Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase

Abstract

Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mitochondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids. We cloned two different, full-length tobacco cDNA sequences by complementation of the protoporphyrin-IX-accumulating Escherichia coli hemG mutant from heme auxotrophy. The two sequences show similarity to the recently published Arabidopsis PPOX, Bacillus subtilis hemY, and to mammalian sequences encoding protoporphyrinogen IX oxidase. One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the second cDNA encodes a protein of 504 amino acid residues. Both deduced protein sequences share 27.2% identical amino acid residues. The first in vitro translated protoporphyrinogen IX oxidase could be translocated to plastids, and the approximately 53-kDa mature protein was detected in stroma and membrane fraction. The second enzyme was targeted to mitochondria without any detectable reduction in size. Localization of both enzymes in subcellular fractions was immunologically confirmed. Steady-state RNA analysis indicates an almost synchronous expression of both genes during tobacco plant development, greening of young seedlings, and diurnal and circadian growth. The mature plastidal and the mitochondrial isoenzyme were overexpressed in E. coli. Bacterial extracts containing the recombinant mitochondrial enzyme exhibit high protoporphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide. The data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochondria.

Figure 1

Sequence alignment of the tobacco protoporphyrinogen oxidases I (NTPPXI) and II (NTPPXII) with the homologous enzymes of the A. thaliana (ATHPPOX), B. subtilis (BACHEMY), and mouse (MUSPO). The glycine-rich motif is underlined. The positions of identical amino acid residues in all sequences are indicated by asterisks, and the positions of well conserved residues are indicated by dots.

Figure 2

Posttranslational uptake of in vitro synthesized precursor proteins. ³⁵S-labeled precursor of protoporphyrinogen IX oxidases I (PPX I) and II (PPX II) were synthesized by in vitro transcription/translation (TLS) and offered to intact pea chloroplasts (Left) or mitochondria (Right). (Left) Following the import reaction, plastids were either analyzed directly (PL) or after treatment with proteases (PL_P). Plastids were reisolated and fractionated into stroma (S) and membrane (M). (Right) Aliquots of the translation products (TLS) and of mitochondria after the import assay were incubated with (+) and without (−) proteinase K (PK) and applied to polyacrylamide gels. In both experiments, coproporphyrinogen oxidase (CPO) as a plastidal marker and a nuclear-encoded mitochondrial subunit of NADH:ubiquinone oxidoreductase (76 kDa) was included as a reference.

Figure 3

Immunological determination of the subcellular localization of the protoporphyrinogen IX oxidase isoforms. Proteins from total leaf extract (E), purified mitochondria (Mt), and plastids before (Pl) and after separation into stroma (S) and membrane (M) fraction were subjected to Western blot analysis using monospecific antibodies raised against recombinant plastidal (PPX I) and mitochondrial (PPX II) protoporphyrinogen IX oxidases.

Figure 4

Southern blot analysis of protoporphyrinogen IX oxidases in tobacco. Ten micrograms of genomic DNA was digested with BamHI (B), DraI (D), EcoRV (E), HindIII (H), XbaI (X), and KpnI (K) and subjected to Southern blot hybridization using ³²P-labeled cDNA inserts encoding the plastidal (PPX I) and mitochondrial (PPX II) isoenzymes.

Figure 5

Accumulation of protoporphyrinogen IX oxidase transcripts during leaf development and in roots. Roots and leaves were harvested from 6-week-old tobacco plants including the youngest leaves (–3) and the oldest fully expanded leaf (13). Total RNA was extracted, and 10 μg was subjected to Northern blot analysis using ³²P-labeled cDNA encoding both isoenzymes (PPX I and PPX II). Actin RNA levels were displayed as control for equal loading of RNA samples.

Figure 6

Steady-state levels of protoporphyrinogen IX oxidase transcripts under diurnal and circadian growth conditions. Tobacco plants were grown for 4 weeks in a 12-h/12-h light/dark cycle. Plants were harvested every 4 h starting 1 h after the onset of the light phase. Another set of plants was subsequently transferred to continuous light or darkness for 48 h. Leaf material was collected for the same 4-h time intervals. Total RNA was isolated and used for Northern blot analysis as described in the legend of Fig. 5.

Figure 7

Activity of the recombinant isoform II of protoporphyrinogen IX oxidase. An E. coli clone expressing the mitochondrial protoporphyrinogen IX oxidase (PPX II) was lysed. Enzyme activity of the bacterial homogenate (1) in the presence of 100 nM (2), 1 μM (3), and 10 μM (4) acifluorfen, and endogenous protoporphyrinogen IX oxidase activity of the E. coli strain SG 13009 harboring the plasmid pDS 56 (5) were measured as described in Material and Methods.