Plant hormone jasmonic acid (JA) has been recognized as a signal molecule that is induced by various stresses such as wounding and pathogen attack and mediates defense reactions. We previously identified the JA-early responsive bHLH factor RERJ1, but its biological function in rice plants has been unknown. Here we conducted histochemical analyses using RERJ1 promoter-GUS transgenic plants, and characterized a rerj1-tos17 mutant. The inductive expression of RERJ1 was observed in the defined area to be wounded within 15 min, suggesting that RERJ1 functions at the sites injured by insect herbivory, for example. Transcriptome analysis of JA-treated rerj1-tos17 mutant indicated that JA-inductive expressions of defense-related genes, such as proteinase inhibitors genes, were severely suppressed in the mutant. These results suggest that RERJ1 is induced by JA at injured sites, then functions to transactivate many defense-related genes in rice. Now bioassays for insect resistance are being conducted with the rerj1-tos17 mutant.

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Protease inhibitors (PIs) of plants play crucial role in defense response against herbivores. Although tomato (Solanum lycopersicum) has many PIs in the genome, most of these PIs are not characterized for their responsivity to herbivory and for their transcription factors. In this report, we analyzed a group of tomato PIs that was showed to be coexpressed with transcription factors in the microarray data. The PIs were divided into two subgroups, kunitz- and potato protease inhibitor-type, and the expression of one kunitz-type PI has shown to be induced by jasmonate (JA)-treatment. Expression analysis of methyl-JA treated leaves showed that expression of all PIs was induced and that the correlation of expression levels of each PI was high, suggesting a possibility that these PIs respond to herbivory. We further analyzed MYB- and TCP transcription factors that were included in the PI coexpression module, however, induction of these transcription factors was not observed by the Me-JA treatment. Overexpression of the MYB gene in tomato had no obvious effect on the PI expression. Thus, we concluded that these transcription factors are not likely to involve in the PI expression.

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Mitogen-activated protein kinases (MAPKs) are components of signaling cascades that transduce extracellular stimuli into intracellular signaling. In plants, wounding causes activation of MAPKs and production of jasmonic acid (JA) and ethylene (ET). The tobacco MAPKs WIPK and SIPK are activated by wounding. Activation of WIPK and SIPK is required for wound-induced JA production, and activation of SIPK is required for ET production. To examine whether the regulation of production of JA and ET by WIPK and SIPK is a general mechanism for other plant species, we used mutants (mpk3 and mpk6) of their Arabidopsis orthologs and transgenic plants in which both MPK3 and MPK6 were silenced by RNAi (MPK3/MPK6RNAi). Wound-induced ET production was significantly reduced in mpk6 and MPK3/MPK6RNAi plants. The mpk3, mpk6, and MPK3/MPK6RNAi plants produced the same level of JA as did wild type, suggesting that the functions of MPK3 and MPK6 in the wound response of Arabidopsis are partially different from those of WIPK and SIPK in tobacco. We also report a metabolomics approach to examine how MPK3 and MPK6 regulate plant metabolism during the wound response.

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The mitogen-activated protein kinases (MAPKs) are key molecules of signal transduction responses to various extracellular stimuli. In tobacco, two pathogen- and wound-induced MAPKs, WIPK and SIPK, regulate stress-induced accumulation of jasmonic and salicylic acids. MAPK phosphatases (MKPs) are negative regulators of MAPKs. We have reported that overexpression of NtMKP1, a tobacco calmodulin-binding MKP, compromised wound-induced activation of WIPK and SIPK. In this study, we investigated the changes in the levels of NtMKP1 transcripts and proteins in response to wounding. Quantitative RT-PCR analysis revealed that NtMKP1 mRNA is once decreased and then increased in response to wounding. The level of NtMKP1 protein was investigated by immunoblot analysis; however it was not detected in the wild type plants. Therefore, we used the plants expressing NtMKP1 under Cauliflower mosaic virus 35S promoter. The level of NtMKP1 protein was rapidly decreased, kept at the lower level for several hours and then returned to the basal level after wounding. These results indicated that NtMKP1 is regulated at both transcriptional and post-transcriptional levels.

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Mechanical damage elicits various defense responses in plants. These responses are preceded by a rapid and transient burst of jasmonic acid (JA) which is supposed to act as a key signal molecule in wound-inducible gene activation. JA synthesis and signaling are interlinked by a positive feedback loop whereby jasmonates stimulates their own synthesis. It was shown that some JA biosynthetic genes such as LOX2 and AOS can be induced in COI1-dependent manner after wounding. JA biosynthesis is regulated by the availability of the substrate, α-linolenic acid, liberated from membrane lipids by phospholipases. The DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) gene largely contribute to JA production. The aim of our study was to examine whether the expression of DAD1 and its paralogs is also regulated by JA. We conducted experiments using JA-biosynthesis (aos, opr3) and JA-response mutants (coi1-1, coi1-dad3). Although DAD1 was weakly induced by JA, its expression levels after wounding were significantly reduced in both JA-biosynthesis mutants and even stronger in JA-response mutants. These results suggest that DAD1 may be involved in providing the substrate for self-stimulated JA production.

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ER body is one of ER-related organelles that highly accumulate a specific protein a beta-glucosidase with an ER-retention signal. Functions of ER bodies are still unclear. ER bodies have a limited distribution in Arabidopsis seedlings. They are present in the epidermal cells of cotyledons and hypocotyls (constitutive ER bodies) of young seedlings, and disappear in those of mature tissues. When mature leaves are wounded, ER bodies are induced around the wound site of the leaves (inducible ER bodies) To clarify the function of ER bodies, we compared constitutive ER bodies with inducible ER bodies and both ER bodies are accumulate different beta-glucosidase, constitutibe ER bodies are accumulated PYK10 and inducible ER bodies are accumulated BGLU18 (Plant Cell Physiol.2009) Therefore, the induction of ER bodies might be involved in self-defense of the plants. We report here behavior inducible ER bodies after wounding.

