三宅親弘 (大学院農学研究科生命機能科学専攻)

The ability of P700 oxidation in photosystem I reflects chilling stress tolerance in cucumber.
Ko Takeuchi, Yufen Che, Takeshi Nakano, Chikahiro Miyake, Kentaro Ifuku
Low temperature inhibits photosynthesis and negatively affects plant growth. Cucumber (Cucumis sativus L.) is a chilling-sensitive plant, and its greenhouse production requires considerable energy during the winter. Therefore, a useful stress marker for selecting chilling-tolerant cucumber cultivars is desirable. In this study, we evaluated chilling-stress damage in different cucumber cultivars by measuring photosynthetic parameters. The majority of cultivars showed decreases in the quantum yield of photosystem (PS) II [Fv/Fm and Y(II)] and the quantity of active PS I (Pm) after chilling stress. In contrast, Y(ND)-the ratio of the oxidized state of PSI reaction center chlorophyll P700 (P700+)-differed among cultivars and was perfectly inversely correlated with Y(NA)-the ratio of the non-photooxidizable P700. It has been known that P700+ accumulates under stress conditions and protects plants to suppress the generation of reactive oxygen species. In fact, cultivars unable to induce Y(ND) after chilling stress showed growth retardation with reductions in chlorophyll content and leaf area. Therefore, Y(ND) can be a useful marker to evaluate chilling-stress tolerance in cucumber.
2022年09月, Journal of plant research, 135 (5), 681 - 692, 英語, 国内誌
研究論文（学術雑誌）

NADPH production in dark stages is critical for cyanobacterial photocurrent generation: a study using mutants deficient in oxidative pentose phosphate pathway.
Jiro Hatano, Shoko Kusama, Kenya Tanaka, Ayaka Kohara, Chikahiro Miyake, Shuji Nakanishi, Ginga Shimakawa
Live cyanobacteria and algae integrated onto an extracellular electrode can generate a light-induced current (i.e., a photocurrent). Although the photocurrent is expected to be correlated with the redox environment of the photosynthetic cells, the relationship between the photocurrent and the cellular redox state is poorly understood. Here, we investigated the effect of the reduced nicotinamide adenine dinucleotide phosphate [NADP(H)] redox level of cyanobacterial cells (before light exposure) on the photocurrent using several mutants (Δzwf, Δgnd, and ΔglgP) deficient in the oxidative pentose phosphate (OPP) pathway, which is the metabolic pathway that produces NADPH in darkness. The NAD(P)H redox level and photocurrent in the cyanobacterium Synechocystis sp. PCC 6803 were measured noninvasively. Dysfunction of the OPP pathway led to oxidation of the photosynthetic NADPH pool in darkness. In addition, photocurrent induction was retarded and the current density was lower in Δzwf, Δgnd, and ΔglgP than in wild-type cells. Exogenously added glucose compensated the phenotype of ΔglgP and drove the OPP pathway in the mutant, resulting in an increase in the photocurrent. The results indicated that NADPH accumulated by the OPP pathway before illumination is a key factor for the generation of a photocurrent. In addition, measuring the photocurrent can be a non-invasive approach to estimate the cellular redox level related to NADP(H) pool in cyanobacteria.
2022年08月, Photosynthesis research, 153 (1-2), 113 - 120, 英語, 国際誌
研究論文（学術雑誌）

Suppression of chloroplast triose phosphate isomerase evokes inorganic phosphate-limited photosynthesis in rice
Yuji Suzuki, Keiki Ishiyama, Dong-Kyung Yoon, Yuki Takegahara-Tamakawa, Eri Kondo, Mao Suganami, Shinya Wada, Chikahiro Miyake, Amane Makino
Abstract The availability of inorganic phosphate (Pi) for ATP synthesis is thought to limit photosynthesis at elevated [CO2] when Pi regeneration via sucrose or starch synthesis is limited. We report here another mechanism for the occurrence of Pi-limited photosynthesis caused by insufficient capacity of chloroplast triose phosphate isomerase (cpTPI). In cpTPI-antisense transgenic rice (Oryza sativa) plants with 55%–86% reductions in cpTPI content, CO2 sensitivity of the rate of CO2 assimilation (A) decreased and even reversed at elevated [CO2]. The pool sizes of the Calvin–Benson cycle metabolites from pentose phosphates to 3-phosphoglycerate increased at elevated [CO2], whereas those of ATP decreased. These phenomena are similar to the typical symptoms of Pi-limited photosynthesis, suggesting sufficient capacity of cpTPI is necessary to prevent the occurrence of Pi-limited photosynthesis and that cpTPI content moderately affects photosynthetic capacity at elevated [CO2]. As there tended to be slight variations in the amounts of total leaf-N depending on the genotypes, relationships between A and the amounts of cpTPI were examined after these parameters were expressed per unit amount of total leaf-N (A/N and cpTPI/N, respectively). A/N at elevated [CO2] decreased linearly as cpTPI/N decreased before A/N sharply decreased, owing to further decreases in cpTPI/N. Within this linear range, decreases in cpTPI/N by 80% led to decreases up to 27% in A/N at elevated [CO2]. Thus, cpTPI function is crucial for photosynthesis at elevated [CO2].
Oxford University Press (OUP), 2022年03月04日, Plant Physiology, 188 (3), 1550 - 1562
研究論文（学術雑誌）

The difficulty of estimating the electron transport rate at photosystem I
Riu Furutani, Miho Ohnishi, Yuki Mori, Shinya Wada, Chikahiro Miyake
Springer Science and Business Media LLC, 2021年11月15日, Journal of Plant Research
研究論文（学術雑誌）

Identification of a Novel Mutation Exacerbated the PSI Photoinhibition in pgr5/pgrl1 Mutants; Caution for Overestimation of the Phenotypes in Arabidopsis pgr5-1 Mutant
Shinya Wada, Katsumi Amako, Chikahiro Miyake
PSI photoinhibition is usually avoided through P700 oxidation. Without this protective mechanism, excess light represents a potentially lethal threat to plants. PGR5 is suggested to be a major component of cyclic electron transport around PSI and is important for P700 oxidation in angiosperms. The known Arabidopsis PGR5 deficient mutant, pgr5-1, is incapable of P700 oxidation regulation and has been used in numerous photosynthetic studies. However, here it was revealed that pgr5-1 was a double mutant with exaggerated PSI photoinhibition. pgr5-1 significantly reduced growth compared to the newly isolated PGR5 deficient mutant, pgr5hope1. The introduction of PGR5 into pgr5-1 restored P700 oxidation regulation, but remained a pale-green phenotype, indicating that pgr5-1 had additional mutations. Both pgr5-1 and pgr5hope1 tended to cause PSI photoinhibition by excess light, but pgr5-1 exhibited an enhanced reduction in PSI activity. Introducing AT2G17240, a candidate gene for the second mutation into pgr5-1 restored the pale-green phenotype and partially restored PSI activity. Furthermore, a deficient mutant of PGRL1 complexing with PGR5 significantly reduced PSI activity in the double-deficient mutant with AT2G17240. From these results, we concluded that AT2G17240, named PSI photoprotection 1 (PTP1), played a role in PSI photoprotection, especially in PGR5/PGRL1 deficient mutants.
MDPI AG, 2021年10月26日, Cells, 10 (11), 2884 - 2884
研究論文（学術雑誌）

Photosynthetic Parameters Show Specific Responses to Essential Mineral Deficiencies
Miho Ohnishi, Riu Furutani, Takayuki Sohtome, Takeshi Suzuki, Shinya Wada, Soma Tanaka, Kentaro Ifuku, Daisei Ueno, Chikahiro Miyake
In response to decreases in the assimilation efficiency of CO2, plants oxidize the reaction center chlorophyll (P700) of photosystem I (PSI) to suppress reactive oxygen species (ROS) production. In hydro-cultured sunflower leaves experiencing essential mineral deficiencies, we analyzed the following parameters that characterize PSI and PSII: (1) the reduction-oxidation states of P700 [Y(I), Y(NA), and Y(ND)]; (2) the relative electron flux in PSII [Y(II)]; (3) the reduction state of the primary electron acceptor in PSII, QA (1 − qL); and (4) the non-photochemical quenching of chlorophyll fluorescence (NPQ). Deficiency treatments for the minerals N, P, Mn, Mg, S, and Zn decreased Y(II) with an increase in the oxidized P700 [Y(ND)], while deficiencies for the minerals K, Fe, Ca, B, and Mo decreased Y(II) without an increase in Y(ND). During the induction of photosynthesis, the above parameters showed specific responses to each mineral. That is, we could diagnose the mineral deficiency and identify which mineral affected the photosynthesis parameters.
MDPI AG, 2021年06月23日, Antioxidants, 10 (7), 996 - 996
[査読有り]
研究論文（学術雑誌）
神戸大学リポジトリ（Kernel）へのリンク

Photosynthetic Linear Electron Flow Drives CO2 Assimilation in Maize Leaves.
Ginga Shimakawa, Chikahiro Miyake
Photosynthetic organisms commonly develop the strategy to keep the reaction center chlorophyll of photosystem I, P700, oxidized for preventing the generation of reactive oxygen species in excess light conditions. In photosynthesis of C4 plants, CO2 concentration is kept at higher levels around ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) by the cooperation of the mesophyll and bundle sheath cells, which enables them to assimilate CO2 at higher rates to survive under drought stress. However, the regulatory mechanism of photosynthetic electron transport for P700 oxidation is still poorly understood in C4 plants. Here, we assessed gas exchange, chlorophyll fluorescence, electrochromic shift, and near infrared absorbance in intact leaves of maize (a NADP-malic enzyme C4 subtype species) in comparison with mustard, a C3 plant. Instead of the alternative electron sink due to photorespiration in the C3 plant, photosynthetic linear electron flow was strongly suppressed between photosystems I and II, dependent on the difference of proton concentration across the thylakoid membrane (ΔpH) in response to the suppression of CO2 assimilation in maize. Linear relationships among CO2 assimilation rate, linear electron flow, P700 oxidation, ΔpH, and the oxidation rate of ferredoxin suggested that the increase of ΔpH for P700 oxidation was caused by the regulation of proton conductance of chloroplast ATP synthase but not by promoting cyclic electron flow. At the scale of intact leaves, the ratio of PSI to PSII was estimated almost 1:1 in both C3 and C4 plants. Overall, the photosynthetic electron transport was regulated for P700 oxidation in maize through the same strategies as in C3 plants only except for the capacity of photorespiration despite the structural and metabolic differences in photosynthesis between C3 and C4 plants.
2021年05月05日, International journal of molecular sciences, 22 (9), 英語, 国際誌
研究論文（学術雑誌）

Effects of co-overproduction of Rubisco and chloroplast glyceraldehyde-3-phosphate dehydrogenase on photosynthesis in rice
Yuji Suzuki, Keiki Ishiyama, Ayaka Cho, Yuki Takegahara-Tamakawa, Shinya Wada, Chikahiro Miyake, Amane Makino
Informa UK Limited, 2021年04月17日, Soil Science and Plant Nutrition, 1 - 5
[査読有り]
研究論文（学術雑誌）

Photochemistry of Photosystems II and I in Rice Plants Grown under Different N Levels at Normal and High Temperature
Yuki Takahashi, Shinya Wada, Ko Noguchi, Chikahiro Miyake, Amane Makino, Yuji Suzuki
Abstract Although N levels affect leaf photosynthetic capacity, the effects of N levels on the photochemistry of photosystems II and I (PSII and PSI, respectively) are not well-understood. In the present study, we examined this aspect in rice (Oryza sativa L. ‘Hitomebore’) plants grown under three different N levels at normal or high temperatures that can occur during rice culture and do not severely suppress photosynthesis. At both growth temperatures, the quantum efficiency of PSII [Y(II)] and the fraction of the primary quinone electron acceptor in its oxidized state were positively correlated with the amount of total leaf-N, whereas the quantum yields of non-photochemical quenching and donor-side limitation of PSI [Y(ND)] were negatively correlated with the amount of total leaf-N. These changes in PSII and PSI parameters were strongly correlated with each other. Growth temperatures scarcely affected these relationships. These results suggest that the photochemistry of PSII and PSI is coordinately regulated primarily depending on the amount of total leaf-N. When excess light energy occurs in low N-acclimated plants, oxidation of the reaction center chlorophyll of PSI is thought to be stimulated to protect PSI from excess light energy. It is also suggested that PSII and PSI normally operate at high temperature used in the present study. In addition, as the relationships between Y(II) and Y(ND) were found to be almost identical to those observed in osmotically stressed rice plants, common regulation is thought to be operative when excess light energy occurs due to different causes.
Oxford University Press (OUP), 2021年02月09日, Plant and Cell Physiology
[査読有り]
研究論文（学術雑誌）

Physiological Roles of Flavodiiron Proteins and Photorespiration in the Liverwort Marchantia polymorpha.
Ginga Shimakawa, Hitomi Hanawa, Shinya Wada, Guy T Hanke, Yusuke Matsuda, Chikahiro Miyake
Against the potential risk in oxygenic photosynthesis, that is, the generation of reactive oxygen species, photosynthetic electron transport needs to be regulated in response to environmental fluctuations. One of the most important regulations is keeping the reaction center chlorophyll (P700) of photosystem I in its oxidized form in excess light conditions. The oxidation of P700 is supported by dissipating excess electrons safely to O2, and we previously found that the molecular mechanism of the alternative electron sink is changed from flavodiiron proteins (FLV) to photorespiration in the evolutionary history from cyanobacteria to plants. However, the overall picture of the regulation of photosynthetic electron transport is still not clear in bryophytes, the evolutionary intermediates. Here, we investigated the physiological roles of FLV and photorespiration for P700 oxidation in the liverwort Marchantia polymorpha by using the mutants deficient in FLV (flv1) at different O2 partial pressures. The effective quantum yield of photosystem II significantly decreased at 2kPa O2 in flv1, indicating that photorespiration functions as the electron sink. Nevertheless, it was clear from the phenotype of flv1 that FLV was dominant for P700 oxidation in M. polymorpha. These data suggested that photorespiration has yet not replaced FLV in functioning for P700 oxidation in the basal land plant probably because of the lower contribution to lumen acidification, compared with FLV, as reflected in the results of electrochromic shift analysis.
2021年, Frontiers in plant science, 12, 668805 - 668805, 英語, 国際誌
研究論文（学術雑誌）

Characterization of Light-Enhanced Respiration in Cyanobacteria.
Ginga Shimakawa, Ayaka Kohara, Chikahiro Miyake
In eukaryotic algae, respiratory O2 uptake is enhanced after illumination, which is called light-enhanced respiration (LER). It is likely stimulated by an increase in respiratory substrates produced during photosynthetic CO2 assimilation and function in keeping the metabolic and redox homeostasis in the light in eukaryotic cells, based on the interactions among the cytosol, chloroplasts, and mitochondria. Here, we first characterize LER in photosynthetic prokaryote cyanobacteria, in which respiration and photosynthesis share their metabolisms and electron transport chains in one cell. From the physiological analysis, the cyanobacterium Synechocystis sp. PCC 6803 performs LER, similar to eukaryotic algae, which shows a capacity comparable to the net photosynthetic O2 evolution rate. Although the respiratory and photosynthetic electron transports share the interchain, LER was uncoupled from photosynthetic electron transport. Mutant analyses demonstrated that LER is motivated by the substrates directly provided by photosynthetic CO2 assimilation, but not by glycogen. Further, the light-dependent activation of LER was observed even with exogenously added glucose, implying a regulatory mechanism for LER in addition to the substrate amounts. Finally, we discuss the physiological significance of the large capacity of LER in cyanobacteria and eukaryotic algae compared to those in plants that normally show less LER.
2020年12月31日, International journal of molecular sciences, 22 (1), 英語, 国際誌
研究論文（学術雑誌）

Intrinsic Fluctuations in Transpiration Induce Photorespiration to Oxidize P700 in Photosystem I
Riu Furutani, Amane Makino, Yuij Suzuki, Shinya Wada, Ginga Shimakawa, Chikahiro Miyake
Upon exposure to environmental stress, the primary electron donor in photosystem I (PSI), P700, is oxidized to suppress the production of reactive oxygen species that could oxidatively inactivate the function of PSI. The illumination of rice leaves with actinic light induces intrinsic fluctuations in the opening and closing of stomata, causing the net CO2 assimilation rate to fluctuate. We examined the effects of these intrinsic fluctuations on electron transport reactions. Under atmospheric O2 conditions (21 kPa), the effective quantum yield of photosystem II (PSII) (Y(II)) remained relatively high while the net CO2 assimilation rate fluctuated, which indicates the function of alternative electron flow. By contrast, under low O2 conditions (2 kPa), Y(II) fluctuated. These results suggest that photorespiration primarily drove the alternative electron flow. Photorespiration maintained the oxidation level of ferredoxin (Fd) throughout the fluctuation of the net CO2 assimilation rate. Moreover, the relative activity of photorespiration was correlated with both the oxidation level of P700 and the magnitude of the proton gradient across the thylakoid membrane in 21 kPa O2 conditions. These results show that photorespiration oxidized P700 by stimulating the proton gradient formation when CO2 assimilation was suppressed by stomatal closure.
MDPI AG, 2020年12月12日, Plants, 9 (12), 1761 - 1761
研究論文（学術雑誌）

Overproduction of Chloroplast Glyceraldehyde-3-Phosphate Dehydrogenase Improves Photosynthesis Slightly under Elevated [CO2] Conditions in Rice
Yuji Suzuki, Keiki Ishiyama, Misaki Sugawara, Yuka Suzuki, Eri Kondo, Yuki Takegahara-Tamakawa, Dong-Kyung Yoon, Mao Suganami, Shinya Wada, Chikahiro Miyake, Amane Makino
Abstract Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) limits the regeneration of ribulose 1,5-bisphosphate (RuBP) in the Calvin-Benson cycle. However, it does not always limit the rate of CO2 assimilation. In the present study, the effects of overproduction of GAPDH on the rate of CO2 assimilation under elevated [CO2] conditions, where the capacity for RuBP regeneration limits photosynthesis, were examined in transgenic rice (Oryza sativa). GAPDH activity was increased to 6.3- to 9.1-fold of the wild-type levels by co-overexpression of the GAPDH genes, GAPA and GAPB. In the transgenic rice plants, the rate of CO2 assimilation under elevated [CO2] conditions increased by approximately 10%, whereas that under normal and low [CO2] conditions was not affected. These results indicate that overproduction of GAPDH is effective in improving photosynthesis under elevated [CO2] conditions, although its magnitude was relatively small. However, biomass production of the transgenic rice plants was not greater than that in wild-type plants under elevated [CO2] conditions, although starch content tended to increase marginally.
Oxford University Press (OUP), 2020年12月08日, Plant and Cell Physiology
[査読有り]
研究論文（学術雑誌）

