JXB Advance Access originally published online on March 2, 2008
Journal of Experimental Botany 2008 59(7):1799-1809; doi:10.1093/jxb/ern016
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RESEARCH PAPER |
Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice
1Photobiology and Photosynthesis Research Unit, National Institute of Agrobiological Sciences (NIAS), Kannondai, Tsukuba 305-8602, Japan
2University Farm, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Midoricho, Nishitokyo, Tokyo 188-0002, Japan
¶ To whom correspondence should be addressed. E-mail: mmiyao{at}affrc.go.jp
Received 15 October 2007; Revised 22 December 2007 Accepted 11 January 2008
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Abstract |
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Four enzymes, namely, the maize C4-specific phosphoenolpyruvate carboxylase (PEPC), the maize C4-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C3-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 CO2 assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO2 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 CO2 assimilation, however, was more marked at higher CO2 concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO2 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 C4-like photosynthetic pathway into C3 plants, are discussed.
Key words: C4 photosynthesis, malate dehydrogenase, malic enzyme, overproduction, phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, transgenic rice
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Introduction |
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Most terrestrial plants, including many important crops such as rice (Oryza sativa), wheat (Triticum aestivum), soybean (Glycine max), and potato (Solanum tuberosum) assimilate CO2 through the C3 photosynthetic pathway (the Calvin cycle), and are classified as C3 plants. However, some plants, such as maize (Zea mays) and sugarcane (Saccharum officinarum), possess the C4 photosynthetic pathway in addition to the C3 pathway, and these are classified as C4 plants. The C4 pathway acts to concentrate CO2 at the site of the reactions of the C3 pathway, and thus inhibits photorespiration (Hatch, 1987). This CO2-concentrating mechanism enables C4 plants to achieve high photosynthetic capacity and higher water and nitrogen use efficiencies (Hatch, 1987). Consequently, the transfer of C4 traits to C3 plants is one strategy being adopted for improving the photosynthetic performance of C3 plants.
The C4 pathway consists of three key steps: (i) the initial fixation of CO2 by phosphoenolpyruvate carboxylase (PEPC) to form a C4 acid, (ii) decarboxylation of a C4 acid to release CO2 near the site of the Calvin cycle, and (iii) regeneration of the primary CO2 acceptor phosphoenolpyruvate (PEP) by pyruvate, orthophosphate dikinase (PPDK) (Hatch, 1987). In terrestrial C4 plants, CO2 release from C4 acids and the resulting elevation of cellular CO2 levels take place at a site that is physically separated from the site of initial carboxylation. This separation occurs through the bundle sheath and mesophyll cells in typical/classical C4 plants (Hatch, 1987; Leegood, 2002), and through two distant subcellular compartments in the recently discovered single-cell C4 plants (Edwards et al., 2004). Structural and biochemical features of chloroplasts in these two sites are also different. It has been proposed that such compartmentation and chloroplast differentiation, together with structural adaptation to minimize CO2 diffusion from the CO2 release/elevation site, are essential for C4 photosynthesis. By contrast, in some aquatic plants C4 photosynthesis is accomplished in a single cell without any compartmentation and chloroplast differentiation (Bowes et al., 2002; Leegood, 2002). At least three submerged macroalga, all of which are freshwater monocots, perform such C4 photosynthesis (Bowes et al., 2002). Hydrilla verticillata has been the best documented. It is a facultative C4 plant that shifts from C3 to C4 photosynthesis under low CO2 conditions without any structural modifications of leaf cells. The C4-like pathway of Hydrilla is relatively simple (Fig. 1). Upon the shift to C4 photosynthesis, genes encoding the C4-specific isoforms of PEPC, PPDK, and NADP-malic enzyme (ME) are up-regulated (Rao et al., 2002, 2006; Estavillo et al., 2007). Although the shift is accompanied by the up-regulation of various genes (Rao et al., 2006), protein components required for the operation of the C4-like pathway remain to be solved.
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The recent application of recombinant DNA technology has made considerable progress in molecular engineering of photosynthetic genes. It enabled us to overproduce enzymes involved in the C4 pathway at high levels and in desired locations in the leaves of C3 plants (for a review, see Matsuoka et al., 2001; Miyao, 2003), and a variety of transgenic C3 plants overproducing C4 enzymes have been generated and intensively analysed (for a review, see Häusler et al., 2002; Miyao, 2003; Miyao and Fukayama, 2003). We are attempting to introduce the simplest model of C4 photosynthesis, the C4-like pathway of Hydrilla, into the mesophyll cell of a C3 plant, rice (Fig. 1). Until now, we have produced more than ten sets of different transgenic rice overproducing four C4 enzymes, namely, PEPC, PPDK, NADP-ME, and NADP-malate dehydrogenase (MDH), independently or in combination. In this study, photosynthesis and growth of these transgenic rice plants were analysed and compared. It was found that overproduction of all four enzymes in combination slightly improved photosynthesis, but at the same time caused slight but reproducible stunting of the transgenic plants.