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We used C13 to analyze dynamics of lipid metabolism in Anabaena variabilis M3 and Synechocystis sp. PCC 6803. Mass spectra were analyzed by custom-made software called C13dist and perl scripts. We found highly labeled isotopomers after labeling for 2 hrs. C13 abundance of the labeled population was not significantly changed after conversion of GlcDG to MGDG and desaturation. However, a new pool containing C13 at a low abundance appeared during the chase period, which suggests the presence of an internal precursor pool. The results are, in general, consistent with the direct desaturation as proposed by Sato et al. (1986), but detailed mathematical analysis will reveal hitherto uncharacterized dynamics of lipid metabolism..

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It has been believed that the Calvin cycle composed of eleven enzymes was initially completed in cyanobacteria. In particular, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) as a CO₂-fixing enzyme and phosphoribulokinase (PRK) catalyzing the ribulose-1,5-bisphosphate (RuBP) production are enzymes unique to this cycle. The primitive cyanobacterium Gloeobactor violaceus possesses prk homologue genes, prk2 and 3 in addition of prk1 for a typical cyanobacterial photosynthetic PRK. The Vmax and Km_(Ru5P) values of PRK1 were 200 μmol/min/mg protein and 0.28 mM, respectively, similar to those of typical cyanobacterial PRK. PRK2 did not show activity at all. Interestingly, PRK3 showed significant PRK activity, and the Vmax and Km_{(Ru5P 0.5)} values were 23.5 μmol/min/mg protein and 5 mM, respectively. In phylogenetic tree, G. violaceus PRK3 and PRK homologues from other organisms form a clade distinct from plant and bacterial lineages, suggesting that G. violaceus PRK3 is a novel PRK form. In order to study on a relationship between the novel type of PRK and molecular evolution of the Calvin cycle, we are analyzing G. violaceus PRK3 homologues.

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In acidic soils, aluminum ion causes root growth inhibition, which is due to the inhibition of cell elongation at root apical meristem by aluminum ion. Furthermore, cell elongation inhibition by aluminum accompanies an increase in the production of reactive oxygen species. These responses to aluminum are also observed in actively growing cultured tobacco cells. For cell elongation, water uptake is necessary, and a motive force of water uptake is an increase in osmolality by increases in solutes such as free sugars, inorganic ions and amino acids in vacuole. Invertase localized in vacuole contributes to an increase in osmolality by hydrolysis of sucrose to glucose and fructose. In this study, we examined a possibility that aluminum ion might inhibit invertase activity in vacuole, in cultured cells and roots of tobacco, by measuring invertase activity in cell-free extracts and histochemical staining of invertase activity, respectively. On the contrary, we found that invertase activity in vacuole was enhanced by aluminum in both systems, suggesting that an increase in invertase activity in vacuole may contribute to alleviate the cell elongation inhibition under aluminum stress.

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In transgenic rice plats with an enhancement of Rubisco protein content to 120% of wild-type level, photosynthetic rates under the present ambient [CO₂] and high irradiance conditions were not enhanced. To investigate this reason, the amounts of photosynthetic and other primary metabolites were determined with CE-TOFMS. Leaves were immediately frozen in liquid nitrogen and sampled after their photosynthetic rate reached steady-state. In transgenic plants, the amount of 3-phosphoglycerate as well as fructose-1,6-bisphosphate and sedoheptulose 7-phosphate increased to 1.4-fold of wild-type plants. On the other hand, the amounts of other photosynthetic metabolites including ribulose 1,5-bisphosphate did not differ between these genotypes. The amounts of ATP, ADP, NADPH and NADP⁺ and their ratios were also similar. These results suggest that although the increase in Rubisco protein contents leads to an enhancement of its catalytic reaction in the transgenic rice plants, enzymatic step(s) in RuBP regeneration become bottleneck for photosynthetic rates. Additionally, changes in other carbon and nitrogen metabolism are also discussed.

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Rubisco is the CO₂ fixing enzyme in photosynthesis and the most abundant leaf protein in plants. Rubisco is composed of eight small subunits coded by RBCS in the nuclear genome and eight large subunits encoded by rbcL in the plastome. Although RBCS forms a multigene family, it is not known how each gene contributes to the amount of Rubisco in a leaf. To clarify this, four RBCS genes (OsRBCS2 to 5), which are highly expressed in a leaf, were individually knocked-down in rice (Oryza sativa L.) and their Rubisco contents were determined at different growth stages (seedling, vegetative, and reproductive stages). Rubisco content declined by 20-30% in RBCS2- and 3- knock-down transformants and by 5-20% in OsRBCS4- and 5- knock-down transformants. Similar trends were observed irrespective of growth stage. Thus, although the effects of individual knock-down of RBCS multigene family were quantitatively different, all RBCS genes were the determinant for the amount of Rubisco in leaves during the life span of rice plants.

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The small subunit of Rubisco is encoded by RBCS multigene family in the nuclear genome in higher plants. Although there are four members of RBCS genes in Arabidopsis, it is unclear how each gene contributes to the amount of Rubisco in leaves. In this study, we identified Arabidopsis T-DNA insertion mutants of AtRBCS1A (rbcs1a), AtRBCS3B (rbcs3b) and both genes (rbcs1a3b), and analyzed the expression of each RBCS gene and Rubisco content in leaves. In wild-type leaves, the mRNAs of AtRBCS1A, 2B and 3B were highly accumulated. The transcriptions of AtRBCS1A were not detected in rbcs1a and rbcs1a3b leaves. The amount of AtRBCS3B mRNA was about 20% of wild-type levels in rbcs3b and rbcs1a3b. Since the amount of other RBCS mRNAs which T-DNA was not inserted was the same with wild-type levels in each mutant, the total RBCS mRNA levels were significantly lower than wild-types. Rubisco contents declined approximately by 38% in rbcs1a leaves, 20% in rbcs3b and 63% in rbcs1a3b compared to wild-types. These results suggest that AtRBCS1A and AtRBCS3B contribute to Rubisco synthesis in Arabidopsis leaves, and leaf Rubisco content was decreased by suppression of these genes expressions.

