CHARACTERIZATION OF THE GENE FOR PYRUVATE, ORTHOPHOSPHATE DIKINASE FROM RICE, A C3 PLANT, AND A COMPARISON OF STRUCTURE AND EXPRESSION BETWEEN C3 AND C4 GENES FOR THIS PROTEIN

To investigate the molecular changes that might have occurred in genes for pyruvate,orthophosphate dikinase (PPDK) during the evolution of C 4 plants from C3 plants, we isolated a full-length cDNA and the corres ponding gene for a C4-like PPDK from rice, a C3 gramineous plant and c ompared their structures and promoter activities to those of the corre sponding gene from maize, a C4 gramineous plant. As in maize, there ar e at least two ppdk genes in rice and one of them was very similar to the maize C4-type ppdk. The deduced amino acid sequence of the rice PP DK was 88% homologous to the maize C4-type PPDK in the mature peptide region and 56% homologous in the transit peptide region. The C4-like p pdk in rice contained 21 exons, which were interrupted by twenty intro ns, and the positions of the introns were essentially the same as thos e in the gene from maize, with the except in that the gene from rice h ad two extra introns. Such extra introns were also found in the C4-typ e ppdk from a dicot, Flaveria, at the same positions. These results st rongly suggest that the two introns were present in an ancestral gene before the divergence of monocot and dicot plants.The C4-like ppdk in rice contained two functionally independent promoters had generated a larger transcript with the transit peptide region and a smaller transc ript without this region. The unusual dual-promoter system for transcr iption has been conserved in the C4-type ppdk gene from maize, indicat ing that the dual-promoter system is a common feature of ppdk genes in both C3 and C4 plants. The patterns of expression of the two transcri pts in rice were different: the larger transcript was expressed exclus ively in green leaves at a low level whereas the smaller transcript wa s expressed in some reproductive organs at a high level. Essentially t he same patterns of expression were observed in maize, but the level o f expression of the larger transcript in maize green leaves was much h igher than that in green leaves of rice. The promoter activities of th e rice and maize genes for PPDK were examined directly in a transient expression assay in maize mesophyll protoplasts after electroporation with promoter::beta-glucuronidase chimeric genes. The rice promoter fo r the smaller transcript was very active in the protoplasts but the ri ce promoter for the larger transcript had relatively low activity. By contrast, both promoters of the maize gene had high activity. Taken to gether, these results demonstrate that the rice C4-Like ppdk is very s imilar to the maize C4-type ppdk, not only in terms of primary structu re but also in terms of the regulation of expression, with the excepti on that the strength of the maize promoter for the larger transcript i s higher. The results strongly suggest that the genetic alterations re quired to give rise to the C4-type ppdk gene were relatively limited.