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Materials and methods |
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Plant materials
For the production of transgenic rice plants overproducing a single enzyme in the mesophyll cell of rice plants, five different gene constructs were used. They contained the intact maize C4-specific gene for overproduction of PEPC and PPDK (Ku et al., 1999; Fukayama et al., 2001) or the full-length cDNA fused to the rice Cab promoter (Sakamoto et al., 1991) for overproduction of the maize C4-specific NADP-ME, the rice C3-specific isoform (Tsuchida et al., 2001), and the sorghum NADP-MDH. The cDNA clone for the sorghum MDH (accession No. X53453 [GenBank] ) was a generous gift from Professor M Miginiac-Maslow, CNRS/Université Paris-Sud, France. The constructs described above were cloned into a binary vector pIG121Hm containing a hygromycin resistance gene, and introduced into rice plants (cv. Kitaake) via Agrobacterium-mediated gene transfer. Transgenic lines in which the T1 generation exhibited a segregation ratio of around 1:3 (assessed by hygromycin resistance) were chosen for further analysis, and levels of the introduced enzyme protein in the leaves were screened by SDS-PAGE. Protein levels were determined from intensities of protein bands after staining with Coomassie Brilliant Blue R-250 for PEPC and PPDK, or from those after immunoblotting for ME and MDH. Plants with the highest protein level were taken as homozygous and self pollinated. Homogeneities of the level of the introduced enzyme were confirmed in the T2 and T3 generations.
Transgenic rice plants overproducing more than one enzyme were produced by crossing two different single transformants and/or the second gene introduction into transformants. Gene constructs used for the second gene introduction contained the full-length cDNA for the rice C3-specific ME fused to the rice Cab promoter for overproduction of ME alone, or the full-length cDNAs for the rice C3-specific ME and the sorghum MDH, each of which were fused to the rice Cab promoter, for overproduction of MDH and ME together. They were cloned into a binary vector pGTV35Sbar containing the bialaphos resistance gene (a generous gift from Dr F Takaiwa, National Institute of Agrobiological Sciences, Japan) and introduced into rice plants. Homozygous lines with a single insertion site were selected by the segregation ratio (assessed by bialaphos resistance) and the protein level as described above. Transgenic rice plants used in this study are listed in Table 1.
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Maize and sorghum plants were planted in vermiculite. Unless otherwise stated, rice plants were planted in 1/10 000- or 1/5000-are Wagner pots containing 1.0 l or 3.0 l of commercial soil mixture (Bonsol No. 1, Sumitomo Chemicals, Japan). They were grown under natural light conditions in temperature-controlled greenhouses at day and night temperatures of 26 °C and 21 °C, respectively. When indicated, plants were grown in a growth chamber at 26/21 °C day/night cycle with a day period for 14 h under illumination with white light at a photosynthetically active photon flux density (PPFD) of 500–600 µmol m–2 s–1.
Extraction of leaf soluble protein and enzyme assays
Segments of about 3 cm were harvested from the mid-section of the uppermost fully expanded leaves at 11.00–12.00 h and immediately frozen in liquid nitrogen until use. Total leaf soluble protein extracts were prepared as described previously (Tsuchida et al., 2001). For assay of the PPDK activity, 5 mM pyruvate and 2 mM KH2PO4 were included in the extraction buffer (Fukayama et al., 2001).
Enzyme activities were determined spectrophotometrically at 30 °C. The PEPC activity was assayed by the method of Fukayama et al. (2003). The activity assayed at pH 7.5 in the presence of 5 mM glucose-6-phosphate was taken as the maximum activity. The PPDK activity was assayed after activation of the enzyme by an incubation of the protein extract at 25 °C for 1 h (Fukayama et al., 2001). The NADP-ME activity was assayed after desalting the protein extract through a gel filtration column as described previously (protocol 2, Tsuchida et al., 2001).
For NADP-MDH, extraction of leaf soluble protein and the activity assay were performed by the method of Tsuchida et al. (2001). The activity measured immediately after protein extraction was taken as the in vivo activity. The maximal potential activity was measured after incubation under a nitrogen gas stream of the protein extract with 1 mM dithiothreitol and 5 µM recombinant thioredoxin m (a generous gift from Professor T Hisabori and Dr K Motohashi, Tokyo Institute of Technology, Japan) at 25 °C for 40 min.
Analytical procedures
Protein determination and SDS–PAGE were carried out as described previously (Tsuchida et al., 2001). After SDS–PAGE, the gel was stained with Coomassie Brilliant Blue R-250 or subjected to immunoblotting using antisera raised against the sorghum MDH (a generous gift from Professor M Miginiac-Maslow), the maize C4-specific PEPC, the phosphorylated form of the maize C4-specific PEPC (a generous gift from Professor K Izui, Kinki University, Japan) (Ueno et al., 2000), or the recombinant rice C3-specific ME produced in Escherichia coli. Immunoreacted bands were visualized by the alkaline phosphatase reaction (Bio-Rad).
Gas exchange of the uppermost fully expanded leaf was measured with an open gas-exchange system (LI-6400, Li-Cor, NB, USA) as described previously (Fukayama et al., 2003). Measurements were performed at a leaf temperature of 25 °C, 21% O2, a PPFD of 1200 µmol m–2 s–1, and VPD of 1.0–1.2 kPa. The Ci response curve of CO2 assimilation was obtained with a single leaf by decreasing the ambient CO2 concentration (Ca) stepwise from 880 to 45 µl l–1.