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Effects of overexpression of Rubisco activase on photosynthesis were studied using transgenic rice expressing barley Rubisco activase. Western blot and SDS-PAGE analyses showed that the transgenic rice expressed barley Rubisco activase at the level higher than endogenous rice Rubisco activase. The activation state of Rubisco under illumination was higher in transgenic rice than that in non transgenic rice (NT). In addition, the relaxation time for light activation of Rubisco was significantly lower in transgenic rice compared with NT. These findings indicate that the overexpression of barley Rubisco activase could enhance Rubisco activation in rice. However, the CO₂ assimilation rate was decreased in transgenic rice. This decrease in CO₂ assimilation rate was observed in young or matured leaves regardless of supplied N levels. The contents of nitrogen and chlorophyll did not differ between transgenic and NT, whereas Rubisco content was significantly decreased in transgenic rice. These results suggest that the overexpression of Rubisco activase can stimulate Rubisco activation, however, which leads to decrease in the photosynthetic rate by the reduction in Rubisco content.

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We investigated relationship between Φ₂*PFD and J_g in photosynthesis of Synechocystis sp. PCC6803.
Both Φ₂ and J_g were simultaneously estimated from both chl fluorescence yield and O₂ evolution rate analyses.
Both Fm^, and Fs decreased with the increase in intensity of white light (WL), but these did not decrease with red light (RL). These parameters decreased with lowering intensity of green light. The decreases in both Fm^, and Fs were not induced by state transition, respiration, or the Water-Water-Cycle (WWC). We propose that both Fm^, and Fs were regulated by Orange Carotenoid Protein (OCP) which controls energy transfer efficiency from PS2-antenna to the reaction-center of PS2.
Φ₂*PFD/J_g (=K) increased at strong intensity of WL. But K did not change even at strong intensity of RL. These results indicated AEF did not work under the condition where electron-sink was limited. In addition,both V(O₂) and Φ₂ did not change in anoxia. These results showed that WWC activity as AEF was very low under WL, and the increase in K under WL was due to the decrease in efficiency of collecting light energy by OCP system. Our data suggests that Φ₂ can be related to J_g under RL.

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We investigated metabolism of DC using spinach chloroplasts and thylakoid membranes. We detected enzyme activity of the reduction of MG by NADPH in stroma of chloroplasts. In this reaction, NADP⁺ as Hill oxidant was produced by the reduction of DC. To detoxify DC continuously, NADPH must be regenerated. We hypothesized that DC functioned as Hill oxidant. To verify our hypothesis, we added DC to intact spinach chloroplasts. The addition of DC to chloroplasts induced photochemical quenching of Chl fluorescence. The production of Hill oxidant should accompany with O₂-evolution in PS2. But DC induced O₂-uptake and H₂O₂ accumulated. We, furthermore added DC to the illuminated thylakoid membranes and investigated dependence of the DC-dependent O₂-uptake rate (Vo₂) on DC concentration, temperature and light intensity. Maximum Vo₂ reached 210 μmol O₂/mg Chl/h. An optimum temperature for Vo₂ was 40^oC and Vo₂ did not saturate against light intensity. DC-dependent uptake of O₂ was inhibited by DCMU and DBMIB. These results showed that DC photoreduced at PS1donated electorons to O₂ producing H₂O₂.

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The redox state in plant mitochondria is affected by a wide range of environmental factors. Depending on the fluctuation of the redox state, the activity of several mitochondrial proteins may be subjected to redox regulation by the thioredoxin (TRX) system. In this study, we captured the potential TRX target proteins in mitochondria by TRX affinity chromatography, and identified them by proteomics technique. The mitochondrial soluble proteins determined as TRX targets contained enzymes which are involved in a variety of mitochondrial functions, such as TCA cycle, photorespiration, and protein synthesis. In addition, we identified some proteins which are associated with the respiratory electron transport and ATP synthesis in the solubilized membrane fraction. Notably, we revealed the interaction of TRX with alternative oxidase which plays a key role in ensuring cellular redox homeostasis especially under the stressful conditions. We present a list of TRX target proteins in mitochondria and discuss the possible regulatory system of the mitochondrial functions.

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C₄ plants show higher activities of cyclic electron flow around PSI (CEF) than C₃ plants. It is suggested that CEF produce ATP to drive C₄ metabolism. To clarify how CEF enhanced with the development of C₄ pathway during evolutional process from C₃ to C₄ photosynthesis, we investigated CEF activity, expression levels of proteins involved in CEF and thylakoid membrane structure using genus Flaveria containing C₃, C₄, C₃-C₄ intermediate and C₄-like species. NDH activity was higher in C₄-like species than in C₃-C₄ intermediate species, as well as the NDH-H expression level. On the other hand, CEF activity estimated from the P700 oxidation rate was higher in C₄ species than in C₄-like species. Drastic difference in expression level of PGR5 involved in FQR activity and grana stacking ratio of bundle sheath chloroplasts were also observed in those species. These results suggested that the NDH pathway developed earlier than the FQR pathway in C₄ evolution. Taken together with evidence that expression of C₄ metabolic enzymes was already elevated in C₃-C₄ intermediate species, we assume that CEF was enhanced in a later process of development of C₄ cycle.

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Phototrophic purple bacteria produce many kinds of acyclic carotenoids, which are rare in organisms. Some carotenoids are specific to some classes, families or genera, and some are not related to phylogenetic classification. Around half species produce spirilloxanthin as the final product. Others produce different carotenoids, which may be due to modification or deletion of four enzymes of spirilloxanthin synthesis and/or addition of a new enzyme.
Rhodobacterales produces spheroidene and spheroidenone, which might be due to modification of CrtI and additional CrtA. All of four species of Phaeospirillum produce hydroxylycopene (rhodopin) or dihydroxylycopene glucosides, which might be due to inactivity of CrtD and additional glucosyltransferase. All of five species of Roseospira produce 3,4-didehydrorhodopin as major carotenoid, which might be due to low activity of CrtF. Rhodoplanes contains five species. Two species produce spirilloxanthin, and one produces rhodopin as major carotenoid. One produces rhodopin as major carotenoid and tetrahydrospirilloxanthin. These might be due to different characteristics of four enzymes in each species among one genus.

