RESUMEN
Pollen-stigma interactions are essential for pollen germination. The highly regulated process of pollen germination includes pollen adhesion, hydration, and germination on the stigma. However, the internal signaling of pollen that regulates pollen-stigma interactions is poorly understood. KINßγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex which plays important roles in the regulation of plant development. Here, we showed that KINßγ was a cytoplasm- and nucleus-localized protein in the vegetative cells of pollen grains in Arabidopsis. The pollen of the Arabidopsis kinßγ mutant could not germinate on stigma, although it germinated normally in vitro. Further analysis revealed the hydration of kinßγ mutant pollen on the stigma was compromised. However, adding water to the stigma promoted the germination of the mutant pollen in vivo, suggesting that the compromised hydration of the mutant pollen led to its defective germination. In kinßγ mutant pollen, the structure of the mitochondria and peroxisomes was destroyed, and their numbers were significantly reduced compared with those in the wild type. Furthermore, we found that the kinßγ mutant exhibited reduced levels of reactive oxygen species (ROS) in pollen. The addition of H2O2 in vitro partially compensated for the reduced water absorption of the mutant pollen, and reducing ROS levels in pollen by overexpressing Arabidopsis CATALASE 3 resulted in compromised hydration of pollen on the stigma. These results indicate that Arabidopsis KINßγ is critical for the regulation of ROS levels by mediating the biogenesis of mitochondria and peroxisomes in pollen, which is required for pollen-stigma interactions during pollination.
Asunto(s)
Proteínas de Arabidopsis/genética , Germinación/genética , Mitocondrias/genética , Polen/genética , Polinización/genética , Proteínas Serina-Treonina Quinasas/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/biosíntesis , Catalasa/biosíntesis , Catalasa/genética , Flores/genética , Flores/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Proteínas Mutantes/biosíntesis , Proteínas Mutantes/genética , Peroxisomas/genética , Polen/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Agua/metabolismoRESUMEN
BACKGROUND: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, can selectively kill cancer cells with little or no cytotoxicity toward normal human cells and is regarded as a potential relatively safe antitumor drug. However, some cancer cells are resistant to TRAIL-induced apoptosis. Thus, reagents that potentiate TRAIL-induced cytotoxicity are needed. Herein, we investigated whether shikonin, a natural compound from the root of Lithospermum erythrorhizon, can sensitize TRAIL-resistant cells to TRAIL-induced cytotoxicity. RESULTS: The viability of A549 cells, which were resistant to TRAIL, was significantly decreased after treatment with TRAIL followed by shikonin. The underlying mechanisms by which shikonin sensitizes cells to TRAIL-induced cytotoxicity were also examined. Combined treatment with shikonin and TRAIL activated the caspase and JNK pathways, inhibited the STAT3 and AKT pathways, downregulated the expression of Mcl-1, Bcl-2, Bcl-xL, c-FLIP and XIAP and upregulated the expression of Bid. CONCLUSIONS: In conclusion, the results indicated that shikonin sensitized resistant cancer cells to TRAIL-induced cytotoxicity via the modulation of the JNK, STAT3 and AKT pathways, the downregulation of antiapoptotic proteins and the upregulation of proapoptotic proteins.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Naftoquinonas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Células A549 , Muerte Celular/efectos de los fármacos , Sinergismo Farmacológico , Células HEK293 , Humanos , Fosforilación/efectos de los fármacosRESUMEN
Pollen hydration is a critical step that determines pollen germination on the stigma. KINßγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex (SnRK1 complex). In pollen of the Arabidopsis kinßγ mutant, the levels of reactive oxygen species were decreased which lead to compromised hydration of the mutant pollen on the stigma. In this study, we analyzed gene expression in kinßγ mutant pollen by RNA-seq and found the expression of inward shaker K+ channel SPIK was down-regulated in the kinßγ pollen. Furthermore, we showed that the pollen hydration of the Arabidopsis spik mutant was defective on the wild-type stigma, although the mutant pollen demonstrated normal hydration in vitro. Additionally, the defective hydration of spik mutant pollen could not be rescued by the wild-type pollen on the stigma, indicating that the spik mutation deprived the capability of pollen absorption on the stigma. Our results suggest that the Arabidopsis SnRK1 complex regulates SPIK expression, which functions in determining pollen hydration on the stigma.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Polen/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Canales de Potasio de la Superfamilia Shaker/metabolismo , Agua/fisiología , Arabidopsis/genética , Potasio/metabolismo , Análisis de Secuencia de ARNRESUMEN
