Molecular and genetic studies on the restoration of fertility in cytoplasmic male sterile petunia

Cytoplasmic male sterility is caused by novel chimeric mitochondrial genes whose protein products interfere with microsporogenesis. Expression of CMS genes is suppressed by one or more nuclear fertility restorer genes, thereby allowing normal pollen production. The petunia CMS-associated locus called pcf encodes a 19.5 kD protein that is highly expressed in the tapetum of floral buds. A dominant nuclear gene called restorer of fertility (Rf) suppresses CMS by dramatically reducing PCF expression.; Two homologous candidate open reading frames (designated Orf591 and Orf592 based on the number of predicted amino acids) found in a BIBAC clone identified from a genomic screen were each tested for their ability to restore fertility in CMS lines. Targeting assays using GFP fused to a putative targeting signal from Orf592 confirmed that it carries a mitochondrial transit sequence. Agrobacterium-mediated transformation of Petunia CMS lines using Orf592 caused fertility restoration in many primary transformants. A strong correlation in the presence of the transgene, reduced amount of PCF protein expression and fertility restoration was found in the primary transformants (T₀) and in two T₁ populations.; Both Orf591 and Orf592 DNA sequences contain pentatricopeptide (PPR) motifs; hence they were renamed Rf-PPR591 and Rf-PPR592, respectively. Unlike Rf-PPR592, Rf-PPR591 did not restore fertility in over three dozen independent transformants. The recessive allele rf-PPR592 had a 530-nt deletion in its promoter compared to the dominant allele Rf-PPR592 . Substitution of the promoter of Rf-PPR592 with the promoter of rf-PPR592 did not cause restoration in the transformants. Hence, the rf allele's inability to restore fertility is likely due to mutations in the coding region.; Transformation using the 2x35S::Rf-PPR592 transgene produced non-restored plants, probably because 35S promoter is poorly expressed in the tapetal cells. All the transgenes caused occasional abnormal phenotypes resembling meristem function mutants. These altered phenotypes may provide useful clues to the function of ancestral PPR genes or implicate the Rf gene in meristem development. Whether these phenotypes are the result of co-suppression or enhanced expression of the Rf gene or other endogenous genes remains to be determined.