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A balanced distribution of absorbed light energy between photosystem I (PSI) and II (PSII) is required for maximum efficiency in photosynthesis. One of the balancing mechanisms is state transition. The mobile light-harvesting antenna could attach to PSI or PSII. The other proposed mechanism is spillover, where PSII transfer its energy to PSI. Red algae have a large light-harvesting antenna, phycobilisome, which attaches to PSII. Here, partial energy harvested by phycobilisome is transferred to PSI. However, the detail energy transfer pathway is still unknown. Since both PSI and PSII have similar chromophores, excitation photon is absorbed by both photosystems. To examine the balancing mechanism, it is required to observe excitation energy flow after selective excitation of PSI or PSII. PSII reaction center specifically emit delayed fluorescence in the range of 15-60 ns, whereas PSI reaction center and phycobilisome show little fluorescence in this time range. Therefore, the delayed fluorescence spectrum reflects energy migration pattern from PSII. We will discuss the energy balancing mechanisms by means of delayed fluorescence spectra of various red algae.

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Diatoms possess fucoxanthin chlorophyll a/c binding proteins (FCPs) as a light harvesting apparatus. The diatom FCPs have been rather poorly understood compared to light harvesting chlorophyll a/b complexes from higher plants and green algae. Although PSI specific FCPs have been reported, there is very little knowledge with regard to FCPs associated with PSII.
Recently, we succeeded in isolation of oxygen evolving crude PSII particles (crude PSII) from a marine centric diatom, Chaetoceros gracilis (UTEX LB 2658). The crude PSII contained several FCP subunits in addition to the PSII subunits. In this study, we attempted to fractionate the FCP subunits by sucrose density gradient centrifugation after treatment of the crude PSII with Triton X-100. We obtained several FCP fractions of different polypeptide composition. Spectral properties and pigment composition will also be presented for discussion of functional organization of the FCP-based light-harvesting system for PSII in diatoms.

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Dark-operative protochlorophyllide reductase (DPOR) is a nitrogenase-like enzyme consisting of two components L-protein (ChlL) and NB-protein (ChlN-ChlB) and plays a critical role in chlorophyll (Chl) biosynthesis in the dark. All subunits of DPOR are encoded in plastid genome in photosynthetic eukaryotes. RNA editing in chlN and chlB, which is required for the restoration of codons for evolutionarily conserved amino acid residues, was reported in some gymnosperms. Here we report that RNA editing regulates the DPOR activity in black pine Pinus thunbergii. We constructed a series of shuttle vectors to express P. thunbergii chlN-chlB with and without mutations responsible for the RNA editing, and introduced them into the mutants lacking chlB of the cyanobacterium Leptolyngbya boryana. Only the transformants carrying the edited chlN-chlB genes restored the ability of Chl biosynthesis in the dark. Biochemical analysis of the cyanobacterial NB-protein with site-directed mutations responsible for the RNA editing supported the in-vivo complementation result. These results suggested that RNA editing regulates the greening in the dark through the NB-protein activity in P. thunbergii.

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Dark-operative protochlorophyllide oxidoreductase (DPOR) catalyzes the stereo-specific reduction of C17=C18 double bond of protochlorophyllide in the bacteriochlorophyll biosynthesis. DPOR consists of two separable components, L-protein (BchL homodimer) and NB-protein (BchN-BchB heterotetramer), which are homologous to Fe protein and MoFe protein of nitrogenase, respectively. L-protein delivers electrons to the catalytic component NB-protein coupled with the ATP hydrolysis. In the NB-protein the electrons are transferred to protochlorophyllide via a [4Fe-4S] cluster to convert chlorophyllide a. In the previous work, we reported the crystal structure of NB-protein from Rhodobacter capsulatus. Based on the structure and biochemical analysis, we proposed a reaction scheme of the stereo-specific reduction in which Asp274 and the propionate side chain of the substrate donate the protons to C17 and C18, respectively. In this work, site-directed variants and a substrate analogue were used to trap some reaction intermediates. The sequence of proton and electron transfers in the reaction will be discussed.

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Light-dependent protochlorophyllide oxidoreductase (LPOR) catalyzes a unique light-driven reaction that produces chlorophyllide a, the direct precursor of chrolophyll a. A mutant (Δpor) lacking por gene encoding LPOR of cyanobacterium Synechocystis sp. PCC 6803 does not grow under aerobic conditions, while it grows under anaerobic conditions. Therefore, the Δpor mutant would be a suitable strain for in-vivo evaluation system for LPOR variants. A marine cyanobacterium Synechococcus sp. CC 9311 possesses three LPOR-like genes in the genome. Two of them are phylogenetically distant from the group containing LPORs from freshwater cyanobacteria and plants. The three genes were individually introduced into the Δpor mutant for expression, and the growth of the resultant transformants was examined under aerobic conditions. In addition, LPOR activity of the purified proteins expressed in E. coli was examined and K_m values for protochlorophyllide and NADPH were determined for one of them. We also report a result of a cyanobacterial LPOR variant lacking a loop specific for LPOR examined by the Δpor expression system.

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Heme oxygenase (HO) catalyzes the oxygen-dependent cleavage of the porphyrin ring of heme producing biliverdin IXα in the phycobilin biosynthesis. There are two genes, ho1 (sll1184) and ho2 (sll1875), encoding heme oxygenase isoforms in the cyanobacterium Synechocystis sp. PCC 6803. Previous study revealed that a mutant lacking ho1 (Δho1) does not grow under aerobic conditions suggesting that ho1 is essential for growth under aerobic conditions. In this study, we isolated a pseudo-revertant R1 from Δho1 and characterized it. R1 grew slightly slower than wild type under aerobic conditions. The phycocyanin content was almost comparable to that of wild type and the anomalous accumulation of protoporphyrin IX in Δho1 was significantly relieved. RT-PCR indicated that ho2 is expressed in a constitutive manner in R1 while ho2 is repressed under aerobic conditions and induced under low-oxygen conditions in wild type. Thus, it is suggested that the aberrant constitutive expression of ho2 restores the defect in ho1 resulting in the growth recovery of Δho1 under aerobic conditions. To identify the relevant mutation sites, genome analysis of R1 is under progress.

