RESUMEN
The maintenance of genome integrity over cell divisions is critical for plant development and the correct transmission of genetic information to the progeny. A key factor involved in this process is the STRUCTURAL MAINTENANCE OF CHROMOSOME5 (SMC5) and SMC6 (SMC5/6) complex, related to the cohesin and condensin complexes that control sister chromatid alignment and chromosome condensation, respectively. Here, we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the SMC5/6 complex in Arabidopsis (Arabidopsis thaliana). NSE4A is expressed in meristems and accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a mutants produce abnormal seeds, with noncellularized endosperm and embryos that maximally develop to the heart or torpedo stage. This phenotype resembles the defects in cohesin and condensin mutants and suggests a role for all three SMC complexes in differentiation during seed development. By contrast, NSE4B is expressed in only a few cell types, and loss-of-function mutants do not have any obvious abnormal phenotype. In summary, our study shows that the NSE4A subunit of the SMC5-SMC6 complex is essential for DNA damage repair in somatic tissues and plays a role in plant reproduction.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Reparación del ADN , Subunidades de Proteína/metabolismo , Semillas/metabolismo , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Daño del ADN/genética , Reparación del ADN/genética , Duplicación de Gen , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Óvulo Vegetal/genética , Polen/genética , Unión Proteica , Semillas/genética , Transcriptoma/genética , Regulación hacia Arriba/genéticaRESUMEN
Increased cellular ploidy is widespread during developmental processes of multicellular organisms, especially in plants. Elevated ploidy levels are typically achieved either by endoreplication or endomitosis, which are often regarded as modified cell cycles that lack an M phase either entirely or partially. We identified GIGAS CELL1 (GIG1)/OMISSION OF SECOND DIVISION1 (OSD1) and established that mutation of this gene triggered ectopic endomitosis. On the other hand, it has been reported that a paralog of GIG1/OSD1, UV-INSENSITIVE4 (UVI4), negatively regulates endoreplication onset in Arabidopsis thaliana. We showed that GIG1/OSD1 and UVI4 encode novel plant-specific inhibitors of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. These proteins physically interact with APC/C activators, CDC20/FZY and CDH1/FZR, in yeast two-hybrid assays. Overexpression of CDC20.1 and CCS52B/FZR3 differentially promoted ectopic endomitosis in gig1/osd1 and premature occurrence of endoreplication in uvi4. Our data suggest that GIG1/OSD1 and UVI4 may prevent an unscheduled increase in cellular ploidy by preferentially inhibiting APC/C(CDC20) and APC/C(FZR), respectively. Generation of cells with a mixed identity in gig1/osd1 further suggested that the APC/C may have an unexpected role for cell fate determination in addition to its role for proper mitotic progression.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Mitosis , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Alelos , Ciclosoma-Complejo Promotor de la Anafase , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromosomas de las Plantas/genética , Cromosomas de las Plantas/metabolismo , Clonación Molecular , Cotiledón/genética , Cotiledón/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Citocinesis , Inhibidores Enzimáticos/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Prueba de Complementación Genética , Mutación , Células Vegetales/metabolismo , Epidermis de la Planta/citología , Epidermis de la Planta/genética , Epidermis de la Planta/crecimiento & desarrollo , Epidermis de la Planta/metabolismo , Estomas de Plantas/citología , Estomas de Plantas/genética , Estomas de Plantas/crecimiento & desarrollo , Estomas de Plantas/metabolismo , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Ploidias , Polen/crecimiento & desarrollo , Polen/metabolismo , Mapeo de Interacción de Proteínas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Técnicas del Sistema de Dos Híbridos , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
Plant SET domain proteins are known to be involved in the epigenetic control of gene expression during plant development. Here, we report that the Arabidopsis SET domain protein, SDG4, contributes to the epigenetic regulation of pollen tube growth, thus affecting fertilization. Using an SDG4-GFP fusion construct, the chromosomal localization of SDG4 was established in tobacco BY-2 cells. In Arabidopsis, sdg4 knockout showed reproductive defects. Tissue-specific expression analyses indicated that SDG4 is the major ASH1-related gene expressed in the pollen. Immunological analyses demonstrated that SDG4 was involved in the methylation of histone H3 in the inflorescence and pollen grains. The significant reduction in the amount of methylated histone H3 K4 and K36 in sdg4 pollen vegetative nuclei resulted in suppression of pollen tube growth. Our results indicate that SDG4 is capable of modulating the expression of genes that function in the growth of pollen tube by methylation of specific lysine residues of the histone H3 in the vegetative nuclei.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Histonas/metabolismo , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , ADN de Plantas/genética , Genes de Plantas , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Histonas/genética , Lisina/química , Lisina/metabolismo , Metilación , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Plantas Modificadas Genéticamente , Polen/genética , Polen/metabolismo , Homología de Secuencia de Aminoácido , NicotianaRESUMEN
Pollen grains of angiosperm plants represent a good model system for studies of chromatin structure and remodelling factors, but very little is known about the DNA methylation status of particular genes in pollen. In this study, we present an analysis of the DNA methylation patterns of the MROS1 gene, which is expressed in the late phases of pollen development in Silene latifolia (syn. Meladrium album). The genomic sequencing technique revealed similar DNA methylation patterns in leaves, binucleate pollen, and trinucleate pollen. Extremely high DNA methylation levels occurred in the CG dinucleotides of the upstream region (99%), whereas only a low level of CG methylation was observed in the transcribed sequence (7%). Low levels of methylation were also observed in asymmetric sequences (in both regions; 2% methylated). The results obtained in the MROS1 gene are discussed in consequence with the immunohistochemical data showing a hypermethylation of DNA in the vegetative nucleus.
Asunto(s)
Metilación de ADN , ADN de Plantas/química , ADN de Plantas/genética , Proteínas de Plantas/genética , Silene/crecimiento & desarrollo , Silene/genética , Secuencia de Bases , Genes de Plantas , Genoma de Planta , Inmunohistoquímica , Datos de Secuencia Molecular , Polen/química , Polen/genética , Polen/crecimiento & desarrollo , Regiones Promotoras Genéticas , Homología de Secuencia de Ácido Nucleico , Silene/química , Especificidad de la Especie , Distribución TisularRESUMEN
In cultured Bright Yellow-2 tobacco (Nicotiana tabacum) cells, the depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium induces amyloplast development. This differentiation also includes a decrease in cell multiplication, and an increase in cell size. These changes were primarily triggered by the depletion of 2,4-D, and accelerated by the addition of benzyladenine (BA). Three cDNAs were identified whose transcript levels are specifically increased during differentiation of starch-storing cells using the differential display method, and designated as starch-storing cell induced genes (SCI genes). One of these cDNAs, SCI2 encodes a 285 amino acids long protein with a KH RNA-binding domain. A database search revealed that this protein showed similarity to respective domains of mammalian quaking proteins. 2,4-D addition, which can convert starch-storing cells into dividing cells, to starch-storing BY-2 cells, immediately decreases the SCI2 transcript level, suggesting that SCI2 may have some role in starch-storing cell differentiation in BY-2 cells.