RESUMO
Thermoinhibition, or failure of seeds to germinate at warm temperatures, is common in lettuce (Lactuca sativa) cultivars. Using a recombinant inbred line population developed from a lettuce cultivar (Salinas) and thermotolerant Lactuca serriola accession UC96US23 (UC), we previously mapped a quantitative trait locus associated with thermoinhibition of germination to a genomic region containing a gene encoding a key regulated enzyme in abscisic acid (ABA) biosynthesis, 9-cis-EPOXYCAROTENOID DIOXYGENASE4 (NCED4). NCED4 from either Salinas or UC complements seeds of the Arabidopsis thaliana nced6-1 nced9-1 double mutant by restoring germination thermosensitivity, indicating that both NCED4 genes encode functional proteins. Transgenic expression of Salinas NCED4 in UC seeds resulted in thermoinhibition, whereas silencing of NCED4 in Salinas seeds led to loss of thermoinhibition. Mutations in NCED4 also alleviated thermoinhibition. NCED4 expression was elevated during late seed development but was not required for seed maturation. Heat but not water stress elevated NCED4 expression in leaves, while NCED2 and NCED3 exhibited the opposite responses. Silencing of NCED4 altered the expression of genes involved in ABA, gibberellin, and ethylene biosynthesis and signaling pathways. Together, these data demonstrate that NCED4 expression is required for thermoinhibition of lettuce seeds and that it may play additional roles in plant responses to elevated temperature.
Assuntos
Dioxigenases/metabolismo , Germinação , Lactuca/enzimologia , Proteínas de Plantas/metabolismo , Sementes/crescimento & desenvolvimento , Ácido Abscísico/biossíntese , Ácido Abscísico/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Dioxigenases/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Giberelinas/genética , Giberelinas/metabolismo , Heterozigoto , Homozigoto , Temperatura Alta , Lactuca/genética , Lactuca/crescimento & desenvolvimento , Mutação , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Sementes/enzimologia , Sementes/genética , Estresse Fisiológico , Transgenes , Água/metabolismoRESUMO
Development of the germline requires the specification and survival of primordial germ cells (PGCs) in the embryo as well as the maintenance of gamete production during the reproductive life of the adult. These processes appear to be fundamental to all Metazoans, and some components of the genetic pathway regulating germ cell development and function are evolutionarily conserved. In both vertebrates and invertebrates, nanos-related genes, which encode RNA-binding zinc finger proteins, have been shown to play essential and conserved roles during germ cell formation. In Drosophila, maternally supplied nanos is required for survival of PGCs in the embryo, while in adults, nanos is required for the continued production of oocytes by maintaining germline stem cells self-renewal. In mice and zebrafish, nanos orthologs are required for PGC survival during embryogenesis, but a role in adults has not been explored. We show here that nanos1 in zebrafish is expressed in early stage oocytes in the adult female germline. We have identified a mutation in nanos1 using a reverse genetics method and show that young female nanos mutants contain oocytes, but fail to maintain oocyte production. This progressive loss of fertility in homozygous females is not a phenotype that has been described previously in the zebrafish and underlines the value of a reverse genetics approach in this model system.
Assuntos
Oócitos/fisiologia , Oogênese/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/fisiologia , Animais , Feminino , Modelos Biológicos , Oogênese/genética , Proteínas de Ligação a RNA , Células-Tronco/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genéticaAssuntos
Análise Mutacional de DNA/métodos , Endonucleases/metabolismo , Etilnitrosoureia/farmacologia , Mutagênese , Mutação Puntual/genética , Peixe-Zebra/genética , Animais , Pareamento Incorreto de Bases/genética , Criopreservação , DNA/análise , Endonucleases/química , Frequência do Gene , Biblioteca Gênica , Masculino , Espermatozoides/químicaRESUMO
DNA methylation has been implicated in a variety of epigenetic processes, and abnormal methylation patterns have been seen in tumors. Analysis of methylation patterns has traditionally been conducted either by using Southern analysis after cleavage with methyl-sensitive restriction endonucleases or by bisulfite sequencing. However, neither method is practical for analyzing more than a few genes. Here, we describe a simple technique for genome-wide mapping of DNA methylation patterns. Fragmentation by a methyl-sensitive restriction endonuclease is followed by size fractionation and hybridization to microarrays. We demonstrate the utility of this method by characterizing methylation patterns in Arabidopsis methylation mutants. This analysis reveals that CHROMOMETHYLASE3 (CMT3), which was previously shown to maintain CpXpG methylation, preferentially methylates transposons, even when they are present as single copies within the genome. Methylation profiling has potential applications in disease research and diagnostic screening.
