An Optimized Whole-Mount Immunofluorescence Method for Shoot Apices.

The localization of a protein provides important information about its biological functions. The visualization of proteins by immunofluorescence has become an essential approach in cell biology. Here, we describe an easy-to-follow immunofluorescence protocol to localize proteins in whole-mount tissues of maize (Zea mays) and Arabidopsis.

Identification of the maize Mediator CDK8 module and transposon-mediated mutagenesis of .

Mediator is a conserved transcriptional co-activator that links transcription factors bound at enhancer elements to RNA Polymerase II. Mediator-RNA Polymerase II interactions can be sterically hindered by the Cyclin Dependent Kinase 8 (CDK8) module, a submodule of Mediator that acts to repress transcription in response to discrete cellular and environmental cues. The CDK8 module is conserved in all eukaryotes and consists of 4 proteins: CDK8, CYCLIN C (CYCC), MED12, and MED13.

Localization of Chromatin Marks in Arabidopsis Early Embryos.

During early embryo development, profound changes in chromatin structure and regulation take place. It is difficult to study these changes in plant embryos however, largely because of their relative inaccessibility, which impedes the application of current epigenomic and biochemistry protocols. To circumvent this issue and to analyze the epigenetic status of the embryo at both the cellular and subcellular level, we describe here a simple method to immunolocalize chromatin marks in whole mount early Arabidopsis embryos, either within maternal tissues or isolated from seeds.

Zygotic genome activation and imprinting: parent-of-origin gene regulation in plant embryogenesis.

Parent-of-origin dependent gene expression refers to differential activity of alleles inherited from the egg and sperm. In plants, zygotic genome activation (ZGA) and gene imprinting are two examples of this phenomenon, both of which occur during seed development. As its name implies, ZGA is a genome-wide process that occurs in embryos during the first few days after fertilization.

The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes.

Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown.

Non-equivalent contributions of maternal and paternal genomes to early plant embryogenesis.

Zygotic genome activation in metazoans typically occurs several hours to a day after fertilization, and thus maternal RNAs and proteins drive early animal embryo development. In plants, despite several molecular studies of post-fertilization transcriptional activation, the timing of zygotic genome activation remains a matter of debate. For example, two recent reports that used different hybrid ecotype combinations for RNA sequence profiling of early Arabidopsis embryo transcriptomes came to divergent conclusions.

Transcriptional analysis of the Arabidopsis ovule by massively parallel signature sequencing.

The life cycle of flowering plants alternates between a predominant sporophytic (diploid) and an ephemeral gametophytic (haploid) generation that only occurs in reproductive organs. In Arabidopsis thaliana, the female gametophyte is deeply embedded within the ovule, complicating the study of the genetic and molecular interactions involved in the sporophytic to gametophytic transition. Massively parallel signature sequencing (MPSS) was used to conduct a quantitative large-scale transcriptional analysis of the fully differentiated Arabidopsis ovule prior to fertilization.

Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte.

In double fertilization, the vegetative cell of the male gametophyte (pollen) germinates and forms a pollen tube that brings to the female gametophyte two sperm cells that fertilize the egg and central cell to form the embryo and endosperm, respectively. The 5-methylcytosine DNA glycosylase DEMETER (DME), expressed in the central cell, is required for maternal allele demethylation and gene imprinting in the endosperm. By contrast, little is known about the function of DME in the male gametophyte.

Inactivation of a DNA methylation pathway in maize reproductive organs results in apomixis-like phenotypes.

Apomictic plants reproduce asexually through seeds by avoiding both meiosis and fertilization. Although apomixis is genetically regulated, its core genetic component(s) has not been determined yet. Using profiling experiments comparing sexual development in maize (Zea mays) to apomixis in maize-Tripsacum hybrids, we identified six loci that are specifically downregulated in ovules of apomictic plants.

CHR11, a chromatin-remodeling factor essential for nuclear proliferation during female gametogenesis in Arabidopsis thaliana.

Chromatin-remodeling factors regulate the establishment of transcriptional programs during plant development. Although 42 genes encoding members of the SWI2/SNF2 family have been identified in Arabidopsis thaliana, <10 have been assigned a precise function on the basis of a mutant phenotype, and none have been shown to play a specific role during the gametophytic phase of the plant life cycle. A.