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ABSTRACT: The role of Rubisco activase in steady-state and non-steady-state photosynthesis was analyzed in wild-type (Oryza sativa) and transgenic rice that expressed different amounts of Rubisco activase. Below 25°C, the Rubisco activation state and steady-state photosynthesis were only affected when Rubisco activase was reduced by more than 70%. However, at 40°C, smaller reductions in Rubisco activase content were linked to a reduced Rubisco activation state and steady-state photosynthesis. As a result, overexpression of maize Rubisco activase in rice did not lead to an increase of the Rubisco activation state, nor to an increase in photosynthetic rate below 25°C, but had a small stimulatory effect at 40°C. On the other hand, the rate at which photosynthesis approached the steady state following an increase in light intensity was rapid in Rubisco activase-overexpressing plants, intermediate in the wild-type, and slowest in antisense plants at any leaf temperature. In Rubisco activase-overexpressing plants, Rubisco activation state at low light was maintained at higher levels than in the wild-type. Thus, rapid regulation by Rubisco activase following an increase in light intensity and/or maintenance of a high Rubisco activation state at low light would result in a rapid increase in Rubisco activation state and photosynthetic rate following an increase in light intensity. It is concluded that Rubisco activase plays an important role in the regulation of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature.
The Plant Journal 05/2012; 71(6):871-80. · 6.16 Impact Factor
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ABSTRACT: The effects of overexpression of Rubisco activase on photosynthesis were studied in transgenic rice expressing barley or maize Rubisco activase. Immunoblot and SDS-PAGE analyses showed that transgenic lines from both gene constructs expressed the foreign Rubisco activase at high levels. The activation state of Rubisco in transgenic lines was slightly higher than that in non-transgenic plants (NT). In addition, light activation of Rubisco was significantly more rapid in transgenic lines compared with NT. These findings indicate that the overexpression of Rubisco activase can enhance Rubisco activation. However, despite enhanced activation of Rubisco in these transgenic plants, the CO(2) assimilation rate at ambient CO(2) conditions was decreased. This decrease in CO(2) assimilation rate was observed in both young developing and mature leaves independent of nitrogen nutrition. The contents of nitrogen and Chl did not differ significantly between transformants and NT; however, Rubisco content was substantially decreased in transgenic lines. There was no evidence for reduced transcription of RbcS or RbcL in these transgenic lines; in fact, transcript levels were marginally increased compared with NT. These results indicate that the overexpression of Rubisco activase leads to a decrease in Rubisco content, possibly due to post-transcriptional mechanisms.
Plant and Cell Physiology 04/2012; 53(6):976-86. · 4.70 Impact Factor
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ABSTRACT: Rubisco limits photosynthetic CO(2) 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 C(4) 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 CO(2) and slightly lower specificity for CO(2) 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 CO(2) assimilation rates under a range of CO(2) 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.
Plant physiology 06/2011; 156(3):1603-11. · 6.53 Impact Factor
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ABSTRACT: The transfer of C(4) plant traits into C(3) plants has long been a strategy for improving the photosynthetic performance of C(3) plants. The introduction of a pathway mimicking the C(4) photosynthetic pathway into the mesophyll cells of C(3) plants was only a realistic approach when transgenic technology was sufficiently well developed and widely adopted. Here an attempt to introduce a single-cell C(4)-like pathway in which CO(2) capture and release occur in the mesophyll cell, such as the one found in the aquatic plant Hydrilla verticillata (L.f.) Royle, into rice (Oryza sativa L.) is described. Four enzymes involved in this pathway were successfully overproduced in the transgenic rice leaves, and 12 different sets of transgenic rice that overproduce these enzymes independently or in combination were produced and analysed. Although none of these transformants has yet shown dramatic improvements in photosynthesis, these studies nonetheless have important implications for the evolution of C(4) photosynthetic genes and their metabolic regulation, and have shed light on the unique aspects of rice physiology and metabolism. This article summarizes the lessons learned during these attempts to engineer single-cell C(4) rice.
Journal of Experimental Botany 03/2011; 62(9):3021-9. · 5.36 Impact Factor
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ABSTRACT: 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 µmol photon m–2 s–1 at 0% carbon dioxide (CO2), 2% oxygen (O2) and 25°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 [Φ(PSI) × PFD]. In non-treated leaves, increasing in the photon flux density (PFD) enhanced V(O2), Φ(PSI) × PFD and the ratio of oxidized P700 to total P700 [(P700+)/(P700)total]. Φ(PSI) × 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.
Soil Science and Plant Nutrition. 02/2011; 57(1):105-113.
