DNA Gyrase Is Involved in Chloroplast Nucleoid Partitioning

Abstract

DNA gyrase, which catalyzes topological transformation of DNA, plays an essential role in replication and transcription in prokaryotes. Virus-induced gene silencing of NbGyrA or NbGyrB, which putatively encode DNA gyrase subunits A and B, respectively, resulted in leaf yellowing phenotypes in Nicotiana benthamiana. NbGyrA and NbGyrB complemented the gyrA and gyrB temperature-sensitive mutations of Escherichia coli, respectively, which indicates that the plant and bacterial subunits are functionally similar. NbGyrA and NbGyrB were targeted to both chloroplasts and mitochondria, and depletion of these subunits affected both organelles by reducing chloroplast numbers and inducing morphological and physiological abnormalities in both organelles. Flow cytometry analysis revealed that the average DNA content in the affected chloroplasts and mitochondria was significantly higher than in the control organelles. Furthermore, 49,6-diamidino-2-phenylindole staining revealed that the abnormal chloroplasts contained one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma. Pulse-field gel electrophoresis analyses of chloroplasts demonstrated that the sizes and/ or structure of the DNA molecules in the abnormal chloroplast nucleoids are highly aberrant. Based on these results, we propose that DNA gyrase plays a critical role in chloroplast nucleoid partitioning by regulating DNA topology.

pTAC2, -6, and -12 Are Components of the Transcriptionally Active Plastid Chromosome That Are Required for Plastid Gene Expression

“…While a putative DNA polymerase (Kimura et al, 2002) and two subunits of the topoisomerase II (Reece and Maxwell, 1991;Cho et al, 2004) are primarily involved in replication and the four plastid-encoded subunits a, b, b9, and b0 of PEP in transcription in the TAC, the exact role of the other components identified in this fraction is less clear. A Fe-SOD has also been detected in the PEP-A complex of a soluble plastid fraction from mustard Ogrzewalla et al, 2002;Loschelder et al, 2004), together with putative kinases, which we did not find in our preparations.…”

Inactivation of Organellar Glutamyl- and Seryl-tRNA Synthetases Leads to Developmental Arrest of Chloroplasts and Mitochondria in Higher Plants

“…The various GFP fusion constructs and the F 1 ATPase-␥-RFP fusion construct were introduced into protoplasts prepared from Arabidopsis seedlings by polyethylene glycol-mediated transformation (19). Expression of the fusion constructs was monitored at 24 h after transformation by confocal laser scanning microscopy, as described previously (18).…”

“…Chlorophyll autofluorescence was observed using an LP650 (excitation 488 nm, emission 650 nm) optical filter. MitoTracker Green FM (Molecular Probes) staining of protoplasts and 4Ј,6-diamidino-2-phenylindole staining of chloroplasts were carried out, as described previously (18).…”

“…Roughly equal numbers of protoplasts from the VIGS lines were embedded, and pulse-field gel electrophoresis was carried out as described (18,21). The rbcL cDNA was used as a probe.…”

“…VIGS is based on the phenomenon that gene expression is suppressed in a sequence-specific manner by infection with viral vectors carrying host genes (22) and has been proved to be a powerful tool to analyze embryoor seedling-lethal genes (17,18). Using a VIGS screen, we found that gene silencing of the N. benthamiana NbERS and NbSRS genes, encoding isoforms of GluRS and SerRS, respectively, causes severe leaf yellowing and abnormal leaf morphology, while maintaining almost normal plant growth.…”