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The esterifying substituent at the 17²-position of chlorophylls has been less studied, because of its indirect attaching to their π-conjugated skeletons. However, this relatively large substituent plays an important role in constructing photosynthetic apparatuses. The structures and distribution of its biosynthetic intermediates possessing a GG, DHGG, or THGG moiety in bacteriochlorophyll-a were carefully investigated for a purple bacterium, Rhodopseudomonas (Rps.) palustris, which excessively accumulated the intermediates. As the result, these intermediates of BChl-a were apparently localized in different types of photosynthetic apparatuses (LH2, LH4, and RC-LH1). In this study, we analyzed the structures and distribution of the corresponding bacteriopheophytin (BPhe). We successfully isolated the RC from Rps. palustris species for the first time. The composition of BPhe-a having different 17²-ester groups in the RC was analyzed by LC-MS. The content of BPhe-a having a matured phytyl group was larger than that of BChl-a, and BPhe-a having a THGG group was first identified in the RC as a photosynthetically active pigment.

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Green sulfur photosynthetic bacteria have extra-membraneous light-harvesting (LH) systems called chlorosomes. Comparing to usual LHs embedded in cytoplasmic membranes of various classes of photosynthetic organisms, chlorosomes are characterized by the following points: they are large micelle-like nano-architectures and the envelopes consist of glyco/phospholipids containing proteins. In the architectures, a huge number of photosynthetic pigments, estimated to 200,000 chlorophyll pigments per single chlorosomes, are capsulated. The green sulfur photosynthetic bacterium Chlorobium tepidum (currently referred to as Chlorobaculum tepidum), which grows at 45 degrees as its optimum temperature, mainly accumulated unique disaccharide rhamnosylgalactosyldiacylglyceride (RGDG) having a methylene-inserted palmitoleyl group together with the corresponding monosaccharide monogalactosyldiacylglyceride (MGDG). Here, we report changes in the structure and composition of the glycolipids dependent upon temperature and period of the cultivation. These results enable us to discuss the biosynthesis as well as the physiological meaning of unique glycolipids in green sulfur photosynthetic bacteria.

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In the environment where plants grow, there are various abiotic stresses. It has been reported that the most of mRNA is inhibited in plant exposed to these stresses. It means that the expression of transgene is also repressed at the translational level. However, a subset of mRNA escapes such repression. It is reported that 5'UTR has an important role in translational regulation under abiotic stress, and we already identified the 5'UTR involved in preferential translation under heat stress.
In this study, in order to evaluate the translational activity of the 5'UTR, we generated two transformants harboring a reporter GUS gene and preferentially translated or translationally inhibited 5'UTR, and were incubated under normal or heat stress condition, and the translational state of each GUS mRNA was analyzed by polysome/qRT-PCR analysis. As a result, they showed the same profile as their original mRNAs. Additionally, the GUS mRNA was translated normally in the transformant exposed to other stresses. These results indicate that a 5'UTR involved in preferential translation under abiotic stresses could serve as important tool to express a useful gene in plants more effectively.

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Okinawa mozuku (Cladosiphon okamuranus Tokida) is an original strain of brown algae in Okinawa. The super large scale culture of the discoid germiling of this strain was established. The discoid germiling is a spherical microorganism having diameter of 40 - 200 micro meter, and it is very useful to isolate photosynthetic membrane in mild conditions. In brown algal photosynthesis, the major antenna pigment-protein complexes, fucoxanthin-chlorophyll a/c protein (FCP) absorb the photo energy and transfer it to both photosystems I and II. The biochemical and spectroscopic properties of brown algal FCP have been relatively less studied. We have been isolate and purified the FCP from the Okinawa mozuku. The FCP found to exist as a homo- or hetero-trimer with two subunits showing molecular weight of 17.5 and 18.2 kD. We directly determined, for the first time, both chlorophyll (Chl) c₁ and c₂ bound equally to the FCP. The pigment stoichiometry was also determined by using ¹H-NMR as fucoxanthin : Chl a : Chl c₁ : Chl c₂ = 5.5 : 4.5 : 1.1 : 1.0. In this study, we applied the femtosecond time-resolved spectroscopy to clarify the dynamics of the excitation energy transfer in the FCP.

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Jasmonates (JAs) are phytohormones derived from oxygenated fatty acids and regulates a broad range of plant defense and development processes. Here we describe another function of JAs: hypocotyl elongation under various light conditions was suppressed by exogenous methyl jasmonate (MeJA) treatment in Arabidopsis. Mutant analyses suggest that SCFCOI1-mediated proteolysis is required for this function. However, (+)-7-iso-JA-L-Ile was showed weaker effect than MeJA under dark and red light. Under blue light, gibberellin (GA) abolished the jasmonate function on hypocotyl elongation. This result suggests a crosstalk between JA and GA signaling. By screening of EMS mutant seeds, three mutants insensitive to MeJA on hypocotyl elongation, jal1 (jasmonate resistance long hypocotyl 1), jal36 and jal17 were isolated. Further analysis indicates that both jal1 and jal36 have a mutation in phytochrome B gene. These results suggest that jasmonate signaling is partially involved in phytomorphogenesis through phytochrome B and SCFCOI1 dependent pathway mediated by (+)-7-iso-JA-L-Ile and other jasmonate derivatives.

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Most flowering plants have evolved self-incompatibility (SI) system to prevent inbreeding. In Brassica, SI is controlled by the S-locus, which encodes pollen and pistil determinants named SP11 (S locus protein 11) and SRK (S receptor kinase), respectively. The binding of SP11 to SRK induces the autophosphorylation of SRK to initiate the signaling cascades resulting in the rejection of self-pollen. MLPK (M-locus protein kinase), a cytoplasmic protein kinase, was shown to be involved in SI signaling, probably by forming a receptor complex with SRK. However, the direct target of MLPK and its exact role in SI signaling remain unclear.
To search for new interacting partners for MLPK, we performed Split-Ubiquitin Membrane Yeast Two-Hybrid screening, which enabled us to detect protein-protein interaction on plasma membrane. As a result, we identified several candidates for MLPK interactor. Some of these factors are involved in membrane trafficking, suggesting that membrane trafficking might be associated with SI reaction. Now, we are verifying their interactions by BiFC assay.

