In the reaction center (RC) of purple photosynthetic bacteria, the electron-transfer reaction occurs from special-pair bacteriochlorophyll (P), in the order, accessory bacteriochlorophyll (B_A), bacteriopheophytin (H_A) and quinone A.
We analyzed the subpicosecond time-resolved absorption spectra of RC by singular-value decomposition followed by global fitting. Within 5-ps time range, the three kinds of distinguishable SADS spectra were successfully extracted, i.e., in addition to the bleaching of P, (i) a broad transient absorption in shorter-wavelength side from H_A (assigned to P^*); (ii) the bleaching of B_A and a more broadband transient absorption (P⁺B_A^-) and (iii) the bleaching of H_A and a broad transient absorption around 665 nm (P⁺H_A^-). In preliminary analysis, we obtained a shorter lifetime in P^* -> P⁺B_A^- than P⁺B_A^- -> P⁺H_A^- in contrast to the previous report (Holzapfel et al., Chem. Phys. Lett. 160 (1989) 1). We are now trying to obtain further information measuring near-infrared region.

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We have found that coherent excitation, with approximate 30 fs pulses, of the optically-allowed 1B_u⁺(0) and the overlapped, optically-forbidden 1B_u^-(2) or 3A_g^-(1) levels of carotenoids (spheroidene or lycopene) generates stimulated emission from the 1B_u^-(0) and 3A_g^-(0) level [T. Miki, Y. Kakitani, Y. Koyama and H. Nagae, Chem. Phys. Lett. 457 (2008) 222 ]. Also, we have shown that the axial coordination to the Mg atom of bacteriochlorophyll, i.e., the penta-coordinated state, in the presence or absence of additional axial interaction, can be probed by the width of the ²⁵Mg-NMR signal [Y. Kakitani, Y. Koyama, Y. Shimoikada, T. Nakai, H. Utsumi, T. Shimizu and H. Nagae, accepted by Biochemistry].
We are applying these techniques to determine the 1B_u^-(0) and 3A_g^-(0) levels of the above carotenoids, and the axial coordination / interaction of B800 and B850 bacteriochlorophyll a, when bound to the LH2 complexes from Rba. sphaeroides 2.4.1 and Rsp. molischianum.

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A pinky strain was obtained from the culture of Rhodobacter sphaeroides R26.1, a carotenoidless mutant, which we have been depositing in a freezer (hereafter, call these the pinky strain and "R26.1"). The pinky strain started to grow in the culture of "R26.1", when exposed to white light. Carotenoids extracted from the pinky strain were analyzed by an HPLC apparatus equipped with a diode-array detector. The electronic absorption spectra of a set of carotenoids showed that the pinky strain contained several different carotenoids with the number of conjugated double bonds, n=10, 11 and 12, the major component being carotenoids with n=11. This type of carotenoid composition has never been found either in Rhodobacter sphaeroides or in other purple bacteria. We are now trying (1) to determine the structures of carotenoids extracted from the pinky strain, and (2) to sequence the 16S rDNAs from the pinky strain, "R26.1",and the authentic R26.1.

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Microscopic linear dichroism of fluorescence spectra manifested by polarization dependence in excitation was studied on chlorolplasts in Zea mays leaves. There are at least two spectral components in the room temperature microscopic spectra induced by two-photon excitation with near infrared pulsed laser. One is attributable to photosystem II and its associated antenna, the other is photosystem I and antenna. The former component is highly localized in several regions with a submicrometer dimension, the latter is almost homogeneously distributed over the whole chloroplasts. The two components are also clearly distinguished by excitation polarization dependence. It has been also found that appearance of small abnormal regions with a third spectral feature that has been unreported, which probably indicates a transient state of thylakoid membrane. Combination of fluorescence spectromicroscopy with linear dichroism measurements will thus help us improve modeling of thylakoid membrane structure and changes based on optical microscopy of live chloroplasts.

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In the previous study, we discovered a novel NDH-PSI supercomplex in wild-type Arabidopsis plants and its intermediate supercomplex in the NDH mutants lacking NdhL and NdhM. To further investigate the components of two supercomplexes, we excised the bands containing them from BN-PAGE and used them for LTQ-Orbitrap Mass analysis. The result showed that supercomplex included PSI subunits and NDH complex, while only the soluble NDH subcomplex was missing in the intermediate. To study further the physiological function of the supercomples, we are investigating whether the supercomplex formation is essential for the stability and/or activity of the NDH complex.
In addition, our proteomics analysis also identified several NDH subunit candidates including PPL2, NDF1, NDF2, NDF4 and NDF6 which have been reported recently. So far, two novel NDH subunits, including one FKBP domain containing protein and one transmembrane protein, were further confirmed to be novel NDH subunits by genetics approach.

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Mitochondrial respiratory system in illuminated leaves is closely associated with photosynthesis (e.g. redox-shuttling, photorespiration). The redox state of the respiratory chain during photosynthesis is likely to be dynamically modulated through such metabolic networks. Using HPLC, we established novel technique for the concurrent determination of plastoquinone (PQ) and ubiquinone (UQ) redox states. This method allowed us to know the in vivo redox states of PQ and UQ and to discuss the metabolic interaction between chloroplasts and mitochondria in a quantitative context. We demonstrate the in vivo PQ and UQ redox states, with regard to the differences among several plant species and the transient responses during the induction phase of photosynthesis. We also present the effects of the deficiency of mitochondrial alternative oxidase, a unique terminal oxidase in plant mitochondria, in Arabidopsis thaliana.

