Uncovering Male Germline Development in Arabidopsis: The Gametophyte Revealed

As flowering plants go about their business alternating between two generations (the haploid gametophyte and diploid sporophyte), they keep their gametophytes hidden. This is in stark contrast to mosses, whose gametophytes blanket the barks of trees. In flowering plants, the tiny gametophyte consists of a few cells growing entirely inside the sporophyte. Gametophyte initial cells undergo meiosis, giving rise to haploid microspores and megaspores. These spores develop into the male (pollen) and female (embryo sac) gametophytes, respectively, which produce gametes (Twell, 2011). How does the haploid microspore produce the two sperm cells required for double fertilization? Deep within the stamen, the haploid microspore divides asymmetrically, producing a large vegetative cell (which eventually degenerates) and a smaller germ cell, which divides to produce twin sperm cells, an event requiring tight control of cell cycle progression. The male germline-specific MYB transcription factor DUO POLLEN1 (DUO1) integrates the processes of generative cell division and sperm cell differentiation. The regulatory hierarchy in which DUO1 functions and how these regulatory mechanisms are integrated with gamete specification have remained obscure.

Borg et al. (pages 2098–2113) made great strides in elucidating this fundamental process. The authors focused on DUO1-ACTIVATED ZINC FINGER1 (DAZ1) and DAZ2, which are induced by ectopically expressed DUO1 in seedlings and show DUO1-dependent expression in sperm cells (Borg et al., 2011) These genes encode C₂H₂-type zinc finger proteins containing leucine-rich ETHYLENE RESPONSE FACTOR–associated amphiphilic repression (EAR) motifs. The authors began by investigating the expression of DAZ1 and DAZ2 in developing Arabidopsis thaliana pollen by generating transgenic protein fusion lines containing the promoters of these genes fused to fluorescent reporter proteins. Fluorescent signals, which were absent in microspores, first appeared in germ cell nuclei following microspore division, and they increased during development and persisted in mature pollen (exclusively in sperm cell nuclei), as confirmed by RT-PCR. Mutagenesis assays suggested that DUO1 binds to the MYB binding sites in the DAZ1 and DAZ2 promoters, implying a direct role for DUO1 in DAZ1/DAZ2 transcription. This notion is supported by the expression profiles of DUO1, DAZ1, and DAZ2. Maximum levels of DUO1 transcripts were observed in bicellular pollen, and they declined in tricellular and mature pollen, while DAZ1 and DAZ2 transcript levels peaked in tricellular pollen before declining in mature pollen. Mutant analysis revealed that DAZ1 and DAZ2 function redundantly and are required for generative cell division (see figure). Indeed, DAZ1/DAZ2-deficient germ cells appear to skip mitosis and reenter S-phase before anthesis. In addition, DAZ1/DAZ2 promote germ cell division, but not gamete differentiation, in the absence of DUO1, and DAZ1/DAZ2 are required for proper accumulation of the cyclin CYCB1;1, which promotes germ cell mitosis. Interestingly, while germ cells are incompletely differentiated and several DUO1-targeted genes are downregulated in the daz1 daz2 mutant, DUO1 expression is upregulated, suggesting that DAZ1/DAZ2 limit DUO1 accumulation in sperm cells via a feedback loop mechanism, which would facilitate the expression of DUO1-targeted genes during sperm cell development. Further experiments demonstrated that the EAR domains in DAZ1/DAZ2 can function as transcriptional repressors and are essential for germ cell division and fertility. DAZ1/DAZ2 physically interact with the corepressor TOPLESS via their EAR domains, suggesting that these proteins operate in the germline through mechanisms involving TOPLESS-mediated transcriptional repression.

DAZ1 and DAZ2 are essential for generative cell division. Tricellular wild-type (A) and bicellular daz1 daz2 mutant (B) pollen stained with 4′,6-diamidino-2-phenylindole. (Reprinted from Borg et al. [2014], Figure 3.)

Therefore, DUO1 acts as a network trigger in male germline development and sets up and later responds to the DAZ1/DAZ2 node to ensure germ cell division and correct specification of the gametes. DUO1 and DAZ1/2 are highly conserved in angiosperms, suggesting that the DUO1-DAZ1/DAZ2 regulatory module is an ancient developmental feature of the hidden male gametophyte.