efflux via ALMT1, electrophysiological analyses were performed using Xenopus oocytes expressing ALMT1 with desired mutations and truncation in C-terminal domain. Furthermore, Al-activation of chimeric constructs with the C-terminal domain from a homologous ones was analyzed. These results suggested that the three-acidic-amino acids and the whole structure of the C-terminal domain are involved in the Al activation mechanism of ALMT1 protein.

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Arsenic contamination is a major problem in South Asia particularly in Bangladesh and West Bengal. It is important to investigate how the As is transported to rice plants. Arsenite transporter in rice and Arabidopsis has recently been reported. It is also reported in Arabidopsis that arsenate is taken by plants through phosphate transporters. Here we investigated possible roles of phosphate transporter in arsenate transport in rice. There are 13 putative rice phosphate transporters (OsPTs) in rice and their patterns of expression vary in terms of induction by phosphate conditions and tissue specificity. We found that a T-DNA insertion mutant for one of the transporter gene accumulate less As in plant body, suggesting involvement of the phosphate transporter(s) in arsenate transport in rice.

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Aluminum (Al) is the most abundant metal in the Earth's crust, but its trivalent ionic form is highly toxic to all organisms at low concentrations. How Al enters cells has not been elucidated in any organisms. Herein, we report a transporter, Nrat1 (Nramp aluminum transporter 1), specific for trivalent Al ion in rice. Nrat1 belongs to the Nramp (natural resistance-associated macrophage protein) family, but shares a low similarity with other Nramp members. When expressed in yeast, Nrat1 transports trivalent Al ion, but not other divalent ions such as manganese, iron and cadmium, or the Al-citrate complex. Nrat1 is localized at the plasma membranes of all cells of root tips. Knockout of Nrat1 resulted in decreased Al uptake and increased sensitivity to Al, but the tolerance to other metals was not altered. One the other hand, over-expression of Nrat1 led to enhanced Al uptake in rice roots. Expression of Nrat1 is up-regulated by Al in the roots and regulated by a C2H2 zinc transcription factor (ART1). Our results indicate that Nrat1 is a transporter specific for trivalent Al, which is involved in Al tolerance in rice.

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Intake of Cd from rice is a serious concern for human health, however the mechanisms controlling Cd accumulation in rice are poorly understood. Herein, we report a transporter (CASTLE1) specifically responsible for low Cd accumulation in rice shoots. We detected a QTL on chromosome 7, by using a mapping population derived from a cross between a high- (Anjana Dhan) and low-cadmium (Nipponbare) accumulating cultivar, and finally were able to isolate the candidate gene (CASTLE1), belonging to P-type ATPase. Allelic CASTLE1 genes were mainly expressed in the root at a similar level in both accessions. Immunostaining showed the localization at tonoplast and vesicles of all root cells. Knockdown of CASTLE1 in Nipponbare resulted in increased Cd accumulation in the shoots, whereas over-expression of this gene resulted in decreased Cd accumulation. Yeast expression analysis showed that CASTLE1 from Nipponbare showed transport activity for Cd, whereas that from Anjana Dhan did not. These results indicate that functional CASTLE1 in Nipponbare plays a role in sequestrating Cd into the vacuoles and vesicles in the roots, thereby reducing Cd translocation to the upper parts.

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Cadmium (Cd) is a harmful heavy metal. We isolated a gene homologous to Arabidopsis NRAMP1 from a low Cd accumulating plant, Solanum torvum (StNRAMP1). Expression of StNRAMP1 gene was induced by iron deficiency and repressed by Cd treatment. Yeast cells expressing StNRAMP1 were sensitive to Cd and the GFP-fused protein predominantly localized in plasma membrane in onion epidermal cells. These results suggest that StNRAMP1 is an influx iron and Cd transporter. Furthermore, we developed transgenic tobacco plants expressing StNRAMP1 under the control of 35S promoter. Transformants showed intercostal chlorosis even in standard culture condition and the symptoms were more severe in older leaves. Cd content in the newest leaves of transformants was decreased to 20% compared to those of non-transformants when plants were grown hydroponically with 0.1 μM Cd for 5 days. Cd content of second newest leaves with intercostal chlorosis was also decreased while the iron content was 2-times higher than those of non-transformants. These results suggest that overexpression of StNRAMP1 changes the distribution of iron resulting in inhibition of Cd translocation or accumulation in tobacco plant.

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Plants induce iron uptake/utilization system in response to iron deficiency. Recently, we identified the rice transcription factor IDEF1 (IDE-binding Factor 1), which specifically binds the iron deficiency-responsive cis-acting element IDE1. We analyzed the regulation mechanism of IDEF1 in response to iron deficiency using transgenic rice plants with induced or repressed IDEF1 expression. During the early stages of iron deficiency, the majority of known iron uptake/utilization-related genes were positively regulated by IDEF1. In subsequent stages, IDEF1-mediated regulation of these iron uptake/utilization-related genes became less obvious. In turn, expression of several iron deficiency-induced genes encoding late embryogenesis abundant proteins was increasingly regulated by IDEF1 at the subsequent stages. We propose a dual function of IDEF1 for iron deficiency response, namely, (i) the coordinated transactivation of iron utilization-related genes via CATGC-containing IDE1-like elements, especially at the early stage, and (ii) the transactivation of seed maturation-related genes via RY elements, especially during the subsequent stages of iron deficiency.

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Arabidopsis transcription factors DREB1s/CBFs specifically interact with a cis-acting element DRE/CRT/LTRE involved in the expression of low-temperature-, drought- and high-salinity-inducible genes. Expression of the DREB1 genes is kept at very low level and regulated by circadian control, but induced rapidly and significantly by low temperature. In this study, we analyzed transcriptional regulation of the DREB1 genes.
Using a GUS reporter gene fused to a DREB1C promoter, we identified a 65-bp region that contains cis-acting elements that function under both low temperature and circadian controls. We isolated cDNA, encoding PIF7, which specifically binds to the 65-bp region by using the yeast one-hybrid screening. We found that the PIF7 could repress DREB1C expression by transient expression assays with Arabidopsis protoplasts. In the pif7 mutant, DREB1 expression was not repressed under circadian control. These results suggest that the PIF7 functions as a transcriptional repressor for the expression of DREB1C under circadian controls.

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