Chloroplast‐targeted bacterial RecA proteins confer tolerance to chloroplast DNA damage by methyl viologen or UV‐C radiation in tobacco (Nicotiana tabacum) plants

Abstract

The nature and importance of the DNA repair system in the chloroplasts of higher plants under oxidative stress or UV radiation-induced genotoxicity was investigated via gain-of-functional approaches exploiting bacterial RecAs. For this purpose, transgenic tobacco (Nicotiana tabacum) plants and cell suspensions overexpressing Escherichia coli or Pseudomonas aeruginosa RecA fused to a chloroplast-targeting transit peptide were first produced. The transgenic tobacco plants maintained higher amounts of chloroplast DNA compared with wild-type (WT) upon treatments with methyl viologen (MV), a herbicide that generates reactive oxygen species (ROS) in chloroplasts. Consistent with these results, the transgenic tobacco leaves showed less bleaching than WT following MV exposure. Similarly, the MV-treated transgenic Arabidopsis plants overexpressing the chloroplast RecA homologue RECA1 showed weak bleaching, while the recA1 mutant showed opposite results upon MV treatment. In addition, when exposed to UV-C radiation, the dark-grown E. coli RecA-overexpressing transgenic tobacco cell suspensions, but not their WT counterparts, resumed growth and greening after the recovery period under light conditions. Measurements of UV radiation-induced chloroplast DNA damage using DraI assays (Harlow et al. 1994) with the chloroplast rbcL DNA probe and quantitative PCR analyses showed that the transgenic cell suspensions better repaired their UV-C radiation-induced chloroplast DNA lesions compared with WT. Taken all together, it was concluded that RecA-overexpressing transgenic plants are endowed with an increased chloroplast DNA maintenance capacity and enhanced repair activities, and consequently have a higher survival tolerance to genotoxic stresses. These observations are made possible by the functional compatibility of the bacterial RecAs in chloroplasts.

RECA plays a dual role in the maintenance of chloroplast genome stability in Physcomitrella patens

“…Recombination also occurs between large inverted repeats (Kolodner and Tewari, 1979), and chloroplasts are transformed by targeted integration of foreign DNA (Boynton et al, 1988), collectively suggesting the existence of RecArelated active HR in chloroplasts. Recently, some T-DNA mutants of the cpRECA gene AtRECA1, one of three A. thaliana RECA genes, have been shown to exhibit leaf variegation (Rowan et al, 2010) or no visible phenotype (Jeon et al, 2013). The variegated cpRECA mutants also exhibit elevated levels of single-stranded DNA and reduced levels of cpDNA (Rowan et al, 2010).…”

Design and assembly of the 117-kbPhaeodactylum tricornutumchloroplast genome

“…The bacterial-derived recombinase RecA (PHATRDRAFT_54013) is expressed in the nuclear genome but localizes to the chloroplast after translation, where it mediates homologous recombination. Previous studies have demonstrated that disrupting this gene in a variety of distantly related photosynthetic eukaryotes disrupts homologous recombination within the chloroplast (Cerutti et al, 1995;Jeon et al, 2013). This will be necessary to prevent homologous recombination between the endogenous and cloned genomes, which posed challenges during transformation of the Chlamydomonas chloroplast genome (O'Neill et al, 2011).…”

Dynamic Interplay between Nucleoid Segregation and Genome Integrity in Chlamydomonas Chloroplasts

“…This suggests that cpRECA may suppress aberrant recombination between SDRs in cp and play a similar role in maintaining cp genome stability to that of mitochondrial RECA in mitochondrial genome stability (Odahara et al, 2009). After reproduction for several generations, Arabidopsis cpRECA mutants exhibited variegation in leaves and enhanced sensitivity to DSBand reactive oxygen species-inducing agents (Rowan et al, 2010;Jeon et al, 2013). In addition, compared with the wild type, mutants exhibit a reduction in cpDNA copy number, an increase in single-stranded cpDNA, and cpDNA structural alterations (Rowan et al, 2010).…”