RESUMEN
BACKGROUND: Mammalian BLM helicase is involved in DNA replication, DNA repair and homologous recombination (HR). These DNA transactions are associated tightly with cell division and are important for maintaining genome stability. However, unlike in mammals, cell division in higher plants is restricted mainly to the meristem, thus genome maintenance at the meristem is critical. The counterpart of BLM in Arabidopsis (AtRecQ4A) has been identified and its role in HR and in the response to DNA damage has been confirmed. However, the function of AtRecQ4A in the meristem during replication stress has not yet been well elucidated. RESULTS: We isolated the BLM counterpart gene OsRecQl4 from rice and analyzed its function using a reverse genetics approach. Osrecql4 mutant plants showed hypersensitivity to DNA damaging agents and enhanced frequency of HR compared to wild-type (WT) plants. We further analyzed the effect of aphidicolin--an inhibitor of S-phase progression via its inhibitory effect on DNA polymerases--on genome stability in the root meristem in osrecql4 mutant plants and corresponding WT plants. The following effects were observed upon aphidicolin treatment: a) comet assay showed induction of DNA double-strand breaks (DSBs) in mutant plants, b) TUNEL assay showed enhanced DNA breaks at the root meristem in mutant plants, c) a recombination reporter showed enhanced HR frequency in mutant calli, d) propidium iodide (PI) staining of root tips revealed an increased incidence of cell death in the meristem of mutant plants. CONCLUSIONS: These results demonstrate that the aphidicolin-sensitive phenotype of osrecql4 mutants was in part due to induced DSBs and cell death, and that OsRecQl4 plays an important role as a caretaker, maintaining genome stability during DNA replication stress in the rice meristem.
Asunto(s)
Recombinación Homóloga , Meristema/enzimología , Oryza/enzimología , Proteínas de Plantas/genética , RecQ Helicasas/genética , Puntos de Control de la Fase S del Ciclo Celular , Muerte Celular , Roturas del ADN de Doble Cadena , Replicación del ADN , Meristema/citología , Meristema/genética , Mutación , Oryza/citología , Oryza/genética , Proteínas de Plantas/metabolismo , RecQ Helicasas/metabolismoRESUMEN
During homologous recombination (HR)-mediated DNA double-strand break (DSB) repair in eukaryotes, an initial step is the creation of a 3'-single-stranded DNA (ssDNA) overhang via resection of a 5' end. Rad51 polymerizes on this ssDNA to search for a homologous sequence, and the gapped sequence is then repaired using an undamaged homologous DNA strand as template. Recent studies in eukaryotes indicate that resection of the DSB site is promoted by the cooperative action of RecQ helicase family proteins: Bloom helicase (BLM) in mammals or Sgs1 in yeast, and exonuclease 1 (Exo1). However, the role of RecQ helicase and exonuclease during the 5'-resection process of HR in plant cells has not yet been defined. Here, we demonstrate that overexpression of rice proteins OsRecQl4 (BLM counterpart) and/or OsExo1 (Exo1 homolog) can enhance DSB processing, as evaluated by recombination substrate reporter lines in rice. These results could be applied to construct an efficient gene targeting system in rice.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Exodesoxirribonucleasas/genética , Recombinación Homóloga/genética , Oryza/genética , RecQ Helicasas/genética , ADN de Plantas/genética , ADN de Plantas/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Exodesoxirribonucleasas/metabolismo , Genes Reporteros , Marcadores Genéticos , Glucuronidasa , Modelos Moleculares , Oryza/enzimología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , RecQ Helicasas/metabolismo , Plantones/enzimología , Plantones/genéticaRESUMEN
Evidence for the involvement of the nonhomologous end joining (NHEJ) pathway in Agrobacterium-mediated transferred DNA (T-DNA) integration into the genome of the model plant Arabidopsis remains inconclusive. Having established a rapid and highly efficient Agrobacterium-mediated transformation system in rice (Oryza sativa) using scutellum-derived calli, we examined here the involvement of the NHEJ pathway in Agrobacterium-mediated stable transformation in rice. Rice calli from OsKu70, OsKu80 and OsLig4 knockdown (KD) plants were infected with Agrobacterium harboring a sensitive emerald luciferase (LUC) reporter construct to evaluate stable expression and a green fluorescent protein (GFP) construct to monitor transient expression of T-DNA. Transient expression was not suppressed, but stable expression was reduced significantly, in KD plants. Furthermore, KD-Ku70 and KD-Lig4 calli exhibited an increase in the frequency of homologous recombination (HR) compared with control calli. In addition, suppression of OsKu70, OsKu80 and OsLig4 induced the expression of HR-related genes on treatment with DNA-damaging agents. Our findings suggest strongly that NHEJ is involved in Agrobacterium-mediated stable transformation in rice, and that there is a competitive and complementary relationship between the NHEJ and HR pathways for DNA double-strand break repair in rice.
Asunto(s)
ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Recombinación Homóloga , Oryza/genética , Proteínas de Plantas/genética , Transformación Genética , Agrobacterium/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , ADN Helicasas/metabolismo , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN Ligasas/metabolismo , Reparación del ADN , ADN Bacteriano , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
An efficient in vitro protocol has been established for somatic embryogenesis and plantlet conversion of Korean wild ginseng (Panax ginseng Meyer). Wild-type and mutant adventitious roots derived from the ginseng produced calluses on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid and 0.3 mg/L kinetin; 53.3% of the explants formed callus. Embryogenic callus proliferation and somatic embryo induction occurred on MS medium containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid. The induced somatic embryos further developed to maturity on MS medium with 5 mg/L gibberellic acid, and 85% of them germinated. The germinated embryos were developed to shoots and elongated on MS medium with 5 mg/L gibberellic acid. The shoots developed into plants with well-developed taproots on one-third strength Schenk and Hildebrandt basal medium supplemented with 0.25 mg/L 1-naphthaleneacetic acid. When the plants were transferred to soil, about 30% of the regenerated plants developed into normal plants.