Generative Cell Specification Requires Transcription Factors Evolutionarily Conserved in Land Plants

Land plants differentiate germ cells in the haploid gametophyte. In flowering plants, a generative cell is specified as a precursor that subsequently divides into two sperm cells in the developing male gametophyte, pollen. Generative cell specification requires cell-cycle control and microtubule-dependent nuclear relocation (reviewed in [1-3]). However, the generative cell fate determinant and its evolutionary origin are still unknown. In bryophytes, gametophytes produce eggs and sperm in multicellular reproductive organs called archegonia and antheridia, respectively, or collectively called gametangia. Given the monophyletic origin of land plants [4-6], evolutionarily conserved mechanisms may play key roles in these diverse reproductive processes. Here, we showed that a single member of the subfamily VIIIa of basic helix-loop-helix (bHLH) transcription factors in the liverwort Marchantia polymorpha primarily accumulated in the initial cells and controlled their development into gametangia. We then demonstrated that an Arabidopsis thaliana VIIIa bHLH transiently accumulated in the smaller daughter cell after an asymmetric division of the meiosis-derived microspore and was required for generative cell specification redundantly with its paralog. Furthermore, these A. thaliana VIIIa bHLHs were functionally replaceable by the M. polymorpha VIIIa bHLH. These findings suggest the VIIIa bHLH proteins as core regulators for reproductive development, including germ cell differentiation, since an early stage of land plant evolution.