For determination of 
13C, three seedlings of rice plants were transplanted in a plastic box (15.5 cm lengthx6 cm widthx10 cm depth) containing 0.6 l of the soil mixture, and grown in the greenhouses. Middle portions of the leaf blade (about two-thirds in length) were harvested from the uppermost fully expanded leaf (7th leaf) at 13.00–14.00 h on sunny days, immediately frozen in liquid nitrogen, and dried at 80 °C for 2 d. The dried sample was ground to a fine powder using a mortar and pestle, and further dried at 80 °C for 1 d. The 13C content was determined using an elemental analyser (NC2500, Thermoquest, San Jose, CA, USA) and a mass spectrometer (Delta Plus System, Thermoquest) (Sasaki et al., 2005), and the 13C value relative to Pee Dee Belemnite was calculated using a 13CO2/12CO2 gas mixture and flour powder of known 13C values as standards.
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Results |
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Effects of overproduction of a single C4 enzyme on photosynthesis and growth of rice plants
In the leaves of C3 plants, PEPC has an anaplerotic role replenishing the tricarboxylic acid cycle with intermediates, which are withdrawn for nitrate assimilation and the subsequent amino acid synthesis (Melzer and O'Leary, 1987). In accordance with this function, it has previously been observed that overproduction of the maize C4-specific PEPC in rice leaves enhanced respiration in the light (Fukayama et al., 2003). At the same time, the Rubisco activity estimated from the slope of the Ci response curve of CO2 assimilation was reduced (Fukayama et al., 2003). As a result, transgenic rice plants overproducing PEPC showed slightly suppressed CO2 assimilation. Figure 2A compares Ci response curves of transgenic rice plants overproducing different levels of the maize PEPC. It is clearly seen that the suppression was more marked in the transformant with a higher level of PEPC. Elevation of the PEPC activity up to 50-fold over that of non-transgenic rice did not significantly affect the growth of rice plants, while further elevation caused slight stunting. It was reported that overproduction of PEPC enhanced stomatal opening, thereby improving CO2 assimilation of rice plants (Ku et al., 2000). Stomatal responses remained unaffected in our transgenic rice plants overproducing PEPC (Fukayama et al., 2003).
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Overproduction of the maize C4-specific PPDK did not affect CO2 assimilation and growth of rice plants at all, unless the foreign PPDK accumulated above some threshold level (Fukayama et al., 2001). As shown in Fig. 2B, the Ci response curve of the homozygous line PD-332 completely matched with that of non-transgenic rice. The other line PD-259 with a higher PPDK level showed slightly suppressed CO2 assimilation. This effect probably resulted from nitrogen deficiency due to a high nitrogen demand of foreign PPDK as discussed previously (Fukayama et al., 2001). Overproduction of PPDK in addition to PEPC did not affect CO2 assimilation, and the hybrid between PE-2 and PD-332 showed the same Ci response curve as PE-2 (Fig. 2A). A previous study demonstrated that transgenic tobacco plants overproducing the plastidic PPDK from the facultative Crassulacean acid metabolism (CAM) plant Mesembyanthemum crystallinum produced more seeds per seed capsule and heavier seed capsules than non-transgenic plants, and proposed that this effect was ascribable to overproduction of PPDK in seeds (Sheriff et al., 1998). Although the maize PPDK was overproduced in spikelets of our transgenic rice plants (Fukayama et al., 2001), increased grain yield was not always observed.
As demonstrated previously (Tsuchida et al., 2001), overproduction of the maize C4-specific NADP-ME led to enhanced photoinhibition of photosynthesis, leaf chlorophyll bleaching and serious stunting, even when the activity of the maize enzyme was only several fold that of non-transgenic rice. These detrimental effects resulted from an increase in the NADPH/NADP+ ratio in the chloroplast stroma and suppression of photorespiration by depletion of malate, which is exported from the stroma in exchange with 2-oxoglutarate for photorespiration (Tsuchida et al., 2001). Overproduction of the rice C3-specific isoform did not affect photosynthesis and growth of rice plants (data not shown). Overproduction of the sorghum NADP-MDH up to 40-fold wild-type activities had little effect on plant growth (see Fig. 6A).
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Activity regulation of foreign C4 enzymes in rice leaves
The activity of plant-type PEPC is regulated by two different, but interactive mechanisms; one is through various metabolite effectors, and the other is reversible phosphorylation of a conserved serine residue near the N terminus (Vidal and Chollet, 1997). Upon phosphorylation PEPC becomes less sensitive to its feedback inhibitor malate and more sensitive to the activator glucose-6-phosphate, thereby attaining a higher activity (Vidal and Chollet, 1997). The maize C4-specific PEPC overproduced in transgenic rice leaves undergoes activity regulation by protein phosphorylation but in a manner opposite to that observed in the leaves of C4 plants: it is phosphorylated at night and dephosphorylated during the middle part of the day, in just the same way as the rice endogenous enzyme (Fukayama et al., 2001, 2006). One of three rice PEPC kinase genes is responsible for this nocturnal phosphorylation (Fukayama et al., 2006). As shown in Fig. 3, the phosphorylation pattern of the maize PEPC in the transgenic rice leaves remained unaffected by additional overproduction of PPDK alone in PExPD or in combination with ME and MDH in the quadruple transformants, and the maize PEPC was phosphorylated at night in all cases. The level of phosphorylated PEPC at night was slightly higher in Quad-3 and Quad-11 than in PE-2 and PExPD.
