Phosphoenolpyruvate Carboxykinase in the C4 Monocot Urochloa panicoides Is Encoded by Four Differentially Expressed Genes1

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Abstract
Previous screening of a cDNA library of leaf poly(A+) RNA from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C4 monocot, led to the characterization of cDNAs encoding the U. panicoides PCK subunit PCK1. A second PCK sequence, designated PCK2, has now been found by rescreening the library. The deduced PCK2 polypeptide is 626 residues in length, has a predicted molecular mass of 68,686 D, and is 96% identical to the deduced PCK1 sequence. Isolation and characterization of genomic DNA fragments revealed that the PCK1 and PCK2genes are each closely linked to another PCK gene. These additional genes have been designated PCK3 andPCK4, respectively. In each case, the second gene is located upstream and in the same transcriptional orientation as the gene characterized through cDNA analysis. A reverse transcription-polymerase chain reaction assay was used to demonstrate that PCK1 and PCK2 transcripts predominate in leaves, whereas PCK3 andPCK4 transcripts predominate in roots. Moreover, accumulation of PCK1 and PCK2 transcripts is light dependent. Direct N-terminal sequencing of PCK polypeptides purified from leaves demonstrated that PCK2 is produced. These results strongly suggest that PCK1 and PCK2 are involved in the photosynthetic CO2-concentrating mechanism active in U. panicoides.