A reevaluation of rice mitochondrial evolution based on the complete sequence of male-fertile and male-sterile mitochondrial genomes

Abstract

Plant mitochondrial genomes have features that distinguish them radically from their animal counterparts: a high rate of rearrangement, of uptake and loss of DNA sequences, and an extremely low point mutation rate. Perhaps the most unique structural feature of plant mitochondrial DNAs is the presence of large repeated sequences involved in intramolecular and intermolecular recombination. In addition, rare recombination events can occur across shorter repeats, creating rearrangements that result in aberrant phenotypes, including pollen abortion, which is known as cytoplasmic male sterility (CMS). Using next-generation sequencing, we pyrosequenced two rice (Oryza sativa) mitochondrial genomes that belong to the indica subspecies. One genome is normal, while the other carries the wild abortive-CMS. We find that numerous rearrangements in the rice mitochondrial genome occur even between close cytotypes during rice evolution. Unlike maize (Zea mays), a closely related species also belonging to the grass family, integration of plastid sequences did not play a role in the sequence divergence between rice cytotypes. This study also uncovered an excellent candidate for the wild abortive-CMS-encoding gene; like most of the CMS-associated open reading frames that are known in other species, this candidate was created via a rearrangement, is chimeric in structure, possesses predicted transmembrane domains, and coopted the promoter of a genuine mitochondrial gene. Our data give new insights into rice mitochondrial evolution, correcting previous reports.

Figure 1.

Rearrangements in the N and WA-CMS mitochondria confirmed by PCR. Shown are negative images of an ethidium bromide-stained gel loaded with PCR products amplified with primers designed to produce amplicons specifically associated with the rearrangements. Above each bracket are given the rearrangements with the same numbers used in Figures 2 and 3. Each set of PCRs includes three lanes, a negative control (−; no DNA template), the N mitochondrion template (N), and the WA-CMS mitochondrion template (W). The PCRs were replicated four times and stopped at an incremental number of cycles (as shown at left). The left panels contain PCR products of small size amplified with a regular Taq enzyme, while the right panels contain larger amplicons obtained with the herculase enzyme (Stratagene). The primers could not lead to PCR amplification with the Nipponbare mitochondrion as a template, either because they are too distant (2-1, 67 kb; 4-8, 88 kb; 4-13, 277 kb) or in the wrong orientation. At 20 cycles, the PCR products are only detected with the primers specific for each template rearrangement: 2-1, 2-2, and 2-5 for N and 4-3, 4-7, 4-1, 4-8, and 4-13 for WA-CMS. Primers associated with 2-6 and 2-9, the common rearrangements found in both N and WA-CMS, lead to the amplification of PCR products with both templates at 20 cycles. As the number of cycles increases, PCR products become detectable in the lanes where the template used does not carry the rearrangement. At 35 cycles, all the lanes with the presence of a template in the PCR show an amplicon, except for 4-8 with the N template. The PCR at 25 cycles with 4-13 primers did not work, as a PCR product is readily detectable at 20 cycles.

Figure 2.

Linear representation of the N mitochondrial genome. Colinear blocks from Nipponbare were reassembled according to the rearrangements found in the N mitochondrion. At the end of each block, the Nipponbare coordinates are given. Above the coordinates is given the rearrangement number in italics. 2-6 and 2-9 are rearrangements also found in WA-CMS. 2-4, 2-5, and 2-8 are connecting sequences that do not border gaps, whose coordinates are given in red. These positions, like 304, are duplicated, with one copy being part of a rearrangement while the other is not. Contiguous rearrangements are underlined. The color in Nipponbare blocks is similar to the one in Figure 3 and is taken from the original paper by Notsu et al. (2002). Underneath the contiguous sequence, colored blocks show the three large duplicated segments in the N mitochondrion with their respective sizes. The total length of the N mitochondrial genome is 637 kb and is slightly off here because noncontiguous rearrangements are represented as contiguous rearrangements. [See online article for color version of this figure.]

Figure 3.

Linear representation of the WA-CMS mitochondrial genome. Colinear blocks from Nipponbare were reassembled according to the rearrangements found in the WA-CMS mitochondrion. At the end of each block, the Nipponbare coordinates are given. Above the coordinates is given the rearrangement number in italics. 2-6 and 2-9 are rearrangements also found in N. 4-2, 4-11, and 4-13 are connecting sequences that do not border gaps, whose coordinates are given in red. These positions, like 215K, are duplicated, with one copy being part of a rearrangement while the other is not. Contiguous rearrangements are underlined. The color in Nipponbare blocks is similar to the one in Figure 4 and is taken from the original paper by Notsu et al. (2002). Underneath the contiguous sequence, colored blocks show the two duplicated segments in the WA-CMS mitochondrion with their respective sizes. The total length of the N mitochondrial genome is 402 kb and is slightly off here because noncontiguous rearrangements are represented as contiguous rearrangements. [See online article for color version of this figure.]

Figure 4.

Candidate WA-CSM-associated ORFs. A schematic representation of orf126 and orf86d, two ORFs that may cause the male sterility phenotype carried by the WA-CMS mitochondrial genome, shows the parts homologous to known mitochondrial genes. The numbers of codons are given below each coding sequence. Underneath the diagram representing the ORFs is presented the output of the TMHMM server, showing the location and probability associated with the predicted transmembrane domains. The start codon of rpl2 is indicated five codons downstream of the start codon of orf126. [See online article for color version of this figure.]

Figure 5.

orf126 is expressed in inflorescence and leaf of rice plants carrying the WA-CMS mitochondrial genome. The top panel shows RT-PCR amplification of a product specific to orf126; on the left is the –RT control, showing that the amplification in the right panel is due to cDNA and not to contaminating DNA during RNA extraction. The bottom panel shows orf126 PCR amplification from total genomic DNA. Lane 1, IR6888A (WA-CMS); lane 2, IR6888B (N); lane 3, IR62161R (N); lane 4, IR6888A × IR62161R; lane 5, IR68897A (WA-CMS); lane 6, IR68897B (N); lane 7, IR60819R (N); lane 8, IR68897A × IR60819R.

Figure 6.

Expression of mitochondrial genes in the inflorescence of a WA-CMS rice plant relative to orf126. The expression of the mitochondrial genes was measured by quantitative RT-PCR and normalized with orf126, the candidate WA-CMS-associated gene whose value was arbitrarily fixed to 100. The values are averages from three repeated measurements. * P < 0.05, ** P < 0.01.