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The activity of the C4-specific PPDK is strictly regulated by light in C4 plants through protein phosphorylation by the PPDK regulator protein, which catalyses both phosphorylation and dephosphorylation (Burnell and Hatch, 1985). As demonstrated previously (Fukayama et al., 2001), the maize PPDK expressed in transgenic rice leaves undergoes activity regulation by light as in the leaves of C4 plants (Fig. 4). In maize, transgenic rice plants overproducing PPDK, and non-transgenic rice, PPDK was activated at the onset of illumination and deactivated in the dark. In transgenic rice leaves expressing the maize PPDK at very high levels, PPDK failed to be fully activated even after 14 h illumination. The maize PPDK in protein extracts from transgenic rice leaves was activated in vitro by incubation with phosphate and inactivated by ADP, as observed in maize (data not shown).
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The activity of the chloroplastic NADP-MDH is also strictly regulated by light in both C3 and C4 plants through the thioredoxin cascade (Miginiac-Maslow et al., 2000). The sorghum MDH expressed in rice leaves also underwent activity regulation by light. The rate of activation by light in transgenic rice, however, was lower than in C4 plants and non-transgenic rice. In sorghum and non-transgenic rice, the MDH activity reached steady-state within 2 min after the onset of illumination at a PPFD of 1000 µmol m–2 s–1, while it took about 60 min in the homozygous line MD-36, in which maximum MDH activity was 40-fold that of non-transgenic rice (data not shown). The steady-state level of activation of the enzyme was the same in MD-36 and non-transgenic rice, and it was about 50% under full sunlight.
Production of transgenic rice plants overproducing more than one enzyme
As described above, overproduction of the maize C4-specific ME in rice leaves led to serious stunting of plants. Such detrimental effects of the maize enzyme were not mitigated by additional overproduction of the sorghum MDH (Fig. 5A), which takes part in the export of reducing equivalents from the chloroplast (Miginiac-Maslow et al., 2000). Overproduction of all three enzymes together, namely, PEPC, PPDK, and MDH, was not effective in reversing this effect either (Fig. 5B). Therefore, it was subsequently chosen to use the rice C3-specific ME for transformation instead of the maize enzyme. Thus, enzymes introduced in the multiple enzyme transgenics were the maize C4-specific PEPC, the maize C4-specific PPDK, the sorghum MDH, and the rice C3-specific ME, the latter three of which are targeted to the chloroplast (Fig. 1). Multiple enzyme transformants were produced by either crossing two different single enzyme transformants or by the introduction of the second gene construct into transformants, or using a combination of both approaches. All transformants used in this study had a single transgene per haploid.
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During the course of experiments, it was found that very high levels of foreign PPDK expression hampered gene introduction. In transgenic lines in which the PPDK protein level exceeded 6% of total leaf soluble protein, the growth of calli in culture was seriously inhibited and, as a result, the efficiency of gene introduction became one-order of magnitude lower than that of non-transgenic rice. Therefore, in all experiments involving double transformation, PD-332 was used as the transgenic explant source, in which PPDK level was around 2% of total protein, although the transformation efficiency was still reduced. Overproduction of PEPC did not have such an effect.
Two sets of double transformants were first produced by crossing: the PExPD cross using lines PE-2 and PD-332 as parents, and the MDxME cross using MD-36 and ME-4 as parents (Table 1). Another double transformant overproducing PEPC and ME (PE•ME) were produced by the introduction of the rice ME gene construct into PE-2. To produce triple and quadruple transformants, the double transformant PExPD was transformed with the rice ME gene construct and the construct containing the rice ME and sorghum MDH genes in tandem, respectively. All these multiple transformants set seeds normally and their fertile progeny were successfully generated.
Physiological characteristics of multiple transformants
Although overproduction of any of the four enzymes did not affect the growth of rice plants, some lines containing multiple enzymes exhibited stunting. Figure 6 compares culm length and panicle weight after grain filling, parameters commonly used for phenotyping the growth of rice plants. Overproduction of MDH and ME did not significantly affect culm length or total panicle weight. By contrast, overproduction of PEPC with PPDK led to slight decreases in these two parameters. Significant effects on growth were observed in transformants overproducing PEPC with ME, namely, the double transformant PE•ME-104, the triple transformant Trip-73, and the quadruple transformants Quad-3 and Quad-11. In these transformants, both culm length and total panicle weight were smaller than those of their respective control transformants. It is noted that line Quad-11 showed occasional stunting, probably due to some genome alteration caused by cell cultivation, and therefore, its growth parameters tended to be underestimated. The stunting caused by overproduction of PEPC and ME together was more marked during the vegetative growth stage, and overproduction of MDH had some mitigating effects against the stunting (data not shown).
Figure 7 compares the response of CO2 assimilation rate to Ci in transformants containing multiple C4 enzymes. Overproduction of MDH with ME had no detectable effect on CO2 assimilation, and the Ci response curve of the MDxME cross was almost identical to that of non-transgenic rice plants. As described above, PE-2 overproducing PEPC showed slightly lower CO2 assimilation rates than non-transformants (Fig. 2A). Neither overproduction of ME in addition to PEPC in PE•ME-104 nor overproduction of PPDK and ME in addition to PEPC (Trip-73) substantially affected CO2 assimilation.
