An EAR-Dependent Regulatory Module Promotes Male Germ Cell Division and Sperm Fertility in Arabidopsis

Abstract

The production of the sperm cells in angiosperms requires coordination of cell division and cell differentiation. In Arabidopsis thaliana, the germline-specific MYB protein DUO1 integrates these processes, but the regulatory hierarchy in which DUO1 functions is unknown. Here, we identify an essential role for two germline-specific DUO1 target genes, DAZ1 and DAZ2, which encode EAR motif-containing C 2 H 2 -type zinc finger proteins. We show that DAZ1/DAZ2 are required for germ cell division and for the proper accumulation of mitotic cyclins. Importantly, DAZ1/DAZ2 are sufficient to promote G2-to M-phase transition and germ cell division in the absence of DUO1. DAZ1/DAZ2 are also required for DUO1-dependent cell differentiation and are essential for gamete fusion at fertilization. We demonstrate that the two EAR motifs in DAZ1/DAZ2 mediate their function in the male germline and are required for transcriptional repression and for physical interaction with the corepressor TOPLESS. Our findings uncover an essential module in a regulatory hierarchy that drives mitotic transition in male germ cells and implicates gene repression pathways in sperm cell formation and fertility.

SYBR Green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content

“…Analysis of the microscopic images in comparison with previously published TEM images of intact sperm cells within A. thaliana pollen (Owen and Makaroff 1995;Borg et al 2014) revealed that all of the sorted sperms inspected (n = 413) completely lacked the plasma membrane (Fig. 3d).…”

“…Apparently, not only vegetative cell nuclei but also duo1 mutant sperm-like cell nuclei are more fragile than wild-type sperm nuclei during sorting. It is known that mutant cells without the DUO1 pathways fail to differentiate (Borg et al 2014), which is caused by alterations in gene expression. As DUO1 target genes include genes coding for proteins that are localized to the plasma membrane and genes involved in chromatin structure (Borg et al 2011), it is reasonable to assume that duo1 mutant spermlike cells could behave differently from wild-type sperm cells during purification steps.…”

SDG2-Mediated H3K4me3 Is Crucial for Chromatin Condensation and Mitotic Division during Male Gametogenesis in Arabidopsis

“…3). One major regulator of sperm cell identity and cell cycle progression is the transcription factor DUO POLLEN1 (DUO1; Rotman et al, 2005;Borg et al, 2011Borg et al, , 2014. In the loss-of-function mutant duo1, the generative cell fails to divide to generate two sperm cells, and the mutant pollen cannot fertilize the female egg cell or central cell.…”

Cis-Regulatory Elements Determine Germline Specificity and Expression Level of an Isopentenyltransferase Gene in Sperm Cells of Arabidopsis

“…Although a putative GRSF binding site was predicted in the promoter region of DUO1 (Haerizadeh et al, 2006), a truncated version of DUO1 promoter excluding the putative GRSF binding site did not prevent germline-specific expression, nor did it result in constitutive expression (Brownfield et al, 2009a). On the other hand, MYB binding sites were overrepresented in the promoters of DUO1-activated target (DAT) genes (Borg et al, 2011(Borg et al, , 2014. DUO1 directly regulates the expression of DATs through binding to the MYB sites in the promoter regions.…”

“…Presence of GRSF binding sequences in other male gamete-expressed genes suggested widespread control of male gamete gene expression by this functionally conserved sequence (Haerizadeh et al, 2006). DUO1 functions in another way, by directly binding to MYB sites to activate its target genes DUO1-ACTIVATED ZINC FINGER1 (DAZ1) and DAZ2, which encode transacting transcriptional repressors (Rotman et al, 2005;Brownfield et al, 2009a;Borg et al, 2011Borg et al, , 2014. A putative GRSF binding site was predicted in the DUO1 promoter (Haerizadeh et al, 2006).…”