Dynamic Interplay between Nucleoid Segregation and Genome Integrity in Chlamydomonas Chloroplasts

Abstract

The chloroplast (cp) genome is organized as nucleoids that are dispersed throughout the cp stroma. Previously, a cp homolog of bacterial recombinase RecA (cpRECA) was shown to be involved in the maintenance of cp genome integrity by repairing damaged chloroplast DNA and by suppressing aberrant recombination between short dispersed repeats in the moss Physcomitrella patens Here, overexpression and knockdown analysis of cpRECA in the green alga Chlamydomonas reinhardtii revealed that cpRECA was involved in cp nucleoid dynamics as well as having a role in maintaining cp genome integrity. Overexpression of cpRECA tagged with yellow fluorescent protein or hemagglutinin resulted in the formation of giant filamentous structures that colocalized exclusively to chloroplast DNA and cpRECA localized to cp nucleoids in a heterogenous manner. Knockdown of cpRECA led to a significant reduction in cp nucleoid number that was accompanied by nucleoid enlargement. This phenotype resembled those of gyrase inhibitor-treated cells and monokaryotic chloroplast mutant cells and suggested that cpRECA was involved in organizing cp nucleoid dynamics. The cp genome also was destabilized by induced recombination between short dispersed repeats in cpRECA-knockdown cells and gyrase inhibitor-treated cells. Taken together, these results suggest that cpRECA and gyrase are both involved in nucleoid dynamics and the maintenance of genome integrity and that the mechanisms underlying these processes may be intimately related in C. reinhardtii cps.

Figure 1.

Subcellular localization of cpRECA. A, Immunofluorescence visualization of cpRECA. C. reinhardtii wild-type cells were immunolabeled using anti-cpRECA antibody. Fluorescence intensities of colocalized 4′,6-diamino-phenylindole (DAPI) and cpRECA (indicated by the arrow) were determined (at right). B to D, Subcellular localization of cpRECA-YFP or cpRECA (K127R)-YFP. YFP fluorescence (B) and anti-YFP immunofluorescence (C and D) were observed in C. reinhardtii cells ectopically expressing cpRECA-YFP or cpRECA (K127R)-YFP. Cells were stained with DAPI. DIC, Differential interference contrast; N, nucleus. Bars = 5 μm.

Figure 2.

The cp nucleoid number and size in cpRECA-KD cells. A, C. reinhardtii cp nucleoids in cpRECA-KD (a), moc A84 (b), moc G33 (c), and wild-type (d) cells. DNA was stained using SYBR Green I. SYBR Green I (green) and chlorophyll (red) fluorescence signals were observed simultaneously using fluorescence microscopy. N denotes the nucleus, and arrows indicate examples of cp nucleoids (cpN). Bar = 5 μm. B, Histograms of cp nucleoid number per cell in cpRECA-KD and wild-type (WT) cells.

Figure 3.

Effects of gyrase inhibitors on cp nucleoids. Wild-type cells were cultivated in medium containing the gyrase inhibitor nalidixic acid or novobiocin. DNA was stained using SYBR Green I. SYBR Green I (green) and chlorophyll (red) fluorescence signals were observed simultaneously using fluorescence microscopy. N and cpN indicate nucleus and cp nucleoids, respectively. Bar = 5 μm.

Figure 4.

Effects of cpRECA knockdown on cpDNA. A to C, Quantification of CD5, CI12, and CD15 recombination products. qPCR analysis is shown for relative copy numbers of CD5 (A), CI12 (B), and CD15 (C) cpDNA recombination products. Copy numbers were normalized to cp psbB. Data represent averages of three replicates. D, PCR analysis of CD20 recombination products in cpRECA-KD lines using psbB as an amplified internal control. E, qPCR analysis of relative cpDNA psbB copy number, normalized to nuclear Cblp. F, Positions of CD5, CI12, CD15, and CD20 in C. reinhardtii cpDNA. Large inverted repeats are shown with thick black lines. SDRs are indicated with triangles. The cpDNA sequence corresponds to accession NC_005353 when taken clockwise from position 1. Data represent means of three replicates. *, P < 0.01 (compared with the wild type [WT]).

Figure 5.

Chloroplast genome instability in gyrase inhibitor-treated cells. A and B, qPCR analysis of relative CD5 copy numbers in wild-type cells treated with nalidixic acid (A) or novobiocin (B). Copy numbers were normalized to cp psbB. C and D, qPCR analysis of CI12 (C) or CD15 (D) recombination products in cells treated with 1.6 mm nalidixic acid or 400 μm novobiocin. Copy numbers were normalized to psbB. E, PCR analysis of CD20 recombination products in cells treated with nalidixic acid or novobiocin, using psbB as an amplified internal control. WT, The wild type. F and G, qPCR analysis of relative cpDNA psbB copy number, normalized to nuclear Cblp, in wild-type cells treated with nalidixic acid (F) or novobiocin (G). The untreated wild-type ratio was set as 1. Data represent means of three replicates. *, P < 0.01 (compared with the untreated control).