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In contrast to the triple transformants, the quadruple transformants, also overproducing MDH, showed slightly enhanced CO2 assimilation especially at high Ci values (Fig. 7D). The CO2 assimilation rate of Quad-3 was higher than that of the control transformants and, in some plants, it was comparable to or slightly higher than that of non-transformants. Quad-11, a line that showed stunting, also exhibited higher rates of CO2 assimilation than the control transformants. The same results were reproducibly obtained when the pot size was large enough: the enhanced CO2 assimilation was always observed in plants grown in large pots with 3.0 l soil but not in small pots with 1.0 l soil (data not shown). The suppression of CO2 assimilation in PE-2 and PExPD was also influenced by the pot size, being more marked in larger pots.
Carbon isotope composition is often used as a simple test for determining the photosynthetic type, since Rubisco largely discriminates against 13CO2 and C3 plants show much lower 13C values than C4 plants (Farquhar et al., 1989). Figure 8 compares 13C values of leaves of the quadruple transformants. Plants used for these experiments were grown in small plastic boxes placed side by side so that all the plants were exposed to the same air stream conditions in closed greenhouses. Although differences were small, the 13C value was decreased by overproduction of PEPC and PPDK together, and increased by overproduction of all four enzymes. These changes did not result from altered stomatal conductance, since the Ci/Ca ratio remained unaffected by overproduction of these enzymes (Fig. 7D). It is presumed that they reflected some alteration of carbon metabolism in transgenic rice leaves.
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Discussion |
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To determine the effects of overproduction of an enzyme in transgenic plants accurately, it is necessary to examine the correlation of the phenotype of transgenic plants with transgene expression level, preferably at the protein and/or enzyme activity level. Transgenic rice plants with different levels of the introduced enzyme were compared and it was concluded that overproduction of a single C4 enzyme did not improve photosynthesis of rice. Rather, overproduction of the maize PEPC slightly inhibited photosynthesis (Fig. 2A) through stimulation of respiration in the light and reduction of the Rubisco activity (Fukayama et al., 2003). The latter effect might be due to enhanced nitrogen assimilation, which competes with the Calvin cycle for ATP and reducing power. It has been demonstrated in transgenic potato plants that overproduction of an engineered unregulated PEPC doubled the rate of dark respiration but did not substantially affect photosynthetic characteristics (Rademacher et al., 2002). Some groups have claimed that overproduction of the maize PEPC enhanced photosynthesis and increased crop yields of transgenic rice plants (Jiao et al., 2002; Bandyopadhyay et al., 2007). However, in these studies, only one or two lines were examined, and the correlation between the level of PEPC protein and the extent of observed phenotypes was not well established.
Of the combinations of C4 enzymes examined, only overproduction of all the four enzymes had some positive effects on photosynthesis, and only to a limited extent (Fig. 7). The reduction in CO2 assimilation rate by overproduction of PEPC was recovered by overproduction of all three other enzymes and, in some cases, CO2 assimilation rates reached levels higher than those of non-transgenic rice plants (Fig. 7D). Since overproduction of PEPC, PPDK, and ME together did not have such effects (Fig. 7C), the elevation of the MDH activity must be essential for the restoration/enhancement of photosynthesis. We consider that overproduction of MDH is necessary for direct uptake of OAA into the chloroplast and also for the decarboxylation reaction of overproduced ME.
As reported previously, the maize C4-specific ME overproduced in rice leaves raises the NADPH/NADP+ ratio of the stroma and thereby makes photosynthesis susceptible to photoinhibition (Tsuchida et al., 2001). These findings indicate that the reaction of the maize enzyme is directed toward decarboxylation of malate in rice leaves. This would not be the case for the rice C3-specific isoform even when PEPC was overproduced together. As shown in Fig. 7, CO2 assimilation was not at all affected in PE•ME-104 and Trip-73 in which ME was overproduced with PEPC but without MDH, or no symptom of photoinhibition of photosynthesis was observed in these transgenics. As compared with the C4-specific ME, the affinity for NADP+ is one-order of magnitude lower in the C3-specific isoform (Saigo et al., 2004). It is likely that the reaction of overproduced rice enzyme attains equilibrium in the chloroplast under illumination where the NADPH/NADP+ ratio is always high, proceeding towards either decarboxylation or carboxylation direction in response to the NADPH/NADP+ status. When ME is overproduced with MDH, NADPH produced by the decarboxylation reaction of ME can be consumed by MDH, leaving the level of NADP+ unchanged. At the same time MDH promotes import of OAA from the cytosol, supporting direct uptake of the product of overproduced PEPC into the chloroplast. Overproduction of PPDK would also act to support the decarboxylation reaction by ME by reducing the level of pyruvate.
The detrimental effects of C4-specific ME have previously been explained by its high Vm and low Km for malate, which are one-order of magnitude different from the C3-specific isoform (Tsuchida et al., 2001). Kinetic parameters for the C3-specific isoform reported previously have been revised by recent studies using recombinant proteins, which reported that Km for NADP+ differed significantly but either 
or Km for malate did not between two isoforms of maize (Saigo et al., 2004).
