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 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.