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The ε subunit of ATP synthase has been suggested to function as an intrinsic inhibitor of ATPase activity, a regulator of ATP synthesis and a coupling factor. The ε subunit is comprised of N-terminal β sandwich domain and C-terminal helix-turn-helix domain, and the latter domain has been reported to be necessary for the inhibition of ATPase. To understand the physiological role of ε in the enzyme complex, we examined C-terminal truncation mutant of ε of Synechocystis sp. PCC 6803.
We confirmed that only the truncated ε was expressed in the mutant strain by immunoblot analysis. PAM measurement showed uncoupled photosynthetic electron transport activity. The ATPase activity of this mutant strain was higher than that of the wild-type when the assay was carried out using the isolated thylakoid membranes. In contrast, the mutant did not show any difference in growth from wild-type under low light, normal light and light/dark conditions.

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Cyacidioschyzon merolae grows photoautotrophyaclly but not heterotrophycally. Previous studies indicate possibility of utilizing organic compounds. For example, glycerol is known to promote cell growth. We measured effects of addition of organic compounds on the respiration. The respiration rate increased by 30% after adding 0.5% (60 mM) glycerol and 8% after adding 10 mM glucose. Upon addition of 0.5 mM acetic acid, the respiration rates increased temporarily by 50% and then decreased. We will analyze uptake an metabolism of these organic compounds.

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The deduced amino acid sequence of an slr1923 gene of Synechocystis sp. PCC6803 is homologous to archaen F₄₂₀H₂ dehydrogenase, which acts as a soluble subcomplex of NADH dehydrogenase complex I (NDH1). The gene was inactivated by insertion of spectinomycin resistant cassette. Chlorophyll of the mutant is not a normal (3-vinyl) chlorophyll a but a 3,8-divinylchlorophyll a (DV- Chl a). Here we will report the minor but key chlorophylls in the mutant. DV-Phe a and DV-Chl a' were present as the minor pigments, which functions as the primary electron acceptor in PS II and the primary electron donor in PS I. The oxidation potential of DV-Chl a was +0.813 V and almost equal to that of Chl a (+0.805 V).

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In PS I of A. marina, the primary electron donor P740 is a Chl d/d' heterodimer, and the midpoint potential E_m was initially reported as +335 mV, being as much as 100 mV lower than P700 (ca. +430 mV), and hence the Chl d was supposed to have an oxidation potential E_ox lower than that of Chl a. We previously reported that E_ox value of Chl d (+0.88 V ) in acetonitrile was higher than that of Chl a (+0.81 V ). Recently, E_m of P740 was re-examined and found to be +430 mV by Benjamin et al., which is almost equal P700 value, suggesting that the interaction between the special pair chlorophylls (Chl d and d') of P740 might be stronger than that between Chl a and a' of P700, due to the presence of the coordination of formyl group to Mg in P740.

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The regulation of the cyclic electron flow around photosystem I (PSI) was investigated in Arabidopsis using a transient increase in chlorophyll fluorescence after cessation of illumination. The fluorescence signal was absent in air in a mutant lacking the NAD(P)H dehydrogenase (NDH) complex (ndhM), indicating that the signal is due to plastoquinone (PQ) reduction by stromal reductants via the NDH complex. In this plant, the signal was detected in CO₂-free air containing 2% O₂, reflecting ferredoxin (Fd)-dependent electron donation around PSI, which is redundant with NDH-dependent PQ reduction. The post-illumination fluorescence increase was enhanced in a mutant impaired in plastid fructose-1,6-bisphosphate aldolase activity. We showed that the main route for the PSI cyclic electron flow shifts from the NDH-dependent path to the Fd-dependent path in response to sink limitation of linear electron flow.

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Under steady-state photosynthetic conditions in low light, 830-nm absorption (A₈₃₀) was very close to that in darkness, indicating that the primary donor P700 in the reaction center of photosystem I (PSI) was in non-oxidized (reduced) form. However, P700 was not fully oxidized by applying a strong white light pulse. This result indicates the presence of a population of PSI centers with reduced P700 that remains thermodynamically stable during the application of a pulse of saturating light. This population of reduced (and photochemically-) inactive P700 disappeared in intermediate light, but appeared in strong light. To investigate the reason for the accumulation of reduced-inactive P700 in low light, P700 oxidation by a pulse of saturation light was analyzed under various conditions. We concluded that the reduced-inactive P700 in low light was produced as a result of a limitation of electron flow at the acceptor side of PSI.

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The typical dinoflagellate plastid has Chl a, Chl c₂, and peridinin. Like brown alga and diatom, peridinin-containing dinoflagellate was suggested to have acquired its plastid from the red algae through the secondary symbiosis. Plastid genome of the dinoflagellate is consisted of separate plasmid-like molecules encoding single gene. To clarify features of the dinoflagellate PSII core proteins, two dinoflagellate strains and diverse oxygen-evolving photoautotrophs were compared using sequences of psbA/D. Our results determined that 1) due to RNA editing, genomic and complementary DNA encoded different amino acid sequences, 2) the C-terminus of the D1 protein, a critical region in Mn₄Ca-cluster, was conserved, but lacks C-terminal extension involved in systematic assembly of PSII, 3) about 25% (D1) and 20% (D2) of sequence were different between the dinoflagellate and Synechocystis sp. PCC6803. Mutations were localized at the regions involved in the turnover of D1 protein or Q_B function.

