Under drought in the presence of strong light, which is potentially induces photoinhibition, absorption of excess light energy causes over-reduction of electron transport chain in chloroplasts. To investigate the regulation of electron transport under such stress condition, we performed proteomic analysis of thylakoid membrane in drought-resistant wild watermelon. We found that new spots with more acidic pI for the Rieske protein, a component of the cyt b₆f complex, emerged in response to the stress. These new spots were disappeared by relieving the stress. The in vivo and in vitro analyses suggested that the cyt b₆f complex was the major rate-limiting step in the electron transport under stress conditions. We discuss on the possible involvement of the Rieske protein with more acidic pI in the regulation of photosynthetic electron transport activity to avoid photoinhibition of PS I under stress conditions.

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When the excitation energy balance between photosystem I (PSI) and photosystem II (PSII) is disturbed due to the changing light environment, light-harvesting complex II (LHCII) is thought to adjust the energy balance by a mechanism called state transition. Recently, we successfully isolated the protein supercomplexes, so-called PSII-LHCII supercomplex and PSI-LHCI/II supercomplex, from Chlamydomonas reinhardtii and revealed the structural changes and reversible phosphorylated proteins during state transitions. However, direct evidence for such dynamic changes of photosystem protein complexes are still remained to be proved. Therefore, to visualize state transitions in vivo, we applied fluorescence lifetime imaging microscopy technique. First, we established a method to induce state transitions in living C. reinhardtii cells under a microscope and then optimized the imaging parameters for live imaging. By comparing the lifetime data between wild type and several mutant cells, we visualized the spatio-temporal dynamics of chlorophyll fluorescence lifetime reflecting state transitions.

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Higher plant chloroplasts induce large energy dependent non-photochemical quenching (qE) after rapid light-to-dark transition for avoiding photodamage. In Chlamydomonas reinhardtii, however, state transitions are the dominant photoprotection mechanism instead of qE. The qE activity is far less than its maximum capacity observed under stress conditions such as high light and low CO2 concentrations. Chlamydomonas raudensis, which inherently lacks state transition ability, shows high qE as in higher plants, suggesting that the functions of state transitions could suppress qE activity. Dark adapted C. reinhardtii in State 2 shows large cyclic electron flow, though accumulation of proton gradient in the thylakoid lumen is slow. The small proton gradient can be explained by the large proton conductivity at the ATP synthase. We propose a new model of photoprotection, where the phosphorylation/dephosphorylation of the proteins in state transitions affect stromal inorganic phosphate concentration for regulating the ATP synthase conductivity.

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The chloroplast NAD(P)H dehydrogenase(NDH) complex reduces plastoquinones and generate H⁺ gradient using NAD(P)H in photosystem I cyclic electron flow and chlororespiration. The NDH complex is found in land plants and cyanobacteria, and a number of NDH-related genes were characterized, while whole machinery of NDH is not identified yet. Last few years, we have succeeded to identify novel six genes related to NDH activity, NDF1, NDF2, NDF4, NDF5, NDF6 and PPL2, using a co-expression analysis of Arabidopsis. Among NDF genes, NDF2 and NDF5 showed weak homology to each other.
Here we report that Physcomitrella has a single homolog of both NDF2 and NDF5, while both of them are conserved in higher plants. Functional roles of these proteins in NDH activity are investigated in immunoblot analyses in a panel of ndh mutants, using antibodies against NDH related proteins containing NDF2 and NDF5.

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Heat-stress causes serious damages to plant, like as loss of photosynthetic activity and retardation of development. Here, we report that activation of NAD(P)H dehydrogenase is closely related to damage of photosynthetic activity in heat-stressed plants. When Arabidopsis plants were exposed to heat stress at 40C in the dark, Fv/Fm was rapidly decreased within a few hours. This decrease was due to release of oxygen-evolving 33 kDa protein. When NAD(P)H dehydrogenase (NDH)-deficient mutant (ndhO) was heat-treated, almost photosynthetic activity was maintained, suggesting that NDH might be closely related to heat-derived damage.

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We observed severe injury in the leaves of rice seedlings (Akitakomachi) after a few days chilling only when the roots were not chilled (L/H), preceded by the blocking of photosynthetic electron transport from PSII to PSI, whereas no such injury was observed when the whole seedlings were chilled (L/L) (Suzuki et al. 2008). It was, however, unclear if the blocking was the primary cause of the injury, because the photosynthesis was analyzed at 25 degrees C after chilling. In this paper, we analyzed photosynthesis at 9 degrees C during chilling treatments. Under L/L, ETR at PSI was as high as that of non-chilled seedlings. Under L/H, quite low ETR at both PSII and PSI, an over-reduction at PSII, and an over-oxidation at PSI during the short-term photosynthesis were observed. No significant difference was observed between L/L and L/H, in the linear electron transport in the thylakoid preparations.

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Effects of elevated CO2 on synthesis and degradation of Rubisco were examined in rice leaves from emergence to senescence. Rubisco content in control plants rapidly increased during leaf expansion, reached its maximum around full expansion, then declined. In elevated [CO2]-grown plants, Rubisco content was lower throughout lifetime of a leaf. Rubisco synthesis in control plants was active during leaf expansion, but declined just before full expansion, then Rubisco degradation occurred. For the elevated [CO2]-plants, Rubisco synthesis was lower in expanding leaves, but did not differ in senescent leaves. Although a slight stimulation of Rubisco degradation was found during the early stage of senescence, total amount of Rubisco degraded was slightly lower. Thus, a decline in Rubisco content by elevated [CO2] is the result of a repression of Rubisco synthesis during leaf expansion as well as a slight stimulation of Rubisco degradation during the early stage of senescence.

