Nuclear-mediated mitochondrial gene regulation and male fertility in higher plants: Light at the end of the tunnel?

H vigor, or heterosis, in higher plants is often attained through the use of cytoplasmic male sterility (CMS), a maternally inherited trait characterized by the absence of functional pollen. Hybrids of many plants are produced using CMS, wherein a male-sterile line is grown adjacent to a selected male-fertile line, which functions as a pollen source. Seed formed on the male-sterile line is then grown as a hybrid. When the hybrid plant is harvested for its vegetative parts, sugarbeet for instance, it is immaterial that the F1 hybrid is male-sterile. However, in plants where the seed is harvested, it is imperative that the F1 hybrid be male-fertile. Fundamental characteristics of CMS impact on this consideration. Historically, most sources of CMS were discovered as a result of genetic crosses involving normal, malefertile plants, wherein male-sterile plants were observed among the progeny. Lines that allow male sterility to be expressed were thus identified as potentially useful for plant breeding. It is this fact that enables the utilization of CMS: some lines allow CMS to be expressed and some do not. CMS systems are found in over 150 plant species and are usually attributed to chimeric ORFs in the mitochondrial genome. These ORFs encode novel proteins, which often interfere with mitochondrial function and pollen development. Evolution has provided the answer to these aberrant mitochondrial proteins through nuclear-encoded, restoration of fertility, or Rf, genes, which suppress mitochondrial abnormalities associated with male sterility. This suppression allows normal metabolic processes that lead to successful microsporogenesis. In many instances, this suppression is directly associated with Rf-genedependent, mitochondrial RNA modification and concurrent reduction of the CMS-associated protein (1). Although many mitochondrial genes associated with CMS have been characterized, the identification of Rf genes has proven elusive. Only maize Rf2a, a mitochondrial aldehyde dehydrogenase (ALDH), has been identified to date (2, 3). It is in this context that the observations of Bentolila et al. (4) represent a critical advance in the fundamental understanding of posttranscriptional, mitochondrial gene regulation and the restoration of male fertility in higher plants. In this issue of PNAS, Bentolila et al. report the molecular identification of Petunia Rf, the first isolation of a gene that controls the expression of an organellar gene encoding cytoplasmic male sterility. The CMS phenotype of Petunia is due to the expression of a 1.2 kilobase chimeric ORF, designated pcf (petunia CMS-associated fused). The pcf ORF is comprised of a 5 segment of the atp9 gene, parts of the first and second exons of the coxII gene, and unidentified sequences (urfS) (5). In the presence of Rf, the accumulation of pcf-derived transcripts is altered, and the accumulation of the 25-kDa PCF protein is reduced. As demonstrated by Bentolila et al. (4), Petunia Rf encodes a mitochondrially targeted protein almost entirely comprised of 14 repeats of a 35-aa pentatricopeptide repeat (PPR) motif. How then can the molecular identity of Petunia Rf be reconciled with its known effect on the pcf transcript or its encoded mitochondrial protein? There are over 200 genes harboring the PPR-motif, and its related TPR (tetratricopeptide repeat)-motif in the Arabidopsis genome, and two-thirds of these proteins are predicted to be targeted to organelles (6). PPRand TPR-motifs are found in helical-repeat proteins and would be predicted to have protein-binding properties. A good example of this result is the maize nuclearencoded protein, CRP1, which, as part of a multisubunit complex, is required not only for the translation of the chloroplast petA and petD mRNAs but also for the processing of the petD mRNA from a polycistronic transcript (7). The CRP1 sequence includes multiple tandem copies of this motif, and appears to activate a site-specific endonuclease independent of the role it plays in translation. Although TPR-containing proteins, would be predicted to mediate protein— protein interactions, PPR-containing proteins, on average, contain many more repeats that could result in additional ligands (6). Because the width of the central groove of PPRcontaining proteins is sufficient to hold an RNA strand and the positively charged surface at bottom of the groove could bind the phosphate backbone, Small and Peeters (6) suggest the possibility that PPR proteins could also be RNA binding. Taken together, an attractive scenario emerges wherein the multifamily of proteins carrying the PPR motif might participate in recognition and facilitation of events such as RNA editing (6) and endonucleolytic processing (7). In yeast, 50% of the cellular proteins are assembled into complexes comprised of one to five proteins; the other 50% are assembled into complexes with even greater numbers (8). If a particular RF protein is part of an RNA processing complex, and the PPR domain is essential, the determination that Petunia Rf is a member of one