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Plants have mechanisms to alleviate its photoinhibition, for examples, heat-dissipation system as observed in NPQ and electron sinks such as photorespiration and WWC. We found a new system to contribute to the protection of PSII from its inhibition.
Low-light grown tobacco plants (LL-plants) were exposed to high-light conditions. The acclimated plants (LH-plants) were back to low-light conditions (LHL-plants)
Both LH- and LHL-plants showed higher values of NPQ and qP against photosynthetic linear electron flow, compared to LL-plants. The increase in qP, PQ oxidation, did not depend on electron sink activity. The increase in only 15% of qP was due to the increase in NPQ. Thylakoid membranes from LH-plants had a tolerance against photoinhibition of PSII, compared to LL-plants, suggesting a new system, PQ-Oxidation System (POS), which contributes to the alleviation of photodamage and differs from both NPQ and alternative electron sinks, in the acclimation to high light stress.

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Lipid peroxide-derived α,β-unsaturated aldehydes (2-alkenals) are potent electrophiles that can modify proteins and nucleic acids. Transgenic tobaccos overproducing the Arabidopsis 2-alkenal reductase (AER) are tolerant to photooxidative stress. This work aimed at the elucidation of their phototolerance mechanism. Upon illumination of strong light, leaves of wild type (SR1) tobacco accumulated 4-hydroxyhexenal and acrolein, but the AER-tobaccos did not, suggesting that these 2-alkenals were responsible for the damage. Because several enzymes in the Calvin cycle are vulnerable to 2-alkenals but thylakoid electron transport chain is much less sensitive, we infer that the tolerance was due to the larger electron sink capacity in the Calvin cycle that was protected from the toxicity of HHE and acrolein.

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In yeast and animals, a single gene encodes eukaryotic translation initiation factor 6 (eIF6) that is involved in ribosome biogenesis. Here we show the presence of two copies of eIF6-like genes in rice and Arabidopsis genomes, which are referred to as Os-eIF6;1 and Os-eIF6;2, and At-eIF6;1 and At-eIF6;2. We found that the insertion of T-DNA into At-eIF6;1 caused embryonic lethal, but the insertion into At-eIF6;2 did not. The experiment with the yeast eif6 mutant suggested a similar molecular function of yeast eIF6 and At-eIF6;1. RT-PCR analysis revealed that the expression level of At-eIF6;1 was much higher than that of At-eIF6;2, and that the expression of only Os-eIF6;2 was inducible by ammonium nitrate treatment. These results suggest that the eIF6 genes may play distinct roles in individual plant species. The result of preliminary analysis suggested that the At-eIF6;1 promoter was active at the place where cell divisions were actively proceeding.

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Expression of Cystathionine γ-synthase (CGS), which catalyzes the key step of methionine biosynthesis in Arabidopsis thaliana, is regulated at the step of mRNA degradation in response to S-adenosyl-L-methionine (SAM). SAM induces translation arrest in cooperation with MTO1 region of CGS1 nascent peptide, which is followed by mRNA degradation. In order to elucidate this translation arrest mechanism, we analyzed arrested ribosome by chemical footprinting and UV crosslinking.
Consequently, translation arrest-specific signals were found at two sites in 26S rRNA. One is close to ribosomal protein L17, which constitutes narrowest region in the ribosome exit tunnel. This implies ribosomal protein L17 is involved in translation arrest. The other located in the P-site of peptidyl-transferase center and this change may induce translation arrest.
We are currently analyzing MTO1 region targets by introducing azide-tyrosine to MTO1 region as an UV crosslinker. How these changes and MTO1 region induce translation arrest will be discussed.

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In the course of studies on post-transcriptional regulation of the Arabidopsis CGS1 gene, which encodes the key enzyme of methionine biosynthesis, we have found that a specific region of the nascent CGS1 polypeptide acts within the ribosome to cause translational elongation arrest in response to S-adenosylmetionine, which is followed by mRNA degradation. In this study, we addressed the question of whether nascent-peptide-mediated ribosome stalling is generally coupled with mRNA degradation in plants, by using an upstream ORF (uORF) system in which the uORF-encoded nascent peptide causes ribosome stalling at the stop codon of the uORF. Promotion of mRNA decay was observed when uORF peptide-mediated ribosome stalling was induced in Arabidopsis callus culture. Since a uORF-contaning genes can be a target of nonsense-medeated mRNA decay (NMD), we are testing the possibility that NMD is involved in the observed mRNA degradation.

