We evaluated the effect of

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In the light-harvesting system, carotenoids capture light-energy to be the optically-allowed 1B_u⁺ state, and transfer it to the bacteriochlorophyll. The singlet-energy transfer can carry out from not only the 1B_u⁺ state but also the other low-lying optically-forbidden states, e.g., 3A_g⁻, 1B_u⁻ and 2A_g⁻ states. The rates of internal-conversion among these singlet states are too fast to complete the vibrational-relaxation and -redistribution. Since the ordering of the state energies drastically dependent on the conjugation length, internal-conversion pathway should change one carotenoid to another. Kerr-gate fluorescence spectroscopy having high spectral resolution is a powerful technique to determine precise transition energies of such a complicated system. We try to analyze Kerr-gate fluorescence spectra of a set of carotenoids having different conjugation length by simulating the internal conversion with Franck-Condon factor and the rate of vibrational relaxations as parameters and including vibrational redistribution between the C=C and the C-C stretching modes.

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Subpicosecond time-resolved absorption spectra were recorded in the visible region for a set of photosynthetic carotenoids (Cars) having different numbers of conjugated double bonds including neurosporene (n=9), spheroidene (n=10), lycopene (n=11). Anhydrorhodovirin (n=12) and spirilloxanthin (n=13). Singular-value decomposition and subsequent global fitting of the spectral data matrices lead us to a branched kinetic model including both (1) singlet internal conversion in the sequence of 11Bu+ * 11Bu- * 21Ag- * 11Ag-(G), and (2) singlet-to-triplet fission of 11Bu- * 13Ag plus 13Bu, and subsequent triplet internal conversion of 13Ag * 13Bu. With the knowledge of the new internal conversion in Cars, it is very crucial to re-examine the energy transfer pathway from Car to bacteriochlorophyll (BChl) proposed previously.

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In photosynthetic-bacterial antenna complexes(LHC), the

photo energy is transferred from the peripheral LH2 to the

LH1 which surround the reaction-center (RC)and finaly to

the RC. A Carotenoid(Car)in the RC dissipate harmful

triplet bacteriochlorophyll(T₁ BChl)via triplet-

energy transfer. Additionally, the LH1 in the RC-LH1(Core)

complex may restrict the energy transfer to the RC to

prevent T₁ BChl generation. Now we focused on

the Core, which is a primitive photosynthetic-unit, to

investigate the excited-state dynamics of the pigments. The

Core was isolated from Rhodobacter sphaeroides G1C

and confirmed their purity by SDS-PAGE. Time-resolved

absorption spectra of the Core show that triplet-excited

state appears more obvious and the Qy state of BChl which

appears as a result of the energy transfer of

Car→BChl is less populated than those in the LH2.

These phenomena can be explained as a reduction of energy

transferred to the oxidized RC.

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Our project is focused on studies of ultrafast energy transfer from carotenoid to BChl in LH1 complexes. It is known that photosynthetic LH1 complex devoid of carotenoids dissociates into B820 and B780 subunit forms in the presence of detergent. B820 subunit consisting of dimeric BChl a was isolated from carotenoid-less mutant Rhodospirillum rubrum G9 strain using a mild detergent β-OG and purified by ion-exchange chromatography on DEAE-cellulose. The subunits bind carotenoid molecules (e.g. anhydrorhodovibrin) and form carotenoid-containing complex. In the reconstituted complex BChl main absorption peak (Q_y) undergoes a large red-shift and has a maximum at 880 nm, as in the native LH1 antenna. The reconstituted complexes having various carotenoids will be investigated by fluorescence and femto-second absorption spectroscopy.

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In order to reveal the photo-protective functions and the excited-state dynamics of chlorosomes from Chlorobium tepidum was examined by pump-and-probe time-resolved absorption spectroscopy in subpicosecond-to-millisecond time region. Upon excitation using a ∼400 nm, 0.12 ps pulse, the singlet-singlet annihilation reaction as well as a pair of the singlet homofission and the reverse triplet-triplet annihilation reactions were seen as bi-molecular reactions. Upon excitation using a 355 nm, 12 ns pulse, a slow dissociation of chlorosomes into the piggy-back dimers was seen after the excited-state reactions; the chlorosome structure was reorganized within ∼20 ms. Thus, the cylindrical aggregate structure of chlorosomes facilitates efficient radiative energy dissipation either through the initial Q_y stimulated emission or through the delayed Q_y stimulated emission. The excess thermal energy that is produced by the internal-conversion processes seems to trigger an instantaneous shut down of the efficient light-harvesting function of the particular aggregate structure.

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In two Rhodobacter and Rubrivivax, all of the carotenogenesis genes have been cloned and the characteristics of enzymes have been investigated. From these and the chemical structures of carotenoids, we can consider the carotenogenesis pathways and the enzymes in purple photosynthetic bacteria. When one enzyme in the normal spirilloxanthin pathway is lacking or is present with reduced activity, the pathway and the composition will be expected to change. These are named as the unusual spirilloxanthin pathways. When CrtI produces neurosporene, the final product is spheroidene. This is the spheroidene pathway, and the additional CrtA is always present. Accumulation of lycopene is due to reduced activity of CrtC. Rhodopin is accumulated in some species, and this is due to reduced activity of CrtD. In the case of the rhodopinal pathway, CrtD is inactive, and additional enzyme(s) to form aldehyde group is present.

