In plants, each male meiotic product undergoes mitosis, and then one of the resulting cells divides again, yielding a three-celled pollen grain comprised of a vegetative cell and two sperm cells. Several genes have been found to act in this process, and DUO1 (DUO POLLEN 1), a transcription factor, plays a key role in sperm cell formation by activating expression of several germline genes. But how DUO1 itself is activated and how sperm cell formation is initiated remain unknown. To expand our understanding of sperm cell formation, we characterized an ARID (AT-Rich Interacting Domain)-containing protein, ARID1, that is specifically required for sperm cell formation in Arabidopsis. ARID1 localizes within nuclear bodies that are transiently present in the generative cell from which sperm cells arise, coincident with the timing of DUO1 activation. An arid1 mutant and antisense arid1 plants had an increased incidence of pollen with only a single sperm-like cell and exhibited reduced fertility as well as reduced expression of DUO1. In vitro and in vivo evidence showed that ARID1 binds to the DUO1 promoter. Lastly, we found that ARID1 physically associates with histone deacetylase 8 and that histone acetylation, which in wild type is evident only in sperm, expanded to the vegetative cell nucleus in the arid1 mutant. This study identifies a novel component required for sperm cell formation in plants and uncovers a direct positive regulatory role of ARID1 on DUO1 through association with histone acetylation.

Sperm entry triggers central cell division during seed development, but what factors besides the genome are inherited from sperm, and the mechanism by which paternal factors regulate early division events, are not understood. Here we show that sperm-transmitted miR159 promotes endosperm nuclear division by repressing central cell-transmitted miR159 targets. Disruption of paternal miR159 causes approximately half of the seeds to abort as a result of defective endosperm nuclear divisions. In wild-type plants, MYB33 and MYB65, two miR159 targets, are highly expressed in the central cell before fertilization, but both are rapidly abolished after fertilization. In contrast, loss of paternal miR159 leads to retention of MYB33 and MYB65 in the central cell after fertilization. Furthermore, ectopic expression of a miR159-resistant version of MYB33 (mMYB33) in the endosperm significantly inhibits initiation of endosperm nuclear division. Collectively, these results show that paternal miR159 inhibits its maternal targets to promote endosperm nuclear division, thus uncovering a previously unknown paternal effect on seed development.

Both female and male gametophytes harbor companion cells and gametes. MET1, a DNA methyltransferase, is down-regulated in companion cells. However, how MET1 is differentially regulated in gametophytes remains unexplored. ARID1, a transcription factor that is specifically depleted in sperm cells, is occupied by MET1-dependent CG methylation. Here, we show that MET1 confines ARID1 to the vegetative cell of male gametes, but ARID1 conversely represses MET1 in the central cell of female gametes. Compared to the vegetative celllocalization in wild type pollen, ARID1 expands to sperm cells in the met1 mutant. To understand whether MET1-dependent ARID1 inhibition exists during female gametogenesis, we first show that ARID1 is expressed in the megaspore mother cell (MMC), ARID1 but not MET1 is detectable in the central cell at maturity. Interestingly, compared to the absence of MET1 in the central cell and the egg cell of wild type ovules, MET1 significantly accumulates in these two cells in arid1 ovules. Lastly, we show that both ARID1 and MET1 are required for the cell specification of MMC. Collectively, our results uncover a reciprocal dependence between ARID1 and MET1, and provide a clue to further understand how the specification of MMC is likely regulated by DNA methylation.

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Summary Small RNAs are 20–24 nucleotides in length. In plants, small RNAs are classified into microRNAs (miRNAs) and small interfering RNAs (siRNAs), based on their biogenesis and molecular features. In contrast to the extensive knowledge of the roles of small RNAs in sporophytic tissues, the distribution and function of small RNAs in gametophytic cells have been less well studied. However, with the improvement of single‐cell sorting and RNA sequencing technologies, the distribution of small RNAs, especially siRNAs, between sperm cells and the vegetative cell, as well as the function of sperm‐delivered small RNAs during early seed development have been elucidated. This review summarizes work from the past 5 years regarding small RNAs in male gametes, emphasizing the intercellular communication and biological significance of small RNAs in Arabidopsis.