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Bioconvection is a non-equilibrium dynamics displayed by directionally moving microorganisms. As a first step towards understanding of cellular dynamics of bioconvection, I developed a system of inverted bioconvection. I previously reported that the establishment of bioconvection proceeds via several distinct states. In the present study, I developed a method to follow individual cells within crowded cellular layer, and analyzed time-dependent development of cellular dynamics.

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Marine diatoms account for about a quarter of primary production and can grow in a wide range of salt. Although salt is a perturbable factor at the ocean surface, acclimation mechanisms in marine photoautotrophs to low salt environment have not been reported. In this study, low-salt-response was characterized in the marine diatom Phaeodactylum tricornutum. Cells were transferred from standard (0.5M[Na⁺]) to low salt (0.1M[Na⁺]) seawater and growth profile was monitored. RNA was obtained from each acclimation stages, pre-, early-stage- and late-stage accumulations, and a semi-exhaustive transcriptome analysis was carried out using cDNA-amplyfied fragment length polymorphism (cDNA-AFLP) technique. Consequently, 74 cDNA-fragments were detected as low-salt-response genes. Of these, cDNA encodes Heat shock factors, ascorbic acid (AsA) biosynthesis, AsA redox processes, and other reactive oxygen scavenging enzymes were shown to be increased during acclimation to low salt. These results indicate that acclimation to low salt is accompanied by oxidative stresses.

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The promoter of a chloroplastic β-carbonic anhydrase in the marine diatom Phaeodactylum tricornutum (PtCA1) is known to be under controls of CO₂, light, and cAMP. We have already shown that three tandem repeat of a typical mammalian-cAMP-responsive element is critical for CO₂ response of this promoter, Pptca1. This repeat sequence was designated as CO₂/cAMP-responsive element, CCRE. In the present study, we searched and isolated 8 candidates of CREB/ATF superfamily cDNAs (PtbZIPs) from genome of P. tricornutum. These cDNAs were ligated into pET21a vector, and expressed in E. coli BL21(DE3). Of these gene products, proteins designated as PtbZIP2, 3, and 7 were found in soluble fractions. Gel shift assay using CCRE pentamer as a probe revealed that PtbZIP7 specifically bound to CCRE. RT-PCR assays showed that the expression level of PtbZIP7 was higher in air.

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PsbP, an extrinsic subunit of photosystem II (PSII), is a nuclear-encoded protein that optimizes the water-splitting reaction. In addition to PsbP, higher plants have a number of PsbP paralogs that include the PsbP-like 1 (PPL1) protein, a putative ortholog of cyanobacterial PsbP homolog (cyanoP). In our previous study, we characterized an Arabidopsis mutant lacking PPL1 and demonstrated that PSII activity was sensitive to high-intensity light and the recovery of photoinhibited PSII activity was delayed in ppl1 mutant plants (Ishihara et al., Plant Physiology 2007). In order to elucidate how PPL1 is involved in the PSII repair cycle, molecular behaviors of PPL1 were further analyzed using Arabidopsis wild-type (Col-0) and various mutants that have a defect to maintain the normal PSII activities. Localization of PPL1 in the thylakoid membranes was also analyzed.

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Deep supercooling (DSC) ability of the parenchyma cells of xylem (XPCs) is essential for freezing resistance of boreal woody plants in winter. DSC ability is induced during seasonal cold acclimation in XRCs with various physiological changes. In XRCs of larch (Larix kaempferi), changes in gene expression and sugar accumulation have been characterized in association with changes in DSC ability. However, roles of proteins on DSC ability are still unclear. Therefore, we examined to identify proteins that are related to the DSC ability. In parallel with the decrease in DSC ability of larch twigs of winter by artificial deacclimation, decrease in levels of sixteen winter-induced proteins were detected by two-dimensional electrophoresis. These proteins were excised from gel spots, digested with trypsin, and analyzed LC-MALDI MS/MS. Database search based on de novo sequences from MS/MS spectra revealed that some candidate proteins for increasing DSC ability were similar to several proteins.

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Trachycarpus fortunei is probably one of the most cold-hardy palm species. Their leaves tolerate -14C without any injury and show similar levels of cold hardiness throughout the year. Even in mid-summer, they survived -10C or lower without any injury when cooled 2C/h. DTA and NMR micro-imaging revealed that most living cells in Trachycarpus leaves employ deep supercooling (-16 to -22C) as the mechanism of cold hardiness, which allows Trachycarpus leaves to be a good system to study the mechanism of deep supercooling. To elucidate the mechanisms, we investigated the property of extracts from the leaves. The extract works as an anti-ice nucleating agent. The extract can inhibit the ice nucleation activity of ice nucleating bacteria, phenazine and AgI. The extract was further fractionated into Ethyl acetate, water and BuOH fractions. We characterized the activity of these fractions and also attempted to quantify the anti-nucleating activity.