Photoprotection mechanisms under different CO2 regimes during photosynthesis in a green alga Chlorella variabilis.
Yoshifumi Ueno, Ginga Shimakawa, Shimpei Aikawa, Chikahiro Miyake, Seiji Akimoto
Oxygenic photosynthesis converts light energy into chemical energy via electron transport and assimilates CO2 in the Calvin-Benson cycle with the chemical energy. Thus, high light and low CO2 conditions induce the accumulation of electrons in the photosynthetic electron transport system, resulting in the formation of reactive oxygen species. To prevent the accumulation of electrons, oxygenic photosynthetic organisms have developed photoprotection mechanisms, including non-photochemical quenching (NPQ) and alternative electron flow (AEF). There are diverse molecular mechanisms underlying NPQ and AEF, and the corresponding molecular actors have been identified and characterized using a model green alga Chlamydomonas reinhardtii. In contrast, detailed information about the photoprotection mechanisms is lacking for other green algal species. In the current study, we examined the photoprotection mechanisms responsive to CO2 in the green alga Chlorella variabilis by combining the analyses of pulse-amplitude-modulated fluorescence, O2 evolution, and the steady-state and time-resolved fluorescence spectra. Under the CO2-limited condition, ΔpH-dependent NPQ occurred in photosystems I and II. Moreover, O2-dependent AEF was also induced. Under the CO2-limited condition with carbon supplementation, NPQ was relaxed and light-harvesting chlorophyll-protein complex II was isolated from both photosystems. In C. variabilis, the O2-dependent AEF and the mechanisms that instantly convert the light-harvesting functions of both photosystems may be important for maintaining efficient photosynthetic activities under various CO2 conditions.
2020年06月, Photosynthesis research, 144 (3), 397 - 407, 英語, 国際誌
[査読有り]
研究論文（学術雑誌）

Photorespiration Enhances Acidification of the Thylakoid Lumen, Reduces the Plastoquinone Pool, and Contributes to the Oxidation of P700 at a Lower Partial Pressure of CO₂ in Wheat Leaves
Shinya Wada, Yuji Suzuki, Chikahiro Miyake
2020年03月, Plants
[査読有り]
研究論文（学術雑誌）
神戸大学リポジトリ（Kernel）へのリンク

P700 oxidation suppresses the production of reactive oxygen species in photosystem I
Riu Furutani, Kentaro Ifuku, Yuji Suzuki, Ko Noguchi, Ginga Shimakawa, Shinya Wada, Amane Makino, Takayuki Sohtome, Chikahiro Miyake
Elsevier, 2020年, Advances in Botanical Research, 151 - 176
論文集(書籍)内論文

Photorespiration Coupled With CO2 Assimilation Protects Photosystem I From Photoinhibition Under Moderate Poly(Ethylene Glycol)-Induced Osmotic Stress in Rice.
Shinya Wada, Chikahiro Miyake, Amane Makino, Yuji Suzuki
Photorespiration coupled with CO2 assimilation is thought to act as a defense system against photoinhibition caused by osmotic stress. In the present study, we examined whether such a mechanism is operative for the protection of photosystem I (PSI) in rice (Oryza sativa L.) including transgenic plants with decreased and increased Rubisco content (RBCS-antisense and RBCS-sense plants, respectively). All plants were hydroponically grown and moderate osmotic stress was imposed using hydroponic culture solutions containing poly(ethylene glycol) (PEG) at 16% or 20% (w/v) for 2 d. In wild-type plants, the rates of CO2 assimilation (A) were significantly decreased by the PEG treatment, whereas the photorespiration activity estimated from the rates of electron transport in photosystem II (PSII) and A were not affected. The maximal quantum efficiency of PSII (Fv/Fm) and the maximal activity of PSI (Pm) were also not affected. In RBCS-antisense plants, A and the estimated photorespiration activity were considerably lower than those in wild-type plants in the presence or absence of the PEG treatment. Pm and both Fv/Fm and Pm decreased in the 16% PEG-treated and 20% PEG-treated RBCS-antisense plants, respectively. Thus, the decrease in Rubisco content led to the photoinhibition of PSI and PSII, indicating the importance of photorespiration coupled with CO2 assimilation for the protection of PSI from moderate PEG-induced osmotic stress. It was also shown that PSI was more sensitive to osmotic stress than PSII. In the PEG-treated wild-type and RBCS-antisense plants, osmotic-stress responses of the photosynthetic electron transport reactions upstream of PSI led to the oxidation of P700, which is thought to prevent PSI from over-reduction. Although such a defense system operated, it was not sufficient for the protection of PSI in RBCS-antisense plants. In addition, there were no large differences in the parameters measured between wild-type and RBCS-sense plants, as overproduction of Rubisco did not increase photorespiration activity.
2020年, Frontiers in plant science, 11, 1121 - 1121, 英語, 国際誌
[査読有り]
研究論文（学術雑誌）

Oxidation of P700 Induces Alternative Electron Flow in Photosystem I in Wheat Leaves.
Kanae Kadota, Riu Furutani, Amane Makino, Yuji Suzuki, Shinya Wada, Chikahiro Miyake
Oxygen (O2)-evolving photosynthetic organisms oxidize the reaction center chlorophyll, P700, in photosystem I (PSI) to suppress the production of reactive oxygen species. The oxidation of P700 is accompanied by alternative electron flow in PSI (AEF-I), which is not required for photosynthetic linear electron flow (LEF). To characterize AEF-I, we compared the redox reactions of P700 and ferredoxin (Fd) during the induction of carbon dioxide (CO2) assimilation in wheat leaves, using dark-interval relaxation kinetics analysis. Switching on an actinic light (1000 μmol photons m-2 s-1) at ambient CO2 partial pressure of 40 Pa and ambient O2 partial pressure of 21 kPa gradually oxidized P700 (P700+) and enhanced the reduction rate of P700+ (vP700) and oxidation rate of reduced Fd (vFd). The vFd showed a positive linear relationship with an apparent photosynthetic quantum yield of PSII (Y[II]) originating at point zero; the redox turnover of Fd is regulated by LEF via CO2 assimilation and photorespiration. The vP700 also showed a positive linear relationship with Y(II), but the intercept was positive, not zero. That is, the electron flux in PSI included the electron flux in AEF-I in addition to that in LEF. This indicates that the oxidation of P700 induces AEF-I. We propose a possible mechanism underlying AEF-I and its physiological role in the mitigation of oxidative damage.
2019年06月05日, Plants (Basel, Switzerland), 8 (6), 英語, 国際誌
[査読有り]
研究論文（学術雑誌）
神戸大学リポジトリ（Kernel）へのリンク

Growth light environment changes the sensitivity of photosystem I photoinhibition by depending on common wheat cultivars
Daisuke Takagi, HIroaki Ihara, Shigeo Takumi, Chikahiro Miyake
2019年06月, Frontiers in Plant Science, 10, 686, 英語
[査読有り]
研究論文（学術雑誌）

What Quantity of Photosystem I Is Optimum for Safe Photosynthesis?
Ginga Shimakawa, Chikahiro Miyake
PSI has the potential to generate reactive oxygen species and be oxidatively inactivated by the reactive oxygen species. The photo-oxidative damage of PSI (also called PSI photoinhibition) causes the inhibition of the plant growth and is a lethal event for plants. It has been reported that PSI photoinhibition does not occur as long as the reaction-center chlorophyll (P700) remains oxidized, even in excess light conditions. This process is termed P700 oxidation and is supported by various regulatory mechanisms and likely also by the stoichiometric quantities of photosynthetic apparatus. In this study, we assessed how decreased photochemically active PSI in Arabidopsis (Arabidopsis thaliana) affected a variety of photosynthetic parameters, including P700 oxidation. Inactivation of PSI was rapidly and selectively induced by repetitive short-pulse illumination. PSI photoinhibition correlated linearly with decreases in effective quantum yield of PSII and nonphotochemical quenching; however, the photosynthetic CO2 assimilation rate was less affected, as exemplified by ∼50% of the normal CO2 assimilation rate maintained with an 80% loss in PSI photochemical activity. In contrast, effective quantum yield of PSI was enhanced following PSI photoinhibition, mainly owing to a decrease in the electron donor-side limitation of PSI. Based on these results, we propose that the stoichiometric quantity of PSI is optimized to induce P700 oxidation for dissipating excess light energy in PSI, thus avoiding inhibition of photosynthetic CO2 assimilation caused by PSI photoinhibition.
2019年04月, Plant physiology, 179 (4), 1479 - 1485, 英語, 国際誌
研究論文（学術雑誌）

Responses of the Photosynthetic Electron Transport Reactions Stimulate the Oxidation of the Reaction Center Chlorophyll of Photosystem I, P700, under Drought and High Temperatures in Rice
和田慎也, 高木大輔, 三宅親弘, 牧野周, 鈴木雄二
It is of interest how photosynthetic electron transport (PET) reactions respond to excess light energy caused by the combination of drought stress and high temperatures. Since such information is scarcely available for photosystem I (PSI), this question was explored in rice (Oryza sativa L.) plants subjected to drought stress, using culture solutions that contain poly(ethylene glycol) at different concentrations under two day/night temperature regimes. At 27/22 °C (day/night), drought stress led to the oxidation of the reaction center of the chlorophyll of PSI (P700), and also led to decreases in the quantum efficiencies of photosystem II (PSII) and PSI, and a reduction of the primary quinone electron acceptor of PSI. Such drought stress responses were wholly stimulated at 35/30 °C. These parameters were strongly correlated with each other and were minimally affected by temperature. These results indicate that the drought stress responses of the respective PET reactions are closely associated with each other in the oxidization of P700 and that such responses are stimulated at high temperatures. The underlying mechanisms of these phenomena were discussed. While P700 oxidation is thought to suppress reactive oxygen species (ROS) production, PSI photoinhibition was observed under severe stress conditions, implying that P700 oxidation is not sufficient for the protection of PSI under drought stress.
MDPI, 2019年04月, International Journal of Molecular Sciences, 20 (9), 2068, 英語, 国際誌
[査読有り]
研究論文（学術雑誌）
神戸大学リポジトリ（Kernel）へのリンク

Comparative analysis of strategies to prepare electron sinks in aquatic photoautotrophs
Shimakawa Ginga, 村上明男, Niwa Kyosuke, Matsuda Yusuke, Wada Ayumi, MIyake Chikahiro
Springer Science and Business Media LLC, 2019年03月, Photosynthesis Research, 139 (1-3), 401 - 411, 英語
[査読有り]
研究論文（学術雑誌）

Responses of the chloroplast glyoxalase system to high CO2 concentrations.
Ginga SHIMAKAWA, Kentaro IFUKU, Yuji SUZUKI, Amane MAKINO, Kimitsune ISHIZAKI, Hiroshi FUKAYAMA, Ryutaro MORITA, Katsuhiko SAKAMOTO, Akiko NISHI, Chikahiro MIYAKE
Sugar metabolism pathways such as photosynthesis produce dicarbonyls, e.g. methylglyoxal ( MG), which can cause cellular damage. The glyoxalase ( GLX) system comprises two enzymes GLX1 and GLX2, and detoxifies MG; however, this system is poorly understood in the chloroplast, compared with the cytosol. In the present study, we determined GLX1 and GLX2 activities in spinach chloroplasts, which constituted 40% and 10%, respectively, of the total leaf glyoxalase activity. In Arabidopsis thaliana, five GFP-fusion GLXs were present in the chloroplasts. Under high CO2 concentrations, where increased photosynthesis promotes the MG production, GLX1 and GLX2 activities in A. thaliana increased and the expression of AtGLX1-2 and AtGLX2-5 was enhanced. On the basis of these findings and the phylogeny of GLX in oxygenic phototrophs, we propose that the GLX system scavenges MG produced in chloroplasts during photosynthesis.
Japan Society for Bioscience, Biotechnology, and Agrochemistry, 2018年12月, Bioscience, Biotechnology and Biochemistry, 82 (12), 2072 - 2083, 英語
[査読有り]
研究論文（学術雑誌）
神戸大学リポジトリ（Kernel）へのリンク

Changing frequency of fluctuating light reveals the molecular mechanism for P700 oxidation in plant leaves.
Ginga Shimakawa, Chikahiro Miyake
Natural sunlight exceeds the demand of photosynthesis such that it can cause plants to produce reactive oxygen species (ROS), which subsequently cause photo-oxidative damage. Because photosystem I (PSI) is a major source of ROS, plants actively maintain the reaction center chlorophyll of PSI(P700) oxidized under excessive light conditions to alleviate the ROS production. P700 oxidation is universally recognized in photosynthetic organisms as a physiological response to excessive light. However, it is still poorly understood how P700 oxidation is induced in response to fluctuating light with a variety of frequencies. Here, we investigated the relationships of photosynthetic parameters with P700 oxidation in Arabidopsis thaliana under a sine fluctuating light with different frequencies. As the photon flux density of the light increased, P700 was oxidized concurrently with the chlorophyll fluorescence parameter qL unless the electron acceptor side of PSI was limited. Conversely, we did not observe a proportional relationship of non-photochemical quenching with P700 oxidation. The mutant crr-2, which lacks chloroplast NADPH dehydrogenase, was impaired in P700 oxidation during light fluctuation at high, but not low frequency, unlike the pgrl1 mutant deficient in PGR5 and PGRL1 proteins, which could not oxidize P700 during light fluctuation at both high and low frequencies. Taken together, our findings suggested that the changing frequency of fluctuating light reveals the tracking performance of molecular mechanisms underlying P700 oxidation.
2018年07月, Plant direct, 2 (7), e00073, 英語, 国際誌
研究論文（学術雑誌）

Reduction-induced suppression of electron flow (RISE) is relieved by non-ATP-consuming electron flow in Synechococcus elongatus PCC 7942
Ginga Shimakawa, Keiichiro Shaku, Chikahiro Miyake
Photosynthetic organisms oxidize P700 to suppress the production of reactive oxygen species (ROS) in photosystem I (PSI) in response to the lower efficiency of photosynthesis under high light and low CO2 conditions. Previously, we found a positive relationship between reduction of plastoquinone (PQ) pool and oxidation of P700, which we named reduction-induced suppression of electron flow (RISE). In the RISE model, we proposed that the highly reduced state of the PQ pool suppresses Q-cycle turnover to oxidize P700 in PSI. Here, we tested whether RISE was relieved by the oxidation of the PQ pool, but not by the dissipation of the proton gradient (ΔpH) across the thylakoid membrane. Formation of ΔpH can also suppress electron flow to P700, because acidification on the luminal side of the thylakoid membrane lowers oxidation of reduced PQ in the cytochrome b6/f complex. We drove photosynthetic electron transport using H2O2-scavenging peroxidase reactions. Peroxidase reduces H2O2 with electron donors regenerated along the photosynthetic electron transport system, thereby promoting the formation of ΔpH. Addition of H2O2 to the cyanobacterium Synechococcus elongatus PCC 7942 under low CO2 conditions induced photochemical quenching of chlorophyll fluorescence, enhanced NADPH fluorescence and reduced P700. Thus, peroxidase reactions relieved the RISE mechanism, indicating that P700 oxidation can be induced only by the reduction of PQ to suppress the production of ROS in PSI. Overall, our data suggest that RISE regulates the redox state of P700 in PSI in cooperation with ΔpH regulation.
Frontiers Media S.A., 2018年05月07日, Frontiers in Microbiology, 9 (MAY), 英語
[査読有り]
研究論文（学術雑誌）
神戸大学リポジトリ（Kernel）へのリンク

Antimycin A inhibits cytochrome b559-mediated cyclic electron flow within photosystem II
Daisuke Takagi, Kentaro Ifuku, Taishi Nishimura, Chikahiro Miyake
Springer Nature, 2018年05月, Photosynthesis Research, 139 (1-3), 487 - 498, 英語
[査読有り]
研究論文（学術雑誌）

Respiratory terminal oxidases alleviate photo-oxidative damage in photosystem I during repetitive short-pulse illumination in Synechocystis sp. PCC 6803
Ginga Shimakawa, Chikahiro Miyake
Oxygenic phototrophs are vulnerable to damage by reactive oxygen species (ROS) that are produced in photosystem I (PSI) by excess photon energy over the demand of photosynthetic CO2 assimilation. In plant leaves, repetitive short-pulse (rSP) illumination produces ROS to inactivate PSI. The production of ROS is alleviated by oxidation of the reaction center chlorophyll in PSI, P700, during the illumination with the short-pulse light, which is supported by flavodiiron protein (FLV). In this study, we found that in the cyanobacterium Synechocystis sp. PCC 6803 P700 was oxidized and PSI was not inactivated during rSP illumination even in the absence of FLV. Conversely, the mutant deficient in respiratory terminal oxidases was impaired in P700 oxidation during the illumination with the short-pulse light to suffer from photo-oxidative damage in PSI. Interestingly, the other cyanobacterium Synechococcus sp. PCC 7002 could not oxidize P700 without FLV during rSP illumination. These data indicate that respiratory terminal oxidases are critical to protect PSI from ROS damage during rSP illumination in Synechocystis sp. PCC 6803 but not Synechococcus sp. PCC 7002.
Springer Netherlands, 2018年03月08日, Photosynthesis Research, 1 - 10, 英語
[査読有り]
研究論文（学術雑誌）

Oxidation of P700 Ensures Robust Photosynthesis.
Ginga Shimakawa, Chikahiro Miyake
In the light, photosynthetic cells can potentially suffer from oxidative damage derived from reactive oxygen species. Nevertheless, a variety of oxygenic photoautotrophs, including cyanobacteria, algae, and plants, manage their photosynthetic systems successfully. In the present article, we review previous research on how these photoautotrophs safely utilize light energy for photosynthesis without photo-oxidative damage to photosystem I (PSI). The reaction center chlorophyll of PSI, P700, is kept in an oxidized state in response to excess light, under high light and low CO2 conditions, to tune the light utilization and dissipate the excess photo-excitation energy in PSI. Oxidation of P700 is co-operatively regulated by a number of molecular mechanisms on both the electron donor and acceptor sides of PSI. The strategies to keep P700 oxidized are diverse among a variety of photoautotrophs, which are evolutionarily optimized for their ecological niche.
2018年, Frontiers in plant science, 9, 1617 - 1617, 英語, 国際誌
研究論文（学術雑誌）

Effects of genetic manipulation of the activity of photorespiration on the redox state of photosystem I and its robustness against excess light stress under CO₂-limited conditions in rice
Shinya Wada, Yuji Suzuki, Daisuke Takagi, Chikahiro Miyake, Amane Makino
Under CO2-limited conditions such as during stomatal closure, photorespiration is suggested to act as a sink for excess light energy and protect photosystem I (PSI) by oxidizing its reaction center chlorophyll P700. In this study, this issue was directly examined with rice (Oryza sativa L.) plants via genetic manipulation of the amount of Rubisco, which can be a limiting factor for photorespiration. At low [CO2] of 5 Pa that mimicked stomatal closure condition, the activity of photorespiration in transgenic plants with decreased Rubisco content (RBCS-antisense plants) markedly decreased, whereas the activity in transgenic plants with overproduction of Rubisco (RBCS-sense plants) was similar to that in wild-type plants. Oxidation of P700 was enhanced at [CO2] of 5 Pa in wild-type and RBCS-sense plants. PSI was not damaged by excess light stress induced by repetitive saturated pulse-light (rSP) in the presence of strong steady-state light. On the other hand, P700 was strongly reduced in RBCS-antisense plants at [CO2] of 5 Pa. PSI was also damaged by rSP illumination. These results indicate that oxidation of P700 and the robustness of PSI against excess light stress are hampered by the decreased activity of photorespiration as a result of genetic manipulation of Rubisco content. It is also suggested that overproduction of Rubisco does not enhance photorespiration as well as CO2 assimilation probably due to partial deactivation of Rubisco.
2018年, Photosynthesis Research, 137 (3), 431 - 441, 英語, 国際誌
[査読有り]
研究論文（学術雑誌）

PROTON GRADIENT REGULATION 5 supports linear electron flow to oxidize photosystem I
Daisuke Takagi, Chikahiro Miyake
2018年, Physiologia Plantaruim, 英語
[査読有り]
研究論文（学術雑誌）

