研究者データベース

田中 亮一(タナカ リヨウイチ)
低温科学研究所 共同研究推進部
教授

基本情報

所属

  • 低温科学研究所 共同研究推進部

職名

  • 教授

学位

  • 博士(理学)(京都大学)
  • 修士(理学)(京都大学)

ホームページURL

J-Global ID

研究キーワード

  • シロイヌナズナ   ラン藻   テトラピロール   葉緑体   クロロフィル   植物生理学   分子生物学   Plant Physiology   

研究分野

  • ライフサイエンス / 植物分子、生理科学
  • ライフサイエンス / 分子生物学

職歴

  • 2021年04月 - 現在 北海道大学 低温科学研究所 生物環境部門 教授
  • 2010年04月 - 2021年03月 - 低温科学研究所生物環境部門 准教授
  • 2007年 - 2010年 低温科学研究所低温基礎科学部門 助教
  • 1998年 - 2007年 低温科学研究所低温基礎科学部門 助手
  • 1998年 - 2007年 Research Associate,The Institute of Low Temperature Science
  • 1997年 - 1998年 Institute fuer Pflanzengenetik und Kulturpflanzenforschung 研究員
  • 1997年 - 1998年 Researcher

学歴

  •         - 1997年   京都大学   理学研究科
  •         - 1997年   京都大学
  •         - 1994年   京都大学   理学研究科
  •         - 1994年   京都大学
  •         - 1992年   京都大学   理学部
  •         - 1992年   京都大学

所属学協会

  • 日本生物工学会   日本植物学会   日本光合成学会   日本植物生理学会   Japanese Society of Plant Physiologists   

研究活動情報

論文

  • Ryo Tachibana, Ayumi Yamagami, Shino Miyagi, Miki Nakazawa-Miklasevica, Minami Matsui, Masaaki Sakuta, Ryouichi Tanaka, Tadao Asami, Takeshi Nakano
    Bioscience, biotechnology, and biochemistry 86 8 1041 - 1048 2022年07月22日 
    Brassinosteroids (BRs), a kind of phytohormone, have various biological activities such as promoting plant growth, increasing stress resistance, and chloroplast development. Though BRs have been known to have physiological effects on chloroplast, the detailed mechanism of chloroplast development and chlorophyll biosynthesis in BR signaling remains unknown. Here we identified a recessive pale green Arabidopsis mutant, Brz-insensitive-pale green1 (bpg1), which was insensitive to promoting of greening by BR biosynthesis-specific inhibitor Brz in the light. BPG1 gene encoded chlorophyll biosynthesis enzyme, 3, 8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), and bpg1 accumulated divinyl chlorophylls. Chloroplast development was suppressed in bpg1. Brz dramatically increased the expression of chlorophyll biosynthesis enzyme genes, including BPG1. These results suggest that chlorophyll biosynthesis enzymes are regulated by BR signaling in the aspect of gene expression and BPG1 plays an important role in regulating chloroplast development.
  • Hanaki Maeda, Koharu Takahashi, Yoshifumi Ueno, Kei Sakata, Akari Yokoyama, Kozue Yarimizu, Fumiyoshi Myouga, Kazuo Shinozaki, Shin-Ichiro Ozawa, Yuichiro Takahashi, Ayumi Tanaka, Hisashi Ito, Seiji Akimoto, Atsushi Takabayashi, Ryouichi Tanaka
    Journal of plant research 135 2 361 - 376 2022年03月 
    The assembly process of photosystem II (PSII) requires several auxiliary proteins to form assembly intermediates. In plants, early assembly intermediates comprise D1 and D2 subunits of PSII together with a few auxiliary proteins including at least ONE-HELIX PROTEIN1 (OHP1), OHP2, and HIGH-CHLOROPHYLL FLUORESCENCE 244 (HCF244) proteins. Herein, we report the basic characterization of the assembling intermediates, which we purified from Arabidopsis transgenic plants overexpressing a tagged OHP1 protein and named the OHP1 complexes. We analyzed two major forms of OHP1 complexes by mass spectrometry, which revealed that the complexes consist of OHP1, OHP2, and HCF244 in addition to the PSII subunits D1, D2, and cytochrome b559. Analysis of chlorophyll fluorescence showed that a major form of the complex binds chlorophyll a and carotenoids and performs quenching with a time constant of 420 ps. To identify the localization of the auxiliary proteins, we solubilized thylakoid membranes using a digitonin derivative, glycodiosgenin, and separated them into three fractions by ultracentrifugation, and detected these proteins in the loose pellet containing the stroma lamellae and the grana margins together with two chlorophyll biosynthesis enzymes. The results indicated that chlorophyll biosynthesis and assembly may take place in the same compartments of thylakoid membranes. Inducible suppression of the OHP2 mRNA substantially decreased the OHP2 protein in mature Arabidopsis leaves without a significant reduction in the maximum quantum yield of PSII under low-light conditions, but it compromised the yields under high-light conditions. This implies that the auxiliary protein is required for acclimation to high-light conditions.
  • Koki Fukura, Ayumi Tanaka, Ryouichi Tanaka, Hisashi Ito
    Journal of Plant Physiology 266 153535 - 153535 2021年11月 [査読有り]
  • Haruka Suehiro, Ryouichi Tanaka, Hisashi Ito
    Archives of Microbiology 203 6 3565 - 3575 2021年08月
  • Michiki Aso, Renon Matsumae, Ayumi Tanaka, Ryouichi Tanaka, Atsushi Takabayashi
    Plant and Cell Physiology 62 3 436 - 446 2021年07月31日 
    Abstract Land plants evolved from a single group of streptophyte algae. One of the key factors needed for adaptation to a land environment is the modification in the peripheral antenna systems of photosystems (PSs). Here, the PSs of Mesostigma viride, one of the earliest-branching streptophyte algae, were analyzed to gain insight into their evolution. Isoform sequencing and phylogenetic analyses of light-harvesting complexes (LHCs) revealed that M. viride possesses three algae-specific LHCs, including algae-type LHCA2, LHCA9 and LHCP, while the streptophyte-specific LHCB6 was not identified. These data suggest that the acquisition of LHCB6 and the loss of algae-type LHCs occurred after the M. viride lineage branched off from other streptophytes. Clear-native (CN)-polyacrylamide gel electrophoresis (PAGE) resolved the photosynthetic complexes, including the PSI–PSII megacomplex, PSII–LHCII, two PSI–LHCI–LHCIIs, PSI–LHCI and the LHCII trimer. Results indicated that the higher-molecular weight PSI–LHCI–LHCII likely had more LHCII than the lower-molecular weight one, a unique feature of M. viride PSs. CN-PAGE coupled with mass spectrometry strongly suggested that the LHCP was bound to PSII–LHCII, while the algae-type LHCA2 and LHCA9 were bound to PSI–LHCI, both of which are different from those in land plants. Results of the present study strongly suggest that M. viride PSs possess unique features that were inherited from a common ancestor of streptophyte and chlorophyte algae.
  • Ying Chen, Wataru Yamori, Ayumi Tanaka, Ryouichi Tanaka, Hisashi Ito
    Plant Science 307 110902 - 110902 2021年06月
  • Shinsa Kameo, Michiki Aso, Ryo Furukawa, Renon Matsumae, Makio Yokono, Tomomichi Fujita, Ayumi Tanaka, Ryouichi Tanaka, Atsushi Takabayashi
    Plant and Cell Physiology 62 2 348 - 355 2021年05月11日 
    Abstract Native polyacrylamide gel electrophoresis (PAGE) is a powerful technique for protein complex separation that retains both their activity and structure. In photosynthetic research, native-PAGE is particularly useful given that photosynthetic complexes are generally large in size, ranging from 200 kD to 1 MD or more. Recently, it has been reported that the addition of amphipol A8-35 to solubilized protein samples improved protein complex stability. In a previous study, we found that amphipol A8-35 could substitute sodium deoxycholate (DOC), a conventional electrophoretic carrier, in clear-native (CN)-PAGE. In this study, we present the optimization of amphipol-based CN-PAGE. We found that the ratio of amphipol A8-35 to α-dodecyl maltoside, a detergent commonly used to solubilize photosynthetic complexes, was critical for resolving photosynthetic machinery in CN-PAGE. In addition, LHCII dissociation from PSII–LHCII was effectively prevented by amphipol-based CN-PAGE compared with that of DOC-based CN-PAGE. Our data strongly suggest that majority of the PSII–LHCII in vivo forms C2S2M2 at least in Arabidopsis and Physcomitrella. The other forms might appear owing to the dissociation of LHCII from PSII during sample preparation and electrophoresis, which could be prevented by the addition of amphipol A8-35 after solubilization from thylakoid membranes. These results suggest that amphipol-based CN-PAGE may be a better alternative to DOC-based CN-PAGE for the study of labile protein complexes.
  • Debayan Dey, Dipanjana Dhar, Helena Fortunato, Daichi Obata, Ayumi Tanaka, Ryouichi Tanaka, Soumalee Basu, Hisashi Ito
    Computational and Structural Biotechnology Journal 19 5333 - 5347 2021年 [査読有り][通常論文]
  • Xueyun Hu, Ting Jia, Stefan Hörtensteiner, Ayumi Tanaka, Ryouichi Tanaka
    Plant Science 290 110314 - 110314 2020年01月 [査読有り]
  • Ryo Furukawa, Michiki Aso, Tomomichi Fujita, Seiji Akimoto, Ryouichi Tanaka, Ayumi Tanaka, Makio Yokono, Atsushi Takabayashi
    Journal of Plant Research Springer Science and Business Media {LLC} 2019年11月 [査読有り][通常論文]
  • Lim H, Tanaka A, Tanaka R, Ito H
    Plant & cell physiology 2019年08月 [査読有り][通常論文]
  • Fumiyoshi Myouga, Kaori Takahashi, Ryoichi Tanaka, Noriko Nagata, Anett Z. Kiss, Christiane Funk, Yuko Nomura, Hirofumi Nakagami, Stefan Jansson, Kazuo Shinozaki
    Plant Physiology 176 3 2277 - 2291 2018年03月01日 [査読有り][通常論文]
     
    The cellular functions of two Arabidopsis (Arabidopsis thaliana) one-helix proteins, OHP1 and OHP2 (also named LIGHT-HARVESTING-LIKE2 [LIL2] and LIL6, respectively, because they have sequence similarity to light-harvesting chlorophyll a/b-binding proteins), remain unclear. Tagged null mutants of OHP1 and OHP2 (ohp1 and ohp2) showed stunted growth with pale-green leaves on agar plates, and these mutants were unable to grow on soil. Leaf chlorophyll fluorescence and the composition of thylakoid membrane proteins revealed that ohp1 deletion substantially affected photosystem II (PSII) core protein function and led to reduced levels of photosystem I core proteins however, it did not affect LHC accumulation. Transgenic ohp1 plants rescued with OHP1-HA or OHP1-Myc proteins developed a normal phenotype. Using these tagged OHP1 proteins in transgenic plants, we localized OHP1 to thylakoid membranes, where it formed protein complexes with both OHP2 and High Chlorophyll Fluorescence244 (HCF244). We also found PSII core proteins D1/D2, HCF136, and HCF173 and a few other plant-specific proteins associated with the OHP1/OHP2-HCF244 complex, suggesting that these complexes are early intermediates in PSII assembly. OHP1 interacted directly with HCF244 in the complex. Therefore, OHP1 and HCF244 play important roles in the stable accumulation of PSII.
  • Yukako Kato, Makio Yokono, Seiji Akimoto, Atsushi Takabayashi, Ayumi Tanaka, Ryouichi Tanaka
    PLANT AND CELL PHYSIOLOGY 58 11 2026 - 2039 2017年11月 [査読有り][通常論文]
     
    Light-harvesting-like (LIL) proteins are a group of proteins that share a consensus amino acid sequence with light-harvesting Chl-binding (LHC) proteins. We hypothesized that they might be involved in photosynthesis-related processes. In order to gain a better understanding of a potential role in photosynthesis-related processes, we examined the most recently identified LIL protein, LIL8/PSB33. Recently, it was suggested that this protein is an auxiliary PSII core protein which is involved in organization of the PSII supercomplex. However, we found that the majority of LIL8/PSB33 was localized in stroma lamellae, where PSI is predominant. Moreover, the PSI antenna sizes measured under visible light were slightly increased in the lil8 mutants which lack LIL8/PSB33 protein. Analysis of fluorescence decay kinetics and fluorescence decay-associated spectra indicated that energy transfer to quenching sites within PSI was partially hampered in these mutants. On the other hand, analysis of the steady-state fluorescence spectra in these mutants indicates that a population of LHCII is energetically disconnected from PSII. Taken together, we suggest that LIL8/PSB33 is involved in the fine-tuning of light harvesting and/or energy transfer around both photosystems.
  • Xueyun Hu, Yukako Kato, Akihiro Sumida, Ayumi Tanaka, Ryouichi Tanaka
    PLANT JOURNAL 90 2 235 - 248 2017年04月 [査読有り][通常論文]
     
