Hiroshi Fukayama

High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants

Using an Agrobacterium-mediated transformation system, we have introduced the intact gene of maize phosphoenolpyruvate carboxylase (PEPC), which catalyzes the initial fixation of atmospheric CO2 in C4 plants into the C3 crop rice. Most transgenic rice plants showed high-level expression of the maize gene; the activities of PEPC in leaves of some transgenic plants were two- to threefold higher than those in maize, and the enzyme accounted for up to 12% of the total leaf soluble protein. RNA gel blot and Southern blot analyses showed that the level of expression of the maize PEPC in transgenic rice plants correlated with the amount of transcript and the copy number of the inserted maize gene. Physiologically, the transgenic plants exhibited reduced O2 inhibition of photosynthesis and photosynthetic rates comparable to those of untransformed plants. The results demonstrate a successful strategy for installing the key biochemical component of the C4 pathway of photosynthesis in C3 plants.

Phosphoenolpyruvate carboxylase intrinsically located in the chloroplast of rice plays a crucial role in ammonium assimilation

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 Vmax 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.

Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice

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.

Functional Incorporation of Sorghum Small Subunit Increases the Catalytic Turnover Rate of Rubisco in Transgenic Rice

Rubisco limits photosynthetic CO2 fixation because of its low catalytic turnover rate (kcat) 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 kcat Rubisco from the C4 plant sorghum (Sorghum bicolor) significantly enhances kcat 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 kcat than in nontransgenic rice. Rubisco from transgenic lines showed a higher Km 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 Jmax/Vcmax 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.

Activity regulation and physiological impacts of maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants

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.

Lessons from engineering a single-cell C4 photosynthetic pathway into rice

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.

Overexpression of Rubisco Activase Decreases the Photosynthetic CO2 Assimilation Rate by Reducing Rubisco Content in Rice Leaves

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.

Metabolic consequences of overproduction of phosphoenolpyruvate carboxylase in C3 plants.

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.

Unusual Small Subunit That Is Not Expressed in Photosynthetic Cells Alters the Catalytic Properties of Rubisco in Rice

Rice utilizes different type of Rubisco small subunit in nonphotosynthetic cells. Rubisco small subunits (RbcSs) are encoded by a nuclear multigene family in plants. Five RbcS genes, OsRbcS1, OsRbcS2, OsRbcS3, OsRbcS4, and OsRbcS5, have been identified in rice (Oryza sativa). Among them, the amino acid sequence of OsRbcS1 differs notably from those of other rice RbcSs. Phylogenetic analysis showed that OsRbcS1 is genetically distant from other rice RbcS genes and more closely related to RbcS from a fern and two woody plants. Reverse transcription-PCR and promoter β-glucuronidase analyses revealed that OsRbcS1 was not expressed in leaf blade, a major photosynthetic organ in rice, but was expressed in leaf sheath, culm, anther, and root central cylinder. In leaf blade of transgenic rice overexpressing OsRbcS1 and leaf sheath of nontransgenic rice, OsRbcS1 was incorporated into the Rubisco holoenzyme. Incorporation of OsRbcS1 into Rubisco increased the catalytic turnover rate and Km for CO2 of the enzyme and slightly decreased the specificity for CO2, indicating that the catalytic properties were shifted to those of a high-activity type Rubisco. The CO2 assimilation rate at low CO2 partial pressure was decreased in overexpression lines but was not changed under ambient and high CO2 partial pressure compared with nontransgenic rice. Although the Rubisco content was increased, Rubisco activation state was decreased in overexpression lines. These results indicate that the catalytic properties of Rubisco can be altered by ectopic expression of OsRbcS1, with substantial effects on photosynthetic performance in rice. We believe this is the first demonstration of organ-specific expression of individual members of the RbcS gene family resulting in marked effects on Rubisco catalytic activity.

Soil and Water Warming Accelerates Phenology and Down-Regulation of Leaf Photosynthesis of Rice Plants Grown Under Free-Air CO2 Enrichment (FACE)

To enable prediction of future rice production in a changing climate, we need to understand the interactive effects of temperature and elevated [CO2] (E[CO2]). We therefore examined if the effect of E[CO2] on the light-saturated leaf photosynthetic rate (Asat) was affected by soil and water temperature (NT, normal; ET, elevated) under open-field conditions at the rice free-air CO2 enrichment (FACE) facility in Shizukuishi, Japan, in 2007 and 2008. Season-long E[CO2] (+200 µmol mol−1) increased Asat by 26%, when averaged over two years, temperature regimes and growth stages. The effect of ET (+2°C) on Asat was not significant at active tillering and heading, but became negative and significant at mid-grain filling; Asat in E[CO2]–ET was higher than in ambient [CO2] (A[CO2])–NT by only 4%. Photosynthetic down-regulation at E[CO2] also became apparent at mid-grain filling; Asat compared at the same [CO2] in the leaf cuvette was significantly lower in plants grown in E[CO2] than in those grown in A[CO2]. The additive effects of E[CO2] and ET decreased Asat by 23% compared with that of A[CO2]–NT plants. Although total crop nitrogen (N) uptake was increased by ET, N allocation to the leaves and to Rubisco was reduced under ET and E[CO2] at mid-grain filling, which resulted in a significant decrease (32%) in the maximum rate of ribulose-1,5-bisphosphate carboxylation on a leaf area basis. Because the change in N allocation was associated with the accelerated phenology in E[CO2]–ET plants, we conclude that soil and water warming accelerates photosynthetic down-regulation at E[CO2].