Light-harvesting strategy during CO2-dependent photosynthesis in the green alga Chlamydomonas reinhardtii
Y. Ueno, G. Shimakawa, C. Miyake, S. Akimoto
2018年, J. Phys. Chem. Lett., 9, 1028 - 1033, 英語
[査読有り]
研究論文（学術雑誌）

A Carbon Dioxide Limitation-Inducible Protein, ColA, Supports the Growth of Synechococcus sp PCC 7002
Ginga Shimakawa, Satoru Watanabe, Chikahiro Miyake
A limitation in carbon dioxide (CO2), which occurs as a result of natural environmental variation, suppresses photosynthesis and has the potential to cause photo-oxidative damage to photosynthetic cells. Oxygenic phototrophs have strategies to alleviate photo-oxidative damage to allow life in present atmospheric CO2 conditions. However, the mechanisms for CO2 limitation acclimation are diverse among the various oxygenic phototrophs, and many mechanisms remain to be discovered. In this study, we found that the gene encoding a CO2 limitation-inducible protein, ColA, is required for the cyanobacterium Synechococcus sp. PCC 7002 (S. 7002) to acclimate to limited CO2 conditions. An S. 7002 mutant deficient in ColA (Delta colA) showed lower chlorophyll content, based on the amount of nitrogen, than that in S. 7002 wild-type (WT) under ambient air but not high CO2 conditions. Both thermoluminescence and protein carbonylation detected in the ambient air grown cells indicated that the lack of ColA promotes oxidative stress in S. 7002. Alterations in the photosynthetic O-2 evolution rate and relative electron transport rate in the short-term response, within an hour, to CO2 limitation were the same between the WT and Delta colA. Conversely, these photosynthetic parameters were mostly lower in the long-term response of a few days in Delta colA than in the WT. These data suggest that ColA is required to sustain photosynthetic activity for living under ambient air in S. 7002. The unique phylogeny of ColA revealed diverse strategies to acclimate to CO2 limitation among cyanobacteria.
MDPI AG, 2017年12月, MARINE DRUGS, 15 (12), 英語
[査読有り]
研究論文（学術雑誌）
神戸大学リポジトリ（Kernel）へのリンク

Diversity of strategies for escaping reactive oxygen species production within photosystem I among land plants: P700 oxidation system is prerequisite for alleviating photoinhibition in photosystem I
Daisuke Takagi, Kimitsune Ishizaki, Hitomi Hanawa, Tomohito Mabuchi, Ginga Shimakawa, Hiroshi Yamamoto, Chikahiro Miyake
In land plants, photosystem I (PSI) photoinhibition limits carbon fixation and causes growth defects. In addition, recovery from PSI photoinhibition takes much longer than PSII photoinhibition when the PSI core-complex is degraded by oxidative damage. Accordingly, PSI photoinhibition should be avoided in land plants, and land plants should have evolved mechanisms to prevent PSI photoinhibition. However, such protection mechanisms have not yet been identified, and it remains unclear whether all land plants suffer from PSI photoinhibition in the same way. In the present study, we focused on the susceptibility of PSI to photoinhibition and investigated whether mechanisms of preventing PSI photoinhibition varied among land plant species. To assess the susceptibility of PSI to photoinhibition, we used repetitive short-pulse (rSP) illumination, which specifically induces PSI photoinhibition. Subsequently, we found that land plants possess a wide variety of tolerance mechanisms against PSI photoinhibition. In particular, gymnosperms, ferns and mosses/liverworts exhibited higher tolerance to rSP illumination-induced PSI photoinhibition than angiosperms, and detailed analyses indicated that the tolerance of these groups could be partly attributed to flavodiiron proteins, which protected PSI from photoinhibition by oxidizing the PSI reaction center chlorophyll (P700) as an electron acceptor. Furthermore, we demonstrate, for the first time, that gymnosperms, ferns and mosses/liverworts possess a protection mechanism against photoinhibition of PSI that differs from that of angiosperms.
Blackwell Publishing Ltd, 2017年09月01日, Physiologia Plantarum, 161 (1), 56 - 74, 英語
[査読有り]
研究論文（学術雑誌）

Land plants drive photorespiration as higher electron-sink: comparative study of post-illumination transient O-2-uptake rates from liverworts to angiosperms through ferns and gymnosperms
Hitomi Hanawa, Kimitsune Ishizaki, Kana Nohira, Daisuke Takagi, Ginga Shimakawa, Takehiro Sejima, Keiichiro Shaku, Amane Makino, Chikahiro Miyake
In higher plants, the electron-sink capacity of photorespiration contributes to alleviation of photoinhibition by dissipating excess energy under conditions when photosynthesis is limited. We addressed the question at which point in the evolution of photosynthetic organisms photorespiration began to function as electron sink and replaced the flavodiiron proteins which catalyze the reduction of O-2 at photosystem I in cyanobacteria. Algae do not have a higher activity of photorespiration when CO2 assimilation is limited, and it can therefore not act as an electron sink. Using land plants (liverworts, ferns, gymnosperms, and angiosperms) we compared photorespiration activity and estimated the electron flux driven by photorespiration to evaluate its electron-sink capacity at CO2-compensation point. In vivo photorespiration activity was estimated by the simultaneous measurement of O-2-exchange rate and chlorophyll fluorescence yield. All C3-plants leaves showed transient O-2-uptake after actinic light illumination (post-illumination transient O-2-uptake), which reflects photorespiration activity. Post- illumination transient O-2-uptake rates increased in the order from liverworts to angiosperms through ferns and gymnosperms. Furthermore, photorespiration-dependent electron flux in photosynthetic linear electron flow was estimated from post-illumination transient O-2-uptake rate and compared with the electron flux in photosynthetic linear electron flow in order to evaluate the electron-sink capacity of photorespiration. The electron-sink capacity at the CO2-compensation point also increased in the above order. In gymnosperms photorespiration was determined to be the main electron-sink. C3-C4 intermediate species of Flaveria plants showed photorespiration activity, which intermediate between that of C3- and C4-flaveria species. These results indicate that in the first land plants, liverworts, photorespiration started to function as electron sink. According to our hypothesis, the dramatic increase in partial pressure of O-2 in the atmosphere about 0.4 billion years ago made it possible to drive photorespiration with higher activity in liverworts.
WILEY, 2017年09月, PHYSIOLOGIA PLANTARUM, 161 (1), 138 - 149, 英語
[査読有り]
研究論文（学術雑誌）

Chloroplastic ATP synthase builds up proton motive force for preventing reactive oxygen species production in photosystem I
Daisuke Takagi, Amako Katsumi, Masaki Hashiguchi, Hidehiro Fukaki, Kimitsune Ishizaki, Tatsuaki Goh, Yoichiro Fukao, Yosuke Sano, Tetsuya Kurata, Taku Demura, Shinichiro Sawa, Chikahiro Miyake
2017年04月, The Plant Journal, 91, 306 - 324, 英語
[査読有り]
研究論文（学術雑誌）

The Liverwort, Marchantia, Drives Alternative Electron Flow Using a Flavodiiron Protein to Protect PSI
Ginga Shimakawa, Kimitsune Ishizaki, Shigeyuki Tsukamoto, Moeko Tanaka, Takehiro Sejima, Chikahiro Miyake
The diffusion efficiency of oxygen in the atmosphere, like that of CO2, is approximately 104 times greater than that in aqueous environments. Consequently, terrestrial photosynthetic organisms need mechanisms to protect against potential oxidative damage. The liverwort Marchantia polymorpha, a basal land plant, has habitats where it is exposed to both water and the atmosphere. Furthermore, like cyanobacteria, M. polymorpha has genes encoding flavodiiron proteins (FLV). In cyanobacteria, FLVs mediate oxygen-dependent alternative electron flow (AEF) to suppress the production of reactive oxygen species. Here, we investigated whether FLVs are required for the protection of photosynthesis in M. polymorpha. A mutant deficient in the FLV1 isozyme (Delta MpFlv1) sustained photooxidative damage to photosystem I (PSI) following repetitive short-saturation pulses of light. Compared with the wild type (Takaragaike-1), Delta MpFlv1 showed the same photosynthetic oxygen evolution rate but a lower electron transport rate during the induction phase of photosynthesis. Additionally, the reaction center chlorophyll in PSI, P700, was highly reduced in DMpFlv1 but not in Takaragaike-1. These results indicate that the gene product of MpFlv1 drives AEF to oxidize PSI, as in cyanobacteria. Furthermore, FLV-mediated AEF supports the production of a proton motive force to possibly induce the nonphotochemical quenching of chlorophyll fluorescence and suppress electron transport in the cytochrome b(6)/f complex. After submerging the thalli, a decrease in photosystem II operating efficiency was observed, particularly in Delta MpFlv1, which implies that species living in these sorts of habitats require FLV-mediated AEF.
AMER SOC PLANT BIOLOGISTS, 2017年03月, PLANT PHYSIOLOGY, 173 (3), 1636 - 1647, 英語
[査読有り]
研究論文（学術雑誌）
神戸大学リポジトリ（Kernel）へのリンク

Diverse strategies of O-2 usage for preventing photo-oxidative damage under CO2 limitation during algal photosynthesis
Ginga Shimakawa, Yusuke Matsuda, Kensuke Nakajima, Masahiro Tamoi, Shigeru Shigeoka, Chikahiro Miyake
Photosynthesis produces chemical energy from photon energy in the photosynthetic electron transport and assimilates CO2 using the chemical energy. Thus, CO2 limitation causes an accumulation of excess energy, resulting in reactive oxygen species (ROS) which can cause oxidative damage to cells. O-2 can be used as an alternative energy sink when oxygenic phototrophs are exposed to high light. Here, we examined the responses to CO2 limitation and O-2 dependency of two secondary algae, Euglena gracilis and Phaeodactylum tricornutum. In E. gracilis, approximately half of the relative electron transport rate (ETR) of CO2-saturated photosynthesis was maintained and was uncoupled from photosynthesis under CO2 limitation. The ETR showed biphasic dependencies on O-2 at high and low O-2 concentrations. Conversely, in P. tricornutum, most relative ETR decreased in parallel with the photosynthetic O-2 evolution rate in response to CO2 limitation. Instead, non-photochemical quenching was strongly activated under CO2 limitation in P. tricornutum. The results indicate that these secondary algae adopt different strategies to acclimatize to CO2 limitation, and that both strategies differ from those utilized by cyanobacteria and green algae. We summarize the diversity of strategies for prevention of photo-oxidative damage under CO2 limitation in cyanobacterial and algal photosynthesis.
NATURE PUBLISHING GROUP, 2017年01月, SCIENTIFIC REPORTS, 7, 41022, 英語
[査読有り]
研究論文（学術雑誌）
神戸大学リポジトリ（Kernel）へのリンク

Oxidation of P700 in Photosystem I Is Essential for the Growth of Cyanobacteria
Ginga Shimakawa, Keiichiro Shaku, Chikahiro Miyake
The photoinhibition of photosystem I (PSI) is lethal to oxygenic phototrophs. Nevertheless, it is unclear how photodamage occurs or how oxygenic phototrophs prevent it. Here, we provide evidence that keeping P700 (the reaction center chlorophyll in PSI) oxidized protects PSI. Previous studies have suggested that PSI photoinhibition does not occur in the two model cyanobacteria, Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942, when photosynthetic CO2 fixation was suppressed under low CO2 partial pressure even in mutants deficient in flavodiiron protein (FLV), which mediates alternative electron flow. The lack of FLV in Synechococcus sp. PCC 7002 (S. 7002), however, is linked directly to reduced growth and PSI photodamage under CO2-limiting conditions. Unlike Synechocystis sp. PCC 6803 and S. elongatus PCC 7942, S. 7002 reduced P700 during CO2-limited illumination in the absence of FLV, resulting in decreases in both PSI and photosynthetic activities. Even at normal air CO2 concentration, the growth of S. 7002 mutant was retarded relative to that of the wild type. Therefore, P700 oxidation is essential for protecting PSI against photoinhibition. Here, we present various strategies to alleviate PSI photoinhibition in cyanobacteria.
AMER SOC PLANT BIOLOGISTS, 2016年11月, PLANT PHYSIOLOGY, 172 (3), 1443 - 1450, 英語
[査読有り]
研究論文（学術雑誌）
神戸大学リポジトリ（Kernel）へのリンク

Photorespiration provides the chance of cyclic electron flow to operate for the redox-regulation of P700 in photosynthetic electron transport system of sunflower leaves
Daisuke Takagi, Masaki Hashiguchi, Takehiro Sejima, Amane Makino, Chikahiro Miyake
To elucidate the molecular mechanism to oxidize the reaction center chlorophyll, P700, in PSI, we researched the effects of partial pressure of O-2 (pO(2)) on photosynthetic characteristic parameters in sunflower (Helianthus annuus L.) leaves. Under low CO2 conditions, the oxidation of P700 was stimulated; however the decrease in pO(2) suppressed its oxidation. Electron fluxes in PSII [Y(II)] and PSI [Y(I)] showed pO(2)-dependence at low CO2 conditions. H+-consumption rate, estimated from Y(II) and CO2-fixation/photorespiration rates (JgH(+)), showed the positive curvature relationship with the dissipation rate of electrochromic shift signal (V (H) (+) ), which indicates H+-efflux rate from lumen to stroma in chloroplasts. Therefore, these electron fluxes contained, besides CO2-fixation/photorespiration-dependent electron fluxes, non-H+-consumption electron fluxes including Mehler-ascorbate peroxidase (MAP)-pathway. Y(I) that was larger than Y(II) surely implies the functioning of cyclic electron flow (CEF). Both MAP-pathway and CEF were suppressed at lower pO(2), with plastoquinone-pool reduced. That is, photorespiration prepares the redox-poise of photosynthetic electron transport system for CEF activity as an electron sink. Excess Y(II), [Delta Y(II)] giving the curvature relationship with V (H) (+) , and excess Y(I) [Delta CEF] giving the difference between Y(I) and Y(II) were used as an indicator of MAP-pathway and CEF activity, respectively. Although Delta Y(II) was negligible and did not show positive relationship to the oxidation-state of P700, Delta CEF showed positive linear relationship to the oxidation-state of P700. These facts indicate that CEF cooperatively with photorespiration regulates the redox-state of P700 to suppress the over-reduction in PSI under environmental stress conditions.
SPRINGER, 2016年09月, PHOTOSYNTHESIS RESEARCH, 129 (3), 279 - 290, 英語
[査読有り]
研究論文（学術雑誌）

Reduction-Induced Suppression of Electron Flow (RISE) in the Photosynthetic Electron Transport System of Synechococcus elongatus PCC 7942
Keiichiro Shaku, Ginga Shimakawa, Masaki Hashiguchi, Chikahiro Miyake
Accumulation of electrons under conditions of environmental stress produces a reduced state in the photosynthetic electron transport (PET) system and causes the reduction of O-2 by PSI in the thylakoid membranes to produce the reactive oxygen species superoxide radical, which irreversibly inactivates PSI. This study aims to elucidate the molecular mechanism for the oxidation of reaction center Chl of PSI, P700, after saturated pulse (SP) light illumination of the cyanobacterium Synechococcus elongatus PCC 7942 under steady-state photosynthetic conditions. Both P700 and NADPH were transiently oxidized after SP light illumination under CO2-depleted photosynthesis conditions. In contrast, the Chl fluorescence intensity transiently increased. Compared with the wild type, the increase in Chl fluorescence and the oxidations of P700 and NADPH were greatly enhanced in a mutant (Delta flv1/3) deficient in the genes encoding FLAVODIIRON 1 (FLV1) and FLV3 proteins even under high photosynthetic conditions. Furthermore, oxidation of Cyt f was also observed in the mutant. After SP light illumination, a transient suppression of O-2 evolution was also observed in Delta flv1/3. From these observations, we propose that the reduction in the plastquinone (PQ) pool suppresses linear electron flow at the Cyt b(6)/f complex, which we call the reduction-induced suppression of electron flow (RISE) in the PET system. The accumulation of the reduced form of PQ probably suppresses turnover of the Q cycle in the Cyt b(6)/f complex.
OXFORD UNIV PRESS, 2016年07月, PLANT AND CELL PHYSIOLOGY, 57 (7), 1443 - 1453, 英語
[査読有り]
研究論文（学術雑誌）

Superoxide and Singlet Oxygen Produced within the Thylakoid Membranes Both Cause Photosystem I Photoinhibition
Daisuke Takagi, Shigeo Takumi, Masaki Hashiguchi, Takehiro Sejima, Chikahiro Miyake
Photosystem I (PSI) photoinhibition suppresses plant photosynthesis and growth. However, the mechanism underlying PSI photoinhibition has not been fully clarified. In this study, in order to investigate the mechanism of PSI photoinhibition in higher plants, we applied repetitive short-pulse (rSP) illumination, which causes PSI-specific photoinhibition in chloroplasts isolated from spinach leaves. We found that rSP treatment caused PSI photoinhibition, but not PSII photoinhibition in isolated chloroplasts in the presence of O-2. However, chloroplastic superoxide dismutase and ascorbate peroxidase activities failed to protect PSI from its photoinhibition. Importantly, PSI photoinhibition was largely alleviated in the presence of methyl viologen, which stimulates the production of reactive oxygen species (ROS) at the stromal region by accepting electrons from PSI, even under the conditions where CuZn-superoxide dismutase and ascorbate peroxidase activities were inactivated by KCN. These results suggest that the ROS production site, but not the ROS production rate, is critical for PSI photoinhibition. Furthermore, we found that not only superoxide (O-2(-)) but also singlet oxygen (O-1(2)) is involved in PSI photoinhibition induced by rSP treatment. From these results, we suggest that PSI photoinhibition is caused by both O-2(-) and O-1(2) produced within the thylakoid membranes when electron carriers in PSI become highly reduced. Here, we show, to our knowledge, new insight into the PSI photoinhibition in higher plants.
AMER SOC PLANT BIOLOGISTS, 2016年07月, PLANT PHYSIOLOGY, 171 (3), 1626 - 1634, 英語
[査読有り]
研究論文（学術雑誌）
神戸大学リポジトリ（Kernel）へのリンク

Suppression of Chloroplastic Alkenal/One Oxidoreductase Represses the Carbon Catabolic Pathway in Arabidopsis Leaves during Night
Daisuke Takagi, Kentaro Ifuku, Ken-ichi Ikeda, Kanako Ikeda Inoue, Pyoyun Park, Masahiro Tamoi, Hironori Inoue, Katsuhiko Sakamoto, Ryota Saito, Chikahiro Miyake
Lipid-derived reactive carbonyl species (RCS) possess electrophilic moieties and cause oxidative stress by reacting with cellular components. Arabidopsis (Arabidopsis thaliana) has a chloroplast-localized alkenal/one oxidoreductase (AtAOR) for the detoxification of lipid-derived RCS, especially alpha,beta-unsaturated carbonyls. In this study, we aimed to evaluate the physiological importance of AtAOR and analyzed AtAOR (aor) mutants, including a transfer DNA knockout, aor (T-DNA), and RNA interference knockdown, aor (RNAi), lines. We found that both aor mutants showed smaller plant sizes than wild-type plants when they were grown under day/night cycle conditions. To elucidate the cause of the aor mutant phenotype, we analyzed the photosynthetic rate and the respiration rate by gas-exchange analysis. Subsequently, we found that both wildtype and aor (RNAi) plants showed similar CO2 assimilation rates; however, the respiration rate was lower in aor (RNAi) than in wild-type plants. Furthermore, we revealed that phosphoenolpyruvate carboxylase activity decreased and starch degradation during the night was suppressed in aor (RNAi). In contrast, the phenotype of aor (RNAi) was rescued when aor (RNAi) plants were grown under constant light conditions. These results indicate that the smaller plant sizes observed in aor mutants grown under day/night cycle conditions were attributable to the decrease in carbon utilization during the night. Here, we propose that the detoxification of lipid-derived RCS by AtAOR in chloroplasts contributes to the protection of dark respiration and supports plant growth during the night.
AMER SOC PLANT BIOLOGISTS, 2016年04月, PLANT PHYSIOLOGY, 170 (4), 2024 - 2039, 英語
[査読有り]
研究論文（学術雑誌）