    Iron-sulfur (Fe-S) proteins play crucial roles in plastids, participating in photosynthesis and other metabolic pathways. Fe-S clusters are thought to be assembled on a scaffold complex composed of SUFB, SUFC and SUFD proteins. However, several additional proteins provide putative scaffold functions in plastids, and, therefore, the contribution of SUFB, C and D proteins to overall Fe-S assembly still remains unclear. In order to gain insights regarding Fe-S cluster biosynthesis in plastids, we analyzed the complex composed of SUFB, C and D in Arabidopsis by blue native-polyacrylamide gel electrophoresis. Using this approach, a major complex of 170 kDa containing all subunits was detected, indicating that these proteins constitute a SUFBC2D complex similar to their well characterized bacterial counterparts. The functional effects of SUFB, SUFC or SUFD depletion were analyzed using an inducible RNAi silencing system to specifically target the aforementioned components; resulting in a decrease of various plastidic Fe-S proteins including the PsaA/B and PsaC subunits of photosystem I, ferredoxin and glutamine oxoglutarate aminotransferase. In contrast, the knockout of potential Fe-S scaffold proteins, NFU2 and HCF101, resulted in a specific decrease in the PsaA/B and PsaC levels. These results indicate that the functions of SUFB, SUFC and SUFD for Fe-S cluster biosynthesis cannot be replaced by other scaffold proteins and that SUFBC2D, NFU2 and HCF101 are involved in the same pathway for the biogenesis of PSI. Taken together, our results provide in vivo evidence supporting the hypothesis that SUFBC2D is the major, and possibly sole scaffold in plastids.
  • Xueyun Hu, Mike T. Page, Akihiro Sumida, Ayumi Tanaka, Matthew J. Terry, Ryouichi Tanaka
    PLANT JOURNAL 89 6 1184 - 1194 2017年03月 [査読有り][通常論文]
     
    Proteins that contain iron-sulfur (Fe-S) clusters play pivotal roles in various metabolic processes such as photosynthesis and redox metabolism. Among the proteins involved in the biosynthesis of Fe-S clusters in plants, the SUFB subunit of the SUFBCD complex appears to be unique because SUFB has been reported to be involved in chlorophyll metabolism and phytochrome-mediated signaling. To gain insights into the function of the SUFB protein, we analyzed the phenotypes of two SUFB mutants, laf6 and hmc1, and RNA interference (RNAi) lines with reduced SUFB expression. When grown in the light, the laf6 and hmc1 mutants and the SUFB RNAi lines accumulated higher levels of the chlorophyll biosynthesis intermediate Mg-protoporphyrin IX monomethylester (Mg-proto MME), consistent with the impairment of Mg-proto MME cyclase activity. Both SUFC- and SUFD-deficient RNAi lines accumulated the same intermediate, suggesting that inhibition of Fe-S cluster synthesis is the primary cause of this impairment. Dark-grown laf6 seedlings also showed an increase in protoporphyrin IX ( Proto IX), Mg-proto, Mg-proto MME and 3,8-divinyl protochlorophyllide a (DV-Pchlide) levels, but this was not observed in hmc1 or the SUFB RNAi lines, nor was it complemented by SUFB overexpression. In addition, the long hypocotyl in far-red light phenotype of the laf6 mutant could not be rescued by SUFB overexpression and segregated from the pale-green SUFB-deficient phenotype, indicating it is not caused by mutation at the SUFB locus. These results demonstrate that biosynthesis of Fe-S clusters is important for chlorophyll biosynthesis, but that the laf6 phenotype is not due to a SUFB mutation.
  • Nino A. Espinas, Koichi Kobayashi, Yasushi Sato, Nobuyoshi Mochizuki, Kaori Takahashi, Ryouichi Tanaka, Tatsuru Masuda
    FRONTIERS IN PLANT SCIENCE 7 1326  2016年08月 [査読有り][通常論文]
     
    Heme is involved in various biological processes as a cofactor of hemoproteins located in various organelles. In plant cells, heme is synthesized by two isoforms of plastid-localized ferrochelatase. FC1 and FC2. In this study, by characterizing Arabidopsis T-DNA insertional mutants, we showed that the allocation of heme is differentially regulated by ferrochelatase isoforms in plant cells. Analyses of weak (fc1-1) and null (fc1-2) mutants suggest that FC1-producing heme is required for initial growth of seedling development. In contrast, weak (fc2-1) and null (fc2-2) mutants of FC2 showed pale green leaves and retarded growth, indicating that FC2-producing heme is necessary for chloroplast development. During the initial growth stage, FC2 deficiency caused reduction of plastid cytochromes. In addition, although FC2 deficiency marginally affected the assembly of photosynthetic reaction center complexes, it caused relatively larger but insufficient light-harvesting antenna to reaction centers, resulting in lower efficiency of photosynthesis. In the later vegetative growth, however, fc2-2 recovered photosynthetic growth, showing that FC1-producing heme may complement the FC2 deficiency. On the other hand, reduced level of cytochromes in microsomal fraction was discovered in fc1-1, suggesting that FC1-producing heme is mainly allocated to extraplastidic organelles. Furthermore, the expression of FC1 is induced by the treatment of an elicitor flg22 while that of FC2 was reduced, and fo1-abolished the flg22-dependent induction of FC1 expression and peroxidase activity. Consequently, our results clarified that FC2 produces heme for the photosynthetic machinery in the chloroplast, while FC1 is the housekeeping enzyme providing heme cofactor to the entire cell. In addition, FC1 can partly complement FC2 deficiency and is also involved in defense against stressful conditions.
  • Xueyun Hu, Satoru Makita, Silvia Schelbert, Shinsuke Sano, Masanori Ochiai, Tohru Tsuchiya, Shigeaki F. Hasegawa, Stefan Hoertensteiner, Ayumi Tanaka, Ryouichi Tanaka
    PLANT PHYSIOLOGY 167 3 660 - + 2015年03月 [査読有り][通常論文]
     
    Chlorophyllase (CLH) is a common plant enzyme that catalyzes the hydrolysis of chlorophyll to form chlorophyllide, a more hydrophilic derivative. For more than a century, the biological role of CLH has been controversial, although this enzyme has been often considered to catalyze chlorophyll catabolism during stress-induced chlorophyll breakdown. In this study, we found that the absence of CLH does not affect chlorophyll breakdown in intact leaf tissue in the absence or the presence of methyljasmonate, which is known to enhance stress-induced chlorophyll breakdown. Fractionation of cellular membranes shows that Arabidopsis (Arabidopsis thaliana) CLH is located in the endoplasmic reticulum and the tonoplast of intact plant cells. These results indicate that CLH is not involved in endogenous chlorophyll catabolism. Instead, we found that CLH promotes chlorophyllide formation upon disruption of leaf cells, or when it is artificially mistargeted to the chloroplast. These results indicate that CLH is responsible for chlorophyllide formation after the collapse of cells, which led us to hypothesize that chlorophyllide formation might be a process of defense against chewing herbivores. We found that Arabidopsis leaves with genetically enhanced CLH activity exhibit toxicity when fed to Spodoptera litura larvae, an insect herbivore. In addition, purified chlorophyllide partially suppresses the growth of the larvae. Taken together, these results support the presence of a unique binary defense system against insect herbivores involving chlorophyll and CLH. Potential mechanisms of chlorophyllide action for defense are discussed.
  • Kaori Takahashi, Atsushi Takabayashi, Ayumi Tanaka, Ryouichi Tanaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 289 2 987 - 999 2014年01月 [査読有り][通常論文]
     
    The light-harvesting complex (LHC) constitutes the major light-harvesting antenna of photosynthetic eukaryotes. LHC contains a characteristic sequence motif, termed LHC motif, consisting of 25-30 mostly hydrophobic amino acids. This motif is shared by a number of transmembrane proteins from oxygenic photoautotrophs that are termed light-harvesting-like (LIL) proteins. To gain insights into the functions of LIL proteins and their LHC motifs, we functionally characterized a plant LIL protein, LIL3. This protein has been shown previously to stabilize geranylgeranyl reductase (GGR), a key enzyme in phytol biosynthesis. It is hypothesized that LIL3 functions to anchor GGR to membranes. First, we conjugated the transmembrane domain of LIL3 or that of ascorbate peroxidase to GGR and expressed these chimeric proteins in an Arabidopsis mutant lacking LIL3 protein. As a result, the transgenic plants restored phytol-synthesizing activity. These results indicate that GGR is active as long as it is anchored to membranes, even in the absence of LIL3. Subsequently, we addressed the question why the LHC motif is conserved in the LIL3 sequences. We modified the transmembrane domain of LIL3, which contains the LHC motif, by substituting its conserved amino acids (Glu-171, Asn-174, and Asp-189) with alanine. As a result, the Arabidopsis transgenic plants partly recovered the phytol-biosynthesizing activity. However, in these transgenic plants, the LIL3-GGR complexes were partially dissociated. Collectively, these results indicate that the LHC motif of LIL3 is involved in the complex formation of LIL3 and GGR, which might contribute to the GGR reaction.
  • Makoto Kusaba, Ayumi Tanaka, Ryouichi Tanaka
    PHOTOSYNTHESIS RESEARCH 117 1-3 221 - 234 2013年11月 [査読有り][通常論文]
     
    A practical approach to increasing crop yields is to extend the duration of active photosynthesis. Stay-green is a term that is used to describe mutant and transgenic plants or cultivars with the trait of maintaining their leaves for a longer period of time than the wild-type or crosses from which they are derived. Analyzing stay-green genotypes contributes to our understanding of the molecular mechanism regulating leaf senescence which may allow us to extend the duration of active photosynthesis in crop plants. This article summarizes recent studies on stay-green plants and the insights they provide on the mechanism of leaf senescence. Briefly, mutations suppressing ethylene, abscisic acid, brassinosteroid, and strigolactone signal transduction or those activating cytokinin signaling often lead to stay-green phenotypes indicating a complex signaling network regulating leaf senescence. Developmentally regulated transcription factors, including NAC or WRKY family members, play key roles in the induction of leaf senescence and thus alteration in the activity of these transcription factors also result in stay-green phenotypes. Impairment in the enzymatic steps responsible for chlorophyll breakdown also leads to stay-green phenotypes. Some of these genotypes die in the middle of the process of chlorophyll breakdown due to the accumulation of toxic intermediates, while others appear to stay-green but their photosynthetic activity declines in a manner similar to wild-type plants. Alterations in certain metabolic pathways in chloroplasts (e.g., photosynthesis) can lead to a delayed onset of leaf senescence with maintenance of photosynthetic activity longer than wild-type plants, indicating that chloroplast metabolism can also affect the regulatory mechanism of leaf senescence.
  • Xueyun Hu, Ayumi Tanaka, Ryouichi Tanaka
    PLANT METHODS 9 June 19  2013年06月 [査読有り][通常論文]
     
    Background: When conducting plant research, the measurement of photosynthetic pigments can provide basic information on the physiological status of a plant. High-pressure liquid chromatography (HPLC) is becoming widely used for this purpose because it provides an accurate determination of a variety of photosynthetic pigments simultaneously. This technique has a drawback compared with conventional spectroscopic techniques, however, in that it is more prone to structural modification of pigments during extraction, thus potentially generating erroneous results. During pigment extraction procedures with acetone or alcohol, the phytol side chain of chlorophyll is sometimes removed, forming chlorophyllide, which affects chlorophyll measurement using HPLC. Results: We evaluated the artifactual chlorophyllide production during chlorophyll extraction by comparing different extraction methods with wild-type and mutant Arabidopsis leaves that lack the major isoform of chlorophyllase. Several extraction methods were compared to provide alternatives to researchers who utilize HPLC for the analysis of chlorophyll levels. As a result, the following three methods are recommended. In the first method, leaves are briefly boiled prior to extraction. In the second method, grinding and homogenization of leaves are performed at sub-zero temperatures. In the third method, N,N'-dimethylformamide (DMF) is used for the extraction of pigments. When compared, the first two methods eliminated almost all chlorophyllide-forming activity in Arabidopsis thaliana, Glebionis coronaria, Pisum sativum L. and Prunus sargentii Rehd. However, DMF effectively suppressed the activity of chlorophyllase only in Arabidopsis leaves. Conclusion: Chlorophyllide production in leaf extracts is predominantly an artifact. All three methods evaluated in this study reduce the artifactual production of chlorophyllide and are thus suitable for pigment extraction for HPLC analysis. The boiling method would be a practical choice when leaves are not too thick. However, it may convert a small fraction of chlorophyll a into pheophytin a. Although extraction at sub-zero temperatures is suitable for all plant species examined in this study, this method might be complicated for a large number of samples and it requires liquid nitrogen and equipment for leaf grinding. Using DMF as an extractant is simple and suitable with Arabidopsis samples. However, this solvent cannot completely block the formation of chlorophyllide in thicker leaves.
  • Atsushi Takabayashi, Ryosuke Kadoya, Masayoshi Kuwano, Katsunori Kurihara, Hisashi Ito, Ryouichi Tanaka, Ayumi Tanaka
    SPRINGERPLUS 2 1 148  2013年 [査読有り][通常論文]
     