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Rice prolamin are composed of cysteine-poor (CysP) and cysteine-rich (CysR) molecules. CysP prolamins have micro-heterogeneity. esp1 mutation reduces the accumulation of CysP prolamins. To clear the genetic regulation mechanism for the synthesis and accumulation of CysP prolamin, Esp1 gene was isolated and the function was analyzed.
High-resolution linkage map was constructed by using the progeny of the cross between esp1 and "Kasalath". Esp1 gene was mapped within 20kb region on the map and five genes were predicted in the region. Sequencing analysis of genomic DNA indicated that single nucleotide in eRF1 coding region of esp1 mutant substituted. Complementation test, which transforms esp1 mutant with eRF1 gene of WT, demonstrated that the Esp1 gene encoded the eRF1. It is reported that eRF1 participates in the translation of the protein, suggesting that eRF1 in rice endosperm functions at the translation of CysP prolamin.

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Active DNA demethylation in vertebrates may also be mediated by a DNA repair pathway. De novo DNA methylation of specific genes in plants can be guided by 24-nt small interfering RNAs. However, it is not known how demethylases are targeted to specific sequences. We report here the identification of ROS3, an essential regulator of DNA demethylation that contains an RRM (RNA recognition motif) domain and binds small RNAs. Mutant analysis suggests that ROS3 acts in the same genetic pathway as ROS1 to prevent DNA hypermethylation and TGS, and immunostaining shows that ROS3 and ROS1 colocalize in discrete foci dispersed throughout the nucleus. These results demonstrate a critical role for ROS3 in preventing DNA hypermethyation and suggest that DNA demethylation by ROS1 may be guided by RNAs bound to ROS3.

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Genomic imprinting is the epigenetic phenomenon that results in mono-allelic gene expression according to their parent-of-origin. In flowering plants, genomic imprinting was found in the endosperm, and the establishment of imprinting is known to require DNA demethylation of maternal allele, but little is know about how DNA demethylation is regulated.
We isolated alarm clock for FWA imprinting 1 (alac1) mutant that were affected the activation of maternally expressed genes, FWA, MEDEA and FIS2. alac1 exhibits fertilization independent endosperm development like mea and fis2. Furthermore, DNA methylation levels within the promoter region of FWA remained higher in the endosperms of mutants than those in WT. These results indicate ALAC1 controls the establishment of genomic imprinting through DNA demathylation. alac1 encodes chromatin related protein. The relationship of chromatin remodeling and DNA demethylation will be discussed.

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Epigenetic mechanisms involving histone modifications, DNA methylation and siRNA regulate gene silencing and heterochromatin formation in eukaryotes. In Arabidopsis, a RNA-dependent RNA polymerase gene RDR2, DNA methyltransferase genes DRM1, DRM2, CMT3, and a histone deacetylase gene HDA6 are involved in siRNA-mediated epigenetic gene silencing.
We identified the genes that were upregulated in hda6 by tiling-array-based transcriptome analysis. Those genes showed considerable overlap with the mapping sites of siRNA and DNA methylation. Surprisingly, the transcriptome analyses using the mutants of hda6, rdr2 and ddc (triple mutant of drm1, drm2, and cmt3) revealed the upregulated genes in hda6 hardly overlapped with those regulated by RDR2 and DDC. These data suggest that HDA6 regulates the gene silencing independently of RDR2 and DDC.

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In Arabidopsis thaliana, the FLOWERING LOCUS C (FLC) gene plays a central role in the repression of the flowering. Transcription of FLC is regulated by epigenetic mechanisms such as covalent histone modifications and histone variant deposition. Here, we reveal the function of histone H2B deubiquitination on the transcriptional activation of FLC. A mutation in the H2B deubiquitinase, SUP32/UBIQUITIN-SPECIFIC PROTEASE 26 (UBP26), resulted in the reduction of FLC expression and an early-flowering phenotype. In the sup32 mutant, H2B monoubiquitination increased at the FLC locus. Furthermore, H3K36 trimethylation decreased and H3K27 trimethylation increased at the FLC locus in the mutant. H3K36 and H3K27 trimethylation is involved in the activation and repression of FLC transcription, respectively. Thus, SUP32 is required for the transcriptional activation of FLC through H2B deubiquitination, which regulates lysine methylation of histone H3.

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Ubiquitin(Ub)/26S proteasome pathway plays an essential housekeeping role to eliminate the proteins which are damaged or misfolded. It is also essential for aspects of cellular regulation by removing short-lived regulatory proteins as a way to fine-tune homeostasis, adapt to new environments, and redirect growth and development. The 26S proteasome consists of two multisubunit complexes, 20S core particle (CP) and 19S regulatory particle (RP). The RP contains thirteen non-ATPase subunits (RPN) and a ring of six AAA-ATPase subunits (RPT).
We reported RPT2a and RPT5a deficient mutants resulted in enlarged leaves, which are caused by accelerated endoreduplication. Here we show that RPT2bRPT5a double deficient mutant reveals the similar phenotype of mutant in microRNA metabolism. The function of RPTs will be discussed in terms of microRNA biogenesis.

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LORE1 (Lotus Retrotransposon 1) is a Ty3-gypsy retrotranposon family in Lotus japoncius, of which transpositional activity has been indirectly demonstrated by the identifications of the three symbiotic (sym) mutants of which responsible genes were inactivated by LORE1 insertions (Madsen, et al. 2005). This time we report the direct demonstration of transpositional activity of LORE1.
The three sym mutants were isolated from same Ac transposon tagging population, established by introducing maize transposon Ac to L. japonicus accession Gifu by tissue culture mediated transformation procedure (Thykjaer, et al. 1995). Therefore, we inferred that the activation of LORE1 is related to in vitro tissue culture process. Our investigations revealed that LORE1 activation can be triggered via tissue culture process, however the transpositions occur in regenerated intact plants, not during tissue culture. Furthermore, LORE1 was shown to transpose in gametophytic manner. Those data proved the feasibility of establishing gene tagging population of L. japonicus using LORE1.