Post-illumination transient O-2-uptake is driven by photorespiration in tobacco leaves
Takehiro Sejima, Hitomi Hanawa, Ginga Shimakawa, Daisuke Takagi, Yuji Suzuki, Hiroshi Fukayama, Amane Makino, Chikahiro Miyake
This study aims to elucidate the molecular mechanism for the transient increase in the O-2-uptake rate in tobacco (Nicotiana tabacum cv Xanthi) leaves after turning off actinic lights (ALs). The photosynthetic O-2 evolution rate reaches a maximum shortly after the onset of illumination with ALs and then decreases to zero in atmospheric CO2/O-2 conditions. After turning off the ALs, tobacco leaves show a transient increase in the O-2-uptake rate, the post-illumination transient O-2-uptake, and thereafter, the O-2-uptake rate decreases to the level of the dark-respiration rate. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II [Y(II)], maintained a steady-state value distinct from the photosynthetic O-2-evolution rate. In high-[CO2] conditions, the photosynthetic O-2-evolution rate and Y(II) showed a parallel behavior, and the post-illumination transient O-2-uptake was suppressed. On the other hand, in maize leaves (a C4 plant), even in atmospheric CO2/O-2 conditions, Y(II) paralleled the photosynthetic O-2-evolution rate and the post-illumination transient O-2-uptake was suppressed. Hypothesizing that the post-illumination transient O-2-uptake is driven by C3 plant photorespiration in tobacco leaves, we calculated both the ribulose 1,5-bisphosphate carboxylase-and oxygenase-rates (Vc and Vo) from photosynthetic O-2-evolution and the post-illumination transient O-2-uptake rates. These values corresponded to those estimated from simultaneous chlorophyll fluorescence/O-2-exchange analysis. Furthermore, the H+-consumption rate for ATP synthesis in both photosynthesis and photorespiration, calculated from both Vc and Vo that were estimated from chlorophyll fluorescence/CO2-exchange analysis, showed a positive linear relationship with the dissipation rate of the electrochromic shift signal. Thus, these findings support our hypothesis.
WILEY-BLACKWELL, 2016年02月, PHYSIOLOGIA PLANTARUM, 156 (2), 227 - 238, 英語
[査読有り]
研究論文（学術雑誌）

Diversity in photosynthetic electron transport under [CO2]- limitation: the cyanobacterium Synechococcus sp. PCC 7002 and green alga Chlamydomonas reinhardtii drive an O2-dependent alternative electron flow and non-photochemical quenching of chlorophyll f
G. Shimakawa, S. Akimoto, Y. Ueno, A. Wada, K. Shaku, Y. Takahashi, C. Miyake
2016年, Photosynth. Res., 130, 293 - 305, 英語
[査読有り]
研究論文（学術雑誌）

Diversity in photosynthetic electron transport under [CO2]-limitation
Ginga Shimakawa, Seiji Akimoto, Yoshifumi Ueno, Ayumi Wada, Keiichiro Shaku, Yuichiro Takahashi, Chikahiro Miyake
2016年, Photosynthesis Research, 1 - 13, 英語
[査読有り]
研究論文（学術雑誌）

Altered levels of primary metabolites in response to exogenous indole-3-acetic acid in wild type and auxin signaling mutants of Arabidopsis thaliana: A capillary electrophoresis-mass spectrometry analysis
Aya Anegawa, Miwa Ohnishi, Daisuke Takagi, Chikahiro Miyake, Chizuko Shichijo, Kimitsune Ishizaki, Hidehiro Fukaki, Tetsuro Mimura
A comprehensive analysis of the levels of primary metabolites in wild type (WT) and several auxin-signaling mutants namely, tir1, slr and arf7 arf19 of Arabidopsis thaliana has been performed using CE-MS, a technique particularly sensitive for the measurement of polar compounds. We first measured the levels of primary metabolites in shoots and roots, most of the analyzed metabolites were found to be quantitatively and qualitatively comparable in WT and three kinds of mutants (tir1, slr and arf7 arf19). Some amino acids such as GABA, Arg, Orn, Val, Thr, Leu and Ile exhibited a unique pattern of distribution between shoots and roots in both WT and the mutants. On the other hand, the mutant slr showed a quite different pattern of metabolites measured in the present study. Subsequently, the responses of primary metabolites to a short-term (60 min) application of exogenous IAA (10(-7), 10(-8) M) in WT and the mutants were characterized. Due to IAA treatments, some amino acids such as GABA in WT roots and Gly and Ala in WT shoots were altered, but not in the mutants. Gln was altered in slr shoots by 10(-7) M IAA treatment. Levels of G6P from the glycolic pathway were altered in WT roots and those of 2PG, 3PG were altered in tir1 shoots in response to IAA treatments. The levels of succinate in TCA cycle were altered by IAA treatments in WT shoots but not in the mutants. IAA treatment inhibited the respiration in WT roots. The suppression of respiration might account for the IAA-dependent alteration of some metabolites. Difference of auxin responses between WT and auxin-signaling mutants suggests that some metabolic processes are under IAA control.
JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY, 2015年03月, PLANT BIOTECHNOLOGY, 32 (1), 65 - 79, 英語
[査読有り]
研究論文（学術雑誌）

FLAVODIIRON2 and FLAVODIIRON4 Proteins Mediate an Oxygen-Dependent Alternative Electron Flow in Synechocystis sp PCC 6803 under CO2-Limited Conditions
Ginga Shimakawa, Keiichiro Shaku, Akiko Nishi, Ryosuke Hayashi, Hiroshi Yamamoto, Katsuhiko Sakamoto, Amane Makino, Chikahiro Miyake
This study aims to elucidate the molecular mechanism of an alternative electron flow (AEF) functioning under suppressed (CO2-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II [Y(II)], reaches a maximum shortly after the onset of actinic illumination. Thereafter, Y(II) transiently decreases concomitantly with a decrease in the photosynthetic oxygen evolution rate and then recovers to a rate that is close to the initial maximum. These results show that CO2 limitation suppresses photosynthesis and induces AEF. In contrast to the wild type, Synechocystis sp. PCC 6803 mutants deficient in the genes encoding FLAVODIIRON2 (FLV2) and FLV4 proteins show no recovery of Y(II) after prolonged illumination. However, Synechocystis sp. PCC 6803 mutants deficient in genes encoding proteins functioning in photorespiration show AEF activity similar to the wild type. In contrast to Synechocystis sp. PCC 6803, the cyanobacterium Synechococcus elongatus PCC 7942 has no FLV proteins with high homology to FLV2 and FLV4 in Synechocystis sp. PCC 6803. This lack of FLV2/4 may explain why AEF is not induced under CO2-limited photosynthesis in S. elongatus PCC 7942. As the glutathione S-transferase fusion protein overexpressed in Escherichia coli exhibits NADH-dependent oxygen reduction to water, we suggest that FLV2 and FLV4 mediate oxygen-dependent AEF in Synechocystis sp. PCC 6803 when electron acceptors such as CO2 are not available.
AMER SOC PLANT BIOLOGISTS, 2015年02月, PLANT PHYSIOLOGY, 167 (2), 472 - U732, 英語
[査読有り]
研究論文（学術雑誌）
神戸大学リポジトリ（Kernel）へのリンク

Overexpression of flv3 improves photosynthesis in the cyanobacterium Synechocystis sp PCC6803 by enhancement of alternative electron flow
Hasunuma Tomohisa, Matsuda Mami, Senga Youhei, Aikawa Shimpei, Toyoshima Masakazu, Shimakawa Ginga, Miyake Chikahiro, Kondo Akihiko
BioMed Central, 2014年12月31日, BIOTECHNOLOGY FOR BIOFUELS, 7, 493 - 493, 英語
[査読有り]
神戸大学リポジトリ（Kernel）へのリンク

Respiration accumulates Calvin cycle intermediates for the rapid start of photosynthesis in Synechocystis sp. PCC 6803
Ginga Shimakawa, Tomohisa Hasunuma, Akihiko Kondo, Mami Matsuda, Amane Makino, Chikahiro Miyake
We tested the hypothesis that inducing photosynthesis in cyanobacteria requires respiration. A mutant deficient in glycogen phosphorylase ( increment GlgP) was prepared in Synechocystis sp. PCC 6803 to suppress respiration. The accumulated glycogen in Delta GlgP was 250-450% of that accumulated in wild type (WT). The rate of dark respiration in Delta GlgP was 25% of that in WT. In the dark, P700(+) reduction was suppressed in Delta GlgP, and the rate corresponded to that in (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone)-treated WT, supporting a lower respiration rate in increment GlgP. Photosynthetic O-2-evolution rate reached a steady-state value much slower in increment GlgP than in WT. This retardation was solved by addition of d-glucose. Furthermore, we found that the contents of Calvin cycle intermediates in increment GlgP were lower than those in WT under dark conditions. These observations indicated that respiration provided the carbon source for regeneration of ribulose 1,5-bisphosphate in order to drive the rapid start of photosynthesis.
TAYLOR & FRANCIS LTD, 2014年12月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 78 (12), 1997 - 2007, 英語
[査読有り]
研究論文（学術雑誌）

Repetitive Short-Pulse Light Mainly Inactivates Photosystem I in Sunflower Leaves
Takehiro Sejima, Daisuke Takagi, Hiroshi Fukayama, Amane Makino, Chikahiro Miyake
Under field conditions, the leaves of plants are exposed to fluctuating light, as observed in sunfleck. The duration and frequency of sunfleck, which is caused by the canopy being blown by the wind, are in the ranges from 0.2 to 50 s, and from 0.004 to 1 Hz, respectively. Furthermore, > 60% of the sunfleck duration ranges from 0.2 to 0.8 s. In the present research, we analyzed the effects of repetitive illumination by short-pulse (SP) light of sunflower leaves on the photosynthetic electron flow. The duration of SP light was set in the range from 10 to 300 ms. We found that repetitive illumination with SP light did not induce the oxidation of P700 in PSI, and mainly inactivated PSI. Increases in the intensity, duration and frequency of SP light enhanced PSI photoinhibition. PSI photoinhibition required the presence of O-2. The inactivation of PSI suppressed the net CO2 assimilation. On the other hand, the increase in the oxidized state of P700 suppressed PSI inactivation. That is, PSI with a reduced reaction center would produce reactive oxygen species (ROS) by SP light, leading to PSI photodamage. This mechanism probably explains the PSI photodamage induced by constant light.
OXFORD UNIV PRESS, 2014年06月, PLANT AND CELL PHYSIOLOGY, 55 (6), 1184 - 1193, 英語
[査読有り]
研究論文（学術雑誌）

Why don't plants have diabetes? Systems for scavenging reactive carbonyls in photosynthetic organisms
Ginga Shimakawa, Mayumi Suzuki, Eriko Yamamoto, Ryota Saito, Tatsuya Iwamoto, Akiko Nishi, Chikahiro Miyake
In the present paper, we review the toxicity of sugar- and lipid-derived RCs (reactive carbonyls) and the RC-scavenging systems observed in photosynthetic organisms. Similar to heterotrophs, photosynthetic organisms are exposed to the danger of RCs produced in sugar metabolism during both respiration and photosynthesis. RCs such as methylglyoxal and acrolein have toxic effects on the photosynthetic activity of higher plants and cyanobacteria. These toxic effects are assumed to occur uniquely in photosynthetic organisms, suggesting that RC-scavenging systems are essential for their survival. The aldo-keto reductase and the glyoxalase systems mainly scavenge sugar-derived RCs in higher plants and cyanobacteria. 2-Alkenal reductase and alkenal/alkenone reductase catalyse the reduction of lipid-derived RCs in higher plants. In cyanobacteria, medium-chain dehydrogenases/reductases are the main scavengers of lipid-derived RCs.
PORTLAND PRESS LTD, 2014年04月, BIOCHEMICAL SOCIETY TRANSACTIONS, 42, 543 - 547, 英語
[査読有り]
研究論文（学術雑誌）

The Calvin Cycle Inevitably Produces Sugar-Derived Reactive Carbonyl Methylglyoxal During Photosynthesis: A Potential Cause of Plant Diabetes
Daisuke Takagi, Hironori Inoue, Mizue Odawara, Ginga Shimakawa, Chikahiro Miyake
Sugar-derived reactive carbonyls (RCs), including methylglyoxal (MG), are aggressive by-products of oxidative stress known to impair the functions of multiple proteins. These advanced glycation end-products accumulate in patients with diabetes mellitus and cause major complications, including arteriosclerosis and cardiac insufficiency. In the glycolytic pathway, the equilibration reactions between dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (GAP) have recently been shown to generate MG as a by-product. Because plants produce vast amounts of sugars and support the same reaction in the Calvin cycle, we hypothesized that MG also accumulates in chloroplasts. Incubating isolated chloroplasts with excess 3-phosphoglycerate (3-PGA) as the GAP precursor drove the equilibration reaction toward MG production. The rate of oxygen (O-2) evolution was used as an index of 3-PGA-mediated photosynthesis. The 3-PGA- and time-dependent accumulation of MG in chloroplasts was confirmed by HPLC. In addition, MG production increased with an increase in light intensity. We also observed a positive linear relationship between the rates of MG production and O-2 evolution (R = 0.88; P< 0.0001). These data provide evidence that MG is produced by the Calvin cycle and that sugar-derived RC production is inevitable during photosynthesis. Furthermore, we found that MG production is enhanced under high-CO2 conditions in illuminated wheat leaves.
OXFORD UNIV PRESS, 2014年02月, PLANT AND CELL PHYSIOLOGY, 55 (2), 333 - 340, 英語
[査読有り]
研究論文（学術雑誌）

6 高等植物におけるalkenal/one oxidoreductase(AOR)の生理学的解析 : 植物も糖尿病を発症する(関西支部講演会,2013年度各支部会)
齊藤亮太, 伊福健太郎, 池田健一, 井上加奈子, 三宅親弘
一般社団法人日本土壌肥料学会, 2014年, 日本土壌肥料学会講演要旨集, 60 (0), 288 - 288, 日本語
[査読有り]

O-2-dependent large electron flow functioned as an electron sink, replacing the steady-state electron flux in photosynthesis in the cyanobacterium Synechocystis sp PCC 6803, but not in the cyanobacterium Synechococcus sp PCC 7942
Ryosuke Hayashi, Ginga Shimakawa, Keiichiro Shaku, Satoko Shimizu, Seiji Akimoto, Hiroshi Yamamoto, Katsumi Amako, Toshio Sugimoto, Masahiro Tamoi, Amane Makino, Chikahiro Miyake
To determine whether alternative electron flow (AEF) can replace the photosynthetic electron flow in cyanobacteria, we used an open O-2-electrode system to monitor O-2-exchange over a long period. In air-grown Synechocystis sp. PCC 6803 (S. 6803 (WT)), the quantum yield of PSII, Y(II), held even after photosynthesis was suppressed by CO2 shortage. The S. 6803 mutant, deficient in flavodiiron (FLV) proteins 1 and 3, showed the same phenotype as S. 6803(WT). In contrast, Y(II) decreased in Synechococcus sp. PCC 7942 (S. 7942). These results suggest that AEF functioned as the Y(II) in S. 6803 and replaced the photosynthetic electron flux. In contrast, the activity of AEF in S. 7942 was lower. The affinity of AEF for O-2 in S. 6803 did not correspond to those of FLVs in bacteria or terminal oxidases in respiration. AEF might be driven by photorespiration.
TAYLOR & FRANCIS LTD, 2014年, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 78 (3), 384 - 393, 英語
[査読有り]
研究論文（学術雑誌）

Functional Analysis of the AKR4C Subfamily of Arabidopsis thaliana: Model Structures, Substrate Specificity, Acrolein Toxicity, and Responses to Light and [CO2]
Ryota Saito, Ginga Shimakawa, Akiko Nishi, Tatsuya Iwamoto, Katsuhiko Sakamoto, Hiroshi Yamamoto, Katsumi Amako, Amane Makino, Chikahiro Miyake
In Arabidopsis thaliana, the aldo-keto reductase (AKR) family includes four enzymes (The AKR4C subfamily: AKR4C8, AKR4C9, AKR4C10, and AKR4C11). AKR4C8 and AKR4C9 might detoxify sugar-derived reactive carbonyls (RCs). We analyzed AKR4C10 and AKR4C11, and compared the enzymatic functions of the four enzymes. Modeling of protein structures based on the known structure of AKR4C9 found an (alpha/beta)(8)-barrel motif in all four enzymes. Loop structures (A, B, and C) which determine substrate specificity, differed among the four. Both AKR4C10 and AKR4C11 reduced methylglyoxal. AKR4C10 reduced triose phosphates, dihydroxyacetone phosphate (DHAP), and glyceraldehydes 3-phosphate (GAP), the most efficiently of all the AKR4Cs. Acrolein, a lipid-derived RC, inactivated the four enzymes to different degrees. Expression of the AKR4C genes was induced under high-[CO2] and high light, when photosynthesis was enhanced and photosynthates accumulated in the cells. These results suggest that the AKR4C subfamily contributes to the detoxification of sugar-derived RCs in plants.
TAYLOR & FRANCIS LTD, 2013年10月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 77 (10), 2038 - 2045, 英語
[査読有り]
研究論文（学術雑誌）

Acrolein, an alpha,beta-Unsaturated Carbonyl, Inhibits Both Growth and PSII Activity in the Cyanobacterium Synechocystis sp PCC 6803
Ginga Shimakawa, Tatsuya Iwamoto, Tomohito Mabuchi, Ryota Saito, Hiroshi Yamamoto, Katsumi Amako, Toshio Sugimoto, Amane Makino, Chikahiro Miyake
In this study, we sought to determine whether and how an alpha,beta-unsaturated carbonyl, acrolein, can inhibit the growth of the cyanobacterium Synechocystis sp. PCC6803 (S. 6803). Treatment of S. 6803 with 200 mu M acrolein for 3 d significantly and irreversibly inhibited its growth. To elucidate the inhibitory mechanism, we examined the effects of acrolein on photosynthesis. In contrast to dark conditions, the addition of acrolein to S. 6803 under conditions of illumination lowered the CO2-dependent O-2 evolution rate (photosynthetic activity). Furthermore, treatment with acrolein lowered the activity reducing dimethyl benzoquinone in photosystem II (PSII). Acrolein also suppressed the reduction rate for the oxidized form of the reaction center chlorophyll of photosystem I (PSI), P700. These results indicate that acrolein inhibited PSII activity in thylakoid membranes. The addition of 200 mu M acrolein to the illuminated S. 6803 cells gradually increased the steady-state level (Fs) of Chl fluorescence and decreased the quantum yield of PSII. These results suggested that acrolein damaged the acceptor side of PSII. On the other hand, acrolein did not inhibit respiration. From the above results, we gained insight into the metabolism of acrolein and its physiological effects in S. 6803.
TAYLOR & FRANCIS LTD, 2013年08月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 77 (8), 1655 - 1660, 英語
[査読有り]
研究論文（学術雑誌）

