A mitochondrial mutator system in maize

Abstract

The P2 line of maize (Zea mays) is characterized by mitochondrial genome destabilization, initiated by recessive nuclear mutations. These alleles alter copy number control of mitochondrial subgenomes and disrupt normal transfer of mitochondrial genomic components to progeny, resulting in differences in mitochondrial DNA profiles among sibling plants and between parents and progeny. The mitochondrial DNA changes are often associated with variably defective phenotypes, reflecting depletion of essential mitochondrial genes. The P2 nuclear genotype can be considered a natural mutagenesis system for maize mitochondria. It dramatically accelerates mitochondrial genomic divergence by increasing low copy-number subgenomes, by rapidly amplifying aberrant recombination products, and by causing the random loss of normal components of the mitochondrial genomes.

Figure 1.

Individual mtDNAs from P2-line families display multiple RFLPs that differ between sibling plants. mtDNA samples from four siblings (family 4712, lanes 1–4), and from three progeny of the self-pollinated 4712-4 individual (family 96H55, lanes 5–7) were digested with XhoI, and DNA gel blots were hybridized to the cosmid probes N8B1 (left) and N7C9 (right). B37N mtDNA is used for a normal (NB) control.

Figure 2.

Different copy number effects in mtDNAs of P2-inbred plants. A, mtDNA samples of individuals from P2 families 4712 and H55 (as in Fig. 1) were hybridized to the 7.8-kb XhoI fragment from cosmid N7C9. B, The blot was rehybridized to the 3.7-kb BamHI fragment from cosmid N6D6 (rps3/rpl16 probe). C, XhoI-digested DNAs from four P2 sibling plants of family H115 were hybridized with the tRNA-Trp coding region of the n-plasmid (left), and the 1,020-bp PCR probe from cosmid N8B11 as a loading control (right; see “Materials and Methods”).

Figure 3.

Similar mtDNA rearrangements can be detected in P2-inbred and in P2-converted lines. A to C, A DNA gel blot with XhoI-digested mtDNA samples from five siblings of the P2-converted family 5714 was hybridized with the 28S rRNA-containing, 14.2-kb BamHI fragment from cosmid N5F6 (A); 10.5-kb XhoI fragment from cosmid N8B1 (B); and 2.9-kb fragment from cosmid N7E8 (C). The plant 5535-7 was a sibling of the female parent of 5714 plants. D to F, The DNA gel blot with P2-inbred families 4712 and H55 was hybridized to the same set of probes shown in A to C above. The asterisk indicates a 9.7-kb XhoI fragment in NB (Mo17N and B37N) mtDNAs. G, Recombination giving rise to the rec-rps2 sequence. H, DNA gel of the rec-rps2 PCR product, using primers specific to the 5′ flank of rps2A (P5) and the 3′ flank of rps2B (P8).

Figure 4.

Polymorphic mitochondrial transcripts in P2-converted plants. A, RNA samples from ear shoots of two P2-converted plants, 5714-2 and 5714-3, hybridized successively with probes specific to the 5′ UTR of rps2A, 3′ UTR of rps2B, and rps2 ORF (see “Materials and Methods”). B, rps2 isomers in mtDNA and the hybridization probes used. C, Mitochondrial RNA samples from individual 5714 siblings were hybridized with probes for the indicated mitochondrial genes (only mature transcripts are shown). Loading controls are shown by the hybridization with a mitochondrial 18S rRNA probe.

Figure 5.

Segregation analysis of the rec-rps2 amplification. Progeny from a backcross of a Mo17/P2 F1 hybrid (plant 4992-17) with P2 pollen were grown to obtain 4-d-old seedling roots. Total DNA isolated from 14 individual samples was digested with HindIII, and gel blots were hybridized to the PCR-amplified 5′ flank of rps2A (A), 5′ flank of rps2B (B), or one of the flanks of the 0.7-kb repeat (C; see “Materials and Methods”). An additional 2.1-kb HindIII fragment detected with the rps2B 5′ probe is likely to represent an unknown homologous sequence from elsewhere within the mitochondrial genome; (e.g. Hartmann et al., 1994). Plant 5535-22 is a related P2-converted plant after one more generation of backcrossing. D, Progeny from a backcross of another Mo17/P2 F1 hybrid (plant 4992-22) with P2 pollen was analyzed as in A, except that XhoI was used to digest mtDNA. E, Schematic representation of the possible rps2 recombination isomers. Hd, Positions of HindIII sites.

Figure 6.

Cosegregation of amplification events for different mtDNA regions. Siblings from three related families of P2-converted maize plants with CMS-T cytoplasm were analyzed by hybridizing mtDNA gel blots with three probes: A, rps2A (5′ UTR); B, nad9; C, rps3/rpl16. D, Linearized map of the CMS-T mtDNA (Fauron et al., 1990; C.M.-R. Fauron, personal communication) with the hybridization probes indicated.

Figure 7.

P2 siblings with wild-type mtDNA copy number control show differences in mtDNA partitioning. A, mtDNAs from six 5677 P2(A)XP2(B) siblings were probed with the rps2 ORF probe to check the rps2B/rec-rps2 ratio. B, mtDNAs from three of the 5677 siblings, the grandparent 4712-4, and B37N control were probed with cosmids (from left to right) N8E6, N8B11, and N8B1.

Figure 8.

Rapid amplification of an aberrant recombination product in mitochondria from P2-inbred plants. A, The mtDNAs from B37N (NB mtDNA control), 3999-4, and three individual progeny from 3999-7 (sibs 1–3) and from 3999-10 (sibs 4–6) were digested with PstI and probed with the R1-homologous 7-kb PstI fragment. The plants 3999-4, -7, and -10 were sibling plants. B, PCR amplification of the R1/rps13 rearrangement, using primers (P15 and P16) specific for the rearranged 2.1-kb PstI fragment, and the same mtDNAs as in A for templates. C, Schematic representation of the aberrant R1/rps13 recombination product and the recombination site.