It is currently unknown if the C4-like pathway operates in the quadruple transformants. The 13C value of rice leaves was decreased by overproduction of PEPC and PPDK together and increased by overproduction of all four enzymes, as was the CO2 assimilation rate (Fig. 8). Even if the C4-like pathway operates in these plants, it does not seem to act as a CO2 pump in the same way as in C4 plants. As seen in Fig. 7D and E, the increase in the CO2 assimilation rate in the quadruple transformants was larger at higher Ci values, and the CO2 compensation point was not significantly altered. At low Ci where photorespiration predominates over CO2 assimilation, a subtle increase of the CO2 concentration around Rubisco (Cc) could not significantly affect the CO2 assimilation rate. At high Ci, in contrast, photorespiration is largely suppressed and Cc becomes lower than Ci because of active carboxylation by Rubisco (see Evans and von Caemmerer, 1996). Under these conditions, some increase in Cc would be effective in enhancing CO2 assimilation. For the proper functioning of the C4-like pathway it is important that overproduced enzymes constitute a closed cycle so as not to perturb metabolisms of the host plant. Limited export of PEP from the chloroplast, usually found in the leaves of C3 plants, may limit the reaction of PEPC and thereby hamper operation of the cycle.
The present study revealed that overproduction of PEPC and ME together led to the stunting of transgenics (Fig. 6). Since overproduction of either PEPC or ME alone did not lead to appreciable stunting and overproduction of ME in addition to PEPC did not affect photosynthesis (Fig. 7B), it is possible that PEPC and ME together catalyse a futile pathway which wastes metabolic energy.
As described in the Results, PPDK and MDH undergo strict activity regulation by light, and they are completely inactivated in darkness in transgenic rice leaves, while PEPC is active throughout the day. The C3-specific ME may also be active both in the light and in darkness, as judged by its enzyme characteristics. The C4-specific isoform has a pH optimum of around 8 and it is activated by light through the elevation of pH and the increase in an essential cofactor Mg2+ in the stroma (Edwards and Andreo, 1992; Drincovich et al., 2001). In addition, its sensitivity to inhibition by the substrate malate depends on pH: it is inhibited by malate at pH 7 while it becomes insensitive at pH 8, thus exhibiting a high activity only in the light in C4 plants (Edwards and Andreo, 1992). Activity regulation through a change in pH is possible for the C3-specific isoform, for which a pH optimum of 7.8 has been reported for the recombinant maize protein (Saigo et al., 2004). Unlike the C4-specific ME, however, the C3-specific isoform is not inhibited by malate at pH 7 (Drincovich et al., 2001; Edwards and Andreo, 1992), remaining active even in darkness. The level of NADP+ in the stroma in darkness would be high enough to support the decarboxylation reaction of overproduced ME, and moreover, direct uptake of malate is feasible. When PEPC is overproduced together with ME, it raises the level of malate inside the cell so that overproduced ME can support appreciable flux.
The maize PEPC in transgenic rice leaves is phosphorylated and in the more active form during a period from 2 h before the beginning of the dark period until 2 h after the onset of the light period (Fukayama et al., 2003). Therefore, the putative wasteful pathway supported by PEPC and ME seems to be active at night and also for some time after sunrise and before sunset. The stunting caused by overproduction of PEPC with ME would thus be inevitable unless the phosphorylation pattern of PEPC in rice leaves were to be modified. By contrast, stunting seems to be much less marked in other transgenic C3 plants such as barley, cucumber, Arabidopsis, and Lotus japonicus, in which PEPC is phosphorylated in the daytime (H Fukayama et al., unpublished results). Previous studies on transgenic potato and tobacco plants overproducing PEPC together with ME (Lipka et al., 1999; Häusler et al., 2001) did not report stunting of these transformants. Even in rice plants, the stunting might be greatly mitigated when the C3-specific PEPC is used as the transgene source, as this protein is more sensitive to inhibition by malate than the C4-specific isoform (Svensson et al., 2003). In any case, the activity regulation of PEPC is essential for the growth of transgenic C3 plants. As demonstrated previously, overproduction of PEPC almost void of activity regulation leads to serious stunting of transgenic Arabidopsis and potato plants (Rademacher et al., 2002; Chen et al., 2004; for a review see Miyao and Fukayama, 2003).
Taken together, we consider that rice is not good plant material for an approach to introduce the C4-like pathway with respect to increased production. Even so, this study does not eliminate the possibility of improving photosynthesis of C3 plants by this approach. Four homozygous lines of the quadruple transformants used in this study unexpectedly had almost the same activities of MDH and ME (Table 1). It remains to be determined whether photosynthesis could be substantially improved by changing the relative level of expression of the introduced enzymes, as well as the nature of a metabolic pathway(s) operating in the quadruple transformants.
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Acknowledgements |
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The authors are grateful to Professors Myroslawa Miginiac-Maslow, CNRS/University Paris-Sud, France, Katsura Izui, Kinki University, Japan, and Toru Hisabori, Tokyo Institute of Technology, Japan, for their generous gifts of antibodies, the cDNA clone and the recombinant protein. They are also thankful to Emeritus Professor Kuni Ishihara, Tokyo University of Agriculture and Technology, Japan, and to Professors Amane Makino, Tohoku University, Japan, and Hideaki Usuda, Teikyo University, Japan, for their helpful discussion and encouragement throughout the study and to Drs Julie and Robert T Furbank, CSIRO, Australia, for their careful reading of the manuscript. This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Green Technology Project IP-3001) to MM.