Scavenging systems for reactive carbonyls in the cyanobacterium synechocystis sp. PCC 6803
Ginga Shimakawa, Mayumi Suzuki, Eriko Yamamoto, Akiko Nishi, Ryota Saito, Katsuhiko Sakamoto, Hiroshi Yamamoto, Amane Makino, Chikahiro Miyake
To elucidate the scavenging systems of sugar- and lipid-derived reactive carbonyls (RCs) in the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803), we selected proteins from S. 6803 based on amino-acid (AA) sequence similarities with proteins from Arabidopsis thaliana, and characterized the properties of the GST-fusion proteins expressed. Slr0942 catalyzed the aldo-keto reductase (AKR) reaction scavenging mainly sugar-derived RCs, methylglyoxal (MG). Slr1192 is the medium-chain dehydrogenase/redutase (MDR). It catalyzed the AKR reaction scavenging several lipid-derived RCs, acrolein, propionaldehyde, and crotonaldehyde. Slr0315 is a short-chain dehydrogenase/redutase (SDR), and it catalyzed only the reduction of MG in the AKR reaction. Slr0381 catalyzed the conversion of hemithioacetal to S-lactoylglutahione (SLG) in the glyoxalase (GLX) 1 reaction. Sll1019 catalyzed the conversion of SLG to glutathione and lactate in the GLX2 reaction. GLX1 and GLX2 compose the glyoxalase system, which scavenges MG. These enzymes contribute to scavenging sugar- and lipid-derived RCs as scavenging systems.
Japan Society for Bioscience, Biotechnology, and Agrochemistry, 2013年, Bioscience, Biotechnology and Biochemistry, 77 (12), 2441 - 2448, 英語
[査読有り]
研究論文（学術雑誌）

O2-enhanced induction of photosynthesis in rice leaves: The Mehler-ascorbate peroxidase (MAP) pathway drives cyclic electron flow within PSII and cyclic electron flow around PSI.
Miyake C, Suzuki Y, Yamamoto H, Amako K, Makino A
IF = 1.017
TAYLOR & FRANCIS LTD, 2012年12月, Soil Science and Plant Nutrition, 58 (6), 718 - 727, 英語
[査読有り]
研究論文（学術雑誌）

O2 supports 3-phosphoglycerate-dependent O2 evolution in chloroplasts from spinach leaves
Daisuke Takagi, Hiroshi Yamamoto, Katsumi Amako, Amane Makino, Toshio Sugimoto, Chikahiro Miyake
2012年, Soil Science and Plant Nutrition, 58 (4), 462 - 468, 英語
[査読有り]
研究論文（学術雑誌）

O-2 supports 3-phosphoglycerate-dependent O-2 evolution in chloroplasts from spinach leaves
Daisuke Takagi, Hiroshi Yamamoto, Katsumi Amako, Amane Makino, Toshio Sugimoto, Chikahiro Miyake
We tested the hypothesis that the Mehler-ascorbate peroxidase (MAP) pathway supports 3-phosphoglycerate (PGA)-dependent oxygen (O-2) evolution using intact chloroplasts. Lowering O-2 concentration (<1 mu M) suppressed PGA-dependent O-2 evolution rate. High O-2 concentration (about 250 mM) enhanced the electron fluxes in Photosystem II (PSII). Also, high O-2 concentration oxidized both Q(A) in PSII and Cyt f in thylakoid membranes. These results indicated that the MAP pathway stimulated photosynthetic electron transport. Furthermore, electrochromic shift signal was also increased at high O-2 concentration, compared to low O-2 concentration. Non-photochemical quenching of chlorophyll fluorescence was also enhanced at high O-2 concentration. These data support our hypothesis that the MAP pathway functioned in intact chloroplasts and accelerated PGA-dependent O-2 evolution by inducing Delta pH formation to produce and supply adenosine triphosphate (ATP) to the conversion reaction of PGA to glyceraldehyde 3-phosphate through 1,3-diphosphoglycerate in chloroplasts.
TAYLOR & FRANCIS LTD, 2012年, SOIL SCIENCE AND PLANT NUTRITION, 58 (4), 462 - 468, 英語
[査読有り]
研究論文（学術雑誌）

Methylglyoxal functions as Hill oxidant and stimulates the photoreduction of O-2 at photosystem I: a symptom of plant diabetes
Ryota Saito, Hiroshi Yamamoto, Amane Makino, Toshio Sugimoto, Chikahiro Miyake
We elucidated the metabolism of methylglyoxal (MG) in chloroplasts of higher plants. Spinach chloroplasts showed MG-dependent NADPH oxidation because of aldo-keto reductase (AKR) activity. K-m for MG and V-max of AKR activity were 6.5 mM and 3.3 mu mol NADPH (mg Chl)(-1) h(-1), respectively. Addition of MG to illuminated chloroplasts induced photochemical quenching (Qp) of Chl fluorescence, indicating that MG stimulated photosynthetic electron transport (PET). Furthermore, MG enhanced the light-dependent uptake of O-2 into chloroplasts. After illumination of chloroplasts, accumulation of H2O2 was observed. K-m for MG and V-max of O-2 uptake were about 100 mu M and 200 mu mol O-2 (mg Chl)(-1) h(-1), respectively. MG-dependent O-2 uptake was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Under anaerobic conditions, the Qp of Chl fluorescence was suppressed. These results indicate that MG was reduced as a Hill oxidant by the photosystem I (PSI), and that O-2 was reduced to O-2 by the reduced MG. In other words, MG produced in chloroplasts is preferentially reduced by PSI rather than through AKR. This triggers a type of oxidative stress that may be referred to as 'plant diabetes', because it ultimately originates from a common metabolite of the primary pathways of sugar anabolism and catabolism.
WILEY-BLACKWELL, 2011年09月, PLANT CELL AND ENVIRONMENT, 34 (9), 1454 - 1464, 英語
[査読有り]
研究論文（学術雑誌）

Cyclic electron flow around PSI functions in the photoinhibited rice leaves
Satoshi Kubo, Takehiro Masumura, Yuhi Saito, Hiroshi Fukayama, Yuji Suzuki, Toshio Sugimoto, Amane Makino, Katsumi Amako, Chikahiro Miyake
We investigated the regulation mechanism of cyclic electron flow around photosystem I (CEF-PSI) in the rice leaves which suffered from photosystem II (PSII) photoinhibition. High-light (HL) treatment [2000 mu mol photon m-2 s-1 at 0% carbon dioxide (CO2), 2% oxygen (O2) and 25 degrees C] of rice leaves decreased both the maximum quantum efficiency of PSII (Fv/Fm) and the light-dependent O2-evolution rate [V(O2)]. High-light treatment did not affect the relative electron flux in PSI [phi(PSI) x PFD]. In non-treated leaves, increasing in the photon flux density (PFD) enhanced V(O2), phi(PSI) x PFD and the ratio of oxidized P700 to total P700 [(P700+)/(P700)total]. phi(PSI) x PFD continued to increase even after the saturation of V(O2) against PFD. These results suggested that the electrons not used for the major electron sink, photosynthetic carbon reduction-cycle, did turnover in PSI, that is, CEF-PSI functioned at a higher PFD. High-light treatments did not affect the activity of CEF-PSI and increased (P700+)/(P700)total in the lower PFDs, compared to non-treated leaves. The activity of CEF-PSI depends on the amount of oxidized PQ. Photoinhibition of PSII suppressed electron influx from PSII to photosynthetic linear electron transport. The enhanced (P700+)/(P700)total suggested the increase in the ratio of oxidized plastoquinone (PQ) to total PQ, which supported the activity of CEF-PSI in the photoinhibited leaves.
WILEY-BLACKWELL, 2011年02月, SOIL SCIENCE AND PLANT NUTRITION, 57 (1), 105 - 113, 英語
[査読有り]
研究論文（学術雑誌）

Cyclic electron flow around PSI functions in the photoinhibited rice leaves
Satoshi Kubo, Takehiro Masumura, Yuhi Saito, Hiroshi Fukayama, Yuji Suzuki, Toshio Sugimoto, Amane Makino, Katsumi Amako, Chikahiro Miyake
We investigated the regulation mechanism of cyclic electron flow around photosystem I (CEF-PSI) in the rice leaves which suffered from photosystem II (PSII) photoinhibition. High-light (HL) treatment [2000 mu mol photon m-2 s-1 at 0% carbon dioxide (CO2), 2% oxygen (O2) and 25 degrees C] of rice leaves decreased both the maximum quantum efficiency of PSII (Fv/Fm) and the light-dependent O2-evolution rate [V(O2)]. High-light treatment did not affect the relative electron flux in PSI [phi(PSI) x PFD]. In non-treated leaves, increasing in the photon flux density (PFD) enhanced V(O2), phi(PSI) x PFD and the ratio of oxidized P700 to total P700 [(P700+)/(P700)total]. phi(PSI) x PFD continued to increase even after the saturation of V(O2) against PFD. These results suggested that the electrons not used for the major electron sink, photosynthetic carbon reduction-cycle, did turnover in PSI, that is, CEF-PSI functioned at a higher PFD. High-light treatments did not affect the activity of CEF-PSI and increased (P700+)/(P700)total in the lower PFDs, compared to non-treated leaves. The activity of CEF-PSI depends on the amount of oxidized PQ. Photoinhibition of PSII suppressed electron influx from PSII to photosynthetic linear electron transport. The enhanced (P700+)/(P700)total suggested the increase in the ratio of oxidized plastoquinone (PQ) to total PQ, which supported the activity of CEF-PSI in the photoinhibited leaves.
WILEY-BLACKWELL, 2011年02月, SOIL SCIENCE AND PLANT NUTRITION, 57 (1), 105 - 113, 英語
[査読有り]
研究論文（学術雑誌）

Functional Incorporation of Sorghum Small Subunit Increases the Catalytic Turnover Rate of Rubisco in Transgenic Rice.
石川知恵, 畠中知子, 三十尾修司, 三宅親弘, 深山浩
Abstract Rubisco limits photosynthetic CO2 fixation because of its low catalytic turnover rate (k cat) and competing oxygenase reaction. Previous attempts to improve the catalytic efficiency of Rubisco by genetic engineering have gained little progress. Here we demonstrate that the introduction of the small subunit (RbcS) of high k cat Rubisco from the C4 plant sorghum (Sorghum bicolor) significantly enhances k cat of Rubisco in transgenic rice (Oryza sativa). Three independent transgenic lines expressed sorghum RbcS at a high level, accounting for 30%, 44%, and 79% of the total RbcS. Rubisco was likely present as a chimera of sorghum and rice RbcS, and showed 1.32- to 1.50-fold higher k cat than in nontransgenic rice. Rubisco from transgenic lines showed a higher K m for CO2 and slightly lower specificity for CO2 than nontransgenic controls. These results suggest that Rubisco in rice transformed with sorghum RbcS partially acquires the catalytic properties of sorghum Rubisco. Rubisco content in transgenic lines was significantly increased over wild-type levels but Rubisco activation was slightly decreased. The expression of sorghum RbcS did not affect CO2 assimilation rates under a range of CO2 partial pressures. The J max/V cmax ratio was significantly lower in transgenic line compared to the nontransgenic plants. These observations suggest that the capacity of electron transport is not sufficient to support the increased Rubisco capacity in transgenic rice. Although the photosynthetic rate was not enhanced, the strategy presented here opens the way to engineering Rubisco for improvement of photosynthesis and productivity in the future.
Oxford University Press (OUP), 2011年, Plant Physiol., 156 (3), 1603 - 1611, 英語
[査読有り]
研究論文（学術雑誌）

Differences in Rubisco content and its synthesis in leaves at different positions in Eucalyptus globulus seedlings
YUJI SUZUKI, TOMONORI KIHARA-DOI, TETSU KAWAZU, CHIKAHIRO MIYAKE, AMANE MAKINO
The dynamics of ribulose 1 center dot 5-bisphosphate carboxylase/oxygenase (Rubisco) content and turnover during leaf development are not well understood in woody plants. Rubisco synthesis, N influx and the mRNA levels of Rubisco-encoding genes were determined as a function of leaf position in 4.5-month-old Eucalyptus globulus seedlings. Rubisco concentration was slightly higher in the top leaves as leaf expansion progressed and was almost maximal in the uppermost fully expanded leaves. Rubisco concentration remained almost constant in the fully expanded leaves at the top and middle positions and then became slightly low at the lowest positions. Rubisco synthesis was active only in the top leaves. These results suggest that Rubisco turnover rate is low in the middle leaves, leading to the maintenance of Rubisco contents, and that Rubisco degradation primarily occurs in the lowest leaves. Changes in the RBCS and rbcL mRNA levels were roughly parallel with Rubisco synthesis, but N influx was more closely correlated with Rubisco synthesis. These results suggest that N influx rather than the transcript abundance of Rubisco-encoding genes is of primary importance in regulating the rate of Rubisco synthesis. Additionally, expression of RBCS multigene family in E. globulus leaves was discussed.
WILEY, 2010年08月, Plant, Cell and Environment, 33 (8), 1314 - 1323, 英語
[査読有り]
研究論文（学術雑誌）

Acclimation of Tobacco Leaves to High Light Intensity Drives the Plastoquinone Oxidation SystemRelationship Among the Fraction of Open PSII Centers, Non-Photochemical Quenching of Chl Fluorescence and the Maximum Quantum Yield of PSII in the Dark
Chikahiro Miyake, Katsumi Amako, Naomasa Shiraishi, Toshio Sugimoto
Responses of the reductionoxidation level of plasto-quinone (PQ) in the photosynthetic electron transport (PET) system of chloroplasts to growth light intensity were evaluated in tobacco plants. Plants grown in low light (150mol photons m(2) s(1)) (LL plants) were exposed to a high light intensity (1,100mol photons m(2)s(1)) for 1d. Subsequently, the plants exposed to high light (LH plants) were returned back again to the low light condition: these plants were designated as LHL plants. Both LH and LHL plants showed higher values of non-photochemical quenching of Chl fluorescence (NPQ) and the fraction of open PSII centers (qL), and lower values of the maximum quantum yield of PSII in the dark (F(v)F(m)), compared with LL plants. The dependence of qL on the quantum yield of PSII [(PSII)] in LH and LHL plants was higher than that in LL plants. To evaluate the effect of an increase in NPQ and decrease in F(v)F(m) on qL, we derived an equation expressing qL in relation to both NPQ and F(v)F(m), according to the lake model of photoexcitation of the PSII reaction center. As a result, the heat dissipation process, shown as NPQ, did not contribute greatly to the increase in qL. On the other hand, decreased F(v)F(m) did contribute to the increase in qL, i.e. the enhanced oxidation of PQ under photosynthesis-limited conditions. Thylakoid membranes isolated from LH plants, having high qL, showed a higher tolerance against photoinhibition of PSII, compared with those from LL plants. We propose a plastoquinone oxidation system (POS), which keeps PQ in an oxidized state by suppressing the accumulation of electrons in the PET system in such a way as to regulate the maximum quantum yield of PSII.
OXFORD UNIV PRESS, 2009年04月, PLANT AND CELL PHYSIOLOGY, 50 (4), 730 - 743, 英語
[査読有り]
研究論文（学術雑誌）

Metabolic pathway engineering by plastid transformation is a powerful tool for production of compounds in higher plants
Tomohisa Hasunuma, Akihiko Kondo, Chikahiro Miyake
Plastid transformation is a powerful tool for the production of useful compounds in higher plants through metabolic engineering, because it has many advantages over conventional nuclear transformation: high-level foreign protein accumulation, no need for a transit peptide, absence of gene silencing, and convenient transgene stacking in an operon. Plastid transformation has recently yielded remarkable results in the production of highly valued biopharmaceutical proteins and in conferring herbicide and insect resistance. Metabolic pathway engineering by plastid transformation has also produced higher levels of useful compounds than nuclear transformation. Furthermore, recent reports have shown the functional regulation of transgene expression from the plastid genome. In this review, we have focused on the progress of plastid transformation in material production from the aspect of biosynthetic pathway engineering, discussing the issues for future expansion of plastid transformation.
Japanese Society for Plant Cell and Molecular Biology, 2009年03月, Plant Biotechnology, 26 (1), 39 - 46, 英語
[査読有り]
研究論文（学術雑誌）

Knockdown of the PsbP protein does not prevent assembly of the dimeric PSII supercomplexes in tobacco.
Kunio Ido, Kentaro Ifuku, Yumiko Yamamoto, Seiko Ishihara, Akio Murakami, Keiji Takabe, Chikahiro Miyake, Fumihiko Sato
2009年, Biochemica et Biophysica Acta 1787, 873-88, 英語
[査読有り]
研究論文（学術雑誌）

Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering
Tomohisa Hasunuma, Shin-Ichi Miyazawa, Satomi Yoshimura, Yuki Shinzaki, Ken-Ichi Tomizawa, Kazutoshi Shindo, Seon-Kang Choi, Norihiko Misawa, Chikahiro Miyake
The natural pigment astaxanthin has attracted much attention because of its beneficial effects on human health, despite its expensive market price. In order to produce astaxanthin, transgenic plants have so far been generated through conventional genetic engineering of Agrobacterium-mediated gene transfer. The results of trials have revealed that the method is far from practicable because of low yields, i.e. instead of astaxanthin, large quantities of the astaxanthin intermediates, including ketocarotenoids, accumulated in the transgenic plants. In the present study, we have overcome this problem, and have succeeded in producing more than 0.5% (dry weight) astaxanthin (more than 70% of total caroteniods) in tobacco leaves, which turns their green color to reddish brown, by expressing both genes encoding CrtW (beta-carotene ketolase) and CrtZ (beta-carotene hydroxylase) from a marine bacterium Brevundimonas sp., strain SD212, in the chloroplasts. Moreover, the total carotenoid content in the transplastomic tobacco plants was 2.1-fold higher than that of wild-type tobacco. The tobacco transformants also synthesized a novel carotenoid 4-ketoantheraxanthin. There was no significant difference in the size of the aerial part of the plant between the transformants and wild-type plants at the final stage of their growth. The photosynthesis rate of the transformants was also found to be similar to that of wild-type plants under ambient CO(2) concentrations of 1500 mu mol photons m(-2) s(-1) light intensity.
WILEY-BLACKWELL, 2008年09月, PLANT JOURNAL, 55 (5), 857 - 868, 英語
[査読有り]
研究論文（学術雑誌）

Overexpression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene in chloroplast contributes to increment of isoprenoid production
蓮沼誠久, 武野真也, 林俊介, 千代真由美, 馬場健史, 吉村智美, 富澤健一, 福崎英一郎, 三宅親弘
2008年05月, Journal of Bioscience and Biotechnology, Vol 105. No. 5, pp. 518-526, 英語
[査読有り]
研究論文（学術雑誌）

4-Ketoantheraxanthin, a novel carotenoid produced by the combination of the bacterial enzyme b-carotene ketolase CrtW and endogeneous carotenoid biosynthetic enzymes in hihger plants
新藤一敏, 蓮沼誠久, ASAGI Emiko, SANO Aya, HOTTA Eri, MINEMURA Noriko, 三宅親弘, 眞岡孝至, 三沢典彦
2008年03月, Tetrahedron Letters, Vol 49. No. 20, pp. 3294-3296, 英語
[査読有り]
研究論文（学術雑誌）

