RESUMEN
The reproduction toxicity of pubertal exposure to Microcystin-LR (MC-LR) and the underlying mechanism needs to be further investigated. In the current study, pubertal male ICR mice were intraperitoneally injected with 2⯵g/kg MC-LR for four weeks. Pubertal exposure to MC-LR decreased epididymal sperm concentration and blocked spermatogonia proliferation. In-vitro studies found MC-LR inhibited cell proliferation of GC-1 cells and arrested cell cycle in G2/M phase. Mechanistically, MC-LR exposure evoked excessive reactive oxygen species (ROS) and induced DNA double-strand break in GC-1 cells. Besides, MC-LR inhibited DNA repair by reducing PolyADP-ribosylation (PARylation) activity of PARP1. Further study found MC-LR caused proteasomal degradation of SIRT6, a monoADP-ribosylation enzyme which is essential for PARP1 PARylation activity, due to destruction of SIRT6-USP10 interaction. Additionally, MG132 pretreatment alleviated MC-LR-induced SIRT6 degradation and promoted DNA repair, leading to the restoration of cell proliferation inhibition. Correspondingly, N-Acetylcysteine (NAC) pre-treatment mitigated the disturbed SIRT6-USP10 interaction and SIRT6 degradation, causing recovered DNA repair and subsequently restoration of cell proliferation inhibition in MC-LR treated GC-1 cells. Together, pubertal exposure to MC-LR induced spermatogonia cell cycle arrest and sperm count reduction by oxidative DNA damage and simultaneous SIRT6-mediated DNA repair failing. This study reports the effect of pubertal exposure to MC-LR on spermatogenesis and complex mechanism how MC-LR induces spermatogonia cell proliferation inhibition.
Asunto(s)
Toxinas Marinas , Microcistinas , Sirtuinas , Espermatogonias , Animales , Masculino , Ratones , Apoptosis , Proliferación Celular , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN , Toxinas Marinas/metabolismo , Toxinas Marinas/toxicidad , Ratones Endogámicos ICR , Microcistinas/metabolismo , Microcistinas/toxicidad , Semen , Sirtuinas/efectos de los fármacos , Sirtuinas/metabolismo , Espermatogonias/efectos de los fármacos , Espermatogonias/metabolismoRESUMEN
NAC (NAM, ATAF1/2 and CUC2) proteins are plant-specific transcription factors (TFs) that are important in plant abiotic stress responses. In this study we isolated a NAC gene from Capsicum annuum leaves, designated as CaNAC064. We characterized the amino acid sequence of CaNAC064 and found that it contain conserved domains of the NAC family, including a highly conserved N-terminus domain and a highly variable C-terminus domain. Expression analysis showed that the 40C, 400C, salicylic acid (SA) and abscisic acid (ABA) treatments strongly induced the expression of CaNAC064 through silencing of CaNAC064 in pepper and overexpressing in Arabidopsis. CaNAC064-silenced pepper plants exhibited more serious wilting, higher MDA contents and chilling injury index, lower proline content, and more accumulation of ROS in the leaves after cold stress. The CaNAC064-overexpressing Arabidopsis plants exhibited lower MDA content, chilling injury index and relative electrolyte leakage content as compared to WT plants under cold stress. Transcriptional activation activity analysis indicated that CaNAC064 has transcriptional activation activity in the 691-1071 bp key region. We identified 45 proteins that putatively interact with CaNAC064 using the Yeast Two-Hybrid method. According to the Yeast Two-Hybrid and BIFC results, CaNAC064 interacted with low temperature-induced haplo-proteinase proteins in plant cell. These results suggested that CaNAC064 positively modulates plant cold-tolerance, laying the foundation for future investigations into the role of NACs as regulatory proteins of cold tolerance in plants.
Asunto(s)
Capsicum/fisiología , Respuesta al Choque por Frío/genética , Proteínas de Plantas/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Capsicum/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/fisiología , Alineación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Dehydrins play an important role in improving plant resistance to abiotic stresses. In this study, we isolated a dehydrin gene from pepper (Capsicum annuum L.) leaves, designated as CaDHN4. Sub-cellular localization of CaDHN4 was to be found in the nucleus and membrane. To authenticate the function of CaDHN4 in cold- and salt-stress responses and abscisic acid (ABA) sensitivity, we reduced the CaDHN4 expression using virus-induced gene silencing (VIGS), and overexpressed the CaDHN4 in Arabidopsis. We found that silencing of CaDHN4 reduced the growth of pepper seedlings and CaDHN4-silenced plants exhibited more serious wilting, higher electrolyte leakage, and more accumulation of ROS in the leaves compared to pTRV2:00 plants after cold stress, and lower chlorophyll contents and higher electrolyte leakage compared to pTRV2:00 plants under salt stress. However, CaDHN4-overexpressing Arabidopsis plants had higher seed germination rates and post-germination primary root growth, compared to WT plants under salt stress. In response to cold and salt stresses, the CaDHN4-overexpressed Arabidopsis exhibited lower MDA content, and lower relative electrolyte leakage compared to the WT plants. Under ABA treatments, the fresh weight and germination rates of transgenic plants were higher than WT plants. The transgenic Arabidopsis expressing a CaDHN4 promoter displayed a more intense GUS staining than the normal growth conditions under treatment with hormones including ABA, methyl jasmonate (MeJA), and salicylic acid (SA). Our results suggest that CaDHN4 can protect against cold and salt stresses and decrease ABA sensitivity in Arabidopsis.