    Protein-protein interactions are critical for most cellular processes; however, many remain to be identified. Here, to comprehensively identify protein complexes in photosynthetic organisms, we applied the recently developed approach of blue native PAGE (BN-PAGE) coupled with LC-MS/MS to the thylakoid proteins of Arabidopsis thaliana and the whole cell proteins of whole cell proteins of Synechocystis sp. PCC 6803. We identified 245 proteins from the purified Arabidopsis thylakoid membranes and 1,458 proteins from the whole cells of Synechocystis using the method. Next, we generated protein migration profiles that were assessed by plotting the label-free estimations of protein abundances versus migration distance in BN-PAGE. Comparisons between the migration profiles of the major photosynthetic complexes and their band patterns showed that the protein migration profiles were well correlated. Thus, the protein migration profiles allowed us to estimate the molecular size of each protein complex and to identify co-migrated proteins with the proteins of interest by determining the protein pairs that contained peaks in the same gel slice. Finally, we built the protein co-migration database for photosynthetic organisms (PCoM-DB: http://pcomdb.lowtem.hokudai.ac.jp/proteins/top) to make our data publicly accessible online, which stores the analyzed data with a user-friendly interface to compare the migration profiles of proteins of interest. It helps users to find unidentified protein complexes in Arabidopsis thylakoids and Synechocystis cells. The accumulation of the data from the BN-PAGE coupled with LC-MS/MS should reveal unidentified protein complexes and should aid in understanding the adaptation and the evolution of photosynthetic organisms.
  • Saori Nakajima, Hisashi Ito, Ryouichi Tanaka, Ayumi Tanaka
    PLANT PHYSIOLOGY 160 1 261 - 273 2012年09月 [査読有り][通常論文]
     
    Although seeds are a sink organ, chlorophyll synthesis and degradation occurs during embryogenesis and in a manner similar to that observed in photosynthetic leaves. Some mutants retain chlorophyll after seed maturation, and they are disturbed in seed storability. To elucidate the effects of chlorophyll retention on the seed storability of Arabidopsis (Arabidopsis thaliana), we examined the non-yellow coloring1 (nyc1)/nyc1-like (nol) mutants that do not degrade chlorophyll properly. Approximately 10 times more chlorophyll was retained in the dry seeds of the nyc1/nol mutant than in the wild-type seeds. The germination rates rapidly decreased during storage, with most of the mutant seeds failing to germinate after storage for 23 months, whereas 75% of the wild-type seeds germinated after 42 months. These results indicate that chlorophyll retention in the seeds affects seed longevity. Electron microscopic studies indicated that many small oil bodies appeared in the embryonic cotyledons of the nyc1/nol mutant; this finding indicates that the retention of chlorophyll affects the development of organelles in embryonic cells. A sequence analysis of the NYC1 promoter identified a potential abscisic acid (ABA)-responsive element. An electrophoretic mobility shift assay confirmed the binding of an ABA-responsive transcriptional factor to the NYC1 promoter DNA fragment, thus suggesting that NYC1 expression is regulated by ABA. Furthermore, NYC1 expression was repressed in the ABA-insensitive mutants during embryogenesis. These data indicate that chlorophyll degradation is induced by ABA during seed maturation to produce storable seeds.
  • 伊藤 寿, 田中 歩, 田中 亮一
    光合成研究 22 2 98 - 105 日本光合成学会 2012年08月 [査読無し][通常論文]
  • Yasuhito Sakuraba, Salma Balazadeh, Ryouichi Tanaka, Bernd Mueller-Roeber, Ayumi Tanaka
    PLANT AND CELL PHYSIOLOGY 53 3 505 - 517 2012年03月 [査読有り][通常論文]
     
    Leaf senescence is a developmentally and environmentally regulated process which includes global changes in gene expression. Using Arabidopsis as a model, we modified Chl arrangement in photosystems by overexpressing the catalytic domain (the C domain) of chlorophyllide a oxygenase (CAO) fused with the linker domain (the B domain) of CAO and green fluorescent protein (GFP). In these plants (referred to as the BCG plants for the B and C domains of CAO and GFP), the Chl a/b ratio was drastically decreased and Chl b was incorporated into core antenna complexes. The BCG plants exhibited a significant delay of both developmental and dark-induced leaf senescence. The photosynthetic apparatus, CO2 fixation enzymes and the chloroplast structure were lost in wild-type plants during senescence, while BCG plants retained them longer than the wild type. Large-scale quantitative real-time PCR analyses of 1,880 transcription factor (TF) genes showed that 241 TFs are differentially expressed between BCG plants and wild-type plants at senescence, similar to 40% of which are known senescence-associated genes (SAGs). Expression profiling also revealed the down-regulation of a large number of additional non-TF SAGs. In contrast, genes involved in photosynthesis were up-regulated, while those encoding Chl degradation enzymes were down-regulated in BCG plants. These results demonstrate that alteration of pigment composition in the photosynthetic apparatus retards senescence through transcriptional reprogramming.
  • Atsushi Takabayashi, Katsunori Kurihara, Masayoshi Kuwano, Yasuhiro Kasahara, Ryouichi Tanaka, Ayumi Tanaka
    PLANT AND CELL PHYSIOLOGY 52 12 2103 - 2114 2011年12月 [査読有り][通常論文]
     
    The reversible associations between the light-harvesting complexes (LHCs) and the core complexes of PSI and PSII are essential for the photoacclimation mechanisms in higher plants. Two types of Chls, Chl a and Chl b, both function in light harvesting and are required for the biogenesis of the photosystems. Chl b-less plants have been studied to determine the function of the LHCs because the Chl b deficiency has severe effects specific to the LHCs. Previous studies have shown that the amounts of the LHCs, especially the LHCII trimer, were decreased in the mutants; however, it is still unclear whether Chl b is required for the assembly of the LHCs and for the association of the LHCs with PSI and PSII. Here, to reveal the function of Chl b in the LHCs, we investigated the oligomeric states of the LHCs, PSI and PSII in the Arabidopsis Chl b-less mutant. A two-dimensional blue native-PAGE/SDS-PAGE demonstrated that the PSI-LHCI supercomplex was fully assembled in the absence of Chl b, whereas the trimeric LHCII and PSII-LHCII supercomplexes were not detected. The PSI-NAD(P)H dehydrogenase (NDH) supercomplexes were also assembled in the mutant. Furthermore, we detected two forms of monomeric LHC proteins. The faster migrating forms, which were detected primarily in the mutant, were probably apo-LHC proteins, whereas the slower migrating forms were probably the LHC proteins that contained Chl a. These findings increase our understanding of the Chl b function in the assembly of LHCs and the association of the LHCs with PSI, PSII and NDH.
  • Miki Meguro, Hisashi Ito, Atsushi Takabayashi, Ryouichi Tanaka, Ayumi Tanaka
    PLANT CELL 23 9 3442 - 3453 2011年09月 [査読無し][通常論文]
     
    The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation.
  • Ryouichi Tanaka, Ayumi Tanaka
    BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 1807 8 968 - 976 2011年08月 [査読無し][通常論文]
     
    Chlorophyll a and chlorophyll b are the major constituents of the photosynthetic apparatus in land plants and green algae. Chlorophyll a is essential in photochemistry, while chlorophyll b is apparently dispensable for their photosynthesis. Instead, chlorophyll b is necessary for stabilizing the major light-harvesting chlorophyll-binding proteins. Chlorophyll b is synthesized from chlorophyll a and is catabolized after it is reconverted to chlorophyll a. This interconversion system between chlorophyll a and chlorophyll b refers to the chlorophyll cycle. The chlorophyll b levels are determined by the activity of the three enzymes participating in the chlorophyll cycle, namely, chlorophyllide a oxygenase, chlorophyll b reductase, and 7-hydroxymethyl-chlorophyll reductase. This article reviews the recent progress on the analysis of the chlorophyll cycle and its enzymes. In particular, we emphasize the impact of genetic modification of chlorophyll cycle enzymes on the construction and destruction of the photosynthetic machinery. These studies reveal that plants regulate the construction and destruction of a specific subset of light-harvesting complexes through the chlorophyll cycle. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts. (C) 2011 Elsevier B.V. All rights reserved.
  • 田中歩, HU Xueyun, 田中亮一
    生化学 ROMBUNNO.1S16A-4  2011年 [査読無し][通常論文]
  • Tanaka R, Kobayashi K, Masuda T
    The Arabidopsis book 9 e0145  2011年 [査読有り][通常論文]
  • Ryouichi Tanaka, Maxi Rothbart, Seiko Oka, Atsushi Takabayashi, Kaori Takahashi, Masaru Shibata, Fumiyoshi Myouga, Reiko Motohashi, Kazuo Shinozaki, Bernhard Grimm, Ayumi Tanaka
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 107 38 16721 - 16725 2010年09月 [査読無し][通常論文]
     
    The light-harvesting chlorophyll-binding (LHC) proteins are major constituents of eukaryotic photosynthetic machinery. In plants, six different groups of proteins, LHC-like proteins, share a conserved motif with LHC. Although the evolution of LHC and LHC-like proteins is proposed to be a key for the diversification of modern photosynthetic eukaryotes, our knowledge of the evolution and functions of LHC-like proteins is still limited. In this study, we aimed to understand specifically the function of one type of LHC-like proteins, LIL3 proteins, by analyzing Arabidopsis mutants lacking them. The Arabidopsis genome contains two gene copies for LIL3, LIL3:1 and LIL3:2. In the lil3:1/lil3:2 double mutant, the majority of chlorophyll molecules are conjugated with an unsaturated geranylgeraniol side chain. This mutant is also deficient in a-tocopherol. These results indicate that reduction of both the geranylgeraniol side chain of chlorophyll and geranylgeranyl pyrophosphate, which is also an essential intermediate of tocopherol biosynthesis, is compromised in the lil3 mutants. We found that the content of geranylgeranyl reductase responsible for these reactions was severely reduced in the lil3 double mutant, whereas the mRNA level for this enzyme was not significantly changed. We demonstrated an interaction of geranylgeranyl reductase with both LIL3 isoforms by using a split ubiquitin assay, bimolecular fluorescence complementation, and combined blue-native and SDS polyacrylamide gel electrophoresis. We propose that LIL3 is functionally involved in chlorophyll and tocopherol biosynthesis by stabilizing geranylgeranyl reductase.
  • Kazushige Kato, Ryouichi Tanaka, Shinsuke Sano, Ayumi Tanaka, Hideo Hosaka
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 107 38 16649 - 16654 2010年09月 [査読無し][通常論文]
     
    Protoporphyrinogen oxidase (Protox) catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX during the synthesis of tetrapyrrole molecules. Protox is encoded by the hemY gene in eukaryotes and by the hemG gene in many gamma-proteobacteria, including Escherichia coli. It has been suggested that other bacteria possess a yet unidentified type of Protox. To identify a unique bacterial gene encoding Protox, we first introduced the Arabidopsis hemY gene into the genome of the cyanobacterium, Synechocystis sp. PCC6803. We subsequently mutagenized the cells by transposon tagging and screened the tagged lines for mutants that were sensitive to acifluorfen, which is a specific inhibitor of the hemY-type Protox. Several cell lines containing the tagged slr1790 locus exhibited acifluorfen sensitivity. The slr1790 gene encodes a putative membrane-spanning protein that is distantly related to the M subunit of NADH dehydrogenase complex I. We attempted to disrupt this gene in the wild-type background of Synechocystis, but we were only able to obtain heteroplasmic disruptants. These cells accumulated a substantial amount of protoporphyrin IX, suggesting that the slr1790 gene is essential for growth and Protox activity of cells. We found that most cyanobacteria and many other bacteria possess slr1790 homologs. We overexpressed an slr1790 homolog of Rhodobacter sphaeroides in Escherichia coli and found that this recombinant protein possesses Protox activity in vitro. These results collectively demonstrate that slr1790 encodes a unique Protox enzyme and we propose naming the slr1790 gene "hemJ."
  • Nobuyoshi Mochizuki, Ryouichi Tanaka, Bernhard Grimm, Tatsuru Masuda, Michael Moulin, Alison G. Smith, Ayumi Tanaka, Matthew J. Terry
    TRENDS IN PLANT SCIENCE 15 9 488 - 498 2010年09月 [査読無し][通常論文]
     