Deactivation of aquaporins decreases internal conductance to CO(2) diffusion in tobacco leaves grown under long-term drought
Shin-ichi Miyazawa, Satomi Yoshimura, Yuki Shinzaki, Masayoshi Maeshima, Chikahiro Miyake
We compared the diffusion conductance to CO(2) from the intercellular air space to the chloroplasts (internal conductance (g(i))) between tobacco leaves acclimated to long-term drought (drought-acclimated (DA)) and those grown under sufficient irrigation (well-watered (WW)), and analysed the changes in g(i) in relation to the leaf anatomical characteristics and a possible CO(2) transporter, aquaporin. The g(i), which was estimated by combined analyses of CO(2) gas exchange with chlorophyll fluorescence, in the DA plants was approximately half of that in the WW plants. The mesophyll and chloroplast surface areas exposing the intercellular air space, which potentially affect g(i), were not significantly different between the WW and DA plants. The amounts of plasma membrane aquaporins (PIP), immunochemically determined using radish PIP antibodies, were unrelated to g(i). After treatment with HgCl(2), an aquaporin inhibitor, the water permeability of the leaf tissues (measured as the weight loss of fully-turgid leaf disks without the abaxial epidermis in 1 M sorbitol) in WW plants decreased with an increase in HgCl(2) concentration. The g(i) in the WW plants decreased to similar levels to the DA plants when the detached leaflets were fed with 0.5 mM HgCl(2). In contrast, both water permeability and g(i) were insensitive to HgCl(2) treatments in DA plants. These results suggest that deactivation of aquaporins is responsible for the significant reduction in g(i) observed in plants growing under long-term drought.
CSIRO PUBLISHING, 2008年, FUNCTIONAL PLANT BIOLOGY, 35 (7), 553 - 564, 英語
[査読有り]
研究論文（学術雑誌）

Effects of the PsbP knockdown on the photosynthetic electron transfer in Nicotiana tabacum.
Kunio Ido, Kentaro Ifuku, Seiko Ishihara, Yumiko Yamamoto, Chikahiro Miyake, Fumihiko Sato
2008年, Photosynthesis Energy from the Sun, 5, 605 - 608, 英語
研究論文（国際会議プロシーディングス）

Relationship between mesophyll CO2 gas diffusion conductance and leaf plasma-membrane-type aquaporin contents in tobacco plants grown under drought conditions
Shin-Ichi Miyazawa, Yuki Shinzaki, Tomomi Kawasaki, Masayoshi Maeshima, Chikahiro Miyake
OXFORD UNIV PRESS, 2007年, PLANT AND CELL PHYSIOLOGY, 48, S96 - S96, 英語
[査読有り]

Ferredoxin limits cyclic electron flow around PSI (CEF-PSI) in higher plants-stimulation of CEF-PSI enhances non-photochemical quenching of Chl fluorescence in transplastomic tobacco
Yamamoto Hiroshi, Kato Hideki, Shinzaki Yuki, Horiguchi Sayaka, Shikanai Toshiharu, Hase Toshiharu, Endo Tsuyoshi, Nishioka Minori, Makino Amane, Tomizawa Ken-ichi, Miyake Chikahiro
2007年, PLANT AND CELL PHYSIOLOGY, 48, S96
[査読有り]

Ferredoxin limits cyclic electron flow around PSI (CEF-PSI) in higher plants-stimulation of CEF-PSI enhances non-photochemical quenching of Chl fluorescence in transplastomic tobacco
Hiroshi Yamamoto, Hideki Kato, Yuki Shinzaki, Sayaka Horiguchi, Toshiharu Shikanai, Toshiharu Hase, Tsuyoshi Endo, Minori Nishioka, Amane Makino, Ken-ichi Tomizawa, Chikahiro Miyake
We tested the hypothesis that ferredoxin (Fd) limits the activity of cyclic electron flow around PSI (CEF-PSI) in vivo and that the relief of this limitation promotes the non-photochemical quenching (NPQ) of Chl fluorescence. In transplastomic tobacco (Nicotiana tabacum cv Xanthi) expressing Fd from Arabidopsis (Arabidopsis thaliana) in its chloroplasts, the minimum yield (F-o) of Chl fluorescence was higher than in the wild type. F-o was suppressed to the wild-type level upon illumination with far-red light, implying that the transfer of electrons by Fd-quinone oxidoreductase (FQR) from the chloroplast stroma to plastoquinone was enhanced in transplastomic plants. The activity of CEF-PSI became higher in transplastomic than in wild-type plants under conditions limiting photosynthetic linear electron flow. Similarly, the NPQ of Chl fluorescence was enhanced in transplastomic plants. On the other hand, pool sizes of the pigments of the xanthophyll cycle and the amounts of PsbS protein were the same in all plants. All these results supported the hypothesis strongly. We conclude that breeding plants with an NPQ of Chl fluorescence increased by an enhancement of CEF-PSI activity might lead to improved tolerance for abiotic stresses, particularly under conditions of low light use efficiency.
OXFORD UNIV PRESS, 2006年10月, PLANT AND CELL PHYSIOLOGY, 47 (10), 1355 - 1371, 英語
[査読有り]
研究論文（学術雑誌）

Effects of light intensity on cyclic electron flow around PSI and its relationship to non-photochemical quenching of Chl fluorescence in tobacco leaves
Chikahiro Miyake, Sayaka Horiguchi, Amane Makino, Yuki Shinzaki, Hiroshi Yamamoto, Ken-Ichi Tomizawa
We tested the hypothesis that plants grown under high light intensity (HL-plants) had a large activity of cyclic electron flow around PSI (CEF-PSI) compared with plants grown under low light (LL-plants). To evaluate the activity of CEF-PSI, the relationships between photosynthesis rate, quantum yields of both PSII and PSI, and Chl fluorescence parameters were analyzed simultaneously in intact leaves of tobacco plants which had been grown under different light intensities (150 and 1,100 μmol photons m-2 s-1, respectively) and with different amounts of nutrients supplied. HL-plants showed a larger value of non-photochemical quenching (NPQ) of Chl fluorescence at the limited activity of photosynthetic linear electron flow. Furthermore, HL-plants had a larger activity of CEF-PSI than LL-plants. These results suggested that HL-plants dissipated the excess photon energy through NPQ by enhancing the ability of CEF-PSI to induce acidification of the thylakoid lumen. JSPP © 2005.
2005年11月, Plant and Cell Physiology, 46 (11), 1819 - 1830, 英語
[査読有り]
研究論文（学術雑誌）

Cyclic electron flow around photosystem I is essential for photosynthesis.
Yuri Munekage, Mihoko Hashimoto, Chikahiro Miyake, Ken-ichi Tomizawa, Tsuyoshi Endo, Masao Tasaka, Toshiharu Shikanai
Photosynthesis provides at least two routes through which light energy can be used to generate a proton gradient across the thylakoid membrane of chloroplasts, which is subsequently used to synthesize ATP. In the first route, electrons released from water in photosystem II (PSII) are eventually transferred to NADP+ by way of photosystem I (PSI). This linear electron flow is driven by two photochemical reactions that function in series. The cytochrome b6f complex mediates electron transport between the two photosystems and generates the proton gradient (DeltapH). In the second route, driven solely by PSI, electrons can be recycled from either reduced ferredoxin or NADPH to plastoquinone, and subsequently to the cytochrome b6f complex. Such cyclic flow generates DeltapH and thus ATP without the accumulation of reduced species. Whereas linear flow from water to NADP+ is commonly used to explain the function of the light-dependent reactions of photosynthesis, the role of cyclic flow is less clear. In higher plants cyclic flow consists of two partially redundant pathways. Here we have constructed mutants in Arabidopsis thaliana in which both PSI cyclic pathways are impaired, and present evidence that cyclic flow is essential for efficient photosynthesis.
2004年06月03日, Nature, 429 (6991), 579 - 82, 英語, 国際誌
研究論文（学術雑誌）

Water-water cycleの進化と光合成スターターとしての酸素利用
三宅親弘, 牧野周
Japan Society for Bioscience, Biotechnology, and Agrochemistry, 2003年07月, 化学と生物, 41 (7), 478 - 487, 日本語
[査読有り]
研究論文（学術雑誌）

Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts
T Shimaoka, C Miyake, A Yokota
Dehydroascorbate reductase (DHAR) reduces dehydroascorbate (DHA) to ascorbate with glutathione (GSH) as the electron donor. We analyzed the reaction mechanism of spinach chloroplast DHAR, which had a much higher reaction specificity for DHA than animal enzymes, using a recombinant enzyme expressed in Escherichia coli . Kinetic analysis suggested that the reaction proceeded by a bi-uni-uni-uni-ping-pong mechanism, in which binding of DHA to the free, reduced form of the enzyme was followed by binding of GSH. The K (m) value for DHA and the summed K (m) value for GSH were determined to be 53 +/- 12 mum and 2.2 +/- 1.0 mm, respectively, with a turnover rate of 490 +/- 40 s(-1) . Incubation of 10 mum DHAR with 1 mm DHA and 10 mum GSH resulted in stable binding of GSH to the enzyme. Bound GSH was released upon reduction of the GSH-enzyme adduct by 2-mercaptoethanol, suggesting that the adduct is a reaction intermediate. Site-directed mutagenesis indicated that C23 in DHAR is indispensable for the reduction of DHA. The mechanism of catalysis of spinach chloroplast DHAR is proposed.
BLACKWELL PUBLISHING LTD, 2003年03月, EUROPEAN JOURNAL OF BIOCHEMISTRY, 270 (5), 921 - 928, 英語
[査読有り]
研究論文（学術雑誌）

Physiological functions of the water-water cycle and the cyclic electron flow around PSI in rice including RbcS antisense plants
A Makino, C Miyake, A Yokota, A Ushio, T Mae
OXFORD UNIV PRESS, 2003年, PLANT AND CELL PHYSIOLOGY, 44, S14 - S14, 英語
[査読有り]

Stable form of ascorbate peroxidase from the red alga Galdieria partita similar to both chloroplastic and cytosolic isoforms of higher plants
S Kitajima, M Ueda, S Sano, C Miyake, T Kohchi, K Tomizawa, S Shigeoka, A Yokota
Depletion of the electron donor ascorbate causes rapid inactivation of chloroplastic ascorbate peroxidase (APX) of higher plants, while cytosolic APX is stable under such conditions. Here we report the cloning of cDNA from Galdieria partita, a unicellular red alga, encoding a novel type of APX (APX-B). The electrophoretic mobility, K-m values, k(cat) and absorption spectra of recombinant APX-B produced in Escherichia coli were measured. Recombinant APX-B remained active for at least 180 min after depletion of ascorbate. The amino-terminal half of APX-B, which forms the distal pocket of the active site, was richer in amino acid residues conserved in chloroplastic APXs of higher plants rather than cytosolic APXs. In contrast, the sequence of the carboxyl-terminal half, which forms the proximal pocket, was similar to that of the cytosolic isoform. The stability of APX-B might be due to its cytosolic isoform-like structure of the carboxyl-terminal half.
TAYLOR & FRANCIS LTD, 2002年11月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 66 (11), 2367 - 2375, 英語
[査読有り]
研究論文（学術雑誌）

Physiological functions of the water-water cycle (Mehler reaction) and the cyclic electron flow around PSI in rice leaves
Amane Makino, Chikahiro Miyake, Akiho Yokota
Changes in chlorophyll fluorescence, P700+-absorbance and gas exchange during the induction phase and steady state of photosynthesis were simultaneously examined in rice (Oryza sativa L.), including the rbcS antisense plants. The quantum yield of photosystem II (ΦPSII) increased more rapidly than CO2 assimilation in 20% O2. This rapid increase in ΦPSII resulted from the electron flux through the water-water cycle (WWC) because of its dependency on O2. The electron flux of WWC reached a maximum just after illumination, and rapidly generated non-photochemical quenching (NPQ). With increasing CO2 assimilation, the electron flux of WWC and NPQ decreased. In 2% O2, WWC scarcely operated and ΦPSI was always higher than ΦPSII. This suggested that cyclic electron flow around PSI resulted in the formation of NPQ, which remained at higher levels in 2% O2. The electron flux of WWC in the rbcS antisense plants was lower, but these plants always showed a higher NPQ. This was also caused by the operation of the cyclic electron flow around PSI because of a higher ratio of ΦPSI/ΦPSII, irrespective of O2 concentration. The results indicate that WWC functions as a starter of photosynthesis by generating ΔpH across thylakoid membranes for NPQ formation, supplying ATP for carbon assimilation. However, WWC does not act to maintain a high NPQ, and ΦPSII is down-regulated by ΔpH generated via the cyclic electron flow around PSI.
Japanese Society of Plant Physiologists, 2002年09月01日, Plant and Cell Physiology, 43 (9), 1017 - 1026, 英語
[査読有り]
研究論文（学術雑誌）

Characterization of ascorbate peroxidases from unicellular red alga Galdieria partita
S Sano, M Ueda, S Kitajima, T Takeda, S Shigeoka, N Kurano, S Miyachi, C Miyake, A Yokota
Galdieria partita, a unicellular red alga isolated from acidic hot springs and tolerant to sulfur dioxide, has at least two ascorbate peroxidase (APX) isozymes, This was the first report to demonstrate that two isozymes of APX are found in algal cells, Two isozymes were separated from each other at the hydrophobic chromatography step of purification and named APX-A and APX-B after the elution order in the chromatography, APX-B accounted for 85% of the total activity, Both isozymes were purified. APXs from Galdieria were monomers whose molecular weights were about 28,000, similar to stromal APX of higher plants. APX-A cross-reacted with monoclonal antibody raised against APX of Euglena gracilis in immunoblotting, but APX-B did not, although the antibody can recognize all other APXs tested. The amino-terminal sequences of APX-A and -B from Galdieria had some homology with each other but little homology with those from other sources. Their K-m values for ascorbate and hydrogen peroxide were comparable with those of APX from higher plants, Unlike the green algal enzymes, the donor specificities of Galdieria APXs were as high as those of plant chloroplastic APX. On the contrary, these APXs reduced tertiary-butyl hydroperoxide as an electron acceptor as APXs from Euglena and freshwater Chlamydomonas do. The inhibition of APX-A and -B by cyanide and azide, and characteristics of their light absorbance spectra indicated that they were heme peroxidases.
OXFORD UNIV PRESS, 2001年04月, PLANT AND CELL PHYSIOLOGY, 42 (4), 433 - 440, 英語
[査読有り]
研究論文（学術雑誌）

Responses of wild watermelon to drought stress: Accumulation of an ArgE homologue and citrulline in leaves during water deficits
S Kawasaki, C Miyake, T Kohchi, S Fujii, M Uchida, A Yokota
Wild watermelon from the Botswana desert had an ability to survive under severe drought conditions by maintaining its water status (water content and water potential). In the analysis by two-dimensional electrophoresis of leaf proteins, seven spots were newly induced after watering stopped, One with the molecular mass of 40 kilodaltons of the spots was accumulated abundantly. The cDNA encoding for the protein was cloned based on its amino-terminal sequence and the amino acid sequence deduced from the determined nucleotide sequences of the cDNA exhibited homology to the enzymes belong to the ArgE/DapE/Acy1/Cpg2/YscS protein family (including acetylornithine deacetylase, carboxypeptidase and aminoacylase-1). This suggests that the protein is involved in the release of free amino acid by hydrolyzing a peptidic bond. As the drought stress progressed, citrulline became one of the major components in the total free amino acids. Eight days after withholding watering, although the lower leaves wilted significantly, the upper leaves still maintained their water status and the content of citrulline reached about 50% in the total free amino acids. The accumulation of citrulline during the drought stress in wild watermelon is an unique phenomenon in C-3-plants, These results suggest that the drought tolerance of wild watermelon is related to (1) the maintenance of the water status and (2) a metabolic change to accumulate citrulline.
JAPANESE SOC PLANT PHYSIOLOGISTS, 2000年07月, PLANT AND CELL PHYSIOLOGY, 41 (7), 864 - 873, 英語
[査読有り]
研究論文（学術雑誌）

MISC

作物生産における酸化ストレス—障害機構，耐性機構，危険予知による回避—
鈴木雄二, 小林優, 小林佑理子, 大津(大鎌) 直子, 伊福健太郎, 三宅親弘
環境ストレスは作物の生産性を低下させるが，その原因の一つに活性酸素種(ROS)による酸化ストレスがある。作物の生産性低下を防ぐうえで，酸化ストレスによる植物への障害，これに対する耐性機構，さらには酸化ストレスの予知と回避についての可能性，といった点は重要な情報となる。ホウ素欠乏による酸化ストレスの発生機構。ゲノム・トランスクリプトーム解析から見えた根の酸化ストレス応答とその多様性。含硫代謝産物を介した植物の酸化還元制御。光化学系IIからの活性酸素発生を抑制する防御機構の重要性。光呼吸による過剰光エネルギーに対する防御。内在的活性酸素生成を目的としたパルス法の確立と栽培環境診断への応用～P700酸化システムにもとづく活性酸素(ROS)診断の実用化～。
一般社団法人日本土壌肥料学会, 2019年, 日本土壌肥料学雑誌, 90 (4), 273 - 278, 日本語

イネにおけるP700吸光パラメーターによる水ストレス診断
和田慎也, 和田慎也, 鈴木雄二, 鈴木雄二, 高木大輔, 高木大輔, 三宅親弘, 三宅親弘, 牧野周, 牧野周
2018年08月29日, 日本土壌肥料学会講演要旨集, 64, 56, 日本語

Medium-chain dehydrogenase/reductase and aldo-keto reductase scavenge reactive carbonyls in Synechocystis sp. PCC 6803
Ginga Shimakawa, Ayaka Kohara, Chikahiro Miyake
Reactive carbonyls (RCs), which are inevitably produced during respiratory and photosynthetic metabolism, have the potential to cause oxidative damage to photosynthetic organisms. Previously, we proposed a scavenging model for RCs in the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803). In the current study, we constructed mutants deficient in the enzymes medium-chain dehydrogenase/reductase (ΔMDR) and aldo-keto reductase (ΔAKR) to investigate their contributions to RC scavenging in vivo. We found that treatment with the lipid-derived RC acrolein causes growth inhibition and promotes greater protein carbonylation in ΔMDR, compared with the wild-type and ΔAKR. In both ΔMDR and ΔAKR, photosynthesis is severely inhibited in the presence of acrolein. These results suggest that these enzymes function as part of the scavenging systems for RCs in S. 6803 in vivo.
Wiley Blackwell, 2018年03月01日, FEBS Letters, 592 (6), 1010 - 1019, 英語
速報，短報，研究ノート等（学術雑誌）

光化学系IIシトクロムb559におけるアンチマイシンAの影響
高木大輔, 伊福健太郎, 西村太志, 三宅親弘
2018年03月, 第59回日本植物生理学会年会, 日本語
研究発表ペーパー・要旨（全国大会，その他学術会議）

Editorial: How do photosynthetic organisms manage light stress? A tribute to the late professor Kozi Asada
Jun'Ichi Mano, Tsuyoshi Endo, Chikahiro Miyake
2016年, Plant and Cell Physiology, 57 (7), 1351 - 1353
書評論文,書評,文献紹介等

NADPH-dependent alkenal/one oxidoreductase (AOR) supports the growth and protect from oxidative stress in Arabidopsis thaliana
Takagi Daisuke, Kentaro Ifuku, Hironori Inoue, Masahiro Tamoi, Ken-ichi Ikeda, Kanako Ikeda Inoue, Hiroshi Fukayama, Chikahiro Miyake
2015年03月, 第56回日本植物生理学会年会, 日本語
研究発表ペーパー・要旨（全国大会，その他学術会議）