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Cold acclimation (CA), the process of increasing in freezing tolerance, is an important survival mechanism for temperate plants. During CA, lipid and protein composition alters in the plasma membrane (PM) and the cryostability of the membrane increases. These changes are essential for plant survival under freezing conditions. Recently, the presence of sphingolipid/cholesterol-enriched microdomains in the plasma membrane has been proposed but little is known in plant system. To understand the role of microdomains in CA, we have determined their lipid composition. In microdomains, sterols increased and sphingolipids (glucocerebrosides) and phospholipids decreased. In contrast, PM showed no changes in sterols and phospholipids but a decrease in glucocerebrosides. These results indicate that microdomains respond to CA differently from the bulk plasma membrane, suggesting that the specific changes of microdomain lipids may have a specific role in maintaining some functions associated with microdomains at low temperatures, which minimizes freezing injury.

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Some plants are able to induce freezing tolerance by cold acclimation. In this process, abscisic acid (ABA) has an important role in stress signaling. Previous studies using Arabidopsis thaliana suspension-cultured cells reported that induction of freezing tolerance after ABA treatment depended on growth phase of the cells (lag and log phases): ABA-induced increase in freezing tolerance was detected only cells at the lag phase, but not the log phase. To understand the ABA responses at cellular level associated with freezing tolerance induction, gene expression and protein accumulation of dehydrins, stress responsible proteins belonging to LEA (late embryo abundant protein) family, are determined using RT-PCR, 2D-PAGE, and Western blotting with anti-dehydrin antibody. We found that the accumulation of two dehydrins occur only in ABA-treated lag phase cells. These results suggest that these dehydrins may play a role in regulation of freezing tolerance at cellular levels.

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VOZ (Vascular plant One Zink-finger protein) is a one-zinc finger type DNA binding protein, which is highly conserved among higher plants. Arabidopsis harbors two VOZ homologs (VOZ1 and VOZ2). As voz1/voz2 double mutant showed the late-flowering phenotype under long day condition, it was proposed that VOZs are involved in photoperiodic flowering response.
In this work, we show that voz1/voz2 plant has increased freezing tolerance without cold acclimation. This phenotype is consistent with stronger expression of CBF/DREB1-regulated genes. These results suggest that VOZ functions as a negative regulator of genes for cold stress response.

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Environmental stresses influence plants growth and productivity. Previously, we have reported that DREB2A interacts with a cis-acting dehydration responsive element (DRE) and activates expression of many stress responsive genes. To identify the upstream element of DREB2A under heat stress condition we analyze the promoter region of the DREB2A genes using transgenic plants harboring a GUS reporter gene fused various lengths of the DREB2A promoter and found an important sequence containing a heat shock element (HSE) for the expression of DREB2A. As HSFs are known to bind HSE and regulate expression of heat stress-inducible genes we analyzed expression and transactivation activities of 21 HSF-type transcription factors in Arabidopsis and selected some candidates for the regulator of DREB2A. Then, we generated transgenic Arabidopsis overexpressing those HSFs and isolated their knockout mutants. We are currently analyzing expression of the DREB2A gene in those plants.

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In contrast with other Hsfs, class B Hsfs have no activation domain and the function is remain to be elucidate. We show, here, that Arabidopsis HsfB1, encodes a transcriptional repressor that contains novel type of repression domain appeared to be different known EAR-motif repression domain. Ectopically expressed HsfB1 repressed the heat inducible expression of several Hsfs and HSPs in transgenic plants under 28 degrees condition. Transient expression analysis revealed that HsfB1 suppressed the heat inducible promoter activity of those genes. In the double mutant of HsfB1 and its functionally redundant HsfB2B, the expression of heat inducible genes was increased under both in normal and moderate-heat stress (28 degrees) condition. However, in 32 degrees, levels of induction of those gene expressions were similar between hsfb1 hsfb2b and wild-type seedling. These results suggest that the HsfB1 and HsfB2B control the level of heat stress responses to adapt the environmental condition.

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Thellungiella halophila is a halophyte, and its genes have a high sequence identity (90-95% at the cDNA level) to genes of its close relative Arabidopsis thaliana. Thellungiella plants show not only the extreme salinity tolerance, but also freezing and oxidative stress tolerances. Moreover, we have revealed that Thellungiella also shows heat stress tolerance in comparison with Arabidopsis.
Recently, we generated a full-length enriched cDNA library of Thellungiella from various tissues and whole plants treated with salinity-, cold-, freezing-stresses and ABA. To identify the heat tolerant genes in Thellungiella, we are working on FOX hunting (Full-length over expressor gene hunting system) using the library. In this study, we screened heat tolerant transgenic lines from a mini-scale FOX lines of Arabidopsis. Which constitute of 120 genes encoding HSPs, HSFs and osmoprotectant synthesis enzymes.