    Tetrapyrroles such as chlorophyll and heme are co-factors for essential proteins involved in a wide variety of crucial cellular functions. Nearly 2% of the proteins encoded by the Arabidopsis thaliana genome are thought to bind tetrapyrroles, demonstrating their central role in plant metabolism. Although the enzymes required for tetrapyrrole biosynthesis are well characterized, there are still major questions about the regulation of the pathway, and the transport of tetrapyrroles within cells. These issues are important, as misregulation of tetrapyrrole metabolism can lead to severe photo-oxidative stress, and because tetrapyrroles have been implicated in signaling pathways coordinating interactions between plant organelles. In this review, we discuss the cell biology of tetrapyrrole metabolism and its implications for tetrapyrroles as signaling molecules.
  • Yasuhito Sakuraba, Makio Yokono, Seiji Akimoto, Ryouichi Tanaka, Ayumi Tanaka
    PLANT AND CELL PHYSIOLOGY 51 6 1055 - 1065 2010年06月 [査読無し][通常論文]
     
    Chl b is one of the major light-harvesting pigments in land plants. The synthesis of Chl b is strictly regulated in response to light conditions in order to control the antenna size of photosystems. Regulation of Chl b also affects its distribution as it occurs preferentially in the peripheral antenna complexes. However, it has not been experimentally shown how plants respond to environmental conditions when they accumulate excess Chl b. Previously, we produced an Arabidopsis transgenic plant (referred to as the BC plant) in which Chl b biosynthesis was enhanced. In this study, we analyzed the photosynthetic properties and genome-wide gene expression in this plant under high light conditions in order to understand the effects of deregulated Chl b biosynthesis. The energy transfer rates between Chl a molecules in PSII decreased and H(2)O(2) accumulated extensively in the BC plant. Microarray analysis revealed that a group of genes involved in anthocyanin biosynthesis was down-regulated and that another group of genes, reported to be sensitive to H(2)O(2), was up-regulated in the BC plant. We also found that anthocyanin levels were low, which was consistent with the results of the microarray analysis. These results indicate that deregulation of Chl b caused severe photodamage and altered gene expression profiles under strong illumination. The importance of the regulation of Chl b synthesis is discussed in relation to the correct localization of Chl b and gene expression.
  • Masanori Fujii, Yoshinori Takano, Hisaya Kojima, Tamotsu Hoshino, Ryouichi Tanaka, Manabu Fukui
    MICROBIAL ECOLOGY 59 3 466 - 475 2010年04月 [査読無し][通常論文]
     
    "Red snow" refers to red-colored snow, caused by bloom of cold-adapted phototrophs, so-called snow algae. The red snow found in Langhovde, Antarctica, was investigated from several viewpoints. Various sizes of rounded red cells were observed in the red snow samples under microscopy. Pigment analysis demonstrated accumulation of astaxanthin in the red snow. Community structure of microorganisms was analyzed by culture-independent methods. In the analyses of small subunit rRNA genes, several species of green algae, fungus, and various phylotypes of bacteria were detected. The detected bacteria were closely related to psychrophilic or psychrotolerant heterotrophic strains, or sequences detected from low-temperature environments. As predominant lineage of bacteria, members of the genus Hymenobacter were consistently detected from samples obtained in two different years. Nitrogen isotopic compositions analysis indicated that the red snow was significantly N-15-enriched. Based on an estimation of trophic level, it was suggested that primary nitrogen sources of the red snow were supplied from fecal pellet of seabirds including a marine top predator of Antarctica.
  • Tomohiro Nagane, Ayumi Tanaka, Ryouichi Tanaka
    PLANTA 231 4 939 - 949 2010年03月 [査読無し][通常論文]
     
    In plants, chlorophyll is actively synthesized from glutamate in the developmental phase and is degraded into non-fluorescent chlorophyll catabolites during senescence. The chlorophyll metabolism must be strictly regulated because chlorophylls and their intermediate molecules generate reactive oxygen species. Many mechanisms have been proposed for the regulation of chlorophyll synthesis including gene expression, protein stability, and feedback inhibition. However, information on the regulation of chlorophyll degradation is limited. The conversion of chlorophyll b to chlorophyll a is the first step of chlorophyll degradation. In order to understand the regulatory mechanism of this reaction, we isolated a mutant which accumulates 7-hydroxymethyl chlorophyll a (HMChl), an intermediate molecule of chlorophyll b to chlorophyll a conversion, and designated the mutant hmc1. In addition to HMChl, hmc1 accumulated pheophorbide a, a chlorophyll degradation product, when chlorophyll degradation was induced by dark incubation. These results indicate that the activities of HMChl reductase (HAR) and pheophorbide a oxygenase (PaO) are simultaneously down-regulated in this mutant. We identified a mutation in the AtNAP1 gene, which encodes a subunit of the complex for iron-sulfur cluster formation. HAR and PaO use ferredoxin as a reducing power and PaO has an iron-sulfur center; however, there were no distinct differences in the protein levels of ferredoxin and PaO between wild type and hmc1. The concerted regulation of chlorophyll degradation is discussed in relation to the function of AtNAP1.
  • Yasuhito Sakuraba, Ryouichi Tanaka, Akihiro Yamasato, Ayumi Tanaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 284 52 36689 - 36699 2009年12月 [査読無し][通常論文]
     
    Chlorophyll b is one of the major photosynthetic pigments of plants. The regulation of chlorophyll b biosynthesis is important for plants in order to acclimate to changing environmental conditions. In the chloroplast, chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), a Rieske-type monooxygenase. The activity of this enzyme is regulated at the level of protein stability via a feedback mechanism through chlorophyll b. The Clp protease and the N-terminal domain (designated the A domain) of CAO are essential for the regulatory mechanism. In this study, we aimed to identify the specific amino acid residue or the sequence within the A domain that is essential for this regulation. To accomplish this goal, we randomly introduced base substitutions into the A domain and searched for potentially important residues by analyzing 1,000 transformants of Arabidopsis thaliana. However, none of the single amino acid substitutions significantly stabilized CAO. Therefore, we generated serial deletions in the A domain and expressed these deletions in the background of CAO-deficient Arabidopsis mutant. We found that the amino acid sequence (97)QDLLTIMILH(106) is essential for the regulation of the protein stability. We furthermore determined that this sequence induces the destabilization of green fluorescent protein. These results suggest that this sequence serves as a degradation signal that is recognized by proteases functioning in the chloroplast.
  • Yukiko Horie, Hisashi Ito, Makoto Kusaba, Ryouichi Tanaka, Ayumi Tanaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 284 26 17449 - 17456 2009年06月 [査読無し][通常論文]
     
    The light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) is the most abundant membrane protein in green plants, and its degradation is a crucial process for the acclimation to high light conditions and for the recovery of nitrogen (N) and carbon (C) during senescence. However, the molecular mechanism of LHCII degradation is largely unknown. Here, we report that chlorophyll b reductase, which catalyzes the first step of chlorophyll b degradation, plays a central role in LHCII degradation. When the genes for chlorophyll b reductases NOL and NYC1 were disrupted in Arabidopsis thaliana, chlorophyll b and LHCII were not degraded during senescence, whereas other pigment complexes completely disappeared. When purified trimeric LHCII was incubated with recombinant chlorophyll b reductase (NOL), expressed in Escherichia coli, the chlorophyll b in LHCII was converted to 7-hydroxymethyl chlorophyll a. Accompanying this conversion, chlorophylls were released from LHCII apoproteins until all the chlorophyll molecules in LHCII dissociated from the complexes. Chlorophyll-depleted LHCII apoproteins did not dissociate into monomeric forms but remained in the trimeric form. Based on these results, we propose the novel hypothesis that chlorophyll b reductase catalyzes the initial step of LHCII degradation, and that trimeric LHCII is a substrate of LHCII degradation.
  • Yoshito Chikaraishi, Ryouichi Tanaka, Ayumi Tanaka, Naohiko Ohkouchi
    ORGANIC GEOCHEMISTRY 40 5 569 - 573 2009年05月 [査読無し][通常論文]
     
    Deuterium (D) depletion in phytol relative to ambient water as well as other lipids has been widely observed in various biological and geological samples; however, the mechanism for the depletion remains unknown. We have determined the hydrogen isotopic compositions of phytol and its precursors in cucumber cotyledons and have evaluated the fractionation of hydrogen isotopes during phytol biosynthesis. The hydrogen isotopic compositions of geranylgeraniol, dihydrogeranylgeraniol, tetrahydrogeranylgeraniol and phytol are -281 parts per thousand, -302 parts per thousand, -325 parts per thousand and -345 parts per thousand, respectively. The results suggest that significantly D-depleted hydrogen is incorporated stepwise during hydrogenation of geranylgeraniol to phytol. We conclude that hydrogenation is important in controlling D depletion in phytol. (C) 2009 Elsevier Ltd. All rights reserved.
  • Masumi Hirashima, Ryouichi Tanaka, Ayumi Tanaka
    PLANT AND CELL PHYSIOLOGY 50 4 719 - 729 2009年04月 [査読無し][通常論文]
     
    Tetrapyrroles are well-known photosensitizers. In plants, various intermediate molecules of tetrapyrrole metabolism have been reported to induce cell death in a light-dependent manner. In contrast to these reports, we found that pheophorbide a, a key intermediate of chlorophyll catabolism, causes cell death in complete darkness in a transgenic Arabidopsis plant, As-ACD1. In this plant, expression of mRNA for pheophorbide a oxygenase was suppressed by expression of Acd1 antisense RNA; thus, As-ACD1 accumulated an excessive amount of pheophorbide a when chlorophyll breakdown occurred. We observed that when senescence was induced by a continuous dark period, leaves of As-ACD1 plants became dehydrated. By measuring electrolyte leakage, we estimated that 50 of the leaf cells underwent cell death within a 5d period of darkness. Light and electron microscopic observations indicated that the cellular structure had collapsed in a large population of cells. Partially covering a leaf with aluminum foil resulted in light-independent cell death in the covered region and induced bleaching in the uncovered regions. These results indicate that accumulation of pheophorbide a induces cell death under both darkness and illumination, but the mechanisms of cell death under these conditions may differ. We discuss the possible mechanism of light-independent cell death and the involvement of pheophorbide a in the signaling pathway for programmed cell death.
  • 横野牧生, 田中亮一, 田中歩
    低温科学 67 669 - 672 2009年03月31日 [査読無し][通常論文]
  • 田中 歩, 田中 亮一
    植物の生長調節 43 2 95 - 105 植物化学調節学会 2008年12月19日 [査読無し][通常論文]
     
    クロロフィル代謝は60年以上にわたり,多くの研究者の興味を引いてきた.初期は,クロロフィル合成経路と各ステップの反応機構の解明を目指して研究が行われた.それに引き続き,分子遺伝学の発展やゲノム情報を基盤に,酵素とその遺伝子の同定が取り組まれた.クロロフィル分解系は合成系に比べ研究が遅れていたが,近年急速に解明が進んできた.これらの研究によって,クロロフィル代謝経路とそれを担う遺伝子がほぼ全て明らかになった.クロロフィル代謝経路の全容が明らかになるにつれて,クロロフィル代謝が光環境適応や,細胞死,葉緑体シグナル,常緑化など植物細胞の様々な生理現象と強く関連していることが明らかになり,クロロフィル代謝の新しい機能が注目されてきた.本総説では,クロロフィル代謝の概要と,その多機能性について紹介する.
  • Satoshi Kanematsu, Yasuhito Sakuraba, Ayumi Tanaka, Ryouichi Tanaka
    PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES 7 10 1196 - 1205 2008年10月 [査読無し][通常論文]
     