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In bacteriochlorophyll-biosynthesis, bchX, bchY, and bchZ genes have been suggested to be responsible for saturation of RingΙΙ of the porphyrin ring. The amino acid sequence of BchX resembles to that of BchL, a subunit of the enzyme working in RingIV saturation, BchY and BchZ are corresponding to BchN and BchB of this enzyme, respectively. Although BchL, N, and B are known to serve as a complex, it hasn^'t been shown whether BchX, Y, and Z form a complex. We constructed three mutants lacking bchX, bchY, and bchZ, respectively, and analyzed pigments accumulated in these mutants. All of the mutants grown under semi-aerobic conditions accumulated same pigments with a similar ratio. By MALDI-TOF, compounds derived from chlorophyllide a and 3-devinyl-3-hydroxyetyl chlorophyllide a were detected. Significant accumulation of chlorophyllide a derivatives suggested that the saturation of RingΙΙ by BchX, Y, Z precedes hydroxylation of vinyl-group of RingI.

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Chl a is synthesized from chlorophyllide (Chlide) a by esterification of long alcohol chains to its side-chain of ring D, while, BChl a is synthesized from bacteriochlorophyllide a. To convert Chlide a to bacteriochlorophyllide a, two reactions, the reduction of the double bond of ring B and the oxidation of the C3 vinyl group, are necessary. A 3-vinyl bacteriochlorophyllide hydratase which is encoded on bchF gene is seemed to be involved in the latter reaction. We introduced a gene of 3-vinyl bacteriochlorophyllide hydratase, bchF, from purple bacteria Rhodobacter capsulatus into cyanobacteria Synechocystis sp. PCC6803. HPLC analysis revealed that 4 kinds of polar Chl derivatives which are not observed in wild type cells are presented in Synechocystis sp. PCC6803 that express BchF. The absorption spectra of these pigments are very similar and FAB-mass analysis indicated all of the pigments have a same mass-number, suggested these are 3-hydroxyethyl-chlorophyllide a and its stereoisomers.

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The light-independent protochlorophyllide (Pchlide) reductase (DPOR) is the determinant enzyme for the greening in darkness. In the previous study, we have reported the reconstitution of DPOR activity with the purified components, BchL and BchNB. In this report, we have constructed an over-expression system of each component of DPOR with a broad host range vector pJRD215 in Rhodobacter capsulatus to purify the components in an amount enough for further characterization. A pair of plasmids, pYCL10 and pYCNB111, was constructed on pJRD215 to over-express BchL and BchNB, respectively, under the control of the puc promoter. This expression system allowed purifying 100-300 μg of each component from a 500-ml culture. Interestingly, the purified BchNB component showed bright green color, with the absorption spectrum indicating the binding of Pchlide. This feature suggests that the BchNB component provides a binding site and a catalytic center in the DPOR complex.

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Fluorescence of bacteriochlorophylls (BChl) c and a in isolated chlorosomes of Chloroflexus aurantiacus was shown to be controlled by two types of redox-sensitive mechanisms. Fluorescence of the 750 nm Bchl c band decreased as the redox potential of the medium was shifted positive. Aerobic redox titration indicated an apparent Em of 410mV for decrease to 1/5. Decay lifetimes of Bchl c band was decreased from 24 and 480 ps to 9.1 and 227 ps by ferricyanide. The two-step fluorescence quenching is different from the quenching in green sulfur bacterial chlorosomes (1-3) and seems to be evolved in the different growth environments. 1) N V Karapetyan et al. (1980) BBA 593: 254-260.2) J Wang et al. (1990) BBA1015: 457-463. 3) Frigaard et al.(1998) Photosynth.Res 58: 81-90

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Rhodovulum sulfidophilum has cytochrome c₂ and cytochrome c-549 as electron donors to the reaction center, and cytochrome c-549 was suggested to be the main donor. However, cytochrome c-549 appeared only in the stationary phase of growth. Since cytochrome c-549 showed a similar band pattern to that of a membrane-bound electron donor in Rhodobacter capsulatus in SDS-PAGE, it was considered that c-549 was originated from a membrane-bound component by a proteinase cleavage. To investigate if a membrane-bound component is working as the electron donor to the reaction center-bound cytochrome in this species, membranes free from water-soluble components were prepared from spheroplasts. In the prepared membranes, a rapid re-reduction of cytochrome in the reaction center complex was observed after flash-induced oxidation. The re-reduction of cytochrome in membranes free from soluble components strongly suggests that a membrane-bound electron donor is working in this bacterium.

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A chlorosomes-less green filamentous bacterium Roseiflexus castenholzii has a similar photosynthetic reaction center (RC) to Chloroflexus aurantiacus. The pathway of photosynthetic electron transfer to RC in Cfx. aurantiacus has not been known, except for the participation of auracyanin as the electron donor to RC. Spectroscopic measurements in R. castenholzii are easier than in Cfx. aurantiacus due to the lack of chlorosomes.
In the membrane preparations of R. castenholzii, the photooxidized RC-bound cytochrome c was re-reduced after the flash activation, indicating electron donation from membrane-bound proteins. Since an auracyanin-like fraction prepared from the membrane reduced the RC-bound cytochrome c , this protein was suggested to be auracyanin. In the presence of 20μM of HQNO, the re-reduction of the RC-bound cytochrome c in the membrane was inhibited. This result suggests that a quinol-auracyanin oxidoreductase as well as auracyanin are working in the electron transfer pathway to RC in R. castenholzii.