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Many flowering plants possess self-incompatibility (SI) system to prevent inbreeding. In Brassica self-incompatibility, self/non-self recognition is controlled by S-haplotype-specific interaction between the pollen-borne ligand, SP11 (S-locus protein 11), and its stigmatic receptor-kinase, SRK (S receptor kinase). Our genetic analysis of a self-compatible mutant revealed the involvement of a cytoplasmic protein kinase, MLPK (M-locus protein kinase) in the SI signaling. MLPK localizes to the papilla cell membrane and interacts directly with SRK on the plasma membrane to transduce SI signaling, but the regulation mechanism of MLPK kinase activity remains unclear.
To understand the activation mechanism of MLPK, we carried out an in vitro phosphorylation assay using E. coli-expressed recombinant MLPK and SRK protein. Our results indicated that N-terminal region of MLPK was not only the target specifically phosphorylated by SRK kinase domain but also the autoregulatory domain of MLPK itself.

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As a genetic mechanism to prevent self-fertilization, the plants in Solanaceae adopt the S-RNase-based self-incompatibility (SI) system, in which S-ribonuclease (S-RNase) and S-locus F-box (SLF) have been identified as female- and male-specificity determinants, respectively. It was postulated that S-RNase functions as a cytotoxin inhibiting pollen-tube growth, and that SLF specifically detoxifies non-self S-RNases via the ubiquitin-26S-proteasome system, thereby allowing compatible pollinations. However, it remained puzzling how SLF, with much lower allelic sequence diversity than S-RNase, might have the capacity to recognize a large repertoire of non-self S-RNases. We used in vivo functional assays and protein interaction assays to show that in Petunia (Solanaceae), at least three types of divergent SLF proteins (type-1, -2 and -3 SLFs) function as the pollen determinant, each recognizing a subset of non-self S-RNases. Our findings reveal a collaborative non-self recognition system in plants.

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Self-incompatibility (SI) is a genetic mechanism adopted by angiosperms to prevent inbreeding. In the Solanaceae, including Petunia, a ribonuclease, named S-RNase, was shown to be the stylar determinant and function as a cytotoxin specifically inhibits self-pollen tube growth. Recently, we proposed "collaborative non-self recognition" model, in which pollen determinant is comprised of multiple types of F-box proteins, named SLFs, each of which detoxifies a subset of non-self S-RNases. We showed that at least three types of divergent SLF proteins (type-1, -2 and -3 SLFs) function as the pollen determinant, but there remained some non-self S-RNases that were not recognized by these SLFs, suggesting that other types of SLFs should also comprise the pollen determinant. In this study, we explored other types of SLFs in Petunia and found that another type of SLF also functions as an element of pollen determinant. These results further support our "collaborative non-self recognition" model.

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Floral transition, transition from vegetative to reproductive growth, is one of the most important developmental events in flowering plants for reproductive success and is tightly controlled by genetic networks that integrate signals from environmental and endogenous cues. Integration of these signals culminated in the production of a long-distance signal, called florigen, in leaves. Recent functional analyses of the FLOWERING LOCUS T (FT) protein provide the molecular basis of florigen activity in the floral transition process. Here, we report the identification of a novel flowering-time gene, FE, encoding a Myb-related transcription factor. Genetic analysis revealed that fe, ft and fd mutants belong to the same physiological group and share similar genetic interactions with various flowering-related mutants. We will discuss about the current model of the FE function in the floral transition process in Arabidopsis.

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The analysis of a phytochrome deficient mutant revealed that phytochromes repress flowering in rice, a short-day plant by inducing Ghd7, a strong floral repressor, only in the morning under LD. This regulation is required for critical day-length recognition of florigen expression. However, flowering-time phenotypes of single/double phytochrome mutants suggest that each phytochrome can delay flowering by distinct and coordinated molecular actions. To further study the repression of flowering by phytochromes in rice, we examined all single/double phytochrome mutants for gene expression of flowering-time genes.
For example, we examined the role of each phytochrome for Ghd7 induction. Ghd7 was induced in phyA, phyB, phyC, and phyBphyC, but not in phyAphyB, phyAphyC. These suggested that phyA and/or phyBphyC mediate light signals to induce Ghd7 redundantly. In phyB, Ehd1 was highly expressed independent of day-length although the expression of Ghd7 was not changed much. Moreover, in some cases, de-repressed Ehd1 expression patterns were not associated with florigen expression. We here propose a new model to explain possible roles of each phytochrome in photoperiodic flowering of rice.

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Florigen is a plant hormone which was proposed by Chailakhyan in the 1930's. Florigen was proposed to be produced in the leaf after stimulation of day length change and cause floral evocation. Recently, several lines of evidence in plant molecular genetics strongly suggest that Hd3a protein in rice (FT protein in Arabidopsis) is the florigen. Hd3a/FT, as well as the antagonist, RCN/TFL1, belongs to PEBP family which is highly conserved among organism. The molecular function of PEBP in plants, however, remains largely unknown. To understand the molecular function of Hd3a protein complex, in-depth mutation analysis of Hd3a was performed.
Mutant Hd3a proteins were designed on the basis of structural information and tested for the interaction with Hd3a-interacting proteins and for transcriptional activation activity by transient assay in rice suspension cells. Results suggested that Hd3a make complex with rice GF14 and rice FD homolog to activate the target gene expression. Analysis of RCN, by contrast, suggested that RCN make similar protein complex to repress it. Taken together, the molecular mechanism of floral evocation by florigen will be discussed.

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Heading date 3a (Hd3a) protein in rice and its Arabidopsis ortholog FT are recently considered as the likely florigen, the crucial component of a long-distance flowering signal. Hd3a/FT protein is generated at the leaf phloem tissue in flowering-promoting conditions, moves from leaves to apical meristem and causes flowering. In Arabisopsis, FD, a bZIP transcription factor expressed at shoot apex, interacts with FT to promote the expression of floral meristem identity genes. Here we identified at least six FD homologs from rice genome. The functions of these FD homologs are analysed by overexpressing these genes.