    Chlorophyll b is one of the major light-harvesting pigments produced by land plants, green algae and several cyanobacterial species. It is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), which in higher plants consists of three domains, namely, A, B. and C. We previously demonstrated that the C domain exhibits a catalytic function, whereas the A domain destabilizes the CAO protein in the presence of chlorophyll b, thus regulating the cellular level of CAO. In a previous study, we also presented genetic evidence demonstrating the involvement of Clp protease in the destabilization of CAO. In this study, in order to gain further insight into the regulatory mechanism of CAO, we screened for mutants defective ill the control of CAO accumulation. Seeds from an Arabidopsis transgenic plant overexpressing a chimeric protein consisting of the A and B domains of CAO and green fluorescent protein (GFP) were mutagenized by ethyl methane sulfonate. We screened the progenies of the transgenic plants by laser-scanning confocal microscopy, and isolated a total of 66 mutants exhibiting significant GFP fluorescence. By immunoblotting analysis, we confirmed that these mutants accumulated the fusion protein of the N-terminal domains of CAO and GFP at a high level. We further divided these mutants into seven groups by distribution patterns of the fusion protein, and characterized them by pigment and immunoblotting analyses. Based on these analyses, we proposed a model to describe the regulatory mechanism of CAO.
  • Nobuyoshi Mochizuki, Ryouichi Tanaka, Ayumi Tanaka, Tatsuru Masuda, Akira Nagatani
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 39 15184 - 15189 2008年09月 [査読無し][通常論文]
     
    The plastid plays a vital role in various cellular activities within plant cells including photosynthesis and other metabolic pathways. It is believed that the functional status of the plastid is somehow monitored by the nucleus to optimize the expression of genes encoding plastid proteins. The currently dominant model for plastid-derived signaling ("plastid signaling'') proposes that Mg-protoporphyrin IX (MgProto) is a negative signal that represses the expression of a wide range of nuclear genes encoding plastid-localized proteins when plastid development is inhibited. In this study, we have re-evaluated this hypothesis by quantifying the steady-state levels of MgProto (as well as its neighboring intermediates protoporphyrin IX and MgProto monomethyl ester [MgProtoMe]) in Arabidopsis plants with altered plastid signaling responses as monitored by expression of the Lhcb1, RBCS, HEMA1, BAM3 and CA1 genes. In addition, we have examined the correlation between gene expression and MgProto (MgProtoMe) in a range of mutants and conditions in which the steady-state levels of MgProto (MgProtoMe) have been modified. Overall we found that there was no correlation between the steady-state levels of MgProto (MgProtoMe) and Lhcb1 expression or with any of the other genes tested. Taking these results together, we propose that the current model on plastid signaling must be revised.
  • Akihiro Yamasato, Ryouichi Tanaka, Ayumi Tanaka
    BMC PLANT BIOLOGY 8 64  2008年06月 [査読無し][通常論文]
     
    Background: Chlorophyll b is a major photosynthetic pigment in green plants that is synthesized by chlorophyllicle a oxygenase (CAO). The regulation of chlorophyll b biosynthesis is an important determinant for the antenna size of photosystems. Chlorophyll b synthesis is partly regulated on a transcriptional level by the expression of the CAO gene. In addition, the synthesis of chlorophyll b is strictly regulated on a protein level by the stability of the CAO enzyme. CAO consists of three domains, which are sequentially named from the N terminus as the A, B and C domains. The A domain of CAO participates in the regulation of the CAO protein stability. Results: In order to clarify the physiological function of the A domain, we constructed transgenic Arabiclopsis (Arabidopsis thaliana) plants which either overexpressed the complete CAO or a truncated version of CAO lacking the A domain. The transgenic plants overexpressing the Adomain-deleted CAO accumulated an excess amount of chlorophyll b during greening. The transgenic plants which lacked the A domain either died or were obviously retarded when they were exposed to continuous light immediately after etiolation. In addition, the loss of the A domain in CAO impaired another step of chlorophyll biosynthesis, namely the conversion of divinylprotochlorophyllicle a to monovinyl protochlorophyllicle a under dark conditions. Conclusion: The A domain of CAO regulates the level of CAO, and thus prevents the excess accumulation of chlorophyll b. This function of the A domain is especially important during the greening stage of etiolated seedlings. At this stage, the plants are vulnerable to photodamages which could be caused by excessive chlorophyll b accumulation. In addition, cle-regulation of the CAO level affects monovinyl-protochlorophyllicle biosynthesis in darkness by unknown mechanisms. In conclusion, the A domain of CAO is essential in the control of chlorophyll biosynthesis and in the survival of seedlings during cle-etiolation especially under strong illumination.
  • Hisashi Ito, Makio Yokono, Ryouichi Tanaka, Ayumi Tanaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 283 14 9002 - 9011 2008年04月 [査読無し][通常論文]
     
    The vast majority of oxygenic photosynthetic organisms use monovinyl chlorophyll for their photosynthetic reactions. For the biosynthesis of this type of chlorophyll, the reduction of the 8-vinyl group that is located on the B-ring of the macrocycle is essential. Previously, we identified the gene encoding 8-vinyl reductase responsible for this reaction in higher plants and termed it DVR. Among the sequenced genomes of cyanobacteria, only several Synechococcus species contain DVR homologues. Therefore, it has been hypothesized that many other cyanobacteria producing monovinyl chlorophyll should contain a vinyl reductase that is unrelated to the higher plant DVR. To identify the cyanobacterial gene that is responsible for monovinyl chlorophyll synthesis, we developed a bioinformatics tool, correlation coefficient calculation tool, which calculates the correlation coefficient between the distributions of a certain phenotype and genes among a group of organisms. The program indicated that the distribution of a gene encoding a putative dehydrogenase protein is best correlated with the distribution of the DVR-less cyanobacteria. We subsequently knocked out the corresponding gene ( Slr1923) in Synechocystis sp. PCC6803 and characterized the mutant. The knock-out mutant lost its ability to synthesize monovinyl chlorophyll and accumulated 3,8-divinyl chlorophyll instead. We concluded that Slr1923 encodes the vinyl reductase or a subunit essential for monovinyl chlorophyll synthesis. The function and evolution of 8-vinyl reductase genes are discussed.
  • Analysis of the N-terminal domain of chlorophyllide a oxygenase by random mutagenesis. Photosynthesis. Energy from the Sun
    14th International Congress on Photosynthesis 1049 - 1054 2008年 [査読無し][通常論文]
  • Nozomi Nagata, Ryouichi Tanaka, Ayumi Tanaka
    PLANT AND CELL PHYSIOLOGY 48 12 1803 - 1808 2007年12月 [査読無し][通常論文]
     
    In most reviews on Chl biosynthesis, Chl is described as being synthesized via the route involving the reduction of [3,8:-divinyl]-protochlorophyllide a. However, the possibility remains that the conversion of the divinyl form of the Chl intermediate to its monovinyl form takes place at other enzymatic steps. To determine the actual route of Chl biosynthesis, we examined the substrate specificity of the formerly named [3,8-divinyl]-protochlorophyllide a 8-vinyl reductase (DVR) in vitro. In addition, we investigated the accumulation of various Chl intermediates in etiolated seedlings in vivo. Collectively, these studies indicate that [3,8-divinyl]-chlorophyllide a is the major substrate of DVR.
  • Yasuhito Sakuraba, Akihiro Yamasato, Ryouichi Tanaka, Ayumi Tanaka
    PLANT PHYSIOLOGY AND BIOCHEMISTRY 45 10-11 740 - 749 2007年10月 [査読無し][通常論文]
     
    Higher plants acclimate to various light environments by changing the antenna size of a light-harvesting photosystem. The antenna size of a photosystem is partly determined by the amount of chlorophyll b in the light-harvesting complexes. Chlorophyllide a oxygenase (CAO) converts chlorophyll a to chlorophyll b in a two-step oxygenation reaction. In our previous study, we demonstrated that the cellular level of the CAO protein controls accumulation of chlorophyll b. We found that the amino acids sequences of CAO in higher plants consist of three domains (A, B, and C domains). The C domain exhibits a catalytic function, and we demonstrated that the combination of the A and B domains regulates the cellular level of CAO. However, the individual function of each of A and B domain has not been determined yet. Therefore, in the present study we constructed a series of deleted CAO sequences that were fused with green fluorescent protein and overexpressed in a chlorophyll b-less mutant of Arabidopsis thaliana, chI-I, to further dissect functions of A and B domains. Subsequent comparative analyses of the transgenic plants overexpressing B domain containing proteins and those lacking the B domain determined that there was no significant difference in CAO protein levels. These results indicate that the B domain is not involved in the regulation of the CAO protein levels. Taken together, we concluded that the A domain alone is involved in the regulatory mechanism of the CAO protein levels. (C) 2007 Elsevier Masson SAS. All rights reserved.
  • Makoto Kusaba, Hisashi Ito, Ryouhei Morita, Shuichi Iida, Yutaka Sato, Masaru Fujimoto, Shinji Kawasaki, Ryouichi Tanaka, Hirohiko Hirochika, Minoru Nishimura, Ayumi Tanaka
    PLANT CELL 19 4 1362 - 1375 2007年04月 [査読無し][通常論文]
     
    Chlorophyll degradation is an aspect of leaf senescence, which is an active process to salvage nutrients from old tissues. non-yellow coloring1 (nyc1) is a rice (Oryza sativa) stay-green mutant in which chlorophyll degradation during senescence is impaired. Pigment analysis revealed that degradation of not only chlorophylls but also light-harvesting complex II (LHCII) bound carotenoids was repressed in nyc1, in which most LHCII isoforms were selectively retained during senescence. Ultrastructural analysis of nyc1 chloroplasts revealed that large and thick grana were present even in the late stage of senescence, suggesting that degradation of LHCII is required for the proper degeneration of thylakoid membranes. Map-based cloning of NYC1 revealed that it encodes a chloroplast-localized short-chain dehydrogenase/reductase (SDR) with three transmembrane domains. The predicted structure of the NYC1 protein and the phenotype of the nyc1 mutant suggest the possibility that NYC1 is a chlorophyll b reductase. Although we were unable to detect the chlorophyll b reductase activity of NYC1, NOL (for NYC1-like), a protein closely related to NYC1 in rice, showed chlorophyll b reductase activity in vitro. We suggest that NYC1 and NOL encode chlorophyll b reductases with divergent functions. Our data collectively suggest that the identified SDR protein NYC1 plays essential roles in the regulation of LHCII and thylakoid membrane degradation during senescence.
  • Eiki Nakagawara, Yasuhito Sakuraba, Akihiro Yamasato, Ryouichi Tanaka, Ayumi Tanaka
    PLANT JOURNAL 49 5 800 - 809 2007年03月 [査読無し][通常論文]
     
    Chlorophyll b is one of the major light-harvesting pigments in green plants and it is essential for optimal light harvesting. Chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO) which consists of A, B and C domains. Previously, we demonstrated that the C domain alone has a catalytic function, while the A and B domains control the level of CAO protein in response to chlorophyll b accumulation. We hypothesized that the accumulation of chlorophyll b triggers the proteolytic degradation of CAO. In this study, in order to gain further insight into this regulatory mechanism we screened for mutants that have defects in the control of CAO accumulation. Seeds from a transgenic line of Arabidopsis which overexpressed a CAO-GFP fusion were mutagenized and their progenies were screened by laser-scanning confocal microscopy for mutants showing an elevated level of GFP fluorescence. One particular mutant (dca1) exhibited stronger GFP fluorescence and accumulated a GFP-CAO fusion protein at a higher level. Concomitantly, the chlorophyll a to b ratio decreased in this mutant. The mutation in the dca1 mutant was mapped to the ClpC1 gene, thereby indicating that a chloroplast Clp protease is involved in regulating chlorophyll b biosynthesis through the destabilization of CAO protein in response to the accumulation of chlorophyll b.
  • Y. Sakuraba, R. Tanaka, A. Tanaka
    PHOTOSYNTHESIS RESEARCH 91 2-3 252 - 252 2007年02月 [査読無し][通常論文]
  • Ryouichi Tanaka, Ayumi Tanaka
    ANNUAL REVIEW OF PLANT BIOLOGY 58 321 - 346 2007年 [査読有り][通常論文]
     
    Tetrapyrroles play vital roles in various biological processes, including photosynthesis and respiration. Higher plants contain four classes of tetrapyrroles, namely, chlorophyll, heme, siroheme, and phytochromobilin. All of the tetrapyrroles are derived from a common biosynthetic pathway. Here we review recent progress in the research of tetrapyrrole biosynthesis from a cellular biological view. The progress consists of biochemical, structural, and genetic analyses, which contribute to our understanding of how the flow and the synthesis of tetrapyrrole molecules are regulated and how the potentially toxic intermediates of tetrapyrrole synthesis are maintained at low levels. We also describe interactions of tetrapyrrole biosynthesis and other cellular processes including the stay-green events, the cell-death program, and the plastid-to-nucleus signal transduction. Finally, we present several reports on attempts for agricultural and horticultural applications in which the tetrapyrrole biosynthesis pathway was genetically modified.
  • MOHAREKAR S, MOHAREKAR S, MOHAREKAR S, TANAKA R, OGAWA K. I, OGAWA K. I, TANAKA A, TANAKA A, HARA T, HARA T
    Photosynthetica 45 2 259 - 265 2007年 [査読無し][通常論文]
     