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One of the important functions of carotenoids in photosynthetic organisms is protection against photooxidative damage. In this study, we compared photoprotective functions of carotenoid species by using six mutants of purple photosynthetic bacterium, Rubrivivax gelatinosus. Growth analysis under aerobic high-light conditions and viability analysis under artificially generated singlet oxygen showed that neurosporene and spheroidene have rather low photoprotective function. Although lycopene and spirilloxanthin showed significant photoprotective function in the growth analysis, these carotenoids showed relatively low activity in scavenging singlet oxygen. Keto-carotenoids, diketospirilloxanthin and spheroidenone, showed highest protective activity against oxidative damage than other carotenoids. It was suggested that the high protective activity of keto-carotenoid is caused by the presence of keto group, and not by the length of conjugated double bonds.

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The reaction center (RC) of heliobacteria seems to have evolved from an ancestor common with photosystem (PS) I RC of cyanobacteria and plants. However, the reaction of quinone that is obvious in PS I heterodimeric RC has never been evidenced in heliobacterial RC. The RC as well as that of green sulfur bacteria, which is also type-1, are considered to form the symmetrical homodimeric structure. We previously carried out the nanosecond absorption spectroscopy of the primary electron acceptor A₀ at 77K in two different RC preparations from Heliobacterium modesticaldum, and showed that re-oxidation of the photo-oxidized P798⁺ occurs with a t_1/2 = 2 ms, presumably ascribable to the charge recombination between the reduced Fe-S centers and P798⁺. No indication of the quinone function was detected. We here report the F_A- and F_B-like ESR signals in the membrane preparations and discuss their detailed features in terms of the electron transfer mechanism.

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Transient ESR spectra of the membrane fragments of Heliobacterium modesticaldum are observed at X-band (9 GHz). In plant photosystem(PS)I,both P700+ and A1-radicals show symmetric broad ESR spectra with peak-to-peak linewidth of 8-10G statically. ESR spectra after flash excitation show the spin polarized pattern of A/E/A from low magnetic field (E:emission,A:absorption) that are attributed to the P700+A1-. ESR spectrum of P800+ in H.modesticaldum shows lineshape similar to that of P700+. However, the spin-polarized radical pair in H.modesticaldum shows the pattern of E/A/E and seems to be ascribable to the P800+Fx-. We will calculate ESR lineshape based on the model.

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We have prepared two Fds (FdA, B) from Heliobacillus mobilis and determined their N-terminal amino acid sequences. FdB is sensitive to oxygen, its A₃₈₅ value being halved in 2 hours at 4^oC under air. On the other hand, the A₃₈₅ of FdA was not decreased during 20 hours under the above conditions. We have also determined the nucleotide sequences of these Fd genes. The two Fd genes are arranged in tandem. The sequences of FdA and FdB show high similarity with those of clostridial Fds and proteobacterial Fds, respectively. In the presence of Chlorobium tepidum RC and spinach FNR, FdA and FdB supported NADP⁺ photoreduction, but the rates were about 60% and 30% of those supported by C. tepidum Fd, respectively. RC particles were solubilized from membranes of H. mobilis with detergents, and partially purified by sucrose-density ultracentrifugation and by anion-exchange chromatography. The RC preparation photoreduced C. tepidum Fd.

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An anionic glycerolipid, sulfoquinovosyldiacylglycerol (SQDG), is present in the thylakoid membranes of photosynthetic organisms. We previously produced a mutant defective in SQDG synthesis from a cyanobacterium, Synechocystis PCC6803, for the study of the role of SQDG in photosynthesis, demonstrating that SQDG is essential for growth of PCC6803 and has an important role in photosynthesis. In this study, we found that SQDG plays an important function for the maintenance of normal properties of photosystem II (PSII). In contrast, the SQDG-null mutant of Synechococcus PCC7942 we produced could grow without SQDG-supplementation as was reported previously for another SQDG-null mutant of PCC7942 (Güler et al., 1996, J.Biol.Chem. 271, 7501) and showed the normal properties of PSII. Therefore, the difference exists for SQDG requirement of PSII and growth in cyanobacteria. It would provide us with a clue for elucidation of the molecular interaction of SQDG with the PSII complex.

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Glutamine transporter has important roles on the glutamate-glutamine cycle in glutamatergic synapses of mammals. To examine glutamine-mediated regulation system on expression of nitrogen assimilatory genes in rice, we isolated a gene encoding the rat neuronal glutamine transporter (rGlnT) homologue from rice.
One rice cDNA (C10038) encoding rGlnT homologue was found by an EST-database search. This gene (OsGlnT) and its full-length cDNA were isolated by using PCR. The presumed transcribed region of OsGlnT gene, consisted of 6 exons separated by 5 introns, encodes 50 kDa protein, which has 11 hydrophobic membrane-spanning segments. Phylogenetic tree analysis showed that OsGlnT was clustered with mammal glutamine transporters (ATAs and SNs) but was not clustered with any plant amino acid transporters reported previously. OsGlnT transcript constantly accumulated in roots and leaves of rice plants. Further analyses of expression of OsGlnT and function of this gene product are now in progress.