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Plants sense gravity and exhibit tropic response, where amyloplast sedimentation appears to be significant for gravity sensing. Recent live-cell imaging revealed, however, that amyloplasts in endodermal cells show complicated movements toward gravity after 90^o-reorientation probably due to interaction with organelle and/or cytoskeleton. Therefore, it remains obscure what kind of amyloplast movement is required for gravity sensing. To address this issue, we analyzed gravitropism and amyloplast dynamics under hypergravity condition where sedimentation by gravity is more dominant than other movements. Segments of Arabidopsis inflorescence stem that were perpendicularly subjected to hypergravity (10g) for 30 sec exhibited obvious gravitropism. Real-time imaging of amyloplasts during hypergravity was performed using a recently developed centrifuge microscope. Almost all amyloplasts moved toward hypergravity and some of them reached the bottom side of the plasma membrane within 30 sec of 10g. These results suggest that amyloplast sedimentation toward the bottom of endodermal cell is important for gravity sensing.

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Plant acetylcholinesterase (AChE) proteins and genes were purified and cloned from maize and siratro seedlings, which may relate to a signal transduction in the same manner as the animal AChE. In this study, we found rice plant possesses two AChE homologous genes, AChE1 and AChE2, which shared 77% and 66% amino acid sequence identities with maize AChE, respectively. Rice AChE1 and AChE2 were overexpressed and suppressed in rice plants. The transgenic rice plants overexpressed rice AChE1 or AChE2 were exhibited extremely high AChE activities compared with the vector control. The subcellular localizations of two rice AChEs were determined using green fluorescent protein fusions and immunohistochemistry. The two rice AChEs were localized in extracellular spaces. Furthermore, the responses of two rice AChEs to heat and gravity stresses were investigated using transgenic rice plants overexpressed and suppressed two rice AChE genes. (This research was supported by Japan Space Forum.)

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Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through D-galacturonate/L-galactonate as representative intermediates. We report here the identification of a gene encoding aldonolactonase, which catalyzes a penultimate step of the biosynthesis of AsA in Euglena. By a BLAST search, we identified one candidate for the enzyme having significant sequence identity with rat gluconolactonase. The purified recombinant aldonolactonase expressed in E. coli
catalyzed the reversible reaction of L-galactonate and L-galactono-1,4-lactone with zinc ion as a cofactor. The apparent Km values for L-galactonate and L-galactono-1,4-lactone were 1.55 ± 0.3 mM and 1.67 ± 0.39 mM, respectively. The cell growth of Euglena was arrested by silencing the expression of aldonolactonase through RNA interference and then restored to the normal state by supplementation with L-galactono-1,4-lactone. The present results indicate that aldonolactonase is significant for the biosynthesis of AsA in Euglena cells, which predominantly utilize the pathway via D-galacturonate/L-galactonate.

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In Euglena, which lacks catalase, ascorbate peroxidase (APX) is the major H₂O₂-scavenging enzyme. In the present study, to clarify the molecular aspects of the Euglena APX, we have isolated a full-length cDNA clone for the enzyme. The full-length cDNA was cloned by PCR based on the Euglena EST database. Interestingly, the primary structure of the enzyme showed that the enzyme consists of two homologous catalytic domains and 102 amino acids extension at the N-terminal region, which has a typical class II signal for plastid targeting in Euglena. A computer-assisted structure analysis indicated that the enzyme forms an intermolecular dimmer structure. By the cell fractionation analysis, the APX protein had determined in only cytosol fraction, but not in plastid fraction, indicating that Euglena APX became the mature form in cytosol after processing of the precursor.

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Cutinase is a kind of esterases that degrades plant cuticular fatty-acid-polyester "cutin". Cutinases can degrade some polyester plastics as well as cutin, so this enzyme is useful for industrial area. Cutinases have been well investigated in some fungi. It is assumed that fungi use cutinases in their penetration process into plants. In case of plants, cutinase activity was found in pollen grains. Thus, plant cutinase seems to function in pollentube penetration into the stigma. However no plant cutinase genes or proteins have been identified. To identify plant cutinase, we used Arabidopsis thaliana. We have developped a new screening method for isolating a cutinase gene. The screening method and a possible function of the isolated plant cutinase will be discussed.

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We found three types of cysteine protease from spinach leaves, which hydrolyze artificial fluorescent substrate N-succinyl-Leu-Tyr-aminomethylcoumarin (Suc-LY-AMC). They had a maximal activity at pH 5 and we named them SoCP (Spinacia oleracea Cysteine Protease) 1-3. The total activity of these enzymes increased during senescence. Conversion of SoCP2 to SoCP3 was observed and it resulted in a significant change in the molecular weight from monomer to trimer. This change also occurred in vitro, indicating that this conversion occurs not only by senescence, but also by other factors. Highly purified SoCP was a complex consisting of 41-k protein and 14-k protein. By amino acid sequences and cDNA cloning, we revealed that 41-k protein was cysteine protease containing granulin domain and 14-k protein was cystatin, an inhibitor of cysteine protease. In this study, we report on the purification and characterization of SoCP and cystatin, and also their activation and the conversion.

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We have demonstrated that, among 27 types of Arabidopsis Nudix hydrolases (AtNUDX1-27), cytosolic AtNUDX11 and mitochondrial AtNUDX15 hydrolyze CoA and its derivatives (Plant Physiol. 2008, 148: 1412-24, Plant J. in press). AtNUDX15 produced not only mitochondrial protein but also predicted peroxisomal isoform by an alternative splicing. Here we studied the physiological role of AtNUDX11 and 15 using T-DNA insertion mutant of the respective gene (KO Atnudx11 and KO Atnudx15). Activities of CoA pyrophosphohydrolase in KO-Atnudx11 and KO-Atnudx15 mutants were reduced to 80.4 and 46.2%, respectively. There was no difference in growth, flowering, and seed development between the wild-type plants and both mutants. The mutants did now show the inhibition of seed germination and seedling growth in the medium without sucrose and the tolerance against salt (125 mM) which were observed commonly in mutants defective a enzyme involved in CoA biosynthesis. These results suggest that AtNUDX11 and 15 are functionally redundant.

