Analysis of Fluorescent Reporter Activity in the Male Germline During Pollen Development by Confocal Microscopy.

The male germline of flowering plants develops within the vegetative cell of the male gametophyte (pollen). The germline is established by asymmetric division of the microspore to form the generative cell. Mitotic division of the generative cell then produces the two sperm cells required for double fertilization. These differentiate to produce the proteins required for gamete attachment and fusion. An important aspect of understanding germline development is the characterization of germline gene expression. Here, we describe the use of a fluorescent reporter to study germline gene expression in developing pollen to assess the timing and specificity of expression

Identification of Cis-Regulatory Modules that Function in the Male Germline of Flowering Plants.

The male germline of flowering plants develops within the vegetative cell of the male gametophyte and displays a distinct transcriptional profile. Key to understanding the development of this unique cell lineage is determining how gene expression is regulated within germline cells. This knowledge impacts upon our understanding of cell specification, differentiation, and plant fertility. Here, we describe methods to identify cis-regulatory modules (CRMs) that act as key regulatory regions in the promoters of germline-expressed genes. We detail the complimentary techniques of phylogenetic footprinting and the use of fluorescent reporters in pollen for the identification and verification of CRMs

A Conserved cis-Regulatory Module Determines Germline Fate through Activation of the Transcription Factor DUO1 Promoter

The development of the male germline within pollen relies upon the activation of numerous target genes by the transcription factor DUO POLLEN1 (DUO1). The expression of DUO1 is restricted to the male germline and is first detected shortly after the asymmetric division that segregates the germ cell lineage. Transcriptional regulation is critical in controlling DUO1 expression, since transcriptional and translational fusions show similar expression patterns. Here, we identify key promoter sequences required for the germline-specific regulation of DUO1 transcription. Combining promoter deletion analyses with phylogenetic footprinting in eudicots and in Arabidopsis accessions, we identify a cis-regulatory module, Regulatory region of DUO1 (ROD1), which replicates the expression pattern of DUO1 in Arabidopsis (Arabidopsis thaliana). We show that ROD1 from the legume Medicago truncatula directs male germline-specific expression in Arabidopsis, demonstrating conservation of DUO1 regulation among eudicots. ROD1 contains several short conserved cis-regulatory elements, including three copies of the motif DNGTGGV, required for germline expression and tandem repeats of the motif YAACYGY, which enhance DUO1 transcription in a positive feedback loop. We conclude that a cis-regulatory module conserved in eudicots directs the spatial and temporal expression of the transcription factor DUO1 to specify male germline fate and sperm cell differentiation