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Footnotes |
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* Present address: Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan.
Present address: Chiben-Gakuen High School, Gojo, Nara 637-0042, Japan.
Present address: Plant Pathology Department, University of Nebraska-Lincoln, Lincoln, NE 68583-0722, USA.
Present address: Graduate School of Agriculture, Kobe University, Kobe 657-8501, Japan.
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Abbreviations |
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Ca, the ambient CO2 concentration; Ci, the intercellular CO2 concentration;
13C, the relative content of 13C in total carbon atoms; MDH, malate dehydrogenase; ME, malic enzyme; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PPDK, pyruvate, orthophosphate dikinase; PPFD, photosynthetically active photon flux density; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; SD, standard deviation; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis. |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bandyopadhyay A, Datta K, Zhang J, Yang W, Raychaudhuri S, Miyao M, Datta SK. Enhanced photosynthesis rate in genetically engineered indica rice expressing pepc gene cloned from maize. Plant Science (2007) 172:1204–1209.
Bowes G, Rao SK, Estavillo GM, Reiskind JB. C4 mechanisms in aquatic angiosperms: comparisons with terrestrial C4 systems. Functional Plant Biology (2002) 29:379–392.[CrossRef][Web of Science]
Burnell JN, Hatch MD. Light-dark modulation of leaf pyruvate, Pi dikinase. Trends in Biochemical Sciences (1985) 10:288–291.[CrossRef][Web of Science]
Chen L-M, Li K-Z, Miwa T, Izui K. Overexpression of a cyanobacterial phosphoenolpyruvate carboxylase with diminished sensitivity to feedback inhibition in Arabidopsis changes amino acid metabolism. Planta (2004) 219:440–449.[Web of Science][Medline]
Drincovich MF, Casati P, Andreo CS. NADP-malic enzyme from plants: a ubiquitous enzyme involved in different metabolic pathways. FEBS Letters (2001) 490:1–6.[CrossRef][Web of Science][Medline]
Edwards GE, Andreo CS. NADP-malic enzyme from plants. Photochemistry (1992) 31:1845–1857.[CrossRef]
Edwards GE, Franceschi VR, Voznesenskaya EV. Single-cell C4 photosynthesis versus the dual-cell (Kranz) paradigm. Annual Review of Plant Biology (2004) 55:173–196.[CrossRef][Medline]
Estavillo GM, Rao SK, Reiskind JB, Bowes G. Characterization of the NADP malic enzyme gene family in the facultative, single-cell C4 monocot Hydrilla verticillata. Photosynthesis Research (2007) 94:43–57.[CrossRef][Web of Science][Medline]
Evans JR, von Caemmerer S. Carbon dioxide diffusion inside leaves. Plant Physiology (1996) 110:339–346.[CrossRef][Web of Science][Medline]
Farquhar GD, Ehleringer JR, Hubick KT. Carbon isotope discrimination and photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology (1989) 40:503–537.[CrossRef][Web of Science]
Fukayama H, Hatch MD, Tamai T, Tsuchida H, Sudoh S, Furbank RT, Miyao M. Activity regulation and physiological impacts of the maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants. Photosynthesis Research (2003) 77:227–239.[CrossRef][Web of Science][Medline]
Fukayama H, Tamai T, Taniguchi Y, Sullivan S, Miyao M, Nimmo HG. Characterization and functional analysis of phosphoenolpyruvate carboxylase kinase genes in rice. The Plant Journal (2006) 47:258–268.[CrossRef][Web of Science][Medline]
Fukayama H, Tsuchida H, Agarie S, et al. Significant accumulation of C4-specific pyruvate, orthophosphate dikinase in a C3 plant, rice. Plant Physiology (2001) 127:1136–1146.
Hatch MD. C4 photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochimica et Biophysica Acta (1987) 895:81–106.
Häusler RE, Hirsch H-J, Kreuzaler F, Peterhänsel C. Overexpression of C4-cycle enzymes in transgenic C3 plants: a biotechnological approach to improve C3-photosynthesis. Journal of Experimental Botany (2002) 53:591–607.
Häusler RE, Rademacher T, Li J, Lipka V, Fischer KL, Schubert S, Kreuzaler F, Hirsch HJ. Single and double overexpression of C4-cycle genes had differential effects on the pattern of endogenous enzymes, attenuation of photorespiration and on contents of UV protectants in transgenic potato and tobacco plants. Journal of Experimental Botany (2001) 52:1785–1803.