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Phospholipase A₂ (PLA₂) acts on the ester bond at the 2nd position of glycerophospholipids to produce free fatty acids and lysophopholipids. In recent years, as the reports about the gene expression of PLA₂ in plant have been increasing, the attention has been paid to the physiological role of plant PLA₂. However, there is little information on the physiological role of plant PLA₂ compared to that of animal PLA₂. In the rice genome, the four genes with homology to those of animal secretory PLA₂ ( sPLA₂ ) were found. The two of four genes were previously reported expressed in rice. We examined the gene expression levels of the four genes in rice. We found that the four genes were expressed in rice seedlings germinated after one week, and suggested that those genes were functional genes. We are now investigating the gene expression of the four sPLA₂ under various stress condition.

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Plant nitrite reductase (NiR) and sulfite reductase (SiR) have common features in structure and function. Both enzymes are generally distinguished in terms of preferences for nitrite and sulfite as substrates. The Cyanidioschyzon merolae genome encodes two SiR homologs, termed CmSiRA and CmSiRB, but no NiR homolog. As most known SiR have a low NiR activity and CmSiRB is mapped between the genes for nitrate transporter and nitrate reductase, CmSiRB could be a nitrite-reducing enzyme. We characterized catalytic activities of recombinant CmSiRB. The turnover number of nitrite reduction of CmSiRB was relatively high, and the affinity for nitrite was low (Km = >1 mM). The turnover number of sulfite reduction was extremely low, and the affinity for sulfite was very high (Km = <10 μM). This result demonstrates that CmSiRB is a special SiR having reduced activity of sulfite reduction and enhanced activity of nitrite reduction.

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Hsp90 proteins constitute one of the most conserved Hsp families. Many of the identified Hsp90 substrate proteins fall into two classes: transcription factors and signaling kinases. Hsp90 is an emerging therapeutic target for the treatment of cancer since those signaling proteins are associated with cancer cell survival and proliferation. Several small-molecule inhibitors of Hsp90 have shown potent antitumor activity, and are currently in clinical investigation.
The purpose of the present study is to find novel small-molecule compounds that regulate the Hsp90 function. We purified the full-length and various truncated Hsp90 (HtpG) of the cyanobacterium Synechococcus elongatus PCC 7942. We performed chemical array analysis to identify compounds that interact with HtpG. We found that lipopeptide antibiotics bind to HtpG. Next, we investigated effect of these compounds on ATPase activity and chaperone holdase (anti-aggregation) activity of HtpG. They showed no effect on the ATPase activity, but inhibited the holdase activity greatly.

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The genomes of higher plants encode many membrane proteins. Some of the membrane proteins play important roles in biosynthesis and accumulation of useful secondary metabolites. They function as enzymes, catalyzing reaction in biosynthetic pathways, or as transporters of metabolites across membranes. Precise biochemical analyses of these membrane proteins are necessary for elucidating molecular mechanism of biosynthesis and accumulation of useful metabolites. Lately, cell-free protein synthesis systems have been applied to preparation of various membrane proteins for structural and functional analyses. We attempted to synthesize and reconstitute membrane-bound enzymes by wheat germ cell-free protein synthesis system. Cell-free syntheses of enzymes with a membrane-binding domain in their N-termini were performed using wheat-germ extracts in the presence or absence of liposomes. In the case of cofactor-dependent enzymes, optimum concentrations of each cofactor in the reaction mixture for cell-free translation were determined. We have succeeded in cell-free reconstitution of several membrane-bound enzymes.

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Lipid-modified proteins play critical roles in regulation of many cellular functions. Myristoylation is one of well-known lipid modifications of protein. In this process, myristate is enzymatically attached to the N-terminus of target protein. In Arabidopsis, 319 candidates have been predicted as myristoylated protein, but the correlation between protein functions and myristoylation remains unclear. In order to establish efficient in vitro system for preparing myristoylated proteins, we investigated enzymatic activity of protein-myristoylation and its substrate specificity in the wheat-embryo cell-free translation system. By using radio-active myristate, as a substrate, myristoylation of the proteins that had consensus myristoylation sequence was detected in the reaction mixture of cell-free translation for three Arabidopsis proteins. On the other hand, no myristoylation was observed in the proteins that had mutations in the consensus sequence. We thus established simple in vitro detection system for protein-myristoylation based on the wheat cell-free translation technology.

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Protein ubiquitination is implicated in several critical cellular processes. Ubiquitination is mediated by the sequential action of at least three enzymes, the E1, E2 and E3 proteins. Although Arabidopsis genome research estimates over 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrated a novel method for high-throughput in vitro ubiquitination analysis based on wheat cell-free protein synthesis and luminescent detection. The recombinant proteins without purification were used for the assay, and luminescent analysis showed the ubiquitin-conjugation of 29 E2s and a HECT-type E3. Furthermore, this analysis also detected the polyubiquitination of a HECT- and RING-type E3s. Interestingly, these ubiquitinations were carried out without the addition of exogenous E1 and/or E2 proteins, indicating that these enzymes were endogenous to the wheat cell-free system. Based on this study, we are developing the convenient and versatile method to elucidate plant ubiquitination pathway.