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The major form of nitrogen in paddy fields is NH₄⁺, requiring ammonium transporter (AMT) in roots. Also, in plant metabolisms AMTs are required in several processes. To characterize AMTs in rice, two cDNAs with high homology to MEP/AMT2-type AMTs, OsAMT2;1 and OsAMT3;1, were isolated. Expression of OsAMT2;1 in an ammonium-uptake defective yeast mutant showed that this gene encodes functional AMT. OsAMT2;1 was constitutively expressed in both roots and shoots irrespective of the supply of inorganic nitrogen to medium, whereas OsAMT3;1 expression was relatively weak. Database search with amino acid sequence of OsAMT2;1 showed that there are ten putative OsAMT genes, i.e. 3 each for OsAMT1, OsAMT2 and OsAMT3, respectively, and 1 for OsAMT4. Except OsAMT4;1, these genes were expressed in roots and leaf blades. NH₄⁺-inducible accumulation of OsAMT2;2 mRNA was seen in roots, while OsAMT3;3 was expressed following leaf senescence. Expression analyses are now in progress.

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Addition of ammonium to the cultures of Synechococcus sp. PCC7942 reversibly inhibits the activity of nitrate/nitrite transporter (NRT). NRT of the cyanobacterium is an ABC transporter, one of the ATP-binding subunits of which (NrtC) has a large C-terminal domain (R domain) required for the ammonium-promoted inhibition of NRT. To gain insight into the molecular mechanism of the regulation of NRT, we expressed the R domain as a His-tagged protein in E. coli and characterized it biochemically. Equilibrum dialysis experiments showed that the recombinant R domain specifically binds 2-oxoglutarate (2-OG) with a dissociation constant of ~190μM. The intracellular 2-OG concentration of Synechococcus sp. PCC7942 was determinded to be ~150μM in nitrate- or nitrite-grown cells, which was decreased rapidly to ~10μM after addition of ammonium. These results suggested that 2-OG is likely to affect NRT activity by binding to and dissociating from the R domain in vivo.

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Nitrate transporter (NRT) mediates the first step of nitrate assimilation. Plants have two distinct types of NRT; NRT2 type NRT is distributed widely among both procaryotes and eucaryotes, whereas NRT1, which belongs to the peptide transporter family, has been found only in higher plants. To study the structure-function relationships of NRT2, we cloned NRT2 cDNAs from the moss Physcomitrella patens, which is amenable to gene manipulation through homologous recombination. In the phylogenetic tree, the five NRT2 homologs identified from the moss are clustered between green algal and vascular plant NRT2s, with stronger similalities to the plant NRT2s. The serine residues presumed to be involved in regulation of NRT2 are conserved in the moss NRT2 sequences, and mRNA expression was induced by nitrate as in higher plants. These data suggest that P. patens is a good model organism to be used for studies on regulation of plant NRT2.

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Although metabolism of reactive nitrogen species (RNS) plays critical roles in both modulating NO signaling and anti-RNS protection, little is known, especially in plants, of its biochemistry and molecular biology. RNS cytotoxicity is probably mediated by the formation of peroxynitrite, a potent oxidizing and nitrating agent. We showed that the purified recombinant protein of Arabidopsis 2-Cys peroxiredoxin (2CPRX), originally identified as a hydroperoxide-reducing peroxidase, can scavenge peroxynitrite efficiently. Functional expression of the Arabidopsis 2CPRX in the 2CPRX-deficient yeast resulted in complementation of the hypersensitivity of the mutant to RNS and reactive oxygen species. This provides the first in vivo evidence of the protective role against RNS toxicity of 2CPRX from higher eukaryotes. These results demonstrate a new role of plant 2CPRX as a critical determinant of the resistance to RNS, and support the existence of a plant enzymatic system for RNS metabolism.
(Sakamoto et al. Plant J., in press)

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Plants take up atmospheric nitrogen dioxide through stomata, and metabolize it to organic nitrogen compounds such as amino acids. However, it is not clear whether plants can use nitrogen dioxide as an alternative fertilizer. In this study, we addressed whether plants can grow with nitrogen dioxide as a sole nitrogen source.
Wild type and a nitrate reductase-deficient mutant line (E-23) of Nicotiana plumbaginifolia were used. They were grown for 3-6 weeks under natural light at 22^oC in the presence of 4 ppm nitrogen dioxide. There were irrigated either with a medium containing 9.46 mM ammonium succinate or with a medium without nitrogen. It was found that E-23 plants grown with the culture medium lacking nitrogen showed a very similar growth (3-4 times in height) to that of those grown with ammonium succinate medium. This indicates that nitrogen dioxide can serve as an alternative nitrogen fertilizer in this plant species.

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Plants take up nitrogen dioxide (NO₂) and assimilate its nitrogen through a primary nitrate assimilation pathway. However, we found that a part of the nitrogen of NO₂ is converted into compounds in plants whose nitrogen is not recoverable by the Kjeldahl method. We designated this nitrogen as unidentified nitrogen (UN). UN-bearing compounds may include nitro compounds, including nitrated protein-tyrosine. We therefore addressed nitration of protein-tyrosine in plant leaves fumigated with NO₂.
Tobacco plants (Nicotiana tabacum cv. Xanthi) were fumigated with 4 ppm NO₂ for 8 h, and from their leaves protein extracts were prepared. The proteins in the extracts were separated using SDS PAGE and 2-D PAGE and then analyzed immunologically. Spots that react with the antibody were detected, and mass spectroscopic analyses identified them as pathogenesis-related proteins. This result indicates that nitration of protein occurs in response to fumigation of plants with NO₂.

