RESEARCH ARTICLE An evolutionarily conserved NIMA-related kinase directs rhizoid tip growth in the basal land plant Marchantia polymorpha Kento Otani 1 , Kimitsune Ishizaki 2 , Ryuichi Nishihama 3 , Shogo Takatani 1 , Takayuki Kohchi 3 , Taku Takahashi 1 and Hiroyasu Motose 1, * ABSTRACT Tip growth is driven by turgor pressure and mediated by the polarized accumulation of cellular materials. How a single polarized growth site is established and maintained is unclear. Here, we analyzed the function of NIMA-related protein kinase 1 (MpNEK1) in the liverwort Marchantia polymorpha. In the wild type, rhizoid cells differentiate from the ventral epidermis and elongate through tip growth to form hair-like protrusions. In Mpnek1 knockout mutants, rhizoids underwent frequent changes in growth direction, resulting in a twisted and/or spiral morphology. The functional MpNEK1-Citrine protein fusion localized to microtubule foci in the apical growing region of rhizoids. Mpnek1 knockouts exhibited increases in both microtubule density and bundling in the apical dome of rhizoids. Treatment with the microtubule-stabilizing drug taxol phenocopied the Mpnek1 knockout. These results suggest that MpNEK1 directs tip growth in rhizoids through microtubule organization. Furthermore, MpNEK1 expression rescued ectopic outgrowth of epidermal cells in the Arabidopsis thaliana nek6 mutant, strongly supporting an evolutionarily conserved NEK-dependent mechanism of directional growth. It is possible that such a mechanism contributed to the evolution of the early rooting system in land plants. KEY WORDS: Cell polarity, Marchantia polymorpha, NIMA-related kinase, Rhizoid, Tip growth INTRODUCTION Directional cell growth is essential for the development of multicellular organisms. This is especially true for plants, owing to their sessile and autotrophic lifestyle. Plant cells are surrounded by rigid cell walls, which restrict motility; thus, directional cell growth is indispensable for morphogenesis and reproduction in land plants. Plant cell growth is driven by hydraulic turgor pressure, along with localized cell wall deposition and loosening of the cell wall. The turgor pressure causes the cell wall and plasma membrane to stretch, which increases the surface area of the cell. The direction of cell growth is primarily determined by the orientation of cellulose microfibrils in the cell wall, which, in turn, is directed by the cortical microtubule array (Green, 1962; Ledbetter and Porter, 1963). Plant cell growth is categorized as either diffuse or tip growth. Diffuse growth, which occurs in most plant cells, involves the even expansion of the entire cell surface, whereas tip growth, which occurs in specialized cells such as root hairs and rhizoids, results from spatially focused cell expansion, resulting in polarized growth and a filamentous cell morphology. Although tip growth is an evolutionarily conserved process in plants and has been extensively studied in root hairs, pollen tubes and protonemata (Rounds and Bezanilla, 2013), it is not known how a single growth point is established and maintained in this process. Rhizoids are considered to be an early root system in land plant evolution (Jones and Dolan, 2012; Kenrick and Strullu-Derrien, 2014). Formed by the gametophytes of charophytes and seedless land plants, including bryophytes, lycophytes and monilophytes, rhizoids absorb nutrients and attach the gametophyte to a substrate (Jones and Dolan, 2012; Duckett et al., 2014). Although the mechanism of rhizoid development is not well understood, recent studies have revealed that the same regulatory mechanisms mediate the development of rhizoids in bryophytes and root hairs in angiosperms (Proust et al., 2016; Honkanen et al., 2016). The basic helix-loop-helix (bHLH) transcription factors ROOT HAIR DEFECTIVE 6 (RHD6) and RHD6-like (RSL) are conserved in land plants and positively regulate development of polar growing cells, such as caulonema cells, rhizoids and root hairs (Menand et al., 2007; Jang et al., 2011; Proust et al., 2016). Heterologous expression of PpRSL1 from Physcomitrella patens and MpRSL1 from Marchantia polymorpha rescued the loss of root hairs in the Arabidopsis thaliana rhd6 mutant (Menand et al., 2007; Proust et al., 2016). Another family of bHLH transcription factors, Lotus japonicus ROOTHAIRLESS LIKE (LRL), is conserved in streptophytes and participates in the development of rhizoids and root hairs (Tam et al., 2015; Breuninger et al., 2016). In addition, auxin commonly promotes the development of rhizoids and root hairs (Pitts et al., 1998; Sakakibara et al., 2003; Jang et al., 2011; Flores-Sandoval et al., 2015; Kato et al., 2015; Tam et al., 2015; Bowman, 2016). These results demonstrate that a well-conserved molecular mechanism mediates tip growth in land plants. Indeed, forward genetic screening has identified genes required for rhizoid growth and the Arabidopsis orthologs of some of these genes participate in tip growth in root hairs (Honkanen et al., 2016). However, the roles of these genes in rhizoid growth have just started to emerge and the mechanism governing rhizoid tip growth remains to be elucidated. Never in mitosis A (NIMA)-related kinases (NEKs) constitute a family of mitotic kinases that have been shown to control various mitotic events in fungi and animals, such as the G2/M transition, centrosome separation and spindle formation (Osmani et al., 1988; Fry et al., 2012). By contrast, NEK members in plants regulate directional cell growth during interphase (Motose et al., 2008; Sakai et al., 2008; Motose et al., 2011). Consistent with this, NIMA kinase of the filamentous fungus Aspergillus nidulans, a representative member of the NEK family, has been shown to regulate directional Received 12 June 2017; Accepted 23 January 2018 1 Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan. 2 Department of Biology, Graduate School of Science, Kobe University, Kobe 657-8501, Japan. 3 Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan. *Author for correspondence (motose@cc.okayama-u.ac.jp) H.M., 0000-0001-5038-6640 1 © 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev154617. doi:10.1242/dev.154617 DEVELOPMENT