The evolution of C4 plants: acquisition of cis-regulatory sequences in thepromoter of C4-type pyruvate, orthophosphate dikinase gene

In a previous study, we identified the C4-like pyruvate, orthophosphate dik inase gene (Pdk) in the C3 plant rice, with a similar structure to the C4-t ype Pdk in the C4 plant maize. In order to elucidate the differences betwee n C4-type and C4-like Pdk genes in C4 and C3 plants, we have produced chime ric constructs with the P-glucuronidase (GUS) reporter gene under the contr ol of the Pdk promoters. In transgenic rice, both rice and maize promoters directed GUS expression in photosynthetic organs in a light-dependent manne r. However, the maize promoter exhibited a much higher transcriptional acti vity than the rice promoter did. These results indicate that the rice C4-li ke Pdk gene resembles the maize C4-type Pdk gene in terms of regulation of expression. We also tested the activity of the rice promoter in transgenic maize. GUS activity was seen in both photosynthetic and non-photosynthetic organs. Thus, the rice promoter does not confer a strict organ-specific gen e expression, as the maize promoter does. Moreover, the rice promoter direc ted GUS expression not only in mesophyll cells but also in bundle sheath ce lls, whereas the maize promoter directed expression only in mesophyll cells . Taken together, the results obtained from both transgenic maize and rice demonstrate that the rice and maize promoters differ not only quantitativel y, but also qualitatively, in terms of their cell- and organ-specificity. E xperiments with swapped promoters using the rice and maize promoters furthe r demonstrated that a limited sequence region from -330 to -76 of the maize promoter confers light-regulated, high-level expression to the rice promot er in maize mesophyll protoplasts. We conclude the gain of cis-acting eleme nts conferring high-level expression and mesophyll cell specificity was nec essary for establishment of a C4-type Pdk gene during the course of evoluti on from C3 to C4 plants.