Abscisic acid switches cell division modes of asymmetric cell division and symmetric cell division in stem cells of protonemal filaments in the moss Physcomitrium patens

Abstract

Multicellular organisms regulate cell numbers and cell fate by using asymmetric cell division (ACD) and symmetric cell division (SCD) during their development and to adapt to unfavorable environmental conditions. A stem cell self-renews and generates differentiated cells. In plants, various types of cells are produced by ACD or SCD; however, the molecular mechanisms of ACD or SCD and the cell division mode switch are largely unknown. The moss Physcomitrium (Physcomitrella) patens is a suitable model to study plant stem cells due to its simple anatomy. Here, we report the cell division mode switch induced by abscisic acid (ABA) in P. patens. ABA is synthesized in response to abiotic stresses and induces round-shape cells, called brood cells, from cylindrical protonemal cells. Although two daughter cells with distinct sizes were produced by ACD in a protonemal stem cell on ABA-free media, the sizes of two daughter cells became similar with ABA treatment. Actin microfilaments were spatially localized on the apices of apical stem cells in protonemata on ABA-free media, but the polar accumulation was lost under the condition of ABA treatment. Moreover, ABA treatment conferred an identical cell fate to the daughter cells in terms of cell division activity. Collectively, the results indicate ABA may suppress the ACD characteristics but evoke SCD in cells. We also noticed that ABA-induced brood cells not only self-renewed but regenerated protonemal cells when ABA was removed from the media, suggesting that brood cells are novel stem cells that are induced by environmental signals in P. patens.

Keywords: Physcomitrium patens; abscisic acid (ABA); asymmetric cell division (ACD); stem cell; symmetric cell division (SCD).

Figure 1. Ratios of section areas of apical stem cells or side branch initials to basal cells. A: A schematic model of cell division patterns in protonemal cells on ABA-free media. Apical stem cells divide asymmetrically to produce daughter cells, an apical daughter cell and a basal daughter cell. While the apical daughter cell maintains cell division activity, the basal daughter cell, becoming a basal cell, shows reduced cell division activity and it divides up to two times to produce two more daughter cells. B: Protonemata were transferred onto media containing 50 µM ABA. The section areas of apical cells (A), side branch initials (A) and basal cells (B), which were just produced by cell division of the mother cells, were measured and the ratios of the section areas (B/A) were calculated. Values are means±SD of 11–20 independent protonemal filaments. Asterisks indicate significant difference between mock and ABA treatments (*** p<0.001; Student’s t-test). Scale bars=50 µm. Daughter cells with similar sizes were produced under the condition of ABA treatment.

Figure 2. Distribution of an actin marker in apical stem cells of protonemata. Protonemata were transferred onto solid media containing 50 µM ABA. Fluorescence of the actin marker LifeAct-Venus in 3–8 independent chloronemal cells was observed with an inverted microscope at 0, 3 and 5 h after transferring. The same cells were not sequentially observed after transferring but representative images are shown at each time point. Arrowheads indicate accumulation of LifeAct-Venus at the apices of chloronemal cells. Scale bars=50 µm. The polarized accumulation of LifeAct-Venus, which was highly accumulated on the apices of apical stem cells on ABA-free media, was lost within 3 h after 50 µM ABA treatment.

Figure 3. Cell division activity of basal daughter cells after ABA treatment. Protonemata were transferred onto solid media containing 50 µM ABA and cell division was observed at 0, 25 and 96 h. Red dots indicate cells that originated from the basal daughter cell (at 25 and 96 h). Black arrowheads indicate septum of basal daughter cells. Scale bars=50 µm. The basal daughter cell at 0 h (shown by an open red circle) continuously divided to produce 3 daughter cells, indicating maintenance of cell division activity in the basal daughter cells as well as in the apical daughter cells.

Figure 4. A: Chloronemal regeneration from brood cells. Brood cells, which were induced by 50 µM ABA treatment for 10 days, were transferred onto ABA-free media. Chloronemal regeneration from the brood cells was observed at 24 h after transferring. Scale bars=50 µm. B: A schematic model of the cell division mode switching induced by ABA. Protonemata undergo ACD under normal conditions but undergo SCD under stress conditions.