How to express some C ₄ photosynthesis genes at high levels in rice

To investigate the difference between Pdk genes that encode pyruvate, orthophosphate dikinase (PPDK), a Pdk gene homologous to the maize C ₄ -type Pdk gene was isolated from a C ₃ plant, rice, and compared with the maize gene. The primary structures of the genes are essentially the same, except that the rice gene has two additional introns. A transient expression assay of Pdk promoters using maize mesophyll protoplasts showed that the mode of expression of the maize and rice genes differs only in the expression activity of the promoter for the chloroplast-type PPDK: the maize gene was expressed fourfold higher than the rice gene. It was also found that a chimeric gene containing the maize Pdk promoter and a reporter gene led to high expression of the reporter gene in transgenic rice. Based on the above observations, the intact genes from maize encoding enzymes for C ₄ photosynthesis were introduced into rice to increase the activity of the C ₄ enzymes. As expected, the introduction of the maize gene led to high expression of C ₄ enzymes in transgenic rice. The activities of phospoenolpyruvate carboxylase (PEPC) and PPDK increased up to 110- and 40-fold more, respectively, than those of nontransgenic rice. High expression of C ₄ enzymes did not result solely from the high expression activity of the maize gene, since the introduction of a maize PPDK cDNA fused to the maize Pdk promoter or rice Cab promoter did not lead to high expression of PPDK. In some transgenic rice plants carrying the intact maize gene, the level of PPDK protein amounted to 35% of total leaf-soluble protein. The high expression of each C ₄ enzyme altered metabolism slightly but did not seem to increase the photosynthetic efficiency of transgenic rice leaves.