    Arabidopsis thaliana L., chl-I (chlorophyll b-less mutant), gi-1 (GI deficient mutant), cry2-1 (blue-light-photoreceptor CRY2 deficient mutant), and Columbia (Col; wild ecotype) were grown under broad range of irradiances (1) from the beginning of germination and the effect of I on the survival, development, and flowering was studied. Under low and moderate 1 (< 300 mu mol m(-2) s(-1)), flowering time and plant size at flowering showed great variations among chl-1, gi-1, cry2-1, and Col, whereas under higher I (> 500 mu mol m(-2) s(-1)), these characteristics were almost the same. Hence under high I, development and flowering of chl-1, gi-1, cry2-1, and Col converged to almost the same state. Flowering time was negatively correlated with 1, and under high I acclimation in A. thaliana was associated with a decrease in chlorophyll (Chl) content and increases in xanthophyll cycle pool and membrane-bound APX activity (EC 1.11. 1. 11) suggesting that an increase in oxidative stress induces earlier flowering. The plants of gi-1 and cry2-1 survived but Col and chl-l died under 1 000 pmol m(-2) s(-1), showing that mutants deficient in GI or CRY2 are more photo-stress-tolerant than Col and the Chi b-less mutant. Hence high I promotes in plants of Arabidapsis raised from germination till flowering the development and flowering time involving modulation of the photosynthetic apparatus, and this promoting effect is independent of the functions of flower-inducing GI or CRY2 gene. This can be regarded as photo-acclimation of A. thaliana for survival and reproduction under high I.
  • D. Aarti, R. Tanaka, H. Ito, A. Tanaka
    PHOTOCHEMISTRY AND PHOTOBIOLOGY 83 1 171 - 176 2007年01月 [査読無し][通常論文]
     
    Using the vascular plant Cucumis sativus (cucumber) as a model, we studied the effects of high (intense and excess) light upon chlorophyll biosynthesis during de-etiolation. When illuminated with high light (1500-1600 mu E/m(2)/s), ctiolated cucumber cotyledons failed to synthesize chlorophyll entirely. However, upon transfer to low light conditions (40-45 mu E/m(2)/S), chlorophyll biosynthesis and subsequent accumulation resumed following an initial 2-12 h delay. Duration of high light treatment negatively correlated with chlorophyll biosynthetic activity. Specifically, we found that high light severely inhibited 5-aminolevulinic acid (ALA) synthesis. This effect partly could be because of the decrease in protein level of glutamyl-tRNA reductase (GluTR) observed. Protein level of glutamate-1-semialdehyde (GSA-AT) remained unchanged. It was also found that high light did not suppress HEMA I expression. Therefore, we speculated that this significant inhibition of ALA, synthesis might have occurred mainly because of concomitant inactivation of GluTR and/or inhibition of complex formation between GluTR and GSA-AT. Our further observation that both methyl viologen and rose bengal similarly inhibit ALA synthesis under low light conditions suggested that reactive oxygen species (ROS) could be responsible for the inhibition of ALA synthesis in cotyledons exposed to high light conditions.
  • P. D. Aarti, Ryouichi Tanaka, Ayumi Tanaka
    PHYSIOLOGIA PLANTARUM 128 1 186 - 197 2006年09月 [査読無し][通常論文]
     
    We conducted a series of experiments to assess the effects of oxidative stress on chlorophyll biosynthesis in the vascular plant Cucumis sativus (cucumber). Specifically, cucumber cotyledons were treated with 100 mu M methyl viologen (MV) and subsequently exposed to dark (0 mu E m(-2) s(-1)), low light (4045 mu E m(-2) s(-1)), or high light (1500-1600 mu E m(-2) s(-1)). Following treatment, extracts of these samples were subjected to high-performance liquid chromatography (HPLC) to quantitate the accumulation of chlorophyll biosynthetic pathway intermediates. The results of these analyses revealed significant accumulation of Mg-protoporphyrin IX monomethyl ester (Mg-proto IX ME) in green (14-h illuminated) as well as in etiolated cotyledons with MV treatment. These data suggest that MV-induced oxidative stress may have inhibited Mg-proto IX ME cyclase activity. Upon exposure to high light, in the presence or absence of MV, both green and etiolated cotyledons predominantly accumulated protoporphyrin IX (Proto IX). These elevated levels of Proto IX might be attributable to attenuated activity of any or all of the following enzymes: Mg-chelatase, Fe-chelatase and protoporphyrinogen IX oxidase. We also observed that MV-induced oxidative stress impacts on chlorophyll biosynthesis to a greater extent than on photosystem II. These results demonstrate that oxidative stress impedes key steps in chlorophyll biosynthesis by either directly or indirectly inhibiting the activity of these enzymes.
  • Ayumi Tanaka, Ryouichi Tanaka
    CURRENT OPINION IN PLANT BIOLOGY 9 3 248 - 255 2006年06月 [査読有り][通常論文]
     
    Since the 1970s, researchers have proposed several regulatory pathways governing chlorophyll metabolism, but only recently have the underlying molecular mechanisms been elucidated. The recent data indicate that such regulatory systems are more complex than originally anticipated. For instance, the pathways involve a series of protein-protein interactions, including complex formation, the dual localization of enzymes within chloroplasts, and a novel protein degradation mechanism that is triggered by pigments. Furthermore, several lines of evidence suggest that chlorophyll metabolism might not only significantly impact the assembly of photosynthetic machineries but also influence processes such as programmed cell death, the 'stay-green' phenomenon, and chloroplast-nucleus communication.
  • M Hirashima, S Satoh, R Tanaka, A Tanaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 281 22 15385 - 15393 2006年06月 [査読無し][通常論文]
     
    The organization of pigment molecules in photosystems is strictly determined. The peripheral antennae have both chlorophyll a and b, but the core antennae consist of only chlorophyll a in green plants. Furthermore, according to the recent model obtained from the crystal structure of light-harvesting chlorophyll a/b-protein complexes II (LHCII), individual chlorophyll-binding sites are occupied by either chlorophyll a or chlorophyll b. In this study, we succeeded in altering these pigment organizations by introducing a prokaryotic chlorophyll b synthesis gene (chlorophyllide a oxygenase (CAO)) into Arabidopsis. In these transgenic plants (Prochlirothrix hollandica CAO plants), similar to 40% of chlorophyll a of the core antenna complexes was replaced by chlorophyll b in both photosystems. Chlorophyll a/b ratios of LHCII also decreased from 1.3 to 0.8 in PhCAO plants. Surprisingly, these transgenic plants were capable of photosynthetic growth similar to wild type under low light conditions. These results indicate that chlorophyll organizations are not solely determined by the binding affinities, but they are also controlled by CAO. These data also suggest that strict organizations of chlorophyll molecules are not essential for photosynthesis under low light conditions.
  • R Tanaka, A Tanaka
    PHOTOSYNTHESIS RESEARCH 85 3 327 - 340 2005年09月 [査読無し][通常論文]
     
    Land plants change the compositions of light-harvesting complexes (LHC) and chlorophyll (Chl) a/b ratios in response to the variable light environments which they encounter. In this study, we attempted to determine the mechanism which regulates Chl a/b ratios and whether the changes in Chl a/b ratios are essential in regulation of LHC accumulation during light acclimation. We hypothesized that changes in the mRNA levels for chlorophyll a oxygenase (CAO) involved in Chl b biosynthesis are an essential part of light response of Chl a/b ratios and LHC accumulation. We also examined the light-intensity dependent response of CAO-overexpression and wild-type Arabidopsis thaliana plants. When wild-type plants were acclimated from low-light (LL) to high-light (HL) conditions, CAO mRNA levels decreased and the Chl a/b ratio increased. In transgenic plants overexpressing CAO, the Chl a/b ratio remained low under HL conditions; thereby suggesting that changes in the CAO mRNA levels are necessary for those in Chl a/b ratios upon light acclimation. Under HL conditions, the accumulation of Lhcb1, Lhcb3 and Lhcb6 was enhanced in plants overexpressing CAO. On the contrary, in a CAO-deficient mutant, chlorina 1-1, theaccumulation of Lhcb1, Lhcb2, Lhcb3, Lhcb6 and Lhca4 was reduced. In comparison to wild-type, beta-carotene levels were reduced in CAO-overexpressing plants, while they were elevated in chlorina 1-1 mutants. These results imply that the transcriptional control of CAO is a part of the regulatory mechanism for the accumulation of a distinct set of LHC proteins upon light acclimation.
  • S Akimoto, M Yokono, M Ohmae, Yamazaki, I, N Nagata, R Tanaka, A Tanaka, M Mimuro
    CHEMICAL PHYSICS LETTERS 409 4-6 167 - 171 2005年06月 [査読無し][通常論文]
     
    We examined the excitation energy transfer and electron transfer processes in Arabidopsis thaliana thylakoid membranes following replacement of chlorophyll (Ch1) molecules by expression of the vinyl reductase: monovinyl (MV)-Ch1 was replaced with divinyl (DV)-Ch1 and the DV-Ch1 a/DV-Ch1 b ratio was higher than that of control cells. By this replacement, energy transfer within the photosystem (PS) II became faster, whereas the PS II-to-PS I energy transfer was slower. In the DV-Ch1 mutant system, the delayed fluorescence from PS II was not resolved. Alternations of the antenna system and the reaction center are discussed in relation to Ch1 replacement. (c) 2005 Elsevier B.V. All rights reserved.
  • A Yamasato, N Nagata, R Tanaka, A Tanaka
    PLANT CELL 17 5 1585 - 1597 2005年05月 [査読無し][通常論文]
     
    Plants acclimate to variations in light intensity by changing the antenna size of photosystems. This acclimation allows them to undergo efficient photosynthesis and creates a protective strategy to minimize photodamage. Chlorophyll b synthesis by chlorophyllide a oxygenase (CAO) is a key regulatory step in the control of antenna size. Recently, we found that higher plant CAOs consist of three domains (A, B, and C domains) and confirmed that the C domain possesses catalytic function. To investigate the function of the A domain, we fused various combinations of these three domains with green fluorescent protein (GFP) and introduced them into Arabidopsis thaliana. When a full-length CAO-GFP fusion protein was introduced into a chlorophyll b-less chlorina1-1 mutant, chlorophyll b accumulated to almost the same levels as in the chlorophyll b-containing Columbia wild type, but the CAO-GFP could not be detected by immunoblotting. By contrast, when a GFP-C domain fusion was introduced into chlorina1-1 or Columbia wild type, a large amount of GFP-C domain protein accumulated and the chlorophyll a/b ratio decreased drastically from 3.6 to 2.2 in Columbia wild type. When an A domain-GFP was introduced into Columbia wild type, A domain-GFP levels were very low. Conversely, a large amount of the protein accumulated when it was introduced into the chlorina1-1 mutant. These results indicate that the A domain may sense the presence of chlorophyll b and regulate the accumulation of CAO protein in the chloroplasts.
  • N Nagata, R Tanaka, S Satoh, A Tanaka
    PLANT CELL 17 1 233 - 240 2005年01月 [査読無し][通常論文]
     
    Chlorophyll metabolism has been extensively studied with various organisms, and almost all of the chlorophyll biosynthetic genes have been identified in higher plants. However, only the gene for 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), which is indispensable for monovinyl chlorophyll synthesis, has not been identified yet. In this study, we isolated an Arabidopsis thaliana mutant that accumulated divinyl chlorophyll instead of monovinyl chlorophyll by ethyl methanesulfonate mutagenesis. Map-based cloning of this mutant resulted in the identification of a gene (AT5G18660) that shows sequence similarity with isoflavone reductase genes. The mutant phenotype was complemented by the transformation with the wild-type gene. A recombinant protein encoded by AT5G18660 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyllide to monovinyl chlorophyllide, thereby demonstrating that the gene encodes a functional DVR. DVR is encoded by a single copy gene in the A. thaliana genome. With the identification of DVR, finally all genes required for chlorophyll biosynthesis have been identified in higher plants. Analysis of the complete genome of A. thaliana showed that it has 15 enzymes encoded by 27 genes for chlorophyll biosynthesis from glutamyl-tRNA(glu) to chlorophyll b. Furthermore, identification of the DVR gene helped understanding the evolution of Prochlorococcus marinus, a marine cyanobacterium that is dominant in the open ocean and is uncommon in using divinyl chlorophylls. A DVR homolog was not found in the genome of P. marinus but found in the Synechococcus sp WH8102 genome, which is consistent with the distribution of divinyl chlorophyll in marine cyanobacteria of the genera Prochlorococcus and Synechococcus.
  • N Nagata, S Satoh, R Tanaka, A Tanaka
    PLANTA 218 6 1019 - 1025 2004年04月 [査読無し][通常論文]
     