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In Arabidopsis, a transcription factor AtbZIP60 that localizes to the ER membrane and is regulated by proteolysis plays a role in the ER stress response. Additional transcription factor has been considered to be necessary since a T-DNA insertion mutant of AtbZIP60 still showed the ER stress response. In this study, AtbZIP28 possessing a putative transmembrane domain (TMD) was characterized. Truncated forms of AtbZIP28 lacking the C-terminal domain including TMD activated BiP promoters. A fusion protein of green fluorescent protein (GFP) and AtbZIP28 was expressed in Arabidopsis cultured cells. Under non-stress conditions, GFP fluorescence localization almost overlapped with an ER marker; however, ER stress clearly increased GFP fluorescence in the nucleus. Induction of BiP genes were partly suppressed in a T-DNA insertion mutant of AtbZIP28. These observations indicated that AtbZIP28 is an alterative transcription factor involved in the ER stress response.

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The endoplasmic reticulum (ER) body found in Arabidopsis is a spindle-shaped structure that specifically accumulates high levels of PYK10, a beta-glucosidase that bears an ER-retention signal. The molecular mechanisms underlying the formation of the ER body remain obscure. We isolated an ER body-deficient mutant in Arabidopsis seedlings that we termed nai2 (1). The NAI2 gene encodes a member of a unique protein family that is only found in the Brassicaceae. NAI2 localizes to the ER body and a reduction in NAI2 gene expression elongates ER bodies and reduces their numbers. NAI2 deficiency does not affect PYK10 mRNA levels but reduces the level of PYK10 protein, which becomes uniformly diffused throughout the ER. These observations indicate that NAI2 is a key factor that enables ER body formation and the accumulation of PYK10 in ER bodies of Arabidopsis.
(1) K. Yamada et al., (2008) Plant Cell. 20, 2529-2540.

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ER body is an ER (endoplasmic reticulum) derived organelle, which might be responsible for a novel defense system, because it is induced by wounding. ER-localized-type GFP, SP-GFP-HDEL, visualizes ER bodies in addition to the ER network. The fluorescent ER bodies are found in the epidermis cells of cotyledons and roots as spindle-shape structures with major axes of approximately 5 micrometers. To reveal control mechanisms for the ER-body morphology, we established a system for quantitative measurement of the ER-body morphology. By using the quantification system, we identified Arabidopsis mutants having abnormally-shaped ER bodies; long ER body mutants (leb) and short ER body mutants (seb).

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In maturing seeds, storage proteins are synthesized on rough ER as precursors and then are transported to protein storage vacuoles for further processing into mature forms. We previously reported that maigo2 mutant accumulated a large number of MAG2 bodies which are composed of storage protein precursors and ER chaperones within the ER lumen. MAG2 protein might be involved in the exit of storage protein precursors from the ER. In order to clarify mechanism underlying the MAG2-dependent transport, we studied on interacting proteins with MAG2 and found three MAG2-interacting proteins (designated as MIPs). MIP proteins were associated with the ER membrane together with MAG2. mip mutants abnormally accumulated storage protein precursors in their dry seeds. MAG2 body-like structures were also observed in mip seed cells. Our results suggest that MIP proteins cooperate with MAG2, playing a significant role in the transport of storage proteins in maturing seeds.

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Motor protein is essential for not only motive force but regulation and maintenance of membrane trafficking. In animal cell, various motors (mainly kinesins and dynein) are involved in trafficking. Surprisingly, in plant cell, only 2 classes of myosin (plant specific myosin VIII and XI) are involved in. However, they are composed of a lot of members, 4 members exist in myosin VIII and 13 members in myosin XI (Arabidopsis thaliana). Difference in amino acid or domain composition indicates the diversity in cargo binding and motility, regulation.
In order to identify individual functions, all myosin members (full length) were cloned from Arabidopsis. GFP fused myosins were transiently expressed in protoplast of Arabidopsis cultured cell. Live cell imaging revealed various distribution and motility among members. These results showed the wide diversity in plant myosin functions and possible unique and specific mechanisms in plant membrane trafficking.

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Dynamin-related proteins (DRP) are large GTPases that tubulate and pinch off membranes. The Arabidopsis genome has 16 DRPs grouped into 6 subfamilies (DRP1-6). Members of DRP2 subfamily, DRP2A and DRP2B, show similar domain structure to that of dynamin, which is involved in clathrin-coated vesicle (CCV) formation during endocytosis. However, molecular function of DRP2 subfamily is unclear. To investigate whether DRP2 is related to CCV formation during endocytosis, we compared the localizations of fluorescent fusions of DRP2B with those of clathrin light chain around the plasma membrane by variable incidence angle fluorescent microscopy (VIAFM). In our VIAFM images of Arabidopsis cultured cells, the signals of green fluorescent protein-tagged DRP2B (GFP-DRP2B) were co-localized to those of monomeric Kusabira Orange-tagged clathrin light chain (mKO-CLC). Moreover, GFP-DRP2B signals appeared and accumulated to the assembly sites of mKO-CLC. These results suggest that Arabidopsis DRP2 participates in CCV formation during endocytosis.