これまで欠けていた、速度論的評価に基づく、オルタナティブ・エレクトロン・フロー活性の比較と光合成におけるO2の役割～生理的な意味が見える、本丸へ挑む～
高木大輔, 三宅親弘
2014年, 光合成研究, 24, 日本語
[査読有り]
記事・総説・解説・論説等（学術雑誌）

7 RbcL欠損タバコ葉緑体への優良Rubisco-rbcLの導入(東北支部講演会,2012年度各支部会)
鈴木直紀, 関谷卓生, 表谷拓郎, 鈴木雄二, 山本宏, 三宅親弘, 牧野周
一般社団法人日本土壌肥料学会, 2013年09月11日, 日本土壌肥料学会講演要旨集, (59), 253 - 253, 日本語

4-1-13 春コムギ、冬コムギ2品種間における光合成能力の比較(4-1 植物の多量栄養素)
高木大輔, 宅見薫雄, 三宅親弘
一般社団法人日本土壌肥料学会, 2013年09月11日, 日本土壌肥料学会講演要旨集, (59), 58 - 58, 日本語

4-1-14 シロイヌナズナにおけるAKR4C familyの機能解析(4-1 植物の多量栄養素)
齊藤亮太, 嶋川銀河, 西晶子, 坂本克彦, 山本宏, 尼子克己, 杉本敏男, 牧野周, 三宅親弘
一般社団法人日本土壌肥料学会, 2013年09月11日, 日本土壌肥料学会講演要旨集, (59), 58 - 58, 日本語

4-1-12 光合成誘導におけるO_2に依存した電子伝達反応の解析(4-1 植物の多量栄養素)
瀬島健裕, 鈴木雄二, 高木大輔, 山本宏, 杉本敏男, 尼子克己, 牧野周, 三宅親弘
一般社団法人日本土壌肥料学会, 2013年09月11日, 日本土壌肥料学会講演要旨集, (59), 57 - 57, 日本語

4-1-19 RbcL欠損タバコ葉緑体への優良Rubisco-rbcLの導入(4-1 植物の多量栄養素)
関谷卓生, 鈴木直紀, 表谷拓郎, 鈴木雄二, 山本宏, 三宅親弘, 牧野周
一般社団法人日本土壌肥料学会, 2013年09月11日, 日本土壌肥料学会講演要旨集, (59), 60 - 60, 日本語

イネ生葉におけるMAP-pathwayによる光合成誘導メカニズムの解析
瀬島健裕, 鈴木雄二, 高木大輔, 山本宏, 杉本敏男, 尼子克己, 牧野周, 三宅親弘
2013年03月, 第54回日本植物生理学会, 日本語

9-1 イネ生葉におけるO_2に依存した光合成誘導メカニズムの解析(9.植物の多量栄養素,2012年度鳥取大会)
瀬島健裕, 鈴木雄二, 高木大輔, 山本宏, 杉本敏男, 尼子克己, 牧野周, 三宅親弘
一般社団法人日本土壌肥料学会, 2012年09月04日, 日本土壌肥料学会講演要旨集, (58), 53 - 53, 日本語

P19-23 丹波黒(Glycine max)種子成分に及ぼす施肥方法の影響(19.肥料および施肥法,2012年度鳥取大会)
入川草太, 鈴木武志, 三宅親弘, 杉本敏男
一般社団法人日本土壌肥料学会, 2012年09月04日, 日本土壌肥料学会講演要旨集, (58), 142 - 142, 日本語

タバコrbcL遺伝子のポプラrbcL遺伝子への置換によるhybrid Rubiscoの形成
鈴木直紀, 関谷卓生, 表谷拓郎, 鈴木雄二, 山本宏, 三宅親弘, 牧野周
2012年03月, 第53回日本植物生理学会, 日本語

Cyanobacterium Synechocystis sp. PCC6803 におけるO2存性オルタナティブ・エレクトロン・フロー (AEF)の生理的役割
林良祐, 清水聡子, 山本宏, 藍川晋平, 蓮沼誠久, 秋本誠志, 近藤昭彦, 三宅親弘
2012年03月, 第53回日本植物生理学会, 日本語

植物葉緑体メチルグリオキサール代謝メカニズムの解明メチルグリオキサールは内在的酸素光還元メディエーターである
斉藤亮太, 山本宏, 牧野周, 杉本敏男, 三宅親弘
2011年09月, 第75回日本植物学会, 日本語

12 シロイヌナズナを用いたThe Water-Water Cycle機能低下異変体のスクリーニングと候補株の性質 : 光合成の酸素制御因子の同定(関西支部講演会,2010年度各支部会講演要旨)
高木大輔, 杉本敏男, 三宅親弘
一般社団法人日本土壌肥料学会, 2011年08月08日, 日本土壌肥料学会講演要旨集, (57), 329 - 329, 日本語

11 シアノバクテリアでのオルタナティブ・エレクトロン・フロー(AEF)評価系の確立(関西支部講演会,2010年度各支部会講演要旨)
進藤沙織, 林吉祐, 真野陽人, 杉本敏男, 近藤昭彦, 藍川晋平, 蓮沼誠久, 秋本誠志, 三宅親弘
一般社団法人日本土壌肥料学会, 2011年08月08日, 日本土壌肥料学会講演要旨集, (57), 329 - 329, 日本語

10 葉緑体におけるMethylglyoxal解毒機構の解明1 : 植物はなぜ糖尿病にならないのか(関西支部講演会,2010年度各支部会講演要旨)
岩本達弥, 斉藤亮太, 久保智史, 杉本敏男, 伊福健太郎, 佐藤文彦, 牧野周, 三宅親弘
一般社団法人日本土壌肥料学会, 2011年08月, 日本土壌肥料学会講演要旨集, (57), 日本語
研究発表ペーパー・要旨（全国大会，その他学術会議）

高等植物葉緑体での3-ホスホグリセリン酸に依存した光合成における酸素の重要性 ATP/NADPH=1 でさえリニアエレクトロンフローでは満たせられない
高木大輔, 山本宏, 尼子克己, 三宅親弘
2011年, 第75回日本植物学会, 日本語

P9-6 葉位の異なるEucalyptus globulus葉におけるRubiscoの遺伝子発現(ポスター紹介,9.植物の多量栄養素,2010年度北海道大会)
鈴木雄二, 土居(木原) 智仁, 河津哲, 三宅親弘, 牧野周
一般社団法人日本土壌肥料学会, 2010年09月07日, 日本土壌肥料学会講演要旨集, (56), 64 - 64, 日本語

Metabolic turnover analysis by a combination of in vivo C-13-labelling from (CO2)-C-13 and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C-3 photosynthetic pathway in Nicotiana tabacum leaves
Tomohisa Hasunuma, Kazuo Harada, Shin-Ichi Miyazawa, Akihiko Kondo, Eiichiro Fukusaki, Chikahiro Miyake
Understanding of the control of metabolic pathways in plants requires direct measurement of the metabolic turnover rate. Sugar phosphate metabolism, including the Calvin cycle, is the primary pathway in C-3 photosynthesis, the dynamic status of which has not been assessed quantitatively in the leaves of higher plants. Since the flux of photosynthetic carbon metabolism is affected by the CO2 fixation rate in leaves, a novel in vivo C-13-labelling system was developed with (CO2)-C-13 for the kinetic determination of metabolic turnover that was the time-course of the C-13-labelling ratio in each metabolite. The system is equipped with a gas-exchange chamber that enables real-time monitoring of the CO2 fixation rate and a freeze-clamp that excises a labelled leaf concurrently with quenching the metabolic reactions by liquid nitrogen within the photosynthesis chamber. Kinetic measurements were performed by detecting mass isotopomer abundance with capillary electrophoresis-tandem mass spectrometry. The multiple reaction monitoring method was optimized for the determination of each compound for sensitive detection because the amount of some sugar phosphates in plant cells is extremely small. Our analytical system enabled the in vivo turnover of sugar phosphates to be monitored in fresh tobacco (Nicotiana tabacum) leaves, which revealed that the turnover rate of glucose-1-phosphate (G1P) was significantly lower than that of other sugar phosphates, including glucose-6-phosphate (G6P). The pool size of G1P is 12 times lower than that of G6P. These results indicate that the conversion of G6P to G1P is one of the rate-limiting steps in the sugar phosphate pathway.
OXFORD UNIV PRESS, 2010年03月, JOURNAL OF EXPERIMENTAL BOTANY, 61 (4), 1041 - 1051, 英語

シロイヌナズナでのAEF(the Water-Water Cycle and Cyclic Electron Flow around PSI)の強光順化への応答：O2-dependent electron flowはelectron sinkとして機能し、PQ酸化およびNPQ誘導に貢献する
桑原亮, 尼子克己, 山本宏, 鹿内利治, 杉本敏男, 鈴木雄二, 牧野周, 三宅親弘
2010年, 日本農芸化学会2010年度関西支部大会, 日本語

9-22 イネでの、葉緑体光合成電子伝達系の窒素(N)栄養応答(9.植物の多量栄養素,2009年度京都大会)
真野陽人, 渡辺竜馬, 桑原亮, 増村威宏, 斉藤雄飛, 尼子克己, 深山浩, 杉本敏男, 三宅親弘
一般社団法人日本土壌肥料学会, 2009年09月15日, 日本土壌肥料学会講演要旨集, (55), 72 - 72, 日本語

光合成電子伝達反応におけるオルタナティブ・エレクトロン・フロー(The Water-Water CycleおよびCyclic Electron Flow around PSI)を整理してみる--クロロフィル蛍光パラメーターの統一的理解を通して
久保智史, 杉本敏男, 三宅親弘
日本光合成学会, 2009年08月, 光合成研究, 19 (2), 66 - 74, 日本語

10-11 高等植物におけるRubiscoの性能と光合成速度の変異について(10.植物の代謝,2008年度愛知大会)
表谷拓郎, 鈴木雄二, 河津哲, 山本宏, 三宅親弘, 牧野周
一般社団法人日本土壌肥料学会, 2008年09月09日, 日本土壌肥料学会講演要旨集, (54), 99 - 99, 日本語

代謝産物量の定量安定同位体標識を利用した動的代謝プロファイリング (光合成研究法) -- (単離・精製・活性測定)
蓮沼誠久, 原田和生, 三宅親弘, 福崎英一郎
3章単離・精製・活性測定 1. 代謝産物量の定量 b
北海道大学低温科学研究所, 2008年, 低温科学, 67, 169 - 174, 日本語

スクリーニングにより選抜された塩・強光耐性ユーカリの光合成特性解析:–熱散逸機構とその誘導メカニズムについて-
三宅親弘, 宮澤真一, 岩前智子
日本森林学会, 2008年, 日本森林学会大会発表データベース, 119 (0), 276 - 276, 日本語

Effects of the PsbP knockdown on the photosynthetic electron transfer in Nicotiana tabacum.
K. Ido, K. Ifuku, S. Ishihara, Y. Yamamoto, C. Miyake, F. Sato
SPRINGER, 2007年02月, PHOTOSYNTHESIS RESEARCH, 91 (2-3), 192 - 192, 英語
研究発表ペーパー・要旨（国際会議）

Photoinactivation of Ascorbate Peroxidase in Isolated Tobacco Chloroplasts : Galdieria partita APX Maintains the Electron Flux through the Water-Water Cycle in Transplastomic Tobacco Plants
Miyake Chikahiro, Shinzaki Yuki, Nishioka Minori, HORIGUCHI Sayaka, TOMIZAWA Ken-Ichi
Japanese Society of Plant Physiologists, 2006年02月01日, Plant and cell physiology, 47 (2), 200 - 210, 英語

CO_2 Response of Cyclic Electron Flow around PSI (CEF-PSI) in Tobacco Leaves-Relative Electron fluxes through PSI and PSII Determine the Magnitude of Non-photochemical Quenching (NPQ) of Chl Fluorescence
Miyake Chikahiro, Miyata Momoko, Shinzaki Yuki, TOMIZAWA Ken-ichi
Japanese Society of Plant Physiologists, 2005年04月01日, Plant and cell physiology, 46 (4), 629 - 637, 英語

光合成の光誘導過程における律速要因について
佐々木英理子, 廣津直樹, 牧野周, 前忠彦, 三宅親弘
私たちは、光合成の光誘導過程において、water-water cycle (WWC)とPSI循環電子伝達（PSI cyclic）がスターターとして機能していることを報告してきた。ここでは、イネ葉を材料にその誘導過程における律速因子について検討した。暗処理した葉に光を照射すると、炭酸固定反応が進行する前に速やかにNPQが形成され、ATP/ADP比が上昇した。両者の間には非常に高い相関が見られ、WWCとPSI cyclicがΔpH を形成し、ATP生成したものと思われた。5時間以上の暗処理を施すとイネのRubiscoは80%以上がdark-inhibitorにより失活した。また、そのような条件では、光誘導による光合成上昇の応答も著しく悪くなった。Rubiscoのdark-inhibition解除にはATPが必要なことから、次にATP生成とRubiscoの活性発現の関係について調べた。光照射後速やかにATP/ADP比は最大値に達し、続けてRubiscoの活性化（dark-inhibition解除）が認められた。しかし、この応答はCO2同化速度の上昇に対応していなかった。一方、光照射後のRuBPのプールは緩やかに増加を示し、CO2の同化速度の上昇はむしろこのRuBPのプールの増加と高い相関関係にあった。以上の結果より、光合成の光誘導過程の律速因子はRuBPの供給系にあることが推定された。
日本植物生理学会, 2005年, 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 (0), 635 - 635, 英語
研究発表ペーパー・要旨（国際会議）

Enhancement of Cyclic Electron Flow Around PSI at High Light and its Contribution to the Induction of Non-Photochemical Quenching of Chl Fluorescence in Intact Leaves of Tobacco Plants
Miyake Chikahiro, Shinzaki Yuki, Miyata Momoko, TOMIZAWA Ken-ichi
Japanese Society of Plant Physiologists, 2004年10月01日, Plant and cell physiology, 45 (10), 1426 - 1433, 英語

10-7 イネにおけるwater-water cycleとPS I cyclic電子伝達の生理的機能(10.植物の代謝)
牧野周, 前忠彦, 三宅親弘
一般社団法人日本土壌肥料学会, 2003年08月20日, 日本土壌肥料学会講演要旨集, (49), 92 - 92, 日本語

Purification and Characterization of Class-I and Class-II Fructose-1,6-bisphosphate Aldolases from the Cyanobacterium Synechocytis sp. PCC6803
Nakahara Ken, Yamamoto Hiroshi, Miyake Chikahiro, YOKOTA Akiho
Japanese Society of Plant Physiologists, 2003年03月01日, Plant and cell physiology, 44 (3), 326 - 333, 英語

Cyclic Electron Flow within PSII Functions in Intact Chloroplasts from Spinach Leaves
Miyake Chikahiro, Yonekura Kuniaki, Kobayashi Yoshichika, YOKOTA Akiho
Japanese Society of Plant Physiologists, 2002年08月01日, Plant and cell physiology, 43 (8), 951 - 957, 英語

Cyclic Flow of Electrons within PSII in Thylakoid Membranes
Miyake Chikahiro, Yokota Akiho
Japanese Society of Plant Physiologists, 2001年05月, Plant and cell physiology, 42 (5), 508 - 515, 英語

ANALYSIS OF DROUGHT-INDUCED METALLOTHIONEIN GENE IN WILD-TYPE WATERMELON (Citrullus lanatus) :
NISHIMURA Noriyuki, AKASHI Kinya, MIYAKE Chikahiro, KOHCHIi Takayuki, YOKOTA Akiho
Japanese Society of Plant Physiologists, 2001年, Plant and cell physiology, 42, s134, 英語

Hydroxyl Radical Scavenging Activity of Citrulline : Implication for Drought-tolerance of Wild Watermelon :
AKASHI Kinya, MIYAKE Chikahiro, KOHCHI Takayuki, YOKOTA Akiho
Japanese Society of Plant Physiologists, 2001年, Plant and cell physiology, 42, s134, 英語

Alternative electron flow in the leaves of C4 plant, Maize :
Iida Nao, Yokota Akiho, Miyake Chikahiro
Japanese Society of Plant Physiologists, 2001年, Plant and cell physiology, 42, s157, 英語

Physiological functions of the water-water cycle as electron sink and O_2-independent alternative electron flow in PSII :
MIYAKE Chikahiro, YOKOTA Akiho
Japanese Society of Plant Physiologists, 2001年, Plant and cell physiology, 42, s6, 英語

Phenylethylamine-Induced Generation of Reactive Oxygen Species and Ascorbate Free Radicals in Tobacco Suspension Culture: Mechanism for Oxidative Burst Mediating Ca^<2+> Influx
Kawano Tomonori, Pinontoan Reinhard, Uozumi Nobuyuki, MORIMITSU Yasujiro, MIYAKE Chikahiro, ASADA Kozi, MUTO Shoshi
Japanese Society of Plant Physiologists, 2000年11月, Plant and cell physiology, 41 (11), 1259 - 1266, 英語

Purification and Characterization of Chloroplast Dehydroascorbate Reductase from Spinach Leaves
Shimaoka Taise, Yokota Akiho, Miyake Chikahiro
Japanese Society of Plant Physiologists, 2000年10月, Plant and cell physiology, 41 (10), 1110 - 1118, 日本語

Formate protects photosynthetic machinery from photoinhibition
T Shiraishi, EI Fukusaki, C Miyake, A Yokota, A Kobayashi
In plants, environmental adversity often leads to the formation of reactive oxygen species (ROS) because of excess light energy. We found that incubation of rice leaves under high light intensity together with CO2-depleted air seriously damaged photosynthesis; however, the application of potassium formate (2 mM) before the photoinhibitory treatment protected the photosynthesis of the plant from photoinhibition. Further analysis revealed that formate protects PSII, RuBisCO, and FBPase from photoinhibition. Formate is thought to be involved in endogenous radical scavenging and/or in the supply of CO2, derived from the formate, thereby reducing oxidative damage to the photosystems under photoinhibitory conditions.
SOC BIOSCIENCE BIOENGINEERING JAPAN, 2000年06月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 89 (6), 564 - 568, 英語

STRUCTURE/FUNCTION RELATIONSHIPS ESPECIALLY ABOUT FALLOVER PHENOMENON OF RUBISCO FROM Chlamydomonas reinhardtii :
MIZOHATA Eiichi, TOMIZAWA Kenichi, MIYAKE Chikahiro, YOKOTA Akiho
Japanese Society of Plant Physiologists, 2000年, Plant and cell physiology, 41, s113, 英語

