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RECG maintains plastid and mitochondrial genome stability by suppressing extensive recombination between short dispersed repeats.

Odahara M, Masuda Y, Sato M, Wakazaki M, Harada C, Toyooka K, Sekine Y - PLoS Genet. (2015)

Bottom Line: This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants.Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci.These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, College of Science, Rikkyo (St. Paul's) University, Toshima-ku, Tokyo, Japan.

ABSTRACT
Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO) mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8-79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA) instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12-63 bp) in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

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Mitochondrial transcripts in RECG KO plants.A and B. Detailed qRT-PCR analysis of nad7 (A) and nad9 (B) transcripts. Positions of the primers used in the qPCR are schematically represented in the upper parts of the panels. Coding regions are represented by grey boxes. Positions and directions of the primers are shown by arrows. Positions of the repeats involved in the rearrangements between nad7 and nad9 are indicated by triangles. Relative levels of segments of mitochondrial transcripts from nad7 and nad9 were normalized to reference of nuclear gene ST-P 2a transcript. WT was given a value of 1. Slight or no amplification was observed in no reverse-transcription controls (S5G Fig.). The data represent mean of three replicates ± SD. *p<0.01 (versus WT). C. RT-PCR analysis of nad7-nad9 chimeric transcripts. nad7-nad9 chimeric transcripts were amplified using cDNA from WT and RECG KO lines at cycles indicated on the left of the picture. Actin was amplified as an internal control.
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pgen.1005080.g007: Mitochondrial transcripts in RECG KO plants.A and B. Detailed qRT-PCR analysis of nad7 (A) and nad9 (B) transcripts. Positions of the primers used in the qPCR are schematically represented in the upper parts of the panels. Coding regions are represented by grey boxes. Positions and directions of the primers are shown by arrows. Positions of the repeats involved in the rearrangements between nad7 and nad9 are indicated by triangles. Relative levels of segments of mitochondrial transcripts from nad7 and nad9 were normalized to reference of nuclear gene ST-P 2a transcript. WT was given a value of 1. Slight or no amplification was observed in no reverse-transcription controls (S5G Fig.). The data represent mean of three replicates ± SD. *p<0.01 (versus WT). C. RT-PCR analysis of nad7-nad9 chimeric transcripts. nad7-nad9 chimeric transcripts were amplified using cDNA from WT and RECG KO lines at cycles indicated on the left of the picture. Actin was amplified as an internal control.

Mentions: The data presented here showed efficient rearrangements of RECG KO mtDNA at nad7, nad9, atp9, and ccmF loci caused by recombination between short repeats. Quantitative analysis using DNA gel blots revealed that the copy number of normal mtDNA bands (e.g., 1.5 kb of nad7 band and 1.7 kb nad9 band in S5A and B Fig.) in the RECG KO plants decreased to approximately 30%–45% and 35%–50% of WT at nad7 and nad9 loci, respectively. The copy number of normal mtDNA bands at atp9 and ccmF loci did not significantly change (S5C and D Fig.). To investigate the effect of the mtDNA rearrangements on mitochondrial transcripts, we analyzed the transcripts of these loci in the RECG KO plants. Quantitative RT-PCR (qRT-PCR) analysis demonstrated a significant reduction in the levels of transcripts from nad7 and nad9 for some of the primer pairs in the RECG KO mutants (Fig. 7A and B). We found a significant reduction (<10% of WT levels) in the levels of the transcript fragments when the primers were arranged to amplify a segment including a junction of exon 2 and 3 for nad7 or exon 1 and 2 for nad9 (Fig. 7A and B). Because the introns contain repeats involved in the mtDNA rearrangements (Fig. 7A and B), these results suggest that a substantial number of the nad7 and nad9 transcripts exist as chimeric transcripts of nad7 and nad9 and not as individual intact forms. RT-PCR analysis demonstrated the efficient amplification of nad7-nad9 chimeric transcripts from RECG KO mutants (Fig. 7C). We confirmed that the chimeric transcripts were precisely spliced between nad7 exon2 and nad9 exon2 (S5E Fig.). In contrast, similar qRT-PCR analysis demonstrated no significant differences in the levels of transcripts from ccmF and atp9 loci between the WT and RECG KO mutants (S5F Fig.). These results suggest that the efficient rearrangements of some mtDNA loci were associated with a decrease in the normal levels of mtDNA and a significant decrease in the number of the corresponding intact transcripts.


