The petunia fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. To determine whether the pcf gene is expressed at the protein level, we produced antibodies to synthetic peptides specified by the coxII and urfS portions of the pcf gene. Anti-COXII peptide antibodies recognized petunia COXII but no other mitochondrial proteins. Anti-URF-S peptide antibodies recognized a 20-kilodalton protein present in both cytoplasmic male sterile and fertile lines and a protein with an apparent molecular mass of 25 kilodaltons present only in cytoplasmic male sterile lines. The 25-kilodalton protein was found to be synthesized by isolated mitochondria and to fractionate into both the soluble and membrane portions of disrupted mitochondria, whereas the 20-kilodalton protein was found only in the membrane fraction. The abundance of the 25-kilodalton protein was much lower in fertile plants carrying the cytoplasmic male sterile cytoplasm and a single dominant nuclear fertility restorer gene, Rf. Thus, the pcf gene is correlated with cytoplasmic male sterility not only by its co-segregation with the phenotype in somatic hybrids, but also by the modification of its expression at the protein level through the action of a nuclear gene that confers fertility.

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.

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ABSTRACrThe following parameters were found to prolong the time-course of translation by isolated pea (Pisum sativum, cv Progress No. 9) chloroplasts: addition of other amino acids (an effect synergistic with sufficient free Mg"), use of lower light intensities, and additions of inorganic phosphate and ATP. In a chloroplast system which includes these parameters, active translation usually extends to almost an hour. The total amount of leucine incorporated is routinely 60 to 100 nanomoles/milligram chlorophyll and often 200 nanomoles/milligram chlorophyll. Accurate estimation of the amount of amino acid incorporated depends on supplying the labeled amino acid at a concentration sufficient to overcome isotope dilution effects from endogenous pools. Approximately 39 thylakoid and 60 stroma polypeptides were visible on autoradiographs after labeling with I3SImethionine. Label in a few of the polypeptide bands was increased or decreased by specific changes in the reaction conditions. Due to the long period of activity and the large number of labeled products, this chloroplast system should be useful for future studies of chloroplast translation.Isolated, intact chloroplasts have been used to identify products of chloroplast protein synthesis as well as to investigate posttranslational protein transport, processing, assembly, and insertion into membranes. The results of such studies may depend strongly on the characteristics and competence of the in vitro chloroplast systems. For example, in an early in vitro chloroplast system only one soluble product was detected (1), while in more recent work up to 39 thylakoid (13) and 80 soluble (10) polypeptide products have been reported. The movement across the envelope of cytoplasmically synthesized precursors to chloroplast proteins depends on an adequate supply of internal ATP (15). Processing of the chloroplast-synthesized, herbicidebinding protein (6) has been demonstrated to occur in isolated pea (9,17) and lettuce (17) chloroplasts. However, in maize the processing has been seen in vivo but so far not in organello (14) perhaps because some necessary condition(s) has not been identified. Assembly of cytoplasmically synthesized small subunits and chloroplast-synthesized large subunits of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase into a holoenzyme did not occur in isolated chloroplasts until sorbitol, rather than KCI, was used as an osmoticum (2,8). Clearly, increasing the functionality of isolated chloroplasts will increase