Sequencing, processing, and localization of the petunia CMS‐associated mitochondrial protein

Abstract

SummaryThe petunia mitochondrial fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxll, and an unidentified reading frame termed urfS. Pcf transcripts are modified by editing at 11 sites. Codon usage and nearest neighbor analysis suggest that the urfS region is not derived originally from a plant mitochondrial coding region. Although the gene contains an open reading frame coding for a 43 kDa protein, a 25 kDa gene product has previously been identified (Nivison and Hanson, 1989). N-terminal sequencing revealed that the 25 kDa protein is encoded within the urfS portion of pcf and that its actual molecular mass is 19.5 kDa. Through pulsechase labeling of protein in isolated mitochondria, the 25 kDa protein was found to be processed from a 43 kDa precursor protein representing the entire pcf gene sequence. Antibodies to synthetic peptides encoded by the atp9 and coxll portions of pcf recognized petunia ATP9 or COXll but no other mitochondrial proteins on immunoblots. Controlled proteolysis experiments showed that both the 43 kDa precursor and the 25 kDa protein are soluble or loosely associated with membranes. Thus, the 25 kDa protein appears to be the only pcf-encoded protein that accumulates in mitochondria.

A Plant Mitochondrial Sequence Transcribed in Transgenic Tobacco Chloroplasts Is Not Edited

“…Though many mitochondrial DNA-encoded proteins contain membrane-spanning domains, which could conceivably be deleterious when expressed in chloroplasts, coxII exon 2 does not specify such a hydrophobic polypeptide. Most importantly, coxII exon 2 sequences had already been observed to be edited in a disturbed context in a naturally occurring chimeric gene (21). Even when flanked on the 5Ј side by a partial atp9-derived sequence and incomplete coxII exon 1 sequences and on the 3Ј end by an unidentified reading frame, and even though the large group II intron is missing, coxII exon 2 sequences are edited when located in the abnormal petunia pcf gene (Fig.…”

“…Though several mitochondrial sequences in both petunia and maize plants have been shown to be edited in abnormal contexts in naturally occurring chimeric mitochondrial genes (16,21), comparable edited recombined genes have not been detected in chloroplasts. It is possible that editing of chloroplast transcripts is more tightly regulated and has speciesspecific components.…”

Cost of Having the Largest Mitochondrial Genome: Evolutionary Mechanism of Plant Mitochondrial Genome

“…The mitochondrial protein degradation system is also associated with the anther-specific accumulation of the CMS-associated ORF239 polypeptide in the common bean [98]. The petunia CMS-associated PCF protein is trimmed into a smaller polypeptide from a precursor [75]. Therefore, it seems likely that most unique polypeptides are degraded.…”

“…An example is the maize urf13-T gene, which contains two duplicated segments of rrn26, forming an ORF that encodes 115 amino acid residues [74]. A second example is the petunia pcf gene, which contains a duplicated segment of atp9, two duplicated segments of cox2, and a sequence of unknown origin, forming an ORF that encodes 402 amino acid residues [75]. There is no sequence homology between any of the known CMS-associated ORFs except in two cases: orf224, associated with Brassica pol CMS, and orf222, associated with Brassica nap CMS, exhibit 79% homology at the amino acid level [36]; and rice orf79 and sorghum orf107 show some similarity.…”

Interactions of Mitochondrial and Nuclear Genes That Affect Male Gametophyte Development

“…One class of transcripts, those that terminate at -121 before the pcf gene, are reduced in restored lines (Young and Hanson, 1987), and the abundance of the protein products of the pcf gene is greatly reduced . pcf encodes a 45-kD protein that is processed to a 19.5-kD protein that exhibits a mobility of 25 kD on acrylamide gels Nivison et al, 1994). The larger precursor protein appears to be efficiently processed and is present in much lower quantities than is the 19.5-kD protein.…”