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SUMO (small ubiquitin-related modifier) conjugation (sumoylation) to protein substrates is a reversible posttranslational modification that regulates transcription factor activity. The siz1 mutation, impaired in SUMO E3, caused ABA-hypersensitive to seed germination arrest and seedling growth inhibition. Further, expression of ABA-responsive genes was hyper-induced by ABA in siz1 seedlings. abi5 suppressed the ABA sensitivity caused by siz1, revealing an epistatic genetic interaction between SIZ1 and ABI5. A K391R substitution in ABI5 (ABI5^K391R) blocked SIZ1-mediated sumoylation of ABI5 in vitro and in protoplasts, indicating that ABI5 is sumoylated through SIZ1 and that K391 is the principal site for SUMO conjugation. ABI5^K391R expression in abi5 plants resulted in ABA-responsive gene expression, seed germination arrest and primary root growth inhibition greater than that observed in complemented lines (ABI5 expression in abi5). Together, these results demonstrate that SIZ1-dependent sumoylation of ABI5 negatively regulates ABA signaling.

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We are working on Na⁺/H⁺ antiporters from cyanobacteria. We showed that NhaP type Na⁺/H⁺ antiporter from a halotolerant cyanobacterium Aphanothece halophytica (ApNHaP1) exhibited high Na⁺/H⁺ antiporter activity over a wide range of pH. We also showed that ApNapA1 exhibited strongly pH dependent exchange activities. Recently, multi-component Na⁺/H⁺ antiporter (Mrp) was found in a cyanobacterium. We isolated Mrp genes from Synechococcus elongatus PCC 7942 and Aphanothece halophytica. Biochemical and physiological functions of these antiporters were examined and their results will be presented.

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A halophyte Atriplex gmelini possess bladder hairs on the leaf surface. The bladder hairs consist of one- to three-celled stalk and a bladder cell on the top of the stalk, the bladder cells highly concentrating salt under high salinity. Although the bladder hairs are thought to play an important role in salt tolerance, there are few studies on the contribution of bladder hairs to salt tolerance.
In this report, the changes of the morphological and physiological characteristics of the bladder hairs of A. gmelini, grown under several NaCl concentrations, were examined. The light microscopical observation and the localization of Na⁺ by using fluorescent reagent were conducted. The content of ions and a compatible solute, glycinebetaine, were examined. The amount of choline monooxygenase, a key enzyme in glycinebetaine biosynthesis, was also examined.
With the results, the role of bladder hairs under salinity will be discussed.

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In screening of activation-tagged mutants for salt tolerance at cellular levels in Arabidopsis thaliana, we identified 18 potential mutants. In one of them, salt tolerant callus 8 (stc8), a gene for basic helix-loop-helix transcription factor (bHLH106) was found activated regardless of NaCl stress. bHLH106 was highly expressed in calli and salt-inducible. Green fluorescent protein (GFP) fused with bHLH106 has shown that it is located in the nucleus. A knock-out line of bHLH106 was more sensitive to different concentrations of NaCl than the wild-type. The back-transformation has resulted in calli over-expressing bHLH106, which were tolerant to different levels of NaCl stress compared with calli transformed with a blank vector. The over-expression of bHLH106 in differentiated plants has also exhibited tolerance to 100 and 125 mM NaCl. These results indicate that bHLH106 plays an important role in regulation of salt stress in Arabidopsis.

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Thellungiella halophila is a model halophyte with a small plant size, short life cycle, and small genome. Although it is a close relative of Arabidopsis, it shows not only extreme salt tolerance, but also chilling-, freezing-, and oxidative-stress tolerances, suggesting that Thellungiella is a good genomic resource for understanding abiotic stress tolerance. We had generated a full-length cDNA enriched library from various tissues and abiotic stress treated Thellungiella. To elucidate the functions of a large population of Thellungiella genes and to search efficiently for salt tolerance genes, we have been taking advantage of the Full-length cDNA Over-eXpresser gene (FOX) hunting system. We are working on two kinds of FOX hunting, a whole genome FOX hunting and mini-scale FOX huntings using Arabidopsis. In the mini-scale FOX hunting, we extracted 433 genes including abiotic stress inducible genes from the library. We will introduce our recent progress of the mini-scale FOX hunting.

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Regulation of root architecture is a powerful way to adapt to limiting or excessive supplies of nutrients. When roots encounter localized source of nitrate, lateral root growth is stimulated. Studies with Arabidopsis have determined a MADS box transcription factor, ANR1, as a key regulator of this signaling pathway. In this study, we characterized AGL21 which belongs to this gene family. The lateral root length of agl21 mutant was shorter than that of wild-type plant under low-nitrate condition. By contrast, both the numbers and length of lateral roots increased by the over-expression of AGL21. In addition, transgenic plants harboring AGL21 promoter-GFP fusion gene indicated AGL21 is expressed in primary and lateral root tip under low-nitrate conditions. These results suggested that AGL21 is an essential factor to promote lateral root initiation and elongation to adapt with the changes in nitrogen environment.