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ABSTRACT: Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of primary metabolism in bacteria, algae, and vascular plants, and is believed to be cytosolic. Here we show that rice (Oryza sativa L.) has a plant-type PEPC, Osppc4, that is targeted to the chloroplast. Osppc4 was expressed in all organs tested and showed high expression in the leaves. Its expression in the leaves was confined to mesophyll cells, and Osppc4 accounted for approximately one-third of total PEPC protein in the leaf blade. Recombinant Osppc4 was active in the PEPC reaction, showing V(max) comparable to cytosolic isozymes. Knockdown of Osppc4 expression by the RNAi technique resulted in stunting at the vegetative stage, which was much more marked when rice plants were grown with ammonium than with nitrate as the nitrogen source. Comparison of leaf metabolomes of ammonium-grown plants suggested that the knockdown suppressed ammonium assimilation and subsequent amino acid synthesis by reducing levels of organic acids, which are carbon skeleton donors for these processes. We also identified the chloroplastic PEPC gene in other Oryza species, all of which are adapted to waterlogged soil where the major nitrogen source is ammonium. This suggests that, in addition to glycolysis, the genus Oryza has a unique route to provide organic acids for ammonium assimilation that involves a chloroplastic PEPC, and that this route is crucial for growth with ammonium. This work provides evidence for diversity of primary ammonium assimilation in the leaves of vascular plants.
Proceedings of the National Academy of Sciences 03/2010; 107(11):5226-31. · 9.68 Impact Factor
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Plant Production Science - PLANT PROD SCI. 01/2009; 12(3):345-350.
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Yojiro Taniguchi,
Hiroshi Ohkawa,
Chisato Masumoto,
Takuya Fukuda,
Tesshu Tamai,
Kwanghong Lee,
Sizue Sudoh,
Hiroko Tsuchida,
Haruto Sasaki, Hiroshi Fukayama,
Mitsue Miyao
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ABSTRACT: Four enzymes, namely, the maize C(4)-specific phosphoenolpyruvate carboxylase (PEPC), the maize C(4)-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C(3)-specific NADP-malic enzyme (ME), were overproduced in the mesophyll cells of rice plants independently or in combination. Overproduction individually of PPDK, MDH or ME did not affect the rate of photosynthetic CO(2) assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO(2) assimilation in PEPC overproduction lines remained unaffected by overproduction of PPDK, ME or a combination of both, however it was significantly restored by the combined overproduction of PPDK, ME, and MDH to reach levels comparable to or slightly higher than that of non-transgenic rice. The extent of the restoration of CO(2) assimilation, however, was more marked at higher CO(2) concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO(2) inside the chloroplast. Transgenic rice plants overproducing the four enzymes showed slight stunting. Comparison of transformants overproducing different combinations of enzymes indicated that overproduction of PEPC together with ME was responsible for stunting, and that overproduction of MDH had some mitigating effects. Possible mechanisms underlying these phenotypic effects, as well as possibilities and limitations of introducing the C(4)-like photosynthetic pathway into C(3) plants, are discussed.
Journal of Experimental Botany 02/2008; 59(7):1799-809. · 5.36 Impact Factor
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ABSTRACT: Phosphoenolpyruvate carboxylase (PEPC), a key enzyme of primary metabolism of higher plants, is regulated by reversible phosphorylation, which is catalyzed by PEPC kinase (PPCK). Rice has three functional PPCK genes, OsPPCK1, OsPPCK2 and OsPPCK3, all of which have an intron close to the 3' end of the coding region. A novel control mechanism was found for expression of OsPPCK2, namely alternative transcription initiation, and two different transcripts were detected. The four different transcripts of the OsPPCK genes showed different expression patterns. While OsPPCK1 and OsPPCK3 were highly expressed in roots and at low levels in other organs, the two OsPPCK2 transcripts were expressed in all organs. OsPPCK3 was expressed mostly at night, while the long OsPPCK2 transcripts were present in the leaves only in the daytime. Nitrate supplementation of leaves selectively induced expression of both OsPPCK2 transcripts, while phosphate starvation only induced the shorter one. Such diverse expression patterns of OsPPCK genes suggest the importance and variety of strict activity regulation of PEPC in rice. From the correlation between gene expression and the phosphorylation level of PEPC, which was monitored as that of the maize PEPC expressed in transgenic rice plants, it was concluded that the short OsPPCK2 transcripts were expressed in rice leaf mesophyll cells upon nitrogen supplementation and phosphate starvation, whereas OsPPCK3 participated in the nocturnal phosphorylation of PEPC in these cells. Expression of PPCK proteins in rice leaves was detected by immunoblotting using a specific antiserum, and the expression of two different OsPPCK2 proteins derived from alternative transcription initiation was confirmed.