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The evolution of selfing has been regarded as one of the most prevalent evolutionary transitions in flowering plants. The self-incompatibility (SI) system of Brassicaceae consists of male and female specificity genes at the S-locus (SCR/SP11 and SRK, respectively) and SI modifier genes. Despite many studies, the primary mutation responsible for the loss of SI in Arabidopsis thaliana still remained unknown. We found that a disruptive 213-bp inversion in the SCR gene is shared in 95% of European accessions, in contrast to the genome-wide polymorphism pattern. The interspecific crossings using Arabidopsis halleri as a pollen donor revealed that some accessions retain the female SI reaction, suggesting that all female components including SRK are still functional. Moreover, when the 213-bp inversion in SCR was inverted and expressed in transgenic plants, the functional SCR restored the SI reaction. These results indicate that the inversion within SCR is the first mutation disrupting SI. This is consistent with theoretical predictions, in that male mutations enjoy fitness advantages over female mutations because they can spread through both pollen and seeds.

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The FLOWERING LOCUS T (FT ) gene of Arabidopsis thaliana encodes a 20-kDa protein, which induces flowering as a long-range mobile signal. Recent studies show that FT homologs in other plant species are involved in various meristem-associated physiological events, such as tiller bud outgrowth (rice), bud dormancy (poplar and Norway spruce), tuber formation (potato), and compound leaf formation (tomato).
We focused on the lateral branch outgrowth as a physiological event other than flowering in Arabidopsis thaliana, and we compared the lateral branch growth of wild-type plants and that of ft mutants. The ft mutants showed the delay of outgrowth commencement and the decrease of growth rate. We are currently analyzing the expressions of genes including phytohormone biosynthesis and signaling genes, and cell wall-related genes in lateral branches of ft mutants, compared with in those of wild-type plants. The results will be also reported.

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Flowering is the transition from vegetative to reproductive growth in angiosperms. Recently, it was shown that FLOWRING LOCUS T (FT) protein is a main component of florigen, a long-distance flowering signal. In Arabidopsis, transcription of FT is induced in leaves under inductive long days, while FT protein is transported to the shoot apex and interacts with a bZIP transcription factor, FD, to promote flowering. However, genetic analyses have suggested that some factors interacting with FT other than FD is also required for FT function in flowering. It is also likely FT play a role in systemic changes associated with flowering. In order to fully understand the function of FT as a mobile systemic signal, we are searching for novel transcriptional factors interacting with FT. We demonstrated several TCP proteins interact with FT protein by using yeast two-hybrid assay, and confirmed the interaction in plant cells through BiFC assay in tobacco leaves. Possible involvement of TCP in flowering-associated developmental changes through the interaction with FT will be discussed.

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FD, a bZIP transcription factor, preferentially expressed in the shoot apical meristem is required for FT protein to promote flowering. FD and FT interact and act at the SAM to promote flowering. FD contains several possible phosphorylation sites by various protein kinases. A C-terminal RSST motif is a recognition sequence of calcium-dependent protein kinases (CDPKs). Threonine-to-alanine substitution mutant in the RSST motif (T282A mFD) was unable to interact with FT, and expression of T282A mFD by cauliflower mosaic virus 35S promoter failed to rescue delayed-flowering phenotype of fd-1 mutant plants. These results suggest the importance of C-terminal CDPK site in regulation of the FD function. However actual phosphorylation of FD has not been demonstrated.
We demonstrated actual phosphorylation by the protein extracts from the shoot apical meristem of plants prior to flowering. Currently, identification of kinases is in progress and there are a few dozen candidates. Furthermore we are also interested in mechanism of FD-FT protein complex formation. Progress toward understanding the molecular basis of promotion of flowering by FD and FT will be presented.

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Shoot apical meristem (SAM) of plants shows a well-organized spatio-temporal order, despite active cell division and continuous production of aerial tissues. Molecular mechanism of SAM has been extensively studied, and SAM is crucially maintained by the feedback regulation between WUS and CLV. However, dynamic behaviors of SAM are still not understood, and its essential understanding requires analysis as a dynamic system. Therefore, we tried to investigate SAM dynamics using methods of theoretical biology. We constructed a mathematical model including four conditions: WUS-CLV interaction, two-dimensional pattern formation, area expansion by cell division, and PZ induction by CZ. First, we intensively examined patterns generated by the model and their detailed parameter conditions. Observed patterns are divided into six classes according to their shape and proliferation. Obtained results are consistent with many known experimental observations in A. thaliana and other plants. From these findings, we conclude that SAM dynamics is essentially governed by two parameters: stem cell proliferation mode (pattern proliferation) and stem cell containment (spatial restriction strength).

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One of Dof transcription factors of Arabidopsis, Dof5.8, is expressed in provascular cells of leaf primordia. We previously showed that the provascular expression of Dof5.8 is regulated by auxin and MONOPTEROS (MP/ARF5). Here we show functional analysis of Dof5.8. Co-expression of Dof5.8 with MP in Arabidopsis protoplasts resulted in strong suppression of MP-mediated activation of Dof5.8 promoter. Similar suppression was observed when transcriptional activation domain of VP16 was fused to Dof5.8, suggesting that Dof5.8 may function as a transcriptional repressor and represses its own expression. To gain insights into the function of Dof5.8 in planta, we generated transgenic lines overexpressing Dof5.8 in provascular cells. These lines exhibited narrow leaves and lacked the marginal parts of secondary veins and the higher-order veins. Overexpression of Dof5.8 fused to a transcriptional repression domain of SUPERMAN resulted in similar phenotype. These results suggest that Dof5.8 represses the formation of vascular network through its transcriptional repressor activity.

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Stomagen is a cysteine rich peptide that induces stomatal differentiation. Cysteine rich peptides are involved in various processes in plant life: LUREs regulate a pollen tube guidance in Torenia fournieri and NRCs regulate the rhizobium-plant symbiosis in Medicago truncatula.
A physiological function of stomagen is the opposite of that of EPF2, although these peptides belong to the same EPF family. EPF2 overexpression reduced the number of stomata. A similar reduction of stomatal density was observed by an application of recombinant EPF2. We investigated how the physiological difference between stomagen and EPF2 is caused. Replacement of all six cysteine residues by serine made stomagen have no activity, suggesting that the tertiary structure supported by the S-S bonds is important to the function of stomagen. EPF2 has eight cysteines, six of which are conserved in stomagen. We hypothesized the amino-acid sequence that contains EPF2 specific cysteines is responsible for the functional difference between stomagen and EPF2. Domain swapping experiment supported the hypothesis, that is, the stomagen specific sequence gave EPF2 a stomata inducing activity.