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Activity changes of ammonium uptake, assimilation and translocation (UAT) were investigated by growing rice in hydroponic culture ambient (36Pa) or elevated CO₂ (110Pa) using Positron Tracer Emitting Imaging System (PETIS). UAT was measured by ¹³N translocation to the plants from the ¹³NH₄⁺ supplied to the roots was taken up. The UAT per plant weight of 110Pa was higher than 36Pa in nitrogen sufficient culture conditions. 3d-Nitrogen deficiency enhanced the UAT of both 36Pa and 110Pa than the culture conditions. On subsequent experiments, the UAT of 36Pa was down-regulated with following resupply of 2 mM nitrogen for 2h to the 3d-N-starved plants, while the UAT of 110Pa was up-regulated. In case of glutamine treatment instead of nitrogen, the UAT of 36Pa was promoted, but 110Pa UAT was suppressed. Effects of nitrogen status change and elevated CO₂ on ammonium assimilation activity are discussed.

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The comparative induction of non-symbiotic hemoglobins (Hbs) by nitrate, nitrite, and nitric oxide in cultured rice cells were investigated. Rice possesses two Hb genes, ORYsaGLB1a and ORYsaGLB1b. RT-PCR analysis using gene specific primers showed that the transcription of ORYsaGLB1a and ORYsaGLB1b was induced in nitrate treated cells. Maximal induction of ORYsaGLB1a and ORYsaGLB1b mRNA occurred after 8h of treatment with nitrate. In nitrite-supplied cells, the induction of ORYsaGLB1a and ORYsaGLB1b mRNA was more rapid than nitrate treated cells, where ORYsaGLB1a and ORYsaGLB1b was detected 0.5-1 h after the addition of nitrite and increased to a maximum at 4 h. The treatment of rice cells with S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, also resulted in an accumulation of ORYsaGLB1a and ORYsaGLB1b mRNA with a maximum induction at 2-4 h after treatment. The present experiments indicate that nitrate, nitrite, and nitric oxide is an effective inducer for Hbs in cultured rice cells.

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Snare proteins lie at the heart of membrane fusion events in the secretory and endocytic pathways. Development of root nodules on legume plants following interaction with Rhizobium involves a series of unique cellular and molecular events. The nature of this newly formed subcellular compartment and the targeting mechanism to this structure are not known. In order to dissect the process of nodule specific membrane biogenesis, we have screened 12 SNARE-like clones that have high homologies to SNARE genes of yeast from EST clones of Lotus japonicus. Northern blot analysis revealed that two genes expressed at high level in nodule. These two genes are high homology to sed5 gene of yeast. In situ hibridization showed that these genes are localized at the non-infected cells. We try to find the immuno localization of these proteins.

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cDNA macroarray systems were applied to compare the gene expression profile of Fix^- mutant Ljsym75 nodules with that of wild-type Gifu nodules in order to reveal host plant genes related to nitrogen fixation. In total 18,432 non-redundant clones, expressions of 56 genes were reduced and 45 genes were enhanced in sym75 nodules significantly compared with those in Gifu nodules. These results were mostly confirmed by northern blot analysis. Genes of which expression was lower in sym75 nodules were represented by nodulins such as leghemoglobin and Nlj21, enzymes involved in carbon and nitrogen metabolism such as sucrose synthase, phosphoenolpyruvate carboxylase and asparagine synthetase, membrane transport proteins such as aquaporin, pathogenesis-related proteins, and enzymes involved in phytohormone metabolism. On the contrary, enzymes concerning with senescence such as cysteine proteinase and metal binding proteins such as metallothionein were included in those of which expression was higher in sym75 nodules.

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Our previous QTL and linkage analyses showed that a QTL (Pnw1) associated with panicle weight on the main stem of rice was located in the vicinity of marker C777 on chromosome 2. In this case, the alleles from Kasalath contributed to increases in panicle weight. A nearly isogenic line, C-22, was selected in Koshihikari's genetic background. The C-22 confirmed a small segment (50cM), including Pnw1, which was substituted for Kasalath chromosome. In a green house, the C-22 showed more total panicle weight and more panicle number than Koshihikari, particularly when grown with low-nitrogen supply. A high-density genetic map around Pnw1 was constructed with newly 20 markers using 200 segregates. For further linkage analysis, we selected 177 plants that occurred recombination between marker C777 and C10005 using 3385 segregates. Genotyping of these plants and linkage analysis are now in progress.

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In leaves of Alocasia odora, a shade species, respiration rates were lower than those of sun species. These low rates are important for the carbon balance under low-light environments. In vivo activities of alternative oxidase (AOX) were also lower. AOX is not coupled to ATP production. Since the respiratory rates are already low, the low activity of AOX in A. odora leaves could optimise energy production. We analysed what biochemical mechanisms caused low AOX activity in A. odora leaves. A. odora leaves had a large quantity of AOX and most of the AOX protein in A. odora leaves was in the inactive oxidised dimer form under a low light environment. Under photo-oxidative conditions, AOX was found to be in the active reduced dimer form. We hypothesise that AOX prevents over-reduction of the respiratory chain under such an environment.