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To understand the molecular mechanism of Cu tolerance in Arabidopsis, we performed QTL analysis and ecotype studies. One major QTL1 for Cu tolerance in roots explained 52% of the phenotypic variation in Ler/Cvi RILs. The QTL1 regulates Cu translocation capacity and involves a Cu-transporting P_IB-1-type ATPase, HMA5. Complementation test revealed that the Cvi allele is less functional than the Ler allele in Cu tolerance. Yeast complementation assays revealed that N928T of the Cvi-HMA5 allele, which was identified in the tightly conserved domain N(x)₆YN(x)₄P, is a cause of dysfunction of the Cvi-HMA5. Another dysfunctional HMA5 allele was identified in Cu sensitive Chi-2. P626L of Chi-2 was identified in another strictly conserved domain CPC(x)₆P. Polymorphism in HMA5 and Cu-translocating capacity from root to shoot were not significant correlated with Cu-tolerance in root for many Arabidopsis accessions.

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Arabidopsis metal-tolerance protein 1 (AtMTP1) transports zinc into vacuole by Zn²⁺/H⁺ exchanger mechanism. AtMTP1 knockout mutant (mtp1) plants are sensitive to zinc at high concentrations, suggesting an essential role of AtMTP1 in zinc tolerance. In this study, we compared physiological and biochemical properties between the wild-type and mtp1 plants to elucidate mechanism of zinc toxicity and physiological role of AtMTP1 in zinc homeostasis. In 0.5 mM Zn²⁺, roots growth of mtp1 was reversibly inhibited. It may not be due to necrosis of cells. Cell division and elongation were critically suppressed in roots under zinc stress and reactive oxygen species (ROS) were generated in the meristematic zone. ROS may be one of the key factors of zinc toxicity or stress signaling. We will also discuss about the characteristic differences of gene expression, ion valance and cell morphology between wild-type and mtp1 plants under the zinc stress.

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In Synechococcus sp. PCC 7942, expression of smtA gene encoding a metallothionein-like protein, SmtA, is regulated by a transcription factor, SmtB. Excess amount of Zn²⁺ cause the dissociation of SmtB from the operator/promoter region (o/p) of smtA, resulting in the initiation of smtA transcription. In another cyanobacterium, Synechococcus sp. PCC 7002, homologues of SmtA and SmtB appeared to be present from the genome data base. However, the 7002-SmtA is expressed at higher concentrations of Zn²⁺ than the 7942-SmtA, indicating the difference mechanism in response to Zn²⁺. The gel-retardation assay showed that 7942-SmtB formed 4 different complexes with the 7942-o/p, whereas 7002-SmtB formed 6 with the 7002-o/p. It is also found concentrations of Zn²⁺ that inhibits formations of protein-DNA complexes are different between 7942-SmtB and 7002-SmtB. These results suggest that the structural difference of SmtB as well as o/p region are related to specific response to Zn²⁺ in two cyanobacteria.

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Rice requires a large amount of Si for high productivity, especially at the reproductive stage. Recently, we identified three Si transporters in rice. Lsi1 and Lsi2 are involved in the efficient Si uptake by rice roots, while Lsi6 in the xylem unloading of silicic acid. Here we report a novel important role of Lsi6 in Si distribution at rice reproductive stage.
After heading stage, Lsi6 was highly expressed in the nodes in the upper parts of the stem and localized at the xylem parenchyma cells of the large vascular bundles. Knockout of Lsi6 resulted in decreased Si accumulation in the panicles and increased Si accumulation in the flag-leaf. Water-loss from excised immature panicles significantly increased in the knockout line. These results suggest that Lsi6 is involved in xylem unloading of Si in the nodes and is necessary for re-loading onto the vascular tissues toward the panicles.

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HvLsi2 is a homolog of rice efflux silicon transporter OsLsi2 in barley. They share 86% identity. Our previous study showed that HvLsi2 is also able to transport Si out of the cell in Xenopus oocyte. HvLsi2 was mainly expressed in the roots and positive correlation was found between the HvLsi2 expression level and Si uptake in different cultivars. In this study, we further conducted functional analysis of this transporter. To examine the subcellular localization of HvLsi2, we prepared microsome from barley roots and fractionated by sucrose gradient. Western analysis showed that HvLsi2 was in the same fraction as plasma membrane marker HvPIP2;1. Immunostaining showed that HvLsi2 was localized on the endodermis, but did not show polar localization. When HvLsi2 was introduced into rice lsi2 mutant under the control of OsLsi2 promoter, the Si uptake was recovered to the level of wild-type rice. Interestingly, HvLsi2 showed polar localization in rice.

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The physiological studies using deficient mutants of the subunit genes of the heterotrimeric G proteins in plants have suggested that the G proteins were involved in many signaling pathways after perception of external signals. A rice mutant of the α subunit (Gα) gene, d1 showed characteristic phenotypes, such as dwarfism and set small round seeds. Rice Gα may concern with cell division or cell elongation.
We investigated the expression pattern of the Gα gene. First, we constructed the chimera gene which consisted with the promoter of the subunit and the β-glucronidase (GUS) as a reporter gene, namely Gα promoter::GUS and introduced the chimera gene into wild-type rice plants. The GUS expression in transformant expressing Gα promoter::GUS was observed in elongating stage of leaf,internodes,lemma and palea, not in mature stage. These results indicated that the Gα functions at developing tissue.

