RESUMEN
Epigenetic reprogramming is required for proper regulation of gene expression in eukaryotic organisms. In Arabidopsis, active DNA demethylation is crucial for seed viability, pollen function, and successful reproduction. The DEMETER (DME) DNA glycosylase initiates localized DNA demethylation in vegetative and central cells, so-called companion cells that are adjacent to sperm and egg gametes, respectively. In rice, the central cell genome displays local DNA hypomethylation, suggesting that active DNA demethylation also occurs in rice; however, the enzyme responsible for this process is unknown. One candidate is the rice REPRESSOR OF SILENCING1a (ROS1a) gene, which is related to DME and is essential for rice seed viability and pollen function. Here, we report genome-wide analyses of DNA methylation in wild-type and ros1a mutant sperm and vegetative cells. We find that the rice vegetative cell genome is locally hypomethylated compared with sperm by a process that requires ROS1a activity. We show that many ROS1a target sequences in the vegetative cell are hypomethylated in the rice central cell, suggesting that ROS1a also demethylates the central cell genome. Similar to Arabidopsis, we show that sperm non-CG methylation is indirectly promoted by DNA demethylation in the vegetative cell. These results reveal that DNA glycosylase-mediated DNA demethylation processes are conserved in Arabidopsis and rice, plant species that diverged 150 million years ago. Finally, although global non-CG methylation levels of sperm and egg differ, the maternal and paternal embryo genomes show similar non-CG methylation levels, suggesting that rice gamete genomes undergo dynamic DNA methylation reprogramming after cell fusion.
Asunto(s)
ADN Glicosilasas , Metilación de ADN/fisiología , ADN de Plantas , Oryza , Proteínas de Plantas , Polen , Arabidopsis/enzimología , Arabidopsis/genética , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , ADN de Plantas/genética , ADN de Plantas/metabolismo , Oryza/enzimología , Oryza/genética , Óvulo Vegetal/enzimología , Óvulo Vegetal/genética , Desarrollo de la Planta/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polen/enzimología , Polen/genéticaRESUMEN
BACKGROUND: Microspore embryogenesis describes a stress-induced reprogramming of immature male plant gametophytes to develop into embryo-like structures, which can be regenerated into doubled haploid plants after whole genome reduplication. This mechanism is of high interest for both research as well as plant breeding. The objective of this study was to characterize transcriptional changes and regulatory relationships in early stages of cold stress-induced wheat microspore embryogenesis by transcriptome and small RNA sequencing using a highly responsive cultivar. RESULTS: Transcriptome and small RNA sequencing was performed in a staged time-course to analyze wheat microspore embryogenesis induction. The analyzed stages were freshly harvested, untreated uninucleate microspores and the two following stages from in vitro anther culture: directly after induction by cold-stress treatment and microspores undergoing the first nuclear divisions. A de novo transcriptome assembly resulted in 29,388 contigs distributing to 20,224 putative transcripts of which 9,305 are not covered by public wheat cDNAs. Differentially expressed transcripts and small RNAs were identified for the stage transitions highlighting various processes as well as specific genes to be involved in microspore embryogenesis induction. CONCLUSION: This study establishes a comprehensive functional genomics resource for wheat microspore embryogenesis induction and initial understanding of molecular mechanisms involved. A large set of putative transcripts presumably specific for microspore embryogenesis induction as well as contributing processes and specific genes were identified. The results allow for a first insight in regulatory roles of small RNAs in the reprogramming of microspores towards an embryogenic cell fate.
Asunto(s)
Polen/genética , ARN Pequeño no Traducido/genética , Transcriptoma , Triticum/genética , Análisis por Conglomerados , Frío , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , MicroARNs/genética , Proteínas de Plantas/genética , Polen/embriología , Precursores del ARN/genética , ARN de Planta/genética , Análisis de Secuencia de ARN/métodos , Triticum/embriologíaRESUMEN
Hybrid vigor or heterosis results from the combination of genetically distant genomes at fertilization, and as well as being of major commercial importance, it is held to contribute significantly to fitness [1]. Activation of the paternal genome marks the transition from maternal to zygotic control of development, but a reported delay of paternal-genome activation in flowering plants [2-4] and animals [5, 6] excludes heterosis from impacting on very early development. We have analyzed the allele-specific expression of 25 genes after fertilization of the egg in maize and show immediate equivalent parental genomic contribution to the zygote. Every gene expressed before the first cell division of the zygotes showed paternal transcripts. Sequence comparisons indicate that these genes are involved in a range of processes and are distributed throughout the genome. Our findings confirm that some plant species have evolved a strategy to activate the paternal genome immediately after fertilization, in contrast to the situation in other plants and in animals. Such an extensive activation of the paternal genome very early in development is consonant with observations of high levels of heterosis in early hybrid maize embryos [7, 8], indicating a significant impact of this sexual strategy on fitness.