Gene editing is a kind of technologies that makes precise modification to the genome. It can be used to knock out/in and replace the specific DNA fragment, and make accurate gene editing on the genome level. The essence of the technique is the DNA sequence change with use of non homologous end link repair and homologous recombination repair, combined with specific DNA target recognition and endonuclease.This technology has wide range of development prospects and high application value in terms of scientific research, agriculture, medical treatment and other fields. In the field of gene therapy, gene editing technology has achieved cross-time success in cancers such as leukemia, genetic disorders such as hemophilia, thalassemia, multiple muscle nutritional disorders and retrovirus associated infectious diseases such as AIDS and other diseases. The preparation work for new experimental methods and animal models combined with gene editing technology is under rapid development and improvement. Laboratories around the world have also applied gene editing technique in prevention of malaria, organ transplantation, biological pharmaceuticals, agricultural breeding improvement, resurrection of extinct species, and other research areas. This paper summarizes the application and development status of gene editing technique in the above fields, and also preliminarily explores the potential application prospect of the technology in the field of traditional Chinese medicine, and discusses the present controversy and thoughts.
Asunto(s)
Edición Génica , Medicina Tradicional China , AnimalesRESUMEN
ROOT INITIATION DEFECTIVE 1 (RID1) is an Arabidopsis DEAH/RHA RNA helicase. It functions in hypocotyl de-differentiation, de novo meristem formation, and cell specification of the mature female gametophyte (FG). However, it is unclear how RID1 regulates FG development. In this study, we observed that mutations to RID1 disrupted the developmental synchrony and retarded the progression of FG development. RID1 exhibited RNA helicase activity, with a preference for unwinding double-stranded RNA in the 3' to 5' direction. Furthermore, we found that RID1 interacts with GAMETOPHYTIC FACTOR 1 (GFA1), which is an integral protein of the spliceosome component U5 small nuclear ribonucleoprotein (snRNP) particle. Substitution of specific RID1 amino acids (Y266F and T267I) inhibited the interaction with GFA1. In addition, the mutated RID1 could not complement the seed-abortion phenotype of the rid1 mutant. The rid1 and gfa1 mutants exhibited similar abnormalities in pre-mRNA splicing and down-regulated expression of some genes involved in FG development. Our results suggest that an interaction between RID1 and the U5 snRNP complex regulates essential pre-mRNA splicing of the genes required for FG development. This study provides new information regarding the mechanism underlying the FG developmental process.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/crecimiento & desarrollo , Óvulo Vegetal/crecimiento & desarrollo , Factores de Elongación de Péptidos/fisiología , ARN Helicasas/fisiología , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Microscopía Confocal , Óvulo Vegetal/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
The actin-binding protein p57 is a member of mammalian coronin-like proteins. The roles of this protein in phagocytic processes conceivably depend on its interactions with F-actin. Two regions, p57(1-34) and p57(111-204), were previously reported to be actin-binding sites. In this study, we found that the C-terminal region of p57, p57(297-461), also possessed F-actin binding activity. Furthermore, the leucine zipper domain at the C-terminus of p57(297-461) was essential for this actin-binding activity. The F-actin cross-linking assay revealed that the region contained in p57(297-461) was sufficient to cross-link actin filaments. Our results strongly suggested that there was a new actin-binding region at the C-terminus of p57.