Unique changes in mitochondrial genomes associated with reversions of S-type cytoplasmic male sterility in maizemar

Abstract

Cytoplasmic male sterility (CMS) in plants is usually associated with the expression of specific chimeric regions within rearranged mitochondrial genomes. Maize CMS-S plants express high amounts of a 1.6-kb mitochondrial RNA during microspore maturation, which is associated with the observed pollen abortion. This transcript carries two chimeric open reading frames, orf355 and orf77, both unique to CMS-S. CMS-S mitochondria also contain free linear DNA plasmids bearing terminal inverted repeats (TIRs). These TIRs recombine with TIR-homologous sequences that precede orf355/orf77 within the main mitochondrial genome to produce linear ends. Transcription of the 1.6-kb RNA is initiated from a promoter within the TIRs only when they are at linear ends. Reversions of CMS-S to fertility occur in certain nuclear backgrounds and are usually associated with loss of the S plasmids and/or the sterility-associated region. We describe an unusual set of independently recovered revertants from a single maternal lineage that retain both the S plasmids and an intact orf355/orf77 region but which do not produce the 1.6-kb RNA. A 7.3-kb inversion resulting from illegitimate recombination between 14-bp microrepeats has separated the genomic TIR sequences from the CMS-associated region. Although RNAs containing orf355/orf77 can still be detected in the revertants, they are not highly expressed during pollen development and they are no longer initiated from the TIR promoter at a protein-stabilized linear end. They appear instead to be co-transcribed with cytochrome oxidase subunit 2. The 7.3-kb inversion was not detected in CMS-S or in other fertile revertants. Therefore, this inversion appears to be a de novo mutation that has continued to sort out within a single maternal lineage, giving rise to fertile progeny in successive generations.

Figure 1. Organization of the sterility-associated regions in CMS-S mitochondria.

(A) Origin of linear mitochondrial genomes in CMS-S mitochondria. The TIRs of protein-bound plasmids, S1 and S2, recombine with TIR sequences upstream of the chimeric region orf355-orf77, leading to linearization of the genome. The chimeric region is present in two copies called σ-σ' and ψ-ψ' within the “circularized” CMS-S mitochondrial genome. Cox1 is located in σ while cox2 is located upstream of ψ. The direction of transcription for these cox genes is the opposite direction from the orf355 gene. Restriction sites for σ-σ' and ψ-ψ' are indicated: P  =  PstI, H =  HindIII, B = BamHI, X = XhoI. CMS-S genome coordinates are indicated above the lines (343K etc). The box outlines the extent of the 4.2-kb repeat. An S plasmid is represented by a short line with circles at the end (indicating protein caps). ψ* and σ* designate the linearized versions of the mitochondrial genomes after the TIR sequences in the main mtDNA have recombined with the S-plasmid TIR. The plasmid promoter within the TIR initiates transcription of the CMS-associated 1.6-kb RNA only if the chimeric region is present at a linear end . (B) Comparisons of mtDNA from sterile (S) and revertant (Rev) CMS-S plants. Gel blots of mtDNA restriction digests were hybridized with the DIG-labeled orf355, cox1 and cox2 probes. In each panel, all the samples shown were run on the same gel, but the BamHI and PstI lanes were not originally adjacent (indicted by the vertical line). ψ designates the orf355 copy near cox2, whereas σ designates the copy at cox1. The * indicates linear ends. Arrowheads point to a doublet detectable with both the orf355 and cox2 probes in the PstI lanes. On these blots, Rev4 and Rev4n are related revertants (NCS4 striped mutant and a non-striped relative).

Figure 2. Assaying for the presence of S plasmids in sterile and revertant lines.

Mitochondrial DNA after electrophoresis on a 0.8% agarose gel and staining with ethidium bromide. Revertants arising in the Wf9 nuclear background, Rev 1, 2, 3, 6, 8, and 9, have all retained S1 and S2 plasmids. Rev 4, which arose in M825, has lost S plasmids. S  =  CMS-S from B37, m  =  markers. When CMS-S mtDNA is prepared without using proteinase K (-pK), the protein-bound S plasmids, S1 and S2 do not migrate into the gel. A 2.3-kb linear plasmid unrelated to sterility is also seen.