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Protein translocation across biological membranes is one of the most important processes for living of cells. Sec-pathway mediated translocation of unfolded proteins through SecYE translocon is one of the main protein translocation processes. Bacteria and Plants possess SecA, the essential translocation factor. Recently,the relationship between SecA2, non essential translocation factor, and bacterial virulence has been demonstrated. In plants, Sec-pathway is conserved as thylakoid import pathway. However, this pathway has not been studied in detail, and Accessory Sec-pathway has not been discovered in plants.
Cyanidioschyzon merolae, primary red alga, is encoded two secA genes in nucleus and plastid genome. Both secA genes are transcribed and amino acid sequences are highly conserved with other SecAs. Functional analysis of these secA genes might obtain new knowledge of SecA functions and demonstrate Accessory Sec-pathway in plants.We have examined protein interactions between C. merolae SecAs and measured ATPase activities.

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Catalase is a tetrameric heme-containing enzyme that catalyzes the decomposition of H2O2 to water and oxygen in peroxisomes. We have already reported that an internal sequence, Q-K-L, at the positions -13 to -11 from the C-terminus was essential for targeting to peroxisomes, and the N-terminus region of the internal sequence Q-K-L was also necessary for the import of catalase to peroxisomes. The folded structure on the region containing the internal sequence may be important for the import. Catalase binds heme at the N-terminus region. Here, we report that the capacity of heme-binding is required for the import of catalase into peroxisomes. We are investigating the relationship between the heme-binding, and folding structure of catalase on transport to peroxisomes, to clarify the import mechanism of catalase into peroxisomes.

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Flavonoids are usually conjugated with various sugar moieties by UDP-sugar glycosyltransferases (UGT) in a species-specific manner. It is unclear how the sugar-donor specificity of UGTs evolved although it significantly impacts on large structural diversification of phytochemicals. We isolated flavonoid 7-O-glucuronosyltransferases (F7GAT) from different Lamiales plants. All of the F7GATs were members of the UGT88-related cluster and specifically utilized UDP-glucuronic acid (UDPGA). By homology modeling, we identified a unique Arg residue in the PSPG-box of Lamiales F7GATs. Substitution of this Arg with Trp was sufficient to convert the sugar donor-specificity from UDPGA to UDP-glucose, indicating that the Arg recognizes the carboxylate group of UDPGA by interaction of the cationic guanidinium moiety with the anionic carboxylate oxygen of the glucuronic acid. Our results suggest that differentiation of sugar-donor specificity of UGTs occurred locally in specific plant families after establishment of regiospecificity on the sugar acceptor.

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Plants have evolved mechanisms to tolerate toxic levels of Al on acid soils, but the underlying resistance mechanisms remain to be elucidated. Previously, we have showed that OsSTAR1 was essential for Al tolerance in rice. Here, we characterized its orthologue gene AtSTAR1 in Arabidopsis. A knockout mutant of AtSTAR1 showed hypersensitive to Al, but not sensitive to other metals including Cd and La. Complementation test confirmed that AtSTAR1 was responsible for the Al-sensitive phenotype in the mutant. Different from OsSTAR1, which was mainly expressed in the roots, this gene was expressed in the roots as well as the aerial parts. The expression of this gene was relatively constitutive and did not respond to Al or other metals and low pH. AtSTAR1 was located at root tip region and stomata of young leaf. Furthermore, rice OsSTAR1 was able to rescue the Al-inhibited root elongation in atstar1 mutant.

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We recently characterized two MATE genes, HvAACT1 and OsFRDL1, which both are citrate transporter in barley and rice. HvAACT1 is involved in the Al-induced citrate secretion, while OsFRDL1 is involved in Fe translocation from the roots to the shoots. Here we report the function of a homolog of OsFRDL1, OsFRDL4, in rice. We first compared the phenotype between the wild-type and a Tos17 insertion line. Knockout of this gene did not cause chlorosis and decreased concentration of Fe and citrate in the xylem sap, but resulted in decreased Al-induced secretion of citrate from the roots. The protein encoded by this gene was able to transport citrate out of cell. All our results indicate that OsFRDL4 is a citrate transporter, which is involved in the Al-induced citrate secretion in rice. However, different from HvAACT1, which expression is not induced by Al, the expression of OsFRDL4 is greatly induced by Al.

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In a simple calcium (Ca)- medium containing sucrose or glucose (pH 5.0), cultured tobacco cells exhibit an increase in soluble sugar content, which is blocked by aluminum (Al). The sugar content is determined by the balance between sugar uptake and sugar consumption. In this study, the effect of Al on sugar consumption was investigated. After a pulse label with radioactive sucrose, glucose or 3-0-methyl glucose (non metabolizable), cells were resuspended with Ca medium with or without Al, and was monitored the radioactivity retained in the cells. The results suggest that Al enhances the consumption of soluble sugar.