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RNA editing in plant mitochondria alters several hundred C nucleotides to U residues in coding regions of mRNAs. To analyze the biochemical and the specificity parameters of RNA editing in plant mitochondria we have developed a novel in vitro system. The high sensitivity of the mismatch-specific endonuclease thymine glycosylase is employed to allow quantitative evaluation of the in vitro RNA editing products. A pea mitochondrial lysate correctly edits a specific site in the cognate atp9 template. In this contribution we will present the latest results on the biochemical parameters of in vitro RNA editing and of the investigation of the cis-requirements defining an editing site.

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DREB1 is a transcription factor that binds to a drought, salt and low-temperature responsive element, DRE and induces various functional genes that function in stress tolerance in Arabidopsis thaliana. Expression of the DREB1 gene family, DREB1A, 1B and 1C are rapidly induced by low-temperature stress, and at least six motifs are conserved in all three promoters. Promoter analysis of the DREB1C gene showed that 67 bp region from -113 to -47 in the DREB1C promoter was sufficient for low-temperature responsive expression of DREB1C. Two of conserved six motifs among three DREB1 promoters, Box-V containing a G-box and Box-VI were included in this region. Furthermore, gel mobility shift assay revealed that the 67 bp region formed a complex with nuclear extract prepared from non-treated or low-temperature-treated Arabidopsis. Currently, we perform yeast one-hybrid screening uning cDNA library prepared from non-treated Arabidopsis plants.

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DREB2A is a transcriptional factor that functions in drought- and high-salinity-responsive gene expression. This protein specifically binds to DRE/CRT in the promoter region of stress-inducible genes. However, only weak expression of putative target genes induced in transgenic Arabidopsis overexpressing DREB2A. Therefore, the post-translational modification such as phosphorylation seems to be necessary for activation of the DREB2A protein. To characterize activation mechanism of the DREB2A, we carried out domain analysis of the DREB2A protein using Arabidopsis T87 cell protoplasts. The analysis revealed that transcriptional activation domain existed in a region a.a. 253-335 and deletion of a.a. 135-165 or a.a. 318-335 of the DREB2A protein increased expression of a reporter gene. To identify target genes of the DREB2A, we performed microarrray analysis using Arabidopsis overexpressing a constitutive active form of DREB2A.

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A cis-acting promoter element DRE/CRT plays an important role in regulating gene expression in response to drought, salt and cold stress. We have reported that Arabidopsis transcription factors DREBs bind to DRE and control expression of many stress-responsive genes. Overexpression of DREB1A in transgenic Arabidopsis activated the expression of the target stress-responsive genes and resulted in improved stress tolerance. In rice, we isolated cDNAs for DREB homologues, OsDREBs.
We generated transgenic rice overexpressing the DREB or OsDREB genes. These transgenic rice plants revealed drought stress tolerance and contents of free proline in the transgenics are higher than those of control plants. We carried out microarray analysis using the transgenics and identified several target stress-inducible genes of DREB in rice. The promoter regions of these genes contain DRE-related motifs. These results indicate that there is a similar DRE/DREB regulon in the stress-responsible signal transduction in rice.

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A cDNA encoding a DRE (dehydration-responsive element)-binding protein, DREB1A have been isolated using yeast one-hybrid screenig. The DREB1A-transcript-accumulates specifically under low-temperature condition. We have repoted that overexpression of DREB1A transgenic Arabidopsis activated expression of target stress-inducible genes and resulted in improved drought and freezing tolerance. To detect target stress-inducible genes of DREB1A, we performed microarray analysis using two different systems, our 7000 full-length cDNA microarray and 8000 Affymetrix GeneChip, and compared results obtained with the two systems. We identified the direct target genes for DREB1A/CBF3 by 3 criteria as follows after northern blot analysis. (1) Increase level of transcripts in the 35S:DREB1A transgenic-plants under control condition. (2) Significantly increased transcripts from 2 to 10 hours after low temperature treatment. (3) Existence of DRE core motif in the promoter regions. We discuss the functions of the DREB1A target genes and the gene expression network controlled by DREB1A.

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We isolated a recessive mutant slh1 (sensitive to low humidity 1) from Arabidopsis Ds transposant lines. When young plants were transferred to soil, slh1 gradually died with necrotic lesions. When slh1 was directly sowed to soil, plants showed dwarf phenotype with tiny, wilty, and dark green leaves. slh1 could grow and bear fruits at high humidity condition. The serious weakness for drought of slh1 could not be suppressed by exogenous ABA. Microscopy analysis revealed that slh1 dwarfism was caused by decreased cell size. cDNA microarray hybridization monitored global transcriptional changes of defense related genes. Northern blot analysis showed that constitutive expression of marker genes for hypersensitive response. Dead cells and accumulation of callose and autofluorescence in slh1 rosette leaves were detected by trypanblue and anylineblue staining, respectively. These results indicate that slh1 is a mutant which develop HR-like lesions and activate defense responses in the absence of pathogens.