    Chlorophyll b is a photosynthetic antenna pigment found in prochlorophytes and chlorophytes. In chlorophytes, its biosynthesis regulates the photosynthetic antenna size. Chlorophyll b is synthesized from chlorophyll a in a two-step oxygenation reaction by chlorophyllide a oxygenase (CAO). In this study, we first identified the entire sequence of a prochlorophyte CAO gene from Prochlorothrix hollandica to compare it with those from chlorophytes, and we examined the catalytic activity of the gene product. Southern blot analysis showed that the CAO gene is presented in one copy in the P. hollandica genome. The P. hollandica CAO gene (PhCAO) has a coding capacity for 367 amino acids, which is much smaller than that of Arabidopsis thaliana (537 amino acids) and Oryza sativa (542 amino acids) CAO genes. In spite of the small size, PhCAO catalyzed the formation of chlorophyll b. By comparing these sequences, we classified the land-plant sequences into four parts: the N-terminal sequence predicted to be a transit peptide, the successive conserved sequence unique in land plants (A-domain, 134 amino acids), a less-conserved sequence (B-domain, 30 amino acids) and the C-terminal conserved sequence common in chlorophytes and prochlorophytes (C-domain, 337 to 344 amino acids). We demonstrated that the C-domain is sufficient for catalytic activity by transforming the cyanobacterium Synechocystis sp. PCC6803 with the C-domain from A. thaliana. In this paper, the role of the A-domain is discussed in relation to the formation of light-harvesting chlorophyll a/b-protein complexes in land plants.
  • 田中 歩, 平島 真澄, 田中 亮一
    化学と生物 42 2 93 - 98 日本農芸化学会 2004年02月25日 [査読無し][通常論文]
  • Isolation and Characterization of a Gene for Chlorophyllide a Oxygenase from Prochlorothrix hollandica.
    Nozomi Nagata, Ryouichi Tanaka, Soichirou Satoh, Jun Minagawa, Ayumi Tanaka
    Endocytobiosis Cell Res 15 1 321 - 327 2004年 [査読無し][通常論文]
  • R Tanaka, M Hirashima, S Satoh, A Tanaka
    PLANT AND CELL PHYSIOLOGY 44 12 1266 - 1274 2003年12月 [査読無し][通常論文]
     
    Oxygenation of pheophorbide a is a key step in chlorophyll breakdown. Several biochemical studies have implicated that this step was catalyzed by an iron-containing and ferredoxin-dependent monooxygenase, pheophorbide a oxygenase (PaO). It has been proposed that inhibition of its activity arrests the chlorophyll breakdown and leads to the "stay-green" phenotype. We searched the Arabidopsis genome for a possible PaO-encoding gene and hypothesized that it has homology to known iron-containing Rieske-type monooxygenase sequences. We identified three such open reading frames, Tic55, ACD1 and ACD1-Iike. We produced transgenic Arabidopsis plants which expressed antisense RNA as a method to inhibit the expression of these genes. The appearance of these antisense plants were indistinguishable from that of the wild type under illumination. However, after they were kept under darkness for 5 d and again illuminated, the leaves of the antisense ACD1 plants (AsACD1) were bleached. Leaves of AsACD1 accumulated 387 nmol (g FW)(-1) pheophorbide a which corresponded to 60% of chlorophyll a degraded. The rate of decrease in chlorophyll a was not influenced in senesced AsACD1 leaves. These results demonstrated that ACD1 is involved in PaO activity, and its inhibition led to photooxidative destruction of the cell instead of the "stay-green" phenotype.
  • ST Moharekar, SD Lokhande, T Hara, R Tanaka, A Tanaka, PD Chavan
    PHOTOSYNTHETICA 41 2 315 - 317 2003年 [査読無し][通常論文]
     
    With the increase in concentration of applied salicylic acid (SA), chlorophyll (Chl) content decreased significantly in both wheat and moong seedlings. Chl a/b ratio decreased significantly only in wheat and remained constant in moong. On the other hand, total carotenoid (Car) content, size of xanthophyll pool, and de-epoxidation rate increased significantly with an increase in SA concentration in both plant species. Hence SA treatment may induce Car biosynthesis in these plant species, but the increase in the xanthophyll pool and de-epoxidation rate indicates that SA may create oxidative stress the degree of which is different in various plants.
  • R Tanaka, Y Koshino, S Sawa, S Ishiguro, K Okada, A Tanaka
    PLANT JOURNAL 26 4 365 - 373 2001年05月 [査読無し][通常論文]
     
    The light-harvesting efficiency of a photosystem is thought to be largely dependent on its photosynthetic antenna size. It has been suggested that antenna size is controlled by the biosynthesis of chlorophyll b. To verify this hypothesis, we overexpressed the enzyme for chlorophyll b biosynthesis, chlorophyllide a oxygenase (CAO), in Arabidopsis thaliana by transforming the plant with cDNA for CAO under the control of the 35S cauliflower mosaic virus promoter. In the early de-etiolation phase, when the intrinsic CAO expression is very low, the chlorophyll a: b ratio was drastically decreased from 28 to 7.3, indicating that enhancement of chlorophyll b biosynthesis had been successfully achieved. We made the following observations in full-green rosette leaves of transgenic plants. (1) The chlorophyll a: b ratio was reduced from 2.85 to 2.65. (2) The ratio of the peripheral light-harvesting complexes (LHCII) to the core antenna complex (CPa) resolved with the green-gel system increased by 20%. (3) The ratio of the light-harvesting complex II apoproteins (LHCP) to 47-kDa chlorophyll a protein (CP47), which was estimated by the results of immunoblotting, increased by 40%. These results indicated that the antenna size increased by at least 10-20% in transgenic plants, suggesting that chlorophyll b biosynthesis controls antenna size. To the best of our knowledge, this is the first report on enlargement of the antenna size by genetic manipulations.
  • J Papenbrock, HP Mock, R Tanaka, E Kruse, B Grimm
    PLANT PHYSIOLOGY 122 4 1161 - 1169 2000年04月 [査読無し][通常論文]
     
    Magnesium-protoporphyrin IX chelatase (Mg-chelatase) is located at the branchpoint of tetrapyrrole biosynthesis, at which point protoporphyrin IX is distributed for the synthesis of chlorophyll and heme. We investigated the regulatory contribution of Mg-chelatase to the flow of metabolites. In plants, the enzyme complex consists of three subunits, designated CHL D, CHL I, and CHL H. Transgenic tobacco (Nicotiana tabacum) plants expressing antisense RNA for the Mg-chelatase subunit CHL H were analyzed to elucidate further the role of Mg-chelatase in the distribution of protoporphyrin IX into the branched tetrapyrrolic pathway. The transgenic plants displayed a reduced growth rate and chlorophyll deficiency. Both phenotypical properties were correlated with lower Mg-chelatase activity. Unexpectedly, less protoporphyrin IX and heme accumulated, and a decrease in li-aminolevulinate (ALA)-synthesizing capacity and ALA dehydratase activity paralleled the progressive reduction in Mg-chelatase activity in the transformants compared with control plants. The reduced activities of the early enzymatic steps corresponded with lower levels of transcripts encoding glutamyl-tRNA reductase and ALA-dehydratase. The decreased expression and activities of early enzymes in the pathway could be explained by a feedback-controlled mechanism in response to lower Mg-chelatase activity. We discuss intercompartmental signaling that synchronizes the activities of the first steps in tetrapyrrolic metabolism with the late steps for the synthesis of end products.
  • U Oster, R Tanaka, A Tanaka, W Rudiger
    PLANT JOURNAL 21 3 305 - 310 2000年02月 [査読無し][通常論文]
     
    Chlorophyll (Chl) biosynthesis and degradation are the only biochemical processes on Earth that can be directly observed from satellites or other planets. The bulk of the Chls is found in the light-harvesting antenna complexes of photosynthetic organisms. Surprisingly little is known about the biosynthesis of Chl b, which is the second most abundant Chl pigment after Chl a. We describe here the expression and properties of the chlorophyllide a oxygenase gene (CAO) from Arabidopsis thaliana, which is apparently the key enzyme in Chl b biosynthesis. The recombinant enzyme produced in Escherichia coli catalyses an unusual two-step oxygenase reaction that is the 'missing link' in the chlorophyll cycle of higher plants.
  • SATOH Soichirou, TANAKA Ryouichi, MIMURO Mamoru, IKEUCHI Masahiko, TANAKA Ayumi
    Plant and cell physiology 41 0 s98  日本植物生理学会 2000年 [査読無し][通常論文]
  • R Tanaka, U Oster, E Kruse, W Rudiger, B Grimm
    PLANT PHYSIOLOGY 120 3 695 - 704 1999年07月 [査読無し][通常論文]
     
    The enzyme geranylgeranyl reductase (CHL P) catalyzes the reduction of geranylgeranyl diphosphate to phytyl diphosphate. We identified a tobacco (Nicotiana tabacum) cDNA sequence encoding a 52-kD precursor protein homologous to the Arabidopsis and bacterial CHL P. The effects of deficient CHL P activity on chlorophyll (Chl) and tocopherol contents were studied in transgenic plants expressing antisense CHL P RNA. Transformants with gradually reduced Chi P expression showed a delayed growth rate and a pale or variegated phenotype. Transformants grown in high (500 mu mol m(-2) s(-1); HL) and low (70 mu mol photon m(-2) s(-1); LL) light displayed a similar degree of reduced tocopherol content during leaf development, although growth of wild-type plants in HL conditions led to up to a 2-fold increase in tocopherol content. The total Chl content was more rapidly reduced during HL than LL conditions. Up to 58% of the Chl content was esterified with geranylgeraniol instead of phytol under LL conditions. Our results indicate that CHL P provides phytol for both tocopherol and Chl synthesis. The transformants are a valuable model with which to investigate the adaptation of plants with modified tocopherol levels against deleterious environmental conditions.
  • A Tanaka, H Ito, R Tanaka, NK Tanaka, K Yoshida, K Okada
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 95 21 12719 - 12723 1998年10月 [査読有り][通常論文]
     
    Chlorophyll b is an ubiquitous accessory pigment in land plants, green algae, and prochlorophytes. Its biosynthesis plays a key role in the adaptation to various light environments. We isolated six chlorophyll b-less mutants by insertional mutagenesis by using the nitrate reductase or argininosuccinate lyase genes as tags and examined the rearrangement of mutant genomes. We found that an overlapping region of a nuclear genome was deleted in all mutants and that this encodes a protein whose sequence is similar to those of methyl monooxygenases. This coding sequence also contains putative binding domains for a [2Fe-2S] Rieske center and for a mononuclear iron. The results demonstrate that a chlorophyll a oxygenase is involved in chlorophyll b formation. The reaction mechanism of chlorophyll b formation is discussed.
  • R Tanaka, K Yoshida, T Nakayashiki, H Tsuji, H Inokuchi, K Okada, A Tanaka
    PHOTOSYNTHESIS RESEARCH 53 2-3 161 - 171 1997年09月 [査読有り][通常論文]
     
    Accumulation of chlorophylls and heme is primarily controlled at the level of 5-aminolevulinate (ALA) synthesis in higher plants. ALA is formed from glutamate in three enzymatic steps in plants. Among them, the reduction of glutamyl-tRNA(Glu) to glutamate-1-semialdehyde (GSA) is likely to be a regulatory point of ALA synthesis. This reaction is catalyzed by glutamyl-tRNA reductase (GTR), which is encoded by a hemA gene. We have isolated a novel isoform of a hemA cDNA clone from barley (Hordeum vulgare) that is the third member of the hemA gene family. mRNA of this isoform is accumulated primarily in roots, suggesting that the isoform is regulated in an organ-specific manner by the demand for heme synthesis rather than chlorophyll. Phylogenetic analysis was done using the deduced amino acid sequences of hemA isoforms from barley, cucumber and Arabidopsis thaliana. The results indicate that the existing gene families in these plants arose after the divergence of monocotyledonous and dicotyledonous plants.
  • 田中歩, 田中亮一, 伊藤寿, 吉田和市
    蛋白質 核酸 酵素 41 2 136 - 145 共立出版 1996年02月 [査読無し][通常論文]
  • TANAKA R, YOSHIDA K, NAKAYASHIKI T, MASUDA T, TSUJI H, INOKUCHI H, TANAKA A
    Plant Physiology 110 4 1223 - 1230 1996年 [査読無し][通常論文]
     
