Molecular cloning and characterization of a cDNA that encodes protoporphyrinogen oxidase of Arabidopsis thaliana

A cDNA encoding protoporphyrinogen oxidase (PPOX), the last enzyme common to the biosynthetic pathways for chlorophylls and hemes, was obtained from a library of Arabidopsis thaliana cDNA constructed in a lambda vector by screening for complementation of a hemG mutant of Escherichia coli. Extracts of E. coli cells transformed with the Arabidopsis PPOX cDNA had high PPOX activity, and this activity was markedly inhibited by acifluorfen, a specific inhibitor of PPOX. Sequence analysis revealed that the cDNA for Arabidopsis PPOX encodes a protein of 537 amino acids (aa) with a calculated molecular class of 57.7 kDa. The deduced aa sequence exhibited similarity to sequences of PPOX from Bacillus subtilis, mouse, and human. However, the PPOX of Arabidopsis contained a putative leader peptide for import into mitochondria (mt). Southern analysis indicated that the PPOX whose cDNA we cloned is encoded by a single gene in Arabidopsis. Northern blot analysis showed that the level of expression of the gene in Arabidopsis leaves was high, whereas it was low in roots and floral buds. To our knowledge, this is the first report for the cloning of a cDNA for a plant PPOX.