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Photomorphogenic repressor COP1 of Arabidopsis has been shown to repress the activity of transcription factors in the nucleus in the dark. However, light has diminished the nuclear localization of COP1 and abrogate the repressive activities. To modify the intracellular localization of COP1, we introduced mutation in the NLS and fused to GFP gene [GFP-COP1 (Mut)]. In a transient assay in onion epidermal cells, GFP-COP1 was localized to the nucleus but GFP-COP1 (Mut) was localized to cytoplasm. The chimeric genes were introduced into Arabidopsis. In the stable transformants of GFP-COP1 (Mut), opening and expansion of cotyledons were observed but apical hook was not even in complete darkness. However, the GFP-COP1 plants did show the normal skotomorphogenesis and photomorphogenesis in the dark and light, respectively. These suggest that GFP-COP1 (Mut) affects the development of seedlings in the dark. Here, we discuss the relationship between the function and the intracellular localization of COP1.

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An oxygen sensor may play an important role in the N₂-fixing cyanobacterium Anabaena sp. PCC 7120. FixL of rhizobia and DOS of Escherichia coli have a heme PAS domain as an oxygen sensor. We detected in the Anabaena genome a gene alr2428 that encodes a homologous heme PAS domain in addition to various signaling and DNA-binding domains. To analyze this heme PAS domain, we expressed this domain as a His-tagged protein. The purified protein was found to bind a heme. We prepared met, deoxy, O₂-binding and CO-binding forms and measured their absorption spectra and resonance raman spectra. The properties were very similar to those of DOS from E. coli, whose deoxy and met forms have hexacoordinate heme iron. However, no clear sixth ligand to iron was detected in Alr2428, when aligned with DOS from E. coli. Possible structure of the heme PAS domain in Alr2428 will be discussed.

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To analyze cellular responses to ozone, we performed a large-scale analysis of the Arabidopsis transcriptome after the initiation of 12 h of ozone exposure. By using cDNA macroarray, we identified 245 non-redundant genes that were regulated by ozone. Of these, 185 were induced and 60 were suppressed by ozone. The expression of half of the 185 induced genes was controlled by ethylene (ET) signaling, indicating that ozone-induced gene expression was mainly regulated by ET. We studied interactions between the hormonal signaling compounds ET, jasmonic acid (JA), and salicylic acid (SA) in mutants with defects in ET, JA, and SA pathways. Expression analysis revealed that many were regulated by mutual antagonism among these signal molecules. However, many cell rescue/defense genes induced by ET or JA pathways were suppressed by the SA signal, suggesting that the SA pathway acts as a strong antagonist to gene expression induced by ET or JA.

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In many organisms, genes for heat shock proteins are upregulated under sudden increase in temperature and this is essential for accliamtion to heat shock conditions. We analyzed profiles of the gene expression under heat shock conditions in the cyanobacterium Synechocystis sp. PCC 6803 with DNA microarray. As expected, the expression of a number of genes for the known heat shock proteins were enhanced by heat shock. Moreover, a gene encoding a putative histidine kinase (Hik) was also upregulated. Inactivation of the gene for the heat-inducible Hik enhanced levels of expression of the groESL operon even at normal growth temperature, such as 34 degree C, and acquired tolerance against heat shock. These results indicated that the heat-inducible Hik might have repressed the expression of heat shock genes under normal growth conditions and might be involved in the regulation of gene expression under heat shock conditions.

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Exposure of photosynthetic organisms to strong light leads to photoinhibition of photosystem II (PSII). We isolated mutants of histidine kinases (Hiks) in which the extent of photoinhibition was higher than that in wild-type cells. In ΔHik2 cells, photoinhibition was apparently accelerated by hyperosmotic stress with 0.5 M sorbitol due to the inhibition of repair of damaged PSII. Inactivation of Hik16 and Hik33 also enhanced the photoinhibition. ΔHik16 cells were not able to uptake sorbitol under hyperosmotic conditions, whereas ΔHik33 degradated chlorophyll under strong light. Thus, effects of inactivation of Hik2, Hik16 and Hik33 on tolerance of PSII are different. These results suggest that Synechocystis assigns different function to Hik2, Hik16 and Hik33 to tolerate photoinhibition.

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Phosphate is an essential nutrient in all organisms and is utilized for the synthesis of cellular components. We have previously identified histidine kinase Hik7 (sll0337) in Synechocystis sp. PCC 6803 as a sensor of phosphate-limiting conditions. In order to investigate the role of Hik7, we applied DNA microarray technique to analyze the gene expression of wild-type and Hik7-mutant cells in response to phosphate limitation. Incubation of wild-type cells in phosphate-limiting medium for 8 hours induced 28 genes that included alkaline phosphatase, phosphate transporters and genes encoding proteins of unknown functions. Inactivation of Hik7 completely eliminated the expression of all the phosphate limitation-inducible genes. These results suggest that Hik7 is only one sensor for phosphate limitation in this cyanobacterium.

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Activation of wound-induced protein kinase (WIPK), one of the most characterized plant MAPKs, has been implicated in defense responses in tobacco. Despite the important role of WIPK in defense signaling, no endogenous signal responsible for the activation has been identified. We isolated a WIPK-activating substance from TMV-infected tobacco leaves and identified it as a novel labdane-type diterpene. This substance (WAF-1) activated WIPK at nanomolar concentrations.
Quantitative analyses of endogenous WAF-1 revealed that levels increased rapidly in leaves during a hypersensitive response to TMV and after wounding. Exogenously supplied WAF-1 enhanced accumulation of transcripts of defense-related genes. These reuslts suggest that WAF-1 functions as an endogenous signal which activates WIPK and defense-related genes in tobacco.