RECG maintains plastid and mitochondrial genome stability by suppressing extensive recombination between short dispersed repeats.

Odahara M, Masuda Y, Sato M, Wakazaki M, Harada C, Toyooka K, Sekine Y - PLoS Genet. (2015)

Mitochondrial transcripts in RECG KO plants.A and B. Detailed qRT-PCR analysis of nad7 (A) and nad9 (B) transcripts. Positions of the primers used in the qPCR are schematically represented in the upper parts of the panels. Coding regions are represented by grey boxes. Positions and directions of the primers are shown by arrows. Positions of the repeats involved in the rearrangements between nad7 and nad9 are indicated by triangles. Relative levels of segments of mitochondrial transcripts from nad7 and nad9 were normalized to reference of nuclear gene ST-P 2a transcript. WT was given a value of 1. Slight or no amplification was observed in no reverse-transcription controls (S5G Fig.). The data represent mean of three replicates ± SD. *p<0.01 (versus WT). C. RT-PCR analysis of nad7-nad9 chimeric transcripts. nad7-nad9 chimeric transcripts were amplified using cDNA from WT and RECG KO lines at cycles indicated on the left of the picture. Actin was amplified as an internal control.
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Related In: Results  -  Collection

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pgen.1005080.g007: Mitochondrial transcripts in RECG KO plants.A and B. Detailed qRT-PCR analysis of nad7 (A) and nad9 (B) transcripts. Positions of the primers used in the qPCR are schematically represented in the upper parts of the panels. Coding regions are represented by grey boxes. Positions and directions of the primers are shown by arrows. Positions of the repeats involved in the rearrangements between nad7 and nad9 are indicated by triangles. Relative levels of segments of mitochondrial transcripts from nad7 and nad9 were normalized to reference of nuclear gene ST-P 2a transcript. WT was given a value of 1. Slight or no amplification was observed in no reverse-transcription controls (S5G Fig.). The data represent mean of three replicates ± SD. *p<0.01 (versus WT). C. RT-PCR analysis of nad7-nad9 chimeric transcripts. nad7-nad9 chimeric transcripts were amplified using cDNA from WT and RECG KO lines at cycles indicated on the left of the picture. Actin was amplified as an internal control.
Mentions: The data presented here showed efficient rearrangements of RECG KO mtDNA at nad7, nad9, atp9, and ccmF loci caused by recombination between short repeats. Quantitative analysis using DNA gel blots revealed that the copy number of normal mtDNA bands (e.g., 1.5 kb of nad7 band and 1.7 kb nad9 band in S5A and B Fig.) in the RECG KO plants decreased to approximately 30%–45% and 35%–50% of WT at nad7 and nad9 loci, respectively. The copy number of normal mtDNA bands at atp9 and ccmF loci did not significantly change (S5C and D Fig.). To investigate the effect of the mtDNA rearrangements on mitochondrial transcripts, we analyzed the transcripts of these loci in the RECG KO plants. Quantitative RT-PCR (qRT-PCR) analysis demonstrated a significant reduction in the levels of transcripts from nad7 and nad9 for some of the primer pairs in the RECG KO mutants (Fig. 7A and B). We found a significant reduction (<10% of WT levels) in the levels of the transcript fragments when the primers were arranged to amplify a segment including a junction of exon 2 and 3 for nad7 or exon 1 and 2 for nad9 (Fig. 7A and B). Because the introns contain repeats involved in the mtDNA rearrangements (Fig. 7A and B), these results suggest that a substantial number of the nad7 and nad9 transcripts exist as chimeric transcripts of nad7 and nad9 and not as individual intact forms. RT-PCR analysis demonstrated the efficient amplification of nad7-nad9 chimeric transcripts from RECG KO mutants (Fig. 7C). We confirmed that the chimeric transcripts were precisely spliced between nad7 exon2 and nad9 exon2 (S5E Fig.). In contrast, similar qRT-PCR analysis demonstrated no significant differences in the levels of transcripts from ccmF and atp9 loci between the WT and RECG KO mutants (S5F Fig.). These results suggest that the efficient rearrangements of some mtDNA loci were associated with a decrease in the normal levels of mtDNA and a significant decrease in the number of the corresponding intact transcripts.

Bottom Line: This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants.Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci.These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, College of Science, Rikkyo (St. Paul's) University, Toshima-ku, Tokyo, Japan.

ABSTRACT
Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO) mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8-79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA) instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12-63 bp) in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

Show MeSH
Related in: MedlinePlus