The Plant Journal 08/2006; 47(2):258-68. · 6.16 Impact Factor
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ABSTRACT: The aquatic monocot Hydrilla verticillata (L.f.) Royle is a well-documented facultative C4 NADP-malic enzyme species in which the C4 and Calvin cycles operate in the same cell with the specific carboxylases confined to the cytosol and chloroplast, respectively. Several key components had already been characterized at the molecular level, thus the purpose of this study was to begin to identify other, less obvious, elements that may be necessary for a functional single-cell C4 system. Using differential display, mRNA populations from C3 and C4 H. verticillata leaves were screened and expression profiles compared. From this study, 65 clones were isolated and subjected to a customized macroarray analysis; 25 clones were found to be upregulated in C4 leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C4 upregulated genes were identified. Among these one encoded a previously recognized C4 phosphoenolpyruvate carboxylase, and two encoded distinct pyruvate orthophosphate dikinase isoforms, new findings for H. verticillata. Genes that encode a transporter, an aminotransferase and two chaperonins were also upregulated. Twelve false positives, mostly housekeeping genes, were determined from the Northern/semi-quantitative RT-PCR analyses. Sequence data obtained in this study are listed in the dbEST database (DV216698 to DV216767). As a single-cell C4 system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C4 system in a C3 crop species.
Photosynthesis Research 06/2006; 88(2):173-83. · 3.24 Impact Factor
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ABSTRACT: Phosphoenolpyruvate carboxylase (PEPC) was overproduced in the leaves of rice plants by introducing the intact maize C4-specific PEPC gene. Maize PEPC in transgenic rice leaves underwent activity regulation through protein phosphorylation in
a manner similar to endogenous rice PEPC but contrary to that occurring in maize leaves, being downregulated in the light
and upregulated in the dark. Compared with untransformed rice, the level of the substrate for PEPC (phosphoenolpyruvate) was slightly lower and the product (oxaloacetate) was slightly higher in transgenic rice, suggesting that maize
PEPC was functioning even though it remained dephosphorylated and less active in the light. 14CO2 labeling experiments indicated that maize PEPC did not contribute significantly to the photosynthetic CO2 fixation of transgenic rice plants. Rather, it slightly lowered the CO2 assimilation rate. This effect was ascribable to the stimulation of respiration in the light, which was more marked at lower
O2 concentrations. It was concluded that overproduction of PEPC does not directly affect photosynthesis significantly but it
suppresses photosynthesis indirectly by stimulating respiration in the light. We also found that while the steady-state stomatal
aperture remained unaffected over a wide range of humidity, the stomatal opening under non-steady-state conditions was destabilized
in transgenic rice.
Photosynthesis Research 07/2003; 77(2):227-239. · 3.24 Impact Factor
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ABSTRACT: Phosphoenolpyruvate carboxylase (PEPC) has a variety of functions in plants, including a major anaplerotic role in replenishing the tricarboxylic acid cycle with intermediates to meet the demand of carbon skeletons for synthesis of organic acids and amino acids. Various transgenic C3 plants that overproduce PEPC have been produced and analyzed in detail. The results indicate that foreign PEPC is under the control of the regulatory mechanisms intrinsic to the host plant and down-regulated so as not to cause detrimental metabolic effects, although the anaplerotic reaction is slightly enhanced by the foreign PEPC. By use of foreign PEPCs that can avert such regulation, metabolic flow is largely directed toward synthesis of organic acids and amino acids. Observations with transgenic C3 plants also shed light on the interrelation among various metabolic pathways inside the cell.
Archives of Biochemistry and Biophysics 07/2003; 414(2):197-203. · 2.93 Impact Factor
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ABSTRACT: Phosphoenolpyruvate carboxylase (PEPC) was overproduced in the leaves of rice plants by introducing the intact maize C(4)-specific PEPC gene. Maize PEPC in transgenic rice leaves underwent activity regulation through protein phosphorylation in a manner similar to endogenous rice PEPC but contrary to that occurring in maize leaves, being downregulated in the light and upregulated in the dark. Compared with untransformed rice, the level of the substrate for PEPC (phosphoenolpyruvate) was slightly lower and the product (oxaloacetate) was slightly higher in transgenic rice, suggesting that maize PEPC was functioning even though it remained dephosphorylated and less active in the light. (14)CO(2) labeling experiments indicated that maize PEPC did not contribute significantly to the photosynthetic CO(2) fixation of transgenic rice plants. Rather, it slightly lowered the CO(2) assimilation rate. This effect was ascribable to the stimulation of respiration in the light, which was more marked at lower O(2) concentrations. It was concluded that overproduction of PEPC does not directly affect photosynthesis significantly but it suppresses photosynthesis indirectly by stimulating respiration in the light. We also found that while the steady-state stomatal aperture remained unaffected over a wide range of humidity, the stomatal opening under non-steady-state conditions was destabilized in transgenic rice.