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AtRBP1, an RNA-binding protein found in Arabidopsis thaliana, contains two CS-RBDs and is expressed in actively proliferative regions such as root tips, shoot apexes, and calli. We approached the biological roles of AtRBP1 with two methods. First, we generated the transgenic plants whose expression of AtRBP1 gene is suppressed. These transgenic plants showed stunted root growth and depressed salt tolerance. Then we identified RNA sequences bound to AtRBP1. A binding assay using random 50nt RNAs and AtRBP1 expressed in E. coli revealed that UUAGG sequence and its similar sequence can bind to AtRBP1. In order to identify the target RNAs of AtRBP1 in vivo, we immunoprecipitated RNA-AtRBP1 complexes from cell extract of transgenic plants expressing fusion proteins of EGFP and AtRBP1 using anti-GFP antibody. We synthesized cDNAs from RNAs extracted from the immunoprecipitates. After the cloning of cDNAs, the sequences of them were determined. We found some candidate RNAs, which bind to AtRBP1 in vivo. At present, we continue to isolate other candidate RNAs and identify the authentic target RNAs of AtRBP1.

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During development of multicellular organisms including plants and animals, stem cells provide new cells to establish their organs by driving the mitotic cell cycle. The cell division and the stem cell identity formation are tightly regulated to ensure the orchestrated organ development. In spite of their biological importance of stem cell activities, the molecular mechanisms controlling the processes are largely unidentified. Recently, we discovered that the Small ubiquitin related modifier (SUMO) mediated posttranslational modification system is required for the regulation of cell cycle progression and stem cell maintenance. high ploidy (hpy2) mutant lacks proper meristem structure and the cell proliferation activities, suggesting that HPY2, a novel SUMO E3 ligase, may modulate the processes positively. In this study, we analyzed the regulatory mechanisms of the HPY2 expression and the biological processes which HPY2 is involved in. We will discuss about the newly discovered molecular insights of the meristem development especially focusing on the SUMO-mediated processes.

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Maintenance of undifferentiated stem cells is one of the important functions of plant apical meristems. We isolated a novel mutant, named meristem disorganization1-1 (mdo1-1), exhibiting abnormal shoot morphogenesis. The mdo1-1 homozygous plants showed pleiotrophic shoot phenotypes, such as aberrant phyllotaxis and plastochrone, and frequent stem fasciation. In the mutant apical meristems, the canonical layer structure was disrupted and undifferentiated stem cells were not properly maintained, associated with cell death and cell differentiation. The mdo1-1 mutants also showed elevated level of DNA double-strand breaks and expression of DNA damage-induced genes. These results suggest that the enhanced level of DNA damage results in the disorganized meristems in the mdo1-1. Molecular cloning as well as complementation test indicated that the MDO1 gene encodes a relatively small protein found specifically in wide variety of land plants and that, in mdo1-1, tightly conserved amino acid in the orthlogs is substituted. These results, thus, suggest that the MDO1 gene function is crucial for maintenance of undifferentiated stem cells in apical meristems.

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Epidermal cell has diverse functions and differentiates into various specialized cells including trichome and stomatal guard cells. Cuticle layer is formed on the aerial surface of mature epidermal cells. Although WAX INDUCER/SHINE (WIN/SHN) transcription factors have been shown to activate genes for cutin and epidermal wax biosynthesis, other regulators are hardly known. Therefore, we screened transcription factors that induce organ adhesion when CRES-T is applied and found that EPIDERMAL DEFECT1 (EPD1) fused with repression domain (SRDX) induced adhesion of surface of buds and leaves and reduction of the epicuticular wax crystals. Expression analyses revealed that EPD1 regulates the expression of WIN/SHN genes. We further found that epidermal cells of EPD1SRDX plants showed aberrant morphology and the expression of ATML1, a marker gene for epidermal cell, was down-regulated. In addition, the transgenic torenia expressing EPD1SRDX lacked some epidermal cells in petal. Our results suggested that EPD1 regulates not only wax and cutin biosynthesis but also establishment of epidermal cell.

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miR156/157 regulates developmental phase transition by controlling the expression of SBP-box genes, and overexpression of miR156/157 in Arabidopsis caused the retardation of phase transition and the loss of apical dominance. miR156/157 and SBP-box genes are evolutionally conserved across all major lineages of plants. To elucidate the conservation of miR156/157 function between different species, we compared transgenic Arabidopsis and torenias (Torenia fournieri) overexpressing Arabidopsis miR157b. miR157b overexpressing (miR157b-ox) torenia produced many juvenile leaves and reduced apical dominance as well as miR157b-ox Arabidopsis. The fresh weight and the number of leaves per plant increased in miR157b-ox torenias, suggesting that miR157b-ox was useful for increasing of biomass. Expressions of 3 of 6 isolated torenia SBP-box genes were reduced in miR157b-ox torenias. This reduction seemed to be derived from the difference of the target sequence. These results indicate that miR156/157 precisely control the expression of the SBP-box genes by the difference of its expression pattern and the target sequence.

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Many plant species have leaves with flat blade and adaxial-abaxial asymmetric cell differentiation. An Arabidopsis gene FILAMENTOUS FLOWER (FIL) encodes one of transcription factors important for the leaf lamina growth and cell differentiation. It is well characterized that abaxial specific expression of FIL during leaf primordial development is crucial for its function.
A novel Arabidopsis mutant enlarged fil expression domain2 (enf2 ) which we recently isolated shows larger FIL expression domain than that of wild type. It has been suggested from analyses of this mutant that FIL expression pattern is altered when chloroplast gene expression is impaired. It is known that regulation of many nuclear genes to coordinate with chloroplast condition is mediated by GUN1 gene function. Loss of GUN1 function in enf2 mutant background resulted in suppression of the enlargement of FIL expression domain. This indicated that GUN1 is also involved in FIL expression pattern regulation in response to chloroplast condition. Our presentation will be focusing on above results and ongoing analysis about the cell-autonomous and non-cell-autonomous aspects of the chloroplast effect on FIL expression.

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