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WIPK is a protein kinase from tobacco transcriptionally induced by wounding and activation of the hypersensitive response (HR). A yeast two-hybrid screen for proteins interacting with WIPK led to the identification of a new protein, NtWIF. The C-terminus of NtWIF interacts with WIPK in the yeast two-hybrid system as well as in an in vitro pull-down assay. The N-terminus shows high sequence similarity to the DNA binding domain of ARF transcriptional regulators, and the middle region confers transactivation activity to a luciferase reporter gene. Furthermore, a GFP-NtWIF fusion construct localizes to the nucleus in onion epidermal cells, suggesting a role of NtWIF as a transcription factor.
Transgenic tobacco plants constitutively expressing full-length NtWIF were produced. These plants have a drastically changed expression pattern of wound responsive genes. Our results provide evidence for the involvement of NtWIF in a signal transduction pathway from WIPK to the transcriptional control of wound-responsive genes.

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Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes. In plants, MAPK cascades are known to function in responses to various biotic and abiotic stresses. Completion of the Arabidopsis genome-sequencing project has revealed the existence of 20 MAPKs, 10 MAPKKs and 60 MAPKKKs. Here we studied AtMKK3, one of the Arabidopsis MAPKKs, and analyzed which AtMPKs were substrate for AtMKK3. We found that AtMKK3 specifically activate AtMPK8 by in vitro activation assay. AtMPK8 was strongly activated in transgenic plant that over-expressed AtMKK3, suggesting their functional interaction in vivo. Yeast complementation analysis revealed that AtMPK8 function as MAPK. By using yeast two-hybrid system, AtMPK8 was found to interact with calmodulin. Calmodulin was shown to activate AtMPK8. AtMKK3 and AtMPK8 are structurally unique in plants and no significant homologues are found in other eukaryotes. We are analyzing function of AtMKK3-AtMPK8 cascade and a role of calumodulin in the cascade.

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In order to elucidate the mechanism of starch accumulation in the rice endosperm, we have been studying the function of OSK proteins in the developing seed, whose genes are transiently induced in the early developing stage. We partially purified the OSK complex from immature rice seeds and determined the molecular weight of its native form by the gel filtration chromatography. The peak of the activity was eluted around 130kDa, which is similar to yeast Snf1 or rat AMPK. Since Snf1 and AMPK consist of three subunits (α, β and γ), we have searched for interacting subunits of OSK proteins by yeast two-hybrid screening and have isolated three different β subunit homologues (β#15, β#17, β#19). While cDNA sequences of β#15 and β#17 encode the whole polypeptide of β subunit, β#19 is a shorter protein and contains only C-half (ASC domain) of the authentic β subunit.

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Little is known about transcription factors involved in the sugar-signal response in plants. We used microarray of Arabidopsis thaliana for extensive analysis of sugar-induced changes in levels of mRNAs. Among many sugar-regulated genes identified, genes for transcription factors were selected for further analyses. One of the mRNAs that were the most extensively induced by sucrose was AtMYB90 which has been recently shown to regulate the expression of genes for anthocyanin biosynthesis. Although glucose also induced AtMYB90, 2-deoxyglucose or ABA was ineffective. Expression of many genes for bZIP factors was regulated by sucrose, and some of them also responded to ABA, suggesting that they are involved in the ABA-dependent sugar-response pathways. Unlike these, levels of mRNAs for AtbZIP22 and AtbZIP63 changed rapidly after treatment with sucrose, and they did not respond significantly to ABA. In addition, sugar-induced changes in levels of mRNAs for these bZIPs occurred in the presence of cycloheximide.

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Oxidative stress is a major environmental factor for all living organisms. Incubation of the cyanobacterial cells of Synechocystis with 0.25 mM of H₂O₂ causes dramatic changes in the pattern of gene expression. Expression of about 230 genes encoded in the genome was enhanced and 260 genes were repressed by H₂O₂. We also found that two histidine kinases, Hik33 and Hik34, and a putative transcription factor were involved in the regulation of H₂O₂-inducible genes. Hik33 regulated the expression of more than 100 genes and Hik34 regulates that of 20 genes. The transcription factor negatively regulates the expression of a small number of genes. However, the expression of other genes was not regulated by these components. These results suggest that there are at least 4 pathways for signal transduction of oxidative stress in Synechocystis.

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The duckweed Spirodela oligorrhiza grown under Pi-deficient conditions (-P plants) exhibited more than 50-fold higher Pi-transporting activity than did plants grown under Pi-sufficient conditions (+P plants). The Pi-transporting activity of -P plants was inhibited by CCCP, indicating that this Pi transport is energized by the electrochemical proton gradient across PM. From kinetic analyses of Pi transport and western-blot analysis of PM proteins, it was suggested that -P plants would have a high-affinity Pi transporter(s) induced by Pi-depletion. On the contrary, the amount and ATP-hydrolytic and ATP-dependent H⁺-transporting activities of PM H⁺-ATPase did not increase in -P plants. However, kinetic analyses showed that the PM H⁺-ATPase in -P plants had a lower K_m value and higher H⁺/ATP coupling ratio than those in +P plants. Therefore, the significant stimulation of Pi uptake in -P plants may be due mainly to the induction and accumulation of the high-affinity Pi transporter in PM.

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