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Iron is an essencial micronutrient for all the organisms. Iron is incorporated into heme and iron-sulfur cluster, thus works as a cofactor of apoproteins. Previous studies have suggested that biogenesis and transport of mitochondrial iron-sulfur cluster play an important role in whole-cell iron metabolism. To gain a new insight into iron availability in plants, we performed a functional analysis of rice Isu (Iron-sulfur cluster assembly U), OsIsu1. Expressed in yeast cells, OsIsu1 functionally complemented yeast endogenous Isu to maintain mitochondrial iron homeostasis. OsIsu1:GFP localized in mitochondria in onion epidermal cells, like Isu of other species. In Arabidopsis transformed with OsIsu1 promoter:GUS, GUS activity was detected in a vascular bundle and young leaves, which could be active sights of iron transport. Heterologous expression of OsIsu1 in Arabidopsis improved growth under the iron starvation. These results suggest the importance of mitochondrial iron-sulfur cluster biosynthesis on whole-plant iron availability.

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We have investigated the effects of gibberellin (GA) in shoot on the gene expression in root. We have performed microarray analysis of GA-deficient mutant of Arabidopsis thaliana (ga3ox1/ga3ox2), which was grown for 3 weeks, applied by distilled water or GA4 on the shoot, and the root sample was collected 3 days after the hormone treatment. The gene expression level in root was analyzed with or without GA by Real-time PCR and RT-PCR analysis. In this research, we have focused on some metal absorption-related genes which were up-regulated by GA treatment, and compared the gene expression level in root between WT (Col) and GA-deficient mutant. In addition, the gene expression level of some transcription factors was found to be also up-regulated by GA treatment. Those results suggest that GA from shoot affects the gene expression of some metal transporter in root.

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Photosystem I (PSI) is the primary target to be affected by iron deficiency in plant chloroplasts, because of its high iron content. As a result, photooxidative damage to the photosynthetic apparatus occurs under iron-deficient conditions.
In barley, iron deficiency induced the gene of major light-harvesting chlorophyll a/b protein of photosystem II (PSII), Lhcb1. RT-PCR revealed that at least 2 genes of 12 barley Lhcb1 genes were actually up-regulated under a long-term iron-deficient condition. Three barley chlorina mutants which are deficient in Lhcb1 protein to varying degrees were used to clarify the role of Lhcb1 in iron-deficient condition. It was demonstrated that non-photochemical quenching of wild-type plant was increased approximately 2-fold by iron deficiency, whereas that of chlorina mutants were not influenced. Here, we will reveal that the functional model of the Lhcb1-mediated thermal dissipation mechanism in barley grown under iron-deficient condition.

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Expression of AtFRO2 is controlled by promotive signals sent from whole plant to roots under iron deficient condition. On the other hand, the expression of AtbHLH38 is likely regulated by local signals in roots, because the expression is not influenced by leaf-excision. It is showed that AtbHLH38 interacts with AtFIT, and these complex induces the expression of AtFRO2. Therefore, we hypothesize that the long-ditance signal regulated the expression of AtFRO2 via AtFIT which interact with AtbHLH38.
We performed microarray analysis of the iron deficiency response in roots to identify genes which involve in the regulation of iron uptake in roots. Seedlings were grown under iron sufficient or deficient condition, and their roots were harvested 24 hours after all leaves were cut or not cut. The result suggested that 36 genes were influenced by the long distance signals and 43 genes were regulated by the local signals.

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Molybdenum (Mo) is a microelement essential for plant growth. Nitrate reductase, sulfite oxidase, abscisic aldehyde oxydase and xanthine dehydrogenase are Mo-cofactor (MoCo) containing enzymes in plants. Molybdate transporter MOT1 plays an important role in efficient Mo uptake in Arabidopsis thaliana. We performed microarray experiment using A. thaliana wild-type (Col-0) and mot1-1, a T-DNA inserted mutant with reduced Mo accumulation. Plants were grown on solid media with sufficient or deficient Mo for 18 days. We analyzed the metabolic profiles of the plants by GC-TOF/MS and CE-TOF/MS. A number of metabolites and mRNA accumulations were altered significantly by Mo deficiency in mot1-1.

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To understand complex responses of the roots to rhizotoxic ions, we performed comparative transcriptome analysis followed by bioinfomatics characterization. RNA was isolated from the root of Arabidopsis after exposed to aluminum, copper, cadmium and sodium-chloride stress, then analyzed with genome-wide oligo DNA microarray. Our analyses revealed that both general and specific genes were induced in Arabidopsis roots, when exposed to various rhizotoxic ions. Several defense systems, such as ROS production and disturbance of Ca homeostasis, were triggered by all stressors, while specific defense genes (e.g. gene for AtALMT1, Al-activated malate transporter in Al treatment and gene for DREB transcription factor in NaCl treatment) were also induced by individual stressors. Similar studies in different plant species could help to understand resistance mechanisms at molecular mechanism that can be utilized for marker-assisted selection.

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The DREB1A gene of Arabidopsis (AtDREB1A) is an important transcriptional factor that controls many abiotic stress-related genes and confers abiotic stress tolerance on several plant species. From our previously study, AtDREB1A gene conferred abiotic stress tolerance to potato. This result suggests potato also has the system for abiotic stress tolerance controlled by DREB1A. However, the mechanism underlying this system is not understood.
In this study, to elucidate the mechanism of abiotic stress tolerance controlled by DREB1A in potato, we analyzed gene expression profile of non-transgenic potato under abiotic stress condition and that of CaMV35S::AtDREB1A transgenic potato using an Oligo-based DNA microarray. Microarray analysis showed that 98 genes were up-regulated and 19 genes were down-regulated. In addition, to show the correlation between gene expression profile and abiotic stress tolerance in more detail, RT-PCR analyses were also performed.

