| The pcf gene, isolated from the mitochondrial genome of cytoplasmic male sterile Petunia, has been implicated as a cause of the phenotype. The effect of expression of the pcf gene on metabolism in two heterologous systems, Neurospora crassa and Escherichia coli, was examined and the results are reported here. Neurospora crassa cells were transformed with the pcf gene to examine the mechanism of cytoplasmic male sterility in Petunia. Respiration was compared in isolated mitochondria from transgenic and wild type cell lines. Initially, cells expressing PCF showed an increased rate of respiration due to an increase in the alternative oxidase activity. However, over time, respiration in mitochondria from transformed cells became indistinguishable from that of the untransformed progenitors. Neither the protein nor the RNA from the transgene could be detected in these newly “wild-type” cells. DNA blot hybridization analysis indicated that the pcf transgene had become methylated, perhaps quelling expression. Although DNA blot hybridization indicated that the transgene could be markedly demethylated as the result of growth on medium containing 5-azacytidine, cultures could not be maintained and the effect on respiration by the transgene could not be determined.; The effect of the pcf gene was also examined in a second heterologous system through expression in Escherichia coli. The gene was modified for regulated expression and three different chimeric gene constructs were inserted to determine the effect on the growth rates of E. coli. Expression of the entire PCF protein or of the N-terminal portion inhibited growth of E. coli cells. Expression of the C-terminal portion of PCF had no significant effect on the growth of E. coli, even though this is the only part of the protein that can be detected in mitochondria from sterile Petunia. The effect of PCF on respiration in E. coli was examined using respiratory mutants of E. coli and by growth on minimal media. No direct interaction of PCF polypeptides with the respiratory chain subunits could be determined. This pattern of growth raises the possibility that the N-terminal portion of the pcf gene may have a role in abortion of pollen development in CMS plants. |