Determination of the Rate of Photoreduction of O_2 in the Water-Water Cycle in Watermelon Leaves and Enhancement of the Rate by Limitation of Photosynthesis :
Miyake Chikahiro, Yokota Akiho
A study was performed to determine how the electron fluxes for the photosynthetic carbon reduction(PCR) and the photorespiratory carbon oxidation(PCO) cycles affect the photoreduction of O_2 at PSI, which is the limiting step in the water-water cycle. Simultaneous measurements were made of CO_2-gas exchange, transpiration and quantum yield of PSII [Φ(PSII)] using leaves of watermelon(Citrullus lanatus). The total electron flux in PSII [Je(PSII)], as estimated from Φ(PSII), was always larger than the total electron flux required for the PCR and PCO cycles at various partial pressures of CO_2 and O_2 and 1, 100 μmol photons m^<-2>s^<-1>. This observation suggested the existence of an alternative electron flux(Ja). Ja was divided into O_2-dependent [Ja(O_2-depend)] and O_2-independent [Ja(O_2-independ)] components. The magnitude of half Ja(O_2-depend), 7.5 to 9.5 μmol e^-m^<-2>s<-1> and its apparent K_m for O_2, about 8.0 kPa, could be accounted for by the photoreduction of O_2 at PSI either mediated by ferredoxin or catalyzed by monodehydroascorbate reductase. The results indicated that Ja(O_2-depend)was driven by the water-water cycle. A decrease in the intercellular partial pressure of CO_2 from 23 to 5.0 Pa at 21 kPa O_2 enhanced Ja(O_2-depend)by a factor of 1.3. Saturation of the activities of both the PCR and PCO cycles by increasing the photon flux density induced Ja. These results indicate the electron flux in PSII that exceeds the flux required for the PCR and PCO cycles induces the photoreduction of O_2 in the water-water cycle.
Japanese Society of Plant Physiologists, 2000年, Plant and cell physiology, 41 (3), 335 - 343, 英語

Drought responses of the quantum yield of PSII and the activities of active oxygen-scavenging enzymes both in wild and cultivar watermelons :
MIYAKE Chikahiro, YOKOTA Akiho
Japanese Society of Plant Physiologists, 2000年, Plant and cell physiology, 41, s146, 英語

Thioredoxin peroxidase in the Cyanobacterium Synechocystis sp. PCC 6803
H Yamamoto, C Miyake, KJ Dietz, KI Tomizawa, N Murata, A Yokota
The amino acid sequence deduced from the open reading frame designated sll0755 in Synechocystis sp. PCC 6803 is similar to the amino acid sequences of thioredoxin peroxidases from other organisms, In the present study, we found that a recombinant SLL0755 protein that was expressed in Escherichia coli mas able to reduce H2O2 and tertiary butyl hydroperoxide with thioredoxin from E. coli as the electron donor, Targeted disruption of open reading frame sll0755 in Synechocystis sp, PCC 6803 cells completely eliminated the H2O2-dependent and tertiary butyl hydroperoxide-dependent photosynthetic evolution of oxygen and the electron flow in photosystem II. These results indicate that the product of open reading frame sll0755 is a thioredoxin peroxidase whose activities are coupled to the photosynthetic electron transport system in Synechocystis sp. PCC 6803, (C) 1999 Federation of European Biochemical Societies.
ELSEVIER SCIENCE BV, 1999年03月, FEBS LETTERS, 447 (2-3), 269 - 273, 英語

Physiological Fucntions of Photorespiration and Water-Water Cycle in Wild Watermelon Leaves under High Light and Low Partial Pressure of CO_2 conditions and in vivo diagnosis of oxidative stress in chloroplasts
MIYAKE Chikahiro, YOKOTA Akiho
1999年03月, Plant and cell physiology, 40, s119 - s119, 英語

Molecular analysis of drought-tolerant with watermelon from the Botawana desert
KAWASAKI Shinji, MIYAKE Chikahiro, NAKAMURA Chie, FUJII Shinichiro, YOKOTA Akiho
1999年03月, Plant and cell physiology, 40, s91 - s91, 英語

ANALYSIS OF THE RUBISCO OXYGENASE REACTION
OKADA Sachiko, MIYAKE Chikahiro, YOKOTA Akiho
1999年03月, Plant and cell physiology, 40, s116 - s116, 英語

THIOREDOXIN PEROXIDASE IN CYANOBACTERIA, Synechocystis sp. PCC6803 AND Synechococcus sp. PCC7942.
YAMAMOTO Hiroshi, MIYAKE Chikahiro, DIETZ Karl-Josef, TOMIZAWA Ken-ichi, YOKOTA Akiho, MURATA Norio
1998年05月, Plant and cell physiology, 39, S18 - S18, 英語

Purification and molecular properties of ascorbate peroxidase from bovine eye
Naoki Wada, Shigeru Kinoshita, Michinori Matsuo, Katsumi Amako, Chikahiro Miyake, Kozi Asada
Ascorbate peroxidase (APX) is a hydrogen peroxide-scavenging peroxidase which uses ascorbate (AsA) as the specific electron donor. APX has not been isolated in mammals. Ocular tissue contains AsA at high concentrations, and we detected APX activity in bovine retinal pigment epithelium (RPE) and choroid. We purified APX from bovine RPE and choroid by four chromatographic steps. The purified APX was a monomeric hemoprotein with a molecular mass of 43 kDa. The amino acid sequence of the amino-terminal region of the purified APX showed a high degree of homology to that of plants. The primary product of the APX reaction was identified as the monodehydroascorbate radical. The APX showed high specificity for AsA as an electron donor. This is the first isolation and characterization of APX from mammals, and its role in the protection against active species of oxygen in ocular tissue is discussed.
Academic Press Inc., 1998年01月14日, Biochemical and Biophysical Research Communications, 242 (2), 256 - 261, 英語

The FAD-Enzyme Monodehydroascorbate Radical Reductase Mediates Photoproduction of Superoxide Radicals in Spinach Thylakoid Membranes :
Miyake Chikahiro, Schreiber Ulrich, Hormann Henning, Sano Satoshi, Asada Kozi
The photoreduction of dioxygen in spinach thylakoid membranes was enhanced about 10-fold by the FAD-enzyme monodehydroascorbate radical (MDA) reductase at 1 μM. The primary photoreduced product of dioxygen catalyzed by MDA reductase was the superoxide radical, as evidenced by the inhibition of photoreduction of Cyt c by superoxide dismutase. The apparent K_m for dioxygen of the MDA reductase-dependent photoreduction of dioxygen was 100 μM, higher by one order of magnitude than that observed with thylakoid membranes only. Glutathione reductase, ferredoxin-NADP^+ reductase, and glycolate oxidase also mediated the photoproduction of superoxide radicals in thylakoid membranes at rates similar to those with MDA reductase. Among these flavoenzymes, MDA reductase is the most likely mediator stimulating the photoreduction of dioxygen in chloroplasts;its function in the protection from photoinhibition under excess light is discussed.
Japanese Society of Plant Physiologists, 1998年, Plant and cell physiology, 39 (8), 821 - 829, 英語

RESISTANCE TO ENVIRONMENTAL STRESS IN TRANSGENIC TOBACCO OVEREXPRESSING OF CATALASE FROM E. coli IN CHLOROPLASTS
TAKEDA Toru, YOSHIMURA Yukiko, TAMOI Masahiro, SHIKANAI Toshiharu, MIYAKE Chikahiro, SANO Satoshi, TOMIZAWA Ken-ichi, YOKOTA Akiho, SHIGEOKA Shigeru
1997年03月, Plant and cell physiology, 38, s68, 英語

DETECTION OF PHOTOOXIDATIVE STRESS BY MONODEHYDROASCORBATE IN LEAVES
OHNO Chiaki, MANO Junichi, MIYAKE Chikahiro, HEBER Ulrich, ASADA Kozi
1996年03月, Plant and cell physiology, 37, 32 - 32, 英語

Monodehydroascorbate radical reductase-dependent photoreduction of oxygen in chloroplast thylakoids
MIYAKE Chikahiro, SCHREIBER Ulrich, HORMANN Henning, SANO Satoshi, ASADA Kozi
1996年03月, Plant and cell physiology, 37, 79 - 79, 英語

Inactivation Mechanism of Ascorbate Peroxidase at Low Concentrations of Ascorbate; Hydrogen Peroxide Decomposes Compound I of Ascorbate Peroxidase :
Miyake Chikahiro, Asada Kozi
One of the characteristic properties of ascorbate peroxidase (APX), which distinguishes it from guaiacol peroxidase, Cyt c peroxidase and glutathione peroxidase, is the rapid inactivation of the enzyme under conditions where an electron donor is absent. When thylakoid-bound APX (tAPX) in 100 μM ascorbate was diluted 500-fold with an ascorbate-depleted medium, the enzymatic activity was lost with half time of about 15 s. The inactivation of tAPX was suppressed under anaerobic conditions and also by the addition of catalase, but it was unaffected by the addition of superoxide dismutase. These observations suggest that hydrogen peroxide at nanomolar levels, produced by autooxidation of ascorbate at lower than micromolar levels, might participate in the inactivation of tAPX. The participation of hydrogen peroxide was confirmed by the inactivation of tAPX upon incubation with hydrogen peroxide under an-aerobic conditions. In the absence of ascorbate, the heme of the two-electron-oxidized intermediate of tAPX (designated Compound I) is decomposed by hydrogen peroxide. Thus, the instability of Compound I to hydrogen peroxide is responsible for the inactivation of APX when ascorbate is not available for Compound I and the enzyme cannot turnover.
Japanese Society of Plant Physiologists, 1996年, Plant and cell physiology, 37 (4), 423 - 430, 英語

Ferredoxin-Dependent and Antimycin A-Sensitive Reduction of Cyiochrome b-559 by Far-Red Light in Maize Thylakoids : Participation of a Menadiol-Reducible Cytochrome b-559 in Cyclic Electron Flow : ENVIRONMENTAL AND STRESS RESPONSES : MEMBRANES AND BIOENERGETICS :
MIYAKE Chikahiro, SCHREIBER Ulrich, ASADA Kozi
Thylakoids from mesophyll cells of maize showed a high rate of the ferredoxin (Fd)-dependent and antimycin A (AntiA)-sensitive cyclic electron flow as determined by the quenching of 9-aminoacridine fluorescence which indicates the formation of A pH across the membranes. Spectrophotometric survey of the thylakoids showed the reduction of a Cyt having an a-peak at 559 nm [Cyt b-559(Fd)] by far-red light, which depended on Fd and was sensitive to AntiA. Dose dependencies of Fd and AntiA on the photoreduction of Cyt b-559(Fd) were the same as those of the formation of ΔpH. Cyt b-559(Fd) occurred in an oxidized form even in the presence of ascorbate and was reduced by far-red light. In darkness, it was reduced only by menadiol (E_= -10 mV). Thus, Cyt b-559(Fd) was distinguished from Cyt b-559 in the PSII complex by its low redox potential. The present results indicate that Cyt b-559(Fd) mediates electron transfer from Fd to plastoquinone during Fd-dependent cyclic electron flow around PSI.
Japanese Society of Plant Physiologists, 1995年, Plant and cell physiology, 36 (4), 743 - 748, 英語

Photoactivation of the Electron Flow from NADPH to Plastoquinone in Spinach Chloroplasts : GROWTH AND DEVEROPMENT : ENVIRONMENTAL AND STRESS RESPONSES :
MANO Junichi, MIYAKE Chikahiro, SCHREIBER Ulrich, ASADA Kozi
Intact chloroplasts from spinach showed a transient increase in Chl fluorescence after saturating illumination with actinic light and its yield depended on the duration of illumination and the intensity of the actinic light (AL). The increase was partially suppressed when antimycin A was added immediately after termination of the AL. The inhibited fluorescence increase, therefore, reflected the electron flow from the reductant(s) that had accumulated during the actinic illumination to the plastoquinone (PQ) pool via ferredoxin and the antimycin A-sensitive Cyt b-559 [Miyake et al. (1995) Plant Cell Physiol. 36: 743]. Addition of dihydroxyacetone phosphate (DHAP) to chloroplasts caused the enhancement of the increase in fluorescence after AL, which was inhibited by antimycin A. Decay of the transiently raised fluorescence was retarded by 2-heptyl-4-hydroxyquinoline N-oxide and stigmatellin, suggesting that re-oxidation of the reduced PQ pool is coupled with the operation of Q-cycle. Although the activity of the stromal enzyme system that supplies NADPH on addition of DHAP was constant irrespective of light or darkness, the capacity of the intact chloroplasts to show a DHAP-dependent fluorescence increase had a limited lifetime after AL was turned off. This result suggests that the antimycin A-sensitive Cyt b-559 or ferredoxin-NADP reductase is activated by light and deactivated in the dark. In ruptured chloroplasts, the addition of NADPH increased the dark fluorescence yield only in the presence of Fd, which also was inhibited by antimycin A. Thus the photoregulatory mechanism of Cyt b-559 (Fd) in intact chloroplasts appeared to be lost when chloroplasts were ruptured.
Japanese Society of Plant Physiologists, 1995年, Plant and cell physiology, 36 (8), 1589 - 1598, 英語

Ferredoxin-Dependent Photoreduction of the Monodehydroascorbate Radical in Spinach Thylakoids : ENVIRONMENTAL AND STRESS RESPONSES : PROTEINS, ENZYMES AND METABOLISM :
MIYAKE Chikahiro, ASADA Kozi
Thylakoid-bound and stromal ascorbate peroxidases scavenge the hydrogen peroxide that is photoproduced in PSI of chloroplast thylakoids. The primary oxidation product of ascorbate in the reaction catalyzed by ascorbate peroxidase, the monodehydroascorbate (MDA) radical, is photoreduced by thylakoids [Miyake and Asada (1992) Plant Cell Physiol. 33:541]. We have now shown that the photoreduction of MDA radical in spinach thylakoids is largely dependent on ferredoxin (Fd), as determined by the monitoring the MDA radical by electron paramagnetic resonance. Further, the reduced Fd generated by NADPH and Fd-NADP reductase could reduce the MDA radical at a rate of over 10^6 M^<-1> s^<-1>, indicating that the photoreduced Fd in PSI directly reduces the MDA radical to ascorbate. Photoreduction of NADP^+ by spinach thylakoids was suppressed by the MDA radical and conversely that of MDA radical was suppressed by NADP^+, indicating a competition between the MDA radical and NADP^+ for the photoreduced Fd in PSI. The ratio of the rate constant for the photoreduction of MDA radical to that for the photoreduction of NADP^+ was estimated to be more than 30 to 1. Thus, MDA radical is preferentially photoreduced as compared to NADP^+ From these results, we propose that the thylakoid-bound ascorbate peroxidase and the Fd-dependent photoreduction of MDA radical in PSI are the primary system for the scavenging of the hydrogen peroxide that is photoproduced in the thylakoids.
Japanese Society of Plant Physiologists, 1994年, Plant and cell physiology, 35 (4), 539 - 549, 英語

Purification and Molecular Properties of the Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts :
MIYAKE Chikahiro, CAO Wan-Hong, ASADA Kozi
The hydrogen peroxide that is photoproduced in thylakoids is scavenged by the thylakoid-bound ascorbate peroxidase (tAPX) [Miyake and Asada (1992) Plant Cell Physiol. 33: 541]. tAPX was purified from spinach thylakoids to homogeneity as judged by SDS-polyacrylamide gel electrophoresis, and its molecular properties were studied. Spinach tAPX was a monomer with a molecular weight of 40,000, which is about 10.000 higher than that of the stromal ascorbate peroxidase (sAPX) from spinach chloroplasts. tAPX cross-reacted with the antibody raised against sAPX from tea leaves, as determined by Western blotting, which also provided evidence for the higher molecular weight of tAPX from spinach thylakoids than that of tea sAPX. The amino acid sequence of the amino-terminal region of tAPX showed a low degree of homology to those of cylosolic APXs from spinach, pea and Arabidopsis thaliana, but a high degree of homology to that of stromal APX from tea. Thus, the amino-terminal region of tAPX seems not to be a domain required for binding of the enzyme to the thylakoid membranes. tAPX contained protoheme IX, as identified by its pyridine hemochromogen, and gave a Soret peak at 403 nm and 433 nm with an a band at 555 nm in its oxidized and reduced forms, respectively. Resembling sAPX but differing from cytosolic APX, tAPX showed high specificity for ascorbate as the electron donor. tAPX was inhibited by cyanide, thiol-modifying reagents, thiols and several suicide inhibitors, such as hydroxyurea and p-aminophenol.
Japanese Society of Plant Physiologists, 1993年, Plant and cell physiology, 34 (6), 881 - 889, 英語

Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts and Photoreduction of Its Primary Oxidation Product Monodehydroascorbate Radicals in Thylakoids : PROTEINS, ENZYMES AND METABOLISM : MEMBRANES AND BIOENERGETICS :
Miyake Chikahiro, Asada Kozi
Ascorbate peroxidase, a key enzyme for the scavenging of hydrogen peroxide in chloroplasts, was found in a thylakoid-bound form in spinach chloroplasts at comparable activity to that in the stroma. The activity of peroxidase was detectable in the thylakoids only when prepared by an ascorbate-containing medium, and enriched in the stroma thylakoids. The thylakoid enzyme was not released from the membranes by either 2 mM EDTA, 1 M KCl, 2 M NaBr or 2 M NaSCN, but was solubilized by detergents. Enzymatic properties of the thylakoid-bound ascorbate peroxidase were very similar to those of the stromal ascorbate peroxidase. Thylakoid-bound ascorbate peroxidase could scavenge the hydrogen peroxide either added or photoproduced by the thylakoids. No photoreduction of hydrogen peroxide was observed, however, in the thylakoids whose ascorbate peroxidase was inhibited by KCN and thiol reagents or inactivated by the treatment with ascorbate-depletion. The primary oxidation product of ascorbate in a reaction of ascorbate peroxidase, monodehydroascorbate (MDA) radical, was photoreduced in the thylakoids, as detected by the quenching of chlorophyll fluorescence, disappearance of EPR signals of the MDA radicals and the MDA radical-induced oxygen evolution. Thus, ascorbate is photoregenerated in the thylakoids from the MDA radicals produced in a reaction of ascorbate peroxidase for the scavenging of hydrogen peroxide.
Japanese Society of Plant Physiologists, 1992年, Plant and cell physiology, 33 (5), 541 - 553, 英語

Scavenging of Hydrogen Peroxide in Prokaryotic and Eukaryotic Algae: Acquisition of Ascorbate Peroxidase during the Evolution of Cyanobacteria :
Miyake Chikahiro, Michihata Fumi, Asada Kozi
Ascorbate (AsA) peroxidase was found in six species of cyanobacteria among ten species tested. Upon the addition of H_2^<18>O_2 to the cells of AsA peroxidase-containing cyanobacteria, <16>^O_2 derived from water and <18>^O_2 derived from H_2<18>^O_2 Were evolved in the light. The evolution of <16>^O_2 was inhibited by DCMU and did not occur in the dark, but <18>^O_2 was evolved even in the dark or in the presence of DCMU. Similar light-dependent evolution of <16>^O_2 Was observed in the cells of AsA peroxidase-containing Euglena and Chlamydomonas. However, the cells of AsA peroxidase-lacking cyanobacteria evolved only <18>^O_2 in either the light or dark. Furthermore, the quenching of chlorophyll fluorescence induced by hydrogen peroxide was observed only in the cells of the AsA peroxidase-containing Synechocystis 6803, and not in the cells of Anacystis nidulans which lacks AsA peroxidase. Thus, cyanobacteria can be divided into two groups, those that has and those that lacks AsA peroxidase. The first group scavenges hydrogen peroxide with the peroxidase using a photoreductant as the electron donor, and the second group only scavenges hydrogen peroxide with catalase.
Japanese Society of Plant Physiologists, 1991年, Plant and cell physiology, 32 (1), 33 - 43, 英語

講演・口頭発表等

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第10回分子科学討論会2016神戸, 2016年09月, 日本語, 神戸ファッションマート, 国内会議
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第24回「光合成セミナー2016：反応中心と色素系の多様性」, 2016年07月, 日本語, 龍谷大学深草キャンパス, 国内会議
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77 Kにおける蛍光の絶対強度測定 −シアノバクテリアの光応答−
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高等植物におけるalkenal/one oxidoreductase (AOR)の生理学的解析植物も糖尿病を発症する
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口頭発表（一般）

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