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We isolated a high-light inducible NAC transcription factor (HLN) by suppression-subtractive hybridization from Arabidopsis. The HLN protein had a highly conserved NAC DNA binding domain in the N-terminal region and a transmembrane (TM) motif in the C-terminal region. The GFP fused full-length HLN (GFP-HLN) protein was localized in cytosol and nucleus, while the truncated form of HLN without TM motif (HLNΔTM) was only in nucleus, suggesting that HLN is released from membrane and translocated to nucleus under high-light stress conditions.
The transcriptome analysis using the HLN-overexpressing (Ox-HLN) Arabidopsis plants showed that the transcript levels of 166 genes are induced and those of 470 genes are suppressed compared with wild-type plants under high-light stress conditions.

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Abscisic acid (ABA) is a phytohormone that regulates seed dormancy, germination and stress responses in plants. Our group had identified the SNF1-related protein kinase 2 (SnRK2) family as ABA-activated protein kinases which positively regulates ABA signaling. On the other hand, ABI-type protein phosphase 2C, e.g. ABI1 or ABI2, had been known as negative regulators for ABA signaling. Therefore, we supposed that SnRK2 and PP2C have some symmetric relationships each other. To confirm this idea, we checked protein-protein interactions by yeast two-hybrid assay and found several specific interactions between SnRK2 and PP2C. Transient BiFC assay or Co-IP confirmed SnRK2-PP2C interaction in vivo. Furthermore, subcellular localizations or gene expression patterns suggested functional interactions of each SnRK2 and PP2C. We will discuss about the regulatory mechanism of ABA signaling by SnRK2-PP2C complexes.

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Under abiotic stress conditions such as drought and high salinity, abscisic acid (ABA) levels increase in plants, thereby triggering the expression of many genes that function in the stress tolerance. Many ABA-inducible genes contain a conserved cis-element designated ABRE (ABA-responsive element) in their promoter regions. Arabidopsis cDNAs encoding bZIP-type transcription factors referred as ABRE-binding (AREB) proteins or ABRE-binding factors (ABFs) were isolated using the yeast one-hybrid screening method. Among these transcription factors, expression of AREB1, AREB2, and ABF3 was upregulated by ABA, dehydration, and high-salinity stresses in Arabidopsis plants. Based on results of analyses of areb1 areb2 abf3 triple mutant, here we report that these AREB family transcription factors are involved in the ABA and stress signaling. Moreover, taken together with the information on expression patterns of these proteins, we will discuss the role of each AREB transcription factor.

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A histidine kinase, Hik33, in Synechocystis sp. PCC 6803 regulates gene expression under various stress conditions. Generally hisitidine kinases perceive specific stimuli via signal-input domains (SID) located at the N-terminal portions. An SID in Hik33 includes two transmembrane helixes, a periplasmic loop, HAMP and PAS domains. To investigate roles of the SID in the perception of signals, we expressed a chimeric kinase combining the SID from Hik33 with a kinase domain from SphS, which regulates expression of the phoA gene for alkaline phosphatase, in the mutant of SphS. And the expression levels of the phoA gene were determined under standard, chilling or salt conditions. The chimeric kinases expressed the phoA gene under the standard conditions and repressed it under the stress conditions. Removals of the regions of SID from the chimeric sensor indicated the important regions for perception of the signals to regulate the gene expression.

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In plants, under drought stress conditions, ABA levels increase, and then the ABA regulates the expression of many genes that function in the stress tolerance. In the promoters of such ABA-regulated genes, the conserved cis-elements designated ABREs (ABA-responsive elements), which control expression of ABA-responsive genes, have been identified. The Arabidopsis cDNAs encoding bZIP-type transcription factors referred as ABRE-binding (AREB) proteins, were isolated using the yeast one-hybrid screening system. We demonstrated that AREB1 is a transcriptional activator of ABRE-dependent ABA signaling involved in enhancing drought stress tolerance. Moreover, the ABA-dependent multisite phosphorylation of AREB1 by SnRK2 protein kinases has been shown to regulate its own activation. In this study, mainly based on the molecular characterization of SnRK2 protein kinases using SnRK2 mutants, we will discuss the roles of the SnRK2 protein kinases via the activation of AREB1 in ABA and drought stress signaling.

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A phytohormone, abscisic acid (ABA) induces stomatal closure to reduce transpirational water loss via elaborate signal transduction in guard cells. Histidine kinases are known to function in the histidine-to-aspartate phosphorelay signal system. In plants, perception of phytohormones, ethylene and cytokinin, is composed of the signal system. In this study, we reported that gene disruption of an Arabidopsis histidine kinase, AHK5 causes ABA hypersensitivity of guard cells. In the ahk5-1 mutant, ABA-induced stomatal closure was enhanced. Furthermore, inhibitory effect of ABA on inward rectifying potassium channel currents of guard cell plasma membrane was stronger in the ahk5-1 mutant than in wild type. These results suggest that AHK5 functions as a negative regulator of ABA signaling in Arabidopsis guard cells.

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