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Receptor-like kinases comprise a large gene family in Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. We identified a receptor-like cytosolic kinase gene, ABA-HYPERSENSITIVE RECEPTOR-LIKE CYTOSOLIC KINASE1 (ARCK1), which expression is induced during various stresses and ABA treatment. Expression of the ARCK1 promoter:GUS fused gene was detected in cotyledon under normal growth condition. The GUS activity was higher under high salt condition than that under control condition and detected in cotyledon and leaves. The ARCK1-sGFP protein was localized at plasma membrane. We analyzed phenotypes of ARCK1 knockout mutants, arck1-1 and arck1-2. The arck1 mutations did not affect plant morphology under normal growth condition, whereas they germinated slower than the wild type in ABA containing media. These results suggest that ARCK1 is induced by various stresses and might have a role in stress signal transduction pathway in Arabidopsis.

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Cyanobacteria have developed light-harvesting antenna complex phycobilisome. When cells respond to changes in the a viability of various nutrients or to high light exposure, phycobilisome is rapidly degraded. In the cyanobacterium Synechococcus elongatus PCC 7942, it was suggested that the bleaching process is regulated by two-component histidine kinase NblS. To address the molecular mechanisms of the signaling pathway involving NblS, we carried out the yeast two-hybrid analysis. NblS interacted with the response regulators, RpaB and SrrA. Recombinant NblS had the phosphotransfer activity to RpaB and SrrA, respectively. Gel shift assay revealed that RpaB specifically bound to the upstream region of nblA that was induced by bleaching. In addition, either nblS or rpaB defective mutant exhibited the bleaching phenotypes in normal growth condition and the nblA transcripts accumulated in the mutant. These results suggest that the bleaching process is regulated via NblS-RpaB signaling pathway.

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Chloroplasts are involved in the synthesis of stress hormones such as jasmonic acid and production of ROS. However, very little is known about the role of chloroplasts in plant defense responses. We recently found that various biotic (flg22 and chitin) or abiotic stresses (NaCl, sorbitol and H₂O₂) induced the elevation of stromal Ca²⁺ concentration in Arabidopsis. The chloroplast localized putative Ca²⁺ binding protein CAS is involved in the generation of these stromal Ca²⁺ signals. In this study, we found that high salinity-induced chlorosis was accelerated in theCAS knockout mutants. On the other hand, effecter-induced hypersensitive cell death and elicitor flg22- and high osmolarity-induced anthocyanin synthesis were delayed significantly in CAS knockout mutants. Furthermore, we revealed that CAS is involved in the flg22-induced PR1 gene expression (SA pathway). These results suggest that CAS might play a critical role in the promotion of defense responses against these stresses.

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Trichothecenes are a closely related family of phytotoxins produced by a phytopathogenic fungi. Type A trichothecenes, such as T-2 toxin, caused rapid and prolonged activation of MAPKs, and triggered the cell death by activation of an elicitor-like signaling pathway in Arabidopsis. However, a MAP kinase cascade in the response to trichothecenes is unknown. Novel MAPKKK (MKD1) was identified as a subunit of an AtNFXL1 protein complex. We had revealed that AtNFXL1 functions as a negative regulator of the trichothecene-induced defense response. mkd1 mutant growing on a medium without trichothecenes showed no phenotype, whereas a resistant phenotype was observed in T-2 toxin-treated mkd1 mutant compared with wild type. In addition, the expression of MKD1 was induced by application of T-2 toxin. In gel kinase assays showed that the activation of MPK3 and MPK6 by T-2 toxin in mkd1 mutant was decreased compared with wild type.

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Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian NADPH oxidase2, 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 with HEK293T cells, we recently showed that AtrbohC and AtrbohD possessed ROS-producing enzyme activities, which were synergistically activated by ionomycin, a Ca²⁺ ionophore, and calyculin A, a protein phosphatase inhibitor. (Takeda et al., Science, 2008; Ogasawara et al., JBC, 2008). However, functions and activities of the other rbohs are still unknown. To understand the significance and ROS production in plant physiology and its regulation, we have been characterizing all rboh genes using the heterologous expression system. We will report comparative analyses of ROS-producing activities and their regulatory mechanisms of various rboh proteins, and discuss their functional diversity in Arabidopsis.

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Although the functions of some MAP kinases in higher plants were clarified, the functions of group D MAP kinases are obscure. Chlamydomonas reinhardtii, a model alga, encodes five MAP kinases homologs similar to higher plants. MAPK2/4 genes encoded in Chlamydomonas are classified into group D, but the full sequences of the genes were not determined. We have determined each nucleotide sequences of cDNA and genomic DNA, respectively, and have compared the deduced amino acids sequences with those of group D MAP kinases in other algae and higher plants, suggesting conserved domains in group D MAP kinases. The analysis of expression pattern of MAPK2/4 genes suggested that the MAPK2 gene is slightly expressed during normal growth, but the MAPK4 gene isn not not expressed. Now, we are preparing RNAi transgenic algae of each MAP kinases for estimation of the function of each MAP kinases in algae and higher plants.

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