    The first committed step of porphyrin synthesis in higher plants is the reduction of glutamyl-tRNA to glutamate 1-semialdehyde. This reaction is catalyzed by glutamyl-tRNA reductase, which is encoded by hemA genes. Two hemA cDNA clones (hemA1 and hemA2) were obtained from cucumber (Cucumis sativus) cotyledons by the PCR and cDNA library screening. They showed significant homology with published hemA sequences. Southern blot analysis of cucumber genomic DNA revealed that these genes are located at different loci and that there is another gene similar to the hemA genes. Accumulation of hemA1 mRNA was detected primarily in cotyledons and hypocotyls of greening cucumber seedlings, whereas that of hemA2 mRNA was detected in all tissues examined. Illumination of cucumber seedlings increased markedly the accumulation of hemA1 mRNA, but it did not induce remarkable changes in that of hemA2 mRNA. These findings suggest that hemA1 mRNA was accumulated in response to the demand of Chl synthesis in photosynthesizing tissues, whereas hemA2 mRNA was expressed in response to the demand of the synthesis of porphyrins other than chlorophylls.
  • K YOSHIDA, RM CHEN, A TANAKA, H TERAMOTO, R TANAKA, MP TIMKO, H TSUJI
    PLANT PHYSIOLOGY 109 1 231 - 238 1995年09月 [査読有り][通常論文]
     
    Changes in the activity and abundance of NADPH:protochlorophyllide oxidoreductase (NPR) and the abundance of mRNA encoding it were examined during the greening of 5-d-old etiolated cucumber cotyledons under continuous illumination. To measure NPR activity in the extracts from fully greened tissues, we have developed an improved method of assay. Upon exposure of etiolated cotyledons to light, NPR activity decreased rapidly within the first 2 h of exposure. Thereafter, enzymatic activity increased transiently, reaching a submaximum level at 12 h, and decreased slowly. The level of immunodetectable NPR protein followed the same pattern of changes during 96 h of greening as observed for NPR activity. The NPR mRNA in etiolated cotyledons disappeared quickly in the ist h of irradiation. However, the level of mRNA increased thereafter to reach 3-fold or more of the dark level at 12 h and then decreased. The changes in the activity, protein level, and mRNA level after the first rapid decreases corresponded chronologically and nearly paralleled the increase in the rate of chlorophyll accumulation. These findings suggest that the greening of cucumber cotyledons is regulated basically by the level of NPR protein without activation or repression of enzymatic activity and that NPR mRNA increased by light maintains the level of enzyme protein necessary for greening.
  • MIYAMOTO K, TANAKA R, TERAMOTO H, MASUDA T, TSUJI H, INOKUCHI H
    Plant Physiology 105 2 769 - 770 1994年06月 [査読無し][通常論文]
  • T MASUDA, R TANAKA, Y SHIOI, K TAKAMIYA, CG KANNANGARA, H TSUJI
    PLANT AND CELL PHYSIOLOGY 35 2 183 - 188 1994年03月 [査読有り][通常論文]
     
    The mechanism of the stimulatory effect of a cytokinin, namely, benzyladenine (BA), on the synthesis of 5-aminolevulinic acid (ALA) in cucumber cotyledons was studied. The rate of synthesis of ALA by plastids isolated from BA-treated cotyledons was twice that by plastids from untreated controls. Western blot analysis of stromal proteins showed that BA did not affect the level of glutamyl-tRNA synthetase or of glutamate 1-semialdehyde (GSA) aminotransferase. Analysis of free amino acids revealed that BA did not increase the level of glutamate in the stroma. However, the amount of total plastidic RNA was doubled in BA-treated cotyledons. Northern blot analysis showed that the level of plastid TRNA(Glu) was increased by treatment with BA to the same extent as that of another plastid tRNA, reflecting an increase in total plastidic RNA. The rate of formation of glutamyl-tRNA was also doubled in plastids from BA-treated cotyledons. The results indicate that stimulation of the synthesis of ALA by BA is due to an increased level of tRNA(Glu) in plastids.

講演・口頭発表等

  • 伊藤寿, 横野牧生, 田中亮一, 田中歩
    日本植物生理学会年会要旨集 2008年03月
  • Analysis of an Arabidopsis mutant that accumulates 7-hydroxymethyl chlorophyll a  [通常講演]
    T. Nagane, R. Tanaka, A. Tanaka
    PHOTOSYNTHESIS RESEARCH 2007年02月 SPRINGER
  • Examination of the intra-plastid localization of chlorophyllide alpha oxygenase  [通常講演]
    Satoshi Kanematsu, Yasuhito Sakuraba, Ryouichi Tanaka, Ayumi Tanaka
    PLANT AND CELL PHYSIOLOGY 2007年 OXFORD UNIV PRESS
  • Analysis of the Arabidopsis chlorina5 mutant lacking a chloroplast metalloprotease  [通常講演]
    Makoto Sugawara, Ryouichi Tanaka, Shinichiro Sawa, Ayumi Tanaka
    PLANT AND CELL PHYSIOLOGY 2007年 OXFORD UNIV PRESS
  • 永田 望, 田中 亮一, 佐藤 壮一郎, 田中 歩
    日本植物生理学会年会およびシンポジウム 講演要旨集 2005年 日本植物生理学会
     
    クロロフィル<I>a</I>の合成経路は様々な生物で広く研究され、クロロフィル合成を触媒する酵素や遺伝子に関しても多くが同定されている。しかし、その中で高等植物の3,8-ジビニルプロトクロロフィリド<I>a</I> 8-ビニルレダクターゼ(DVR)だけが同定されていない。<br>私たちはEMS処理によってモノビニルクロロフィルではなく、ジビニルクロロフィルを蓄積するシロイヌナズナの変異株を単離した。この変異株のクロロフィル色素がモノビニル型からジビニル型へ変化したのは、変異株がDVR活性を欠損したからであると考えられる。<br>変異株のポジショナルクローニングから同定した遺伝子は、5番染色体上にあり、NADPH依存型酵素のアミノ酸配列に類似していた。同定した野生型の遺伝子を変異株へ導入したところ、変異株の表現系は相補された。また、同定した遺伝子由来の大腸菌発現タンパク質は、ジビニルクロロフィリドからモノビニルクロロフィリドへの還元を触媒する、DVR活性を示した。<br>これらの結果から、シロイヌナズナ変異株の原因遺伝子は、クロロフィル合成系の<I>DVR</I>遺伝子であることが明らかになった。また、シロイヌナズナの<I>DVR</I>遺伝子が同定したことから、私たちは光合成色素としてジビニルクロロフィルを利用する、原核緑藻プロクロロコッカスの進化についても考察する。
  • Analysis of protein levels of chlorophyllide a oxygenase regulated by its own N-terminal domain.  [通常講演]
    A Yamasato, R Tanaka, A Tanaka
    PLANT AND CELL PHYSIOLOGY 2005年 OXFORD UNIV PRESS
  • 佐藤 壮一郎, 平島 真澄, 田中 亮一, 田中 歩
    日本植物生理学会年会およびシンポジウム 講演要旨集 2004年 日本植物生理学会
     
    光合成色素による光エネルギーの捕捉は光合成集光装置によって行われる.光合成集光装置は,中心集光装置と周辺集光装置で構成されている.中心集光装置は,酸素発生型光合成生物では良く保存されており,その構成はどのような環境でも一定である.一方,周辺集光装置は生物種によって多様であり,また環境によってその構成と大きさは変化する.緑色植物においては,中心集光装置はクロロフィル<I>a</I>タンパク質複合体で構成されているのに対して,周辺集光装置は集光性クロロフィル<I>a</I>/<I>b</I>タンパク質複合体(LHC)で構成されている.しかし,周辺集光装置と中心集光装置への異なった色素の選択的分配がどのように決定されているかは不明である.この点を明らかにするため,LHCとの相互作用が示唆されている緑色植物のクロロフィル<I>b</I>合成遺伝子(<I>CAO</I>)のかわりに,LHCと相互作用しないと考えられる原核緑藻<I>Prochlorothrix</I>のCAO(<I>PhCAO</I>)をシロイヌナズナに導入し,色素タンパク質複合体の形成を調べた.その結果,PhCAOを導入した株では,光化学系IおよびIIの中心集光装置にクロロフィル<I>b</I>が取込まれ,クロロフィル<I>a</I>/<I>b</I>タンパク質複合体に転換した。PhCAOを導入した形質転換株は,弱光下では野生型とほぼ同じ形質を示した.これらの結果をもとに,クロロフィルのタンパク質への選択的分配と色素タンパク質の可塑性について議論する.
  • 平島 真澄, 田中 亮一, 佐藤 壮一郎, 田中 歩
    日本植物生理学会年会およびシンポジウム 講演要旨集 2004年 日本植物生理学会
     
    フェオフォルビド<I>a</I>オキシゲナーゼ(PaO)は、クロロフィル分解系において、フェオフォルビド<I>a</I>に酸素を添加し、テトラピロール環を開環する反応を担う酵素である。この酵素の活性が阻害されると、植物は常緑化すると考えられている。また、PaOはフェレドキシンの還元力を利用したモノオキシゲナーゼであり、葉緑体の包膜に存在することが報告されている。我々はシロイヌナズナのゲノムデータベースを調べ、PaO遺伝子候補を3つ選出し、これらの遺伝子に対するアンチセンスRNA形質転換株を作成した。このうち<I>ACD1</I>(<I> Accelerated Cell Death 1</I>)のアンチセンス株(AsACD1株)を暗所で数日間生育させると、野生株ではほとんど蓄積しないフェオフォルビド<I>a</I>の蓄積が見られた。この結果は、<I>ACD1</I>がPaOであることを示唆している。暗所生育下でのこの株のクロロフィルの分解は、野生株とほぼ同程度行われていたことから、PaOの活性阻害は、シロイヌナズナにおいては、常緑化に直接結びつかないと考えられる。また、暗所の後再び連続光下で生育させると、AsACD1株の葉は白く退色し、枯死した。これは蓄積したフェオフォルビド<I>a</I>が光を受けて、活性酸素を発生させたためであると考えられる。
  • Analysis of an Arabidopsis mutant deficient in chlorophyll b accumulation, chlorina5  [通常講演]
    Y Kurata, R Tanaka, S Sawa, A Tanaka
    PLANT AND CELL PHYSIOLOGY 2004年 OXFORD UNIV PRESS
  • Studies in the localization of chloropyllide a oxygenase using green fluorescence protein  [通常講演]
    A Yamasato, N Nagata, R Tanaka, A Tanaka
    PLANT AND CELL PHYSIOLOGY 2003年 OXFORD UNIV PRESS
  • Mechanisms for high light acclimation studied with transgenic arabidopsis plants overexpressing chlorophyllide a oxygenase  [通常講演]
    R Tanaka, M Hirashima, A Tanaka
    PLANT AND CELL PHYSIOLOGY 2003年 OXFORD UNIV PRESS

その他活動・業績

特許

  • クロロフィル生合成に関与する新規ビニル基還元酵素およびその利用
    特開2006-187203

受賞

  • 2016年02月 北海道大学 教育総長賞 奨励賞
     
    受賞者: 田中 亮一

共同研究・競争的資金等の研究課題

  • クロロフィル生合成、光合成の環境適応機構の解析
  • Chlorophyll metabolism

教育活動情報

主要な担当授業

  • 環境応答システム科学特論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : 光合成、クロロフィル代謝
  • 大学院共通授業科目(一般科目):複合領域
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 論文の書き方、プレゼンテーション、生物学
  • 生命科学特別講義Ⅱ(国際)
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : 論文の書き方、プレゼンテーション、生物学
  • 環境分子生物学特論Ⅱ
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 環境科学院
    キーワード : 植物、微生物、環境応答、分子機構、進化、光合成、ストレス耐性 Plants, microorganisms, response to environments, molecular mechanisms, evolution, photosynthesis, resistance to environmental stress
  • 分子生物学基礎論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 環境科学院
    キーワード : 分子生物学,転写,翻訳,微生物の群集構造解析,微生物の呼吸活性,電子顕微鏡観察,遺伝子操作,バイオフィルム形成,植物ストレス耐性,バイオセンサー,昆虫免疫系,昆虫体表脂質, 動物細胞 molecular biology, transcription, translation, bacterial culture, bacterial community structure analysis, bacterial respiration activity, electron microscopic observation, genetic manipulation, biofilm formation, stress tolerance of plants, biosensor, insect immune system, insect body surface lipids, animal cells
  • 環境と人間
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 地球環境、雪氷、大気、海洋、生態系、生物、光合成、耐寒性、宇宙の氷、分子進化、冬眠
  • 一般教育演習(フレッシュマンセミナー)
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 地球環境、環境適応、微生物、植物、昆虫、哺乳類、光合成、冬眠、生態、進化

大学運営

委員歴

  • 2018年01月 - 2020年03月   日本植物生理学会   男女共同参画委員長


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