Figure 3. Analysis of the regions surrounding the two copies of orf355/orf77.

PCR products amplified from ear shoot mtDNA with primer sets listed in Table 1. The predicted PCR products are diagrammed in Figure 6A. (A) Amplification with primer set 1, from a unique region within cox1 (σ) to the right end of the TIR sequence. (B) PCR products with primers from ψ and from the right end of the TIR sequence (primer set 2, Table 1). (C) PCR products with primers from the beginning of orf355 and the unique downstream σ' region (set 3). (D) PCR products with primers from the beginning of orf355 and the unique downstream ψ'region (set 4). (E) PCR products with primers from within cox1 (σ) and from σ' (set 5). (F) PCR products with primers from ψ and from ψ' (set 6).

Figure 4. A mtDNA inversion exists in the Rev1 cytoplasmic revertant.

(A) PCR products amplified from CMS-S (S) and Rev1 mtDNA using primer sets shown in Table 1. Set 3: amplifies from orf355 to σ', set 4: from orf355 to ψ', set 7: from the inverted ψ region (ψΔ) to σ', set 8: from ψΔ to ψ'. (B) Sequence data from the PCR product of primer set 8 shows a 14-bp region (bold), the probable site of recombination, located 33–47 bp before the orf355 start codon (underlined).

Figure 5. Mechanism of the mtDNA inversion.

(A) Diagram of the inversion event in the lineage represented by Rev1, Rev2 and Rev3. A 7.3-kb inversion with breakpoints 1.2 kb after the 3′ end of cox2 (3) and in the region between the TIR and orf355/77 (1; 80 bp after the final nucleotide of the TIR), has separated the intact orf355-orf77 region from its adjacent TIR sequence. Each inversion breakpoint is indicated. This inversion has placed the promoters of cox2 in the same orientation as orf355/orf77, which could allow transcription of the orf355/orf77 region from the cox2 promoters that are now in the same orientation as orf355/orf77 (shown by dotted arrows). (B) Diagram of the second copy of the ψΔ inversion attached to the σ' version of orf355/orf77. (C) Examination of the sequences at the breakpoints revealed a 14-bp repeat (with 13/14 bp identity). The repeats are in opposite orientation within the CMS-S progenitor mitochondrial genotype; thus, recombination between them would lead to the inversion.

Figure 6. Analysis of the organization of the region upstream of orf355/orf77 in revertants with the ψΔ inversion.

(A) Predicted PCR products for primer sets listed in Table 1. PCR products for primer sets 1–6 are shown in Figure 3. (B) PCR of CMS-S (S) mtDNA and two revertants (Rev1 and Rev2). Primer sets 9 and 10 amplify from orf355 into ψΔ, encompassing either the cox2 gene (Set10) or its promoter region (Set 9). Both products are specific for the inverted orientation of the cox2 region present in the two revertants.

Figure 7. All the cytoplasmic revertants lack the sterility-associated 1.6-kb RNA.

Mitochondrial RNAs from “pre-emergent” (A and B) or later “emerging” (C and D) tassels were probed with digoxigenin-labeled orf355 and cox2 probes. (A) The 1.6-kb RNA associated with sterility and the non-CMS-associated 2.8-kb RNA (originating from the ψ region) are not detectable in Rev1 and Rev2 when probed with orf355. A set of larger, ∼5.0, 4.3 and 4.0 kb (arrows), orf355-containing RNAs is detectable in the revertants. (B) A smaller RNA than the 1.6-kb band is detectable in Rev 8, and a slightly larger one is seen in Rev 9. (C) Cyt8 does have the orf355-hybridizing 1.6-kb RNA but it is reduced in amount compared with CMS-S. Cyt8 lacks the 2.8-kb RNA because the ψ region containing the promoter for the 2.8-kb RNA is absent from its genome. Rev3 shows the same set of larger orf355-containing transcripts as does Rev1. (D) The cox2 probe hybridizes to a pair of larger transcripts in Rev1 that are similar in size to the two largest RNAs (∼5.0 and 4.3-kb, arrowheads) detected with orf355.