Mitochondria play a critical role in the normal development of the plant male gametophyte and in the disruption of normal gametophyte development associated with cytoplasmically inherited male sterility (CMS). To investigate the role of mitochondria in these processes, the accumulation of mitochondrial gene transcripts and the accumulation of nuclear gene transcripts encoding mitochondrial proteins were investigated through male gametophyte development in normal maize and through the course of pollen abortion in CMS-S maize. Male gametophytes differing in developmental stage were isolated from male-fertile or male-sterile plants by sucrose density gradient centrifugation. Mature pollen was collected from dehiscent anthers of male-fertile plants. Aborted pollen, which collapsed during starch accumulation, was isolated from emergent tassels of CMS-S malesterile plants. Microspores, developing pollen and mature pollen exhibited striking differences in mitochondrial transcript accumulation. Mature pollen lacked detectable mitochondrial transcripts. Aborted pollen of CMS-S plants contained abundant, intact transcripts of all mitochondrial genes studied, but prematurely degraded transcripts of several nuclear genes. Transcripts of the CMS-S associated mitochondrial open reading frames (orf355 and orf77) were detected from the early stages of microspore development through the aborted pollen stage. The implications of these findings are discussed in terms of the mitochondrial requirements for pollen function and the mechanism of pollen abortion in CMS-S maize.

The restoration of male fertility in the sorghum IS1112 C (A3) male-sterile cytoplasm is through a two-gene gametophytic system involving complementary action of the restoring alleles Rf3 and Rf4. To develop markers suitable for mapping rf4, AFLP technology was applied to bulks of sterile and fertile individuals from a segregating BC(3)F(1) population. Three AFLP markers linked to rf4were identified and subsequently converted to STS/CAPS markers, two of which are co-dominant. Based on a population of 378 BC(1)F(1) individuals, two STS/CAPS markers, LW7 and LW8, mapped to within 5.31 and 3.18 cM, respectively, of rf4, while an STS marker, LW9, was positioned 0.79 cM on the flanking side of rf4. Markers LW8 and LW9 were used to screen sorghum BAC libraries to identify the genomic region encoding rf4. A series of BAC clones shown to represent a genomic region of linkage group E were identified by the rf4-linked markers. A contig of BAC clones flanking the LW9 marker represent seed clones on linkage group E, from which fine mapping of the rf4 locus and chromosome walking can be initiated.

Cytoplasmic male sterility (CMS) is encoded by the plant mitochondrial genome and can be reversed by nuclear restorer-of-fertility(Rf) alleles. In the CMS-S system of maize, reproductive failure and fertility restoration are gametophytic, occurring during the starch-filling stages of pollen development. Transcripts of the CMS-S-associated mitochondrial open reading frames (orf355 and orf77) are present from the early stages of microspore development through the aborted pollen stage. To investigate the molecular basis of fertility restoration, we compared mitochondrial-transcript accumulation in aborting CMS-S pollen and in CMS-S pollen restored to fertility by the Rf3 nuclear allele. In the presence of the Rf3 allele, novel, shorter transcripts of the orf355-orf77, cob and atp6 mitochondrial genes were created, and the relative abundance of larger transcripts was decreased for each of these loci. The altered transcript patterns cosegregated with male fertility conditioned by the Rf3 allele. The novel cob and atp6 transcripts were also observed in leaf-tissues of both normal and S-cytoplasm plants carrying the Rf3 allele. These observations support the hypothesis that the Rf3 allele encodes, or regulates, a modifier of mitochondrial transcript (Mmt) activity that affects both CMS and essential mitochondrial gene transcripts.

Mitochondrial biogenesis and function depend upon the interaction of mitochondrial and nuclear genomes. Forward genetic analysis of mitochondrial function presents a challenge in organisms that are obligated to respire. In the S-cytoplasmic male sterility (CMS-S) system of maize, expression of mitochondrial open reading frames (orf355-orf77) conditions collapse of developing haploid pollen. Nuclear restorer-of-fertility mutations that circumvent pollen collapse are often homozygous lethal. These spontaneous mutations potentially result from disruption of nuclear genes required for mitochondrial gene expression, in contrast to homozygous-viable restorer-of-fertility alleles that function to block or compensate for the expression of mitochondrial CMS genes. Consistent with this hypothesis, the homozygous-lethal restoring allele historically designated RfIII was shown to be recessive in diploid pollen produced by tetraploid CMS-S plants. Accordingly, the symbol for this allele has been changed to restorer-of-fertility  lethal  1 (rfl1). In haploid rfl1 pollen, orf355-orf77 transcripts and mitochondrial transcripts encoding the α-subunit of the ATP synthase (ATPA) were decreased in abundance. Haploid rfl1 pollen failed to accumulate wild-type levels of ATPA protein, indicating that functional requirements for mitochondrial protein accumulation are relaxed in maize pollen. The CMS-S system and rfl mutations therefore allow for the selection of nuclear mutations disrupting mitochondrial biogenesis in a multicellular eukaryote.