Photosynthesis Research 02/2003; 77(2-3):227-39. · 3.24 Impact Factor
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ABSTRACT: The effects of the ratio of Rubisco activase to Rubisco (activase/Rubisco ratio) on light dependent activation of CO2 assimilation were investigated during leaf aging of rice. Changes of photosynthetic CO2 gas exchange rates in relation to step increases of light intensity from two photon flux densities of 60 µmol m−2 s−1 (low initial PFD) and 500 µmol m−2 s−1 (high initial PFD) to saturated PFD of 1 800 µmol m−2 s−1 were measured. These photosynthetic activation processes were considered to be limited by the Rubisco activation rate when analyzed by the relaxation method. The relaxation time of low initial PFD gradually declined from 3 to 33 days after leaf emergence and showed high and negative correlation to the activase/Rubisco ratio. The initial rate of Rubisco activation under low initial PFD linearly correlated to the amounts of Rubisco activase, whereas these were almost constant from 3 to 23 days after leaf emergence. But these correlations could not be recognized in the case of high initial PFD. Moreover, the relaxation times were more sensitive to intercellular CO2 concentration (Ci) under high initial PFD than under low initial PFD, especially, at Ci below 300 µl l−1. These results suggest the involvement of the activase/Rubisco ratio in the photosynthetic activation under relatively low initial PFD, and the limitation of photosynthetic activation under relatively high initial PFD by Rubisco carbamylation during leaf aging of rice.
Physiologia Plantarum 01/2002; 104(4):541 - 548. · 3.11 Impact Factor
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ABSTRACT: The majority of terrestrial plants, including many important crops such as rice, wheat, soybean, and potato, are classified as C3 plants that assimilate atmospheric CO2 directly through the C3 photosynthetic pathway. C4 plants such as maize and sugarcane evolved from C3 plants, acquiring the C4 photosynthetic pathway to achieve high photosynthetic performance and high water- and nitrogen-use efficiencies. The recent application of recombinant DNA technology has made considerable progress in the molecular engineering of C4 photosynthesis over the past several years. It has deepened our understanding of the mechanism of C4 photosynthesis and provided valuable information as to the evolution of the C4 photosynthetic genes. It also has enabled us to express enzymes involved in the C4 pathway at high levels and in desired locations in the leaves of C3 plants for engineering of primary carbon metabolism.
Annual Review of Plant Biology 07/2001; 52:297-314. · 18.71 Impact Factor
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ABSTRACT: Effects of elevated CO2 (68 Pa versus ambient 38 Pa) on gene expression were studied in rice leaves grown in soil medium with three different nitrogen conditions (0, 0.6 and 1.2 g N per 8-L pot) in CO2 controlled chambers. Soluble protein contents were slightly decreased in leaves grown under elevated CO2 regardless of N supplies, whereas the polypeptide profiles of soluble protein analyzed by 2DE using the same amount of protein were totally unchanged between ambient and elevated CO2. In contrast, gene expressions examined by microarray analyses were significantly affected by elevated CO2. Forty-six up-regulated genes (>1.5-fold) and 35 down-regulated genes (<0.68-fold) were identified and these included many signal transduction and transcription regulation related genes. By contrast, the expressions of most of the genes for primary metabolism were not significantly altered. Although changes were small, the expressions of genes for enzymes involved in CO2 fixation (carbonic anhydrase, Rubisco, phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase) were down-regulated, whereas that of genes encoding enzymes for RuBP regeneration (fructose bisphosphate phosphatase, fructose bisphosphate aldolase, sedoheptulose bisphosphate phosphatase and phosphoribulokinase) and starch synthesis (ADP-glucose pyrophosphorylase and starch synthase) were up-regulated under elevated CO2. These results suggest that some sets of genes involved in primary metabolism pathway in the chloroplast are co-regulated by elevated CO2.
Plant Science.
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ABSTRACT: Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50-70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves.
Plant Physiology and Biochemistry 48(10-11):808-12. · 2.84 Impact Factor