Jiao D, Huang X, Li X, Chi W, Kuang T, Zhang Q, Ku MSB, Cho D. Photosynthetic characteristics and tolerance to photo-oxidation of transgenic rice expressing C4 photosynthesis enzymes. Photosynthesis Research (2002) 72:85–93.[CrossRef][Web of Science][Medline]
Ku MSB, Agarie S, Nomura M, Fukayama H, Tsuchida H, Ono K, Hirose S, Toki S, Miyao M, Matsuoka M. High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants. Nature Biotechnology (1999) 17:76–80.[CrossRef][Web of Science][Medline]
Ku MSB, Cho D, Ranade U, Hsu TP, Li X, Jiao DM, Ehleringer J, Miyao M, Matsuoka M. Photosynthetic performance of transgenic rice plants overexpressing maize C4 photosynthesis enzymes. In: Redesigning rice photosynthesis to increase yield—Sheehy JE, Mitchell PL, Hardy B, eds. (2000) Amsterdam: Elsevier Science Publishers. 193–204.
Leegood RC. C4 photosynthesis: principles of CO2 concentration and prospects for its introduction into C3 plants. Journal of Experimental Botany (2002) 53:581–590.
Lipka V, Häusler RE, Rademacher T, Li J, Hirsch H-J, Kreuzaler F. Solanum tuberosum double transgenic expressing phosphoenolpyruvate carboxylase and NADP-malic enzyme display reduced electron requirement for CO2 fixation. Plant Science (1999) 144:93–105.
Matsuoka M, Furbank RT, Fukayama H, Miyao M. Molecular engineering of C4 photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology (2001) 52:297–314.[CrossRef][Web of Science][Medline]
Melzer E, O'Leary MH. Anapleurotic CO2 fixation by phosphoenolpyruvate carboxylase in C3 plants. Plant Physiology (1987) 84:58–60.
Miginiac-Maslow M, Johansson K, Ruelland E, Issakidis-Bourguet E, Schepens I, Goyer A, Lemaire-Chamley M, Jacquot J-P, Le Maréchal P, Deccotignies P. Light-activation of NADP-malate dehydrogenase: a highly controlled process for an optimized function. Physiologia Plantarum (2000) 110:322–329.[CrossRef]
Miyao M, Fukayama H. Metabolic consequences of overproduction of phosphoenolpyruvate carboxylase in C3 plants. Archives of Biochemistry and Biophysics (2003) 414:197–203.[Web of Science][Medline]
Miyao M. Molecular evolution and genetic engineering of C4 photosynthetic enzymes. Journal of Experimental Botany (2003) 54:179–189.
Rademacher T, Häusler RE, Hirsch H-J, Zhang L, Lipka V, Weier D, Kreuzaler F, Peterhänsel C. An engineered phosphoenolpyruvate carboxylase redirects carbon and nitrogen flow in transgenic potato plants. The Plant Journal (2002) 32:25–39.[CrossRef][Web of Science][Medline]
Rao SK, Fukayama H, Reiskind J, Miyao M, Bowes G. Identification of C4 responsive genes in the facultative C4 plant Hydrilla verticillata. Photosynthesis Research (2006) 88:173–183.[CrossRef][Web of Science][Medline]
Rao SK, Magnin NC, Reiskind JB, Bowes G. Photosynthetic and other phosphoenolpyruvate carboxylase isoforms in the single-cell, facultative C4 system of Hydrilla verticillata. Plant Physiology (2002) 130:876–886.
Saigo M, Bologna FP, Maurino VG, Detarsio E, Andreo CS, Drincovich MF. Maize recombinant non-C4 NADP-malic enzyme: a novel dimeric malic enzyme with high specific activity. Plant Molecular Biology (2004) 55:97–107.[CrossRef][Web of Science][Medline]
Sakamoto M, Sanada Y, Tagiri A, Murakami T, Ohashi Y, Matsuoka M. Structure and characterization of a gene for light-harvesting Chl a/b binding protein from rice. Plant and Cell Physiology (1991) 32:385–393.
Sasaki H, Hara T, Ito S, Miura S, Hoque MM, Lieffering M, Kim H-Y, Okada M, Kobayashi K. Seasonal changes in canopy photosynthesis and respiration, and partitioning of photosynthate, in rice (Oryza sativa L.) grown under free-air CO2 enrichment. Plant and Cell Physiology (2005) 46:1704–1712.
Sheriff A, Meyer H, Riedel E, Schmitt JM, Lapke C. The influence of plant pyruvate, orthophosphate dikinase on a C3 plant with respect to the intracellular location of the enzyme. Plant Science (1998) 136:43–57.
Svensson P, Bläsing OE, Westhoff P. Evolution of C4 phosphoenolpyruvate carboxylase. Archives of Biochemistry and Biophysics (2003) 414:180–188.[CrossRef][Web of Science][Medline]
Tsuchida H, Tamai T, Fukayama H, et al. High level expression of C4-specific NADP-malic enzyme in leaves and impairment of photoautotrophic growth of a C3 plant, rice. Plant and Cell Physiology (2001) 42:138–145.
Ueno Y, Imanari E, Emura J, Yoshizawa-Kumagaye K, Nakajima K, Inami K, Shiba T, Sakakibara H, Sugiyama T, Izui K. Immunological analysis of the phosphorylation state of maize C4-form phosphoenolpyruvate carboxylase with specific antibodies raised against a synthetic phosphorylated peptide. The Plant Journal (2000) 21:17–26.[CrossRef][Web of Science][Medline]
Vidal J, Chollet R. Regulatory phosphorylation of C4 PEP carboxylase. Trends in Plant Science (1997) 2:230–237.[CrossRef][Web of Science]
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