A NIMA-related protein kinase, MpNEK1, directs tip growth of rhizoids through microtubule depolymerization in a liverwort Marchantia polymorpha . The Mp nek1 knockouts were shown to develop curly and spiral rhizoids due to the fluctuated direction of growth. Still, physiological roles and mechanisms of MpNEK1-dependent rhizoid tip growth remain to be clarified. Here, we developed novel culture methods to further study rhizoid growth of M. polymorpha , in which plants were grown on vertical plates. We applied the established methods to investigate MpNEK1 function in rhizoid growth. Rhizoids of the wild-type and Mp nek1 plants grew toward the gravity. The aerial rhizoids were longer in Mp nek1 than in the wild type. When the rhizoids were grown on the surface of a cellophane sheet, rhizoid length was comparable between the wild type and Mp nek1 , whereas Mp nek1 developed more rhizoids compared to the wild type. We also applied gellan gum, which is more transparent than agar, to analyze rhizoids grown in the medium. Rhizoids of Mp nek1 displayed defect on entering into the solid medium. These results suggest that Mp nek1 rhizoids have the deficiency in invasive tip growth. Thus, stable directional growth is important for rhizoids to get into the soil to anchor plant body and to adsorb water and nutrients. Collectively, our newly designed growth systems are valuable for analyzing rhizoid growth.

NIMA-related kinases (NEK) regulate various mitotic events in fungi and animals, whereas plant NEKs regulate growth direction of cells and organs. The liverwortMarchantia polymorphahas a single functional MpNEK1gene, whose knockout leads to aberrant growth direction of rhizoids. Its gain-of-function phenotype, however, remains unknown. Here, we generated inducible transgenic lines of MpNEK1, using an estrogen receptor mediated system. Estradiol treatment efficiently increased accumulation of MpNEK1mRNA and overexpression at the protein level was confirmed in the inducible lines of MpNEK1-Citrine. Overexpression of MpNEK1 severely suppressed growth of rhizoids and thalli, eventually causing lethality of juvenile plants. The effect of estradiol was reversible until 3 days, whereas 7-days treatment resulted in irreversible suppression of growth. This severe growth suppression was observed even at the nanomolar level of estradiol. EdU staining clearly indicated elimination of cell proliferation by estradiol-induced MpNEK1. In conclusion, overexpression of MpNEK1 suppresses cell division and elongation, leading to severe growth defects and lethality. Our results imply that the expression of MpNEK1 is tightly regulated at the appropriate level for cell growth. Plant NEK might control cell division as in the case of fungi and animal NEKs.