Actin Reorganization Underlies Phototropin-Dependent Positioning of Nuclei in Arabidopsis Leaf Cells

In epidermal and mesophyll cells of Arabidopsis (Arabidopsis thaliana) leaves, nuclei become relocated in response to strong blue light. We previously reported that nuclear positions both in darkness and in strong blue light are regulated by the blue light receptor phototropin2 in mesophyll cells. Here, we investigate the involvement of phototropin and the actin cytoskeleton in nuclear positioning in epidermal cells. Analysis of geometrical parameters revealed that, in darkness, nuclei were distributed near the center of the cell, adjacent to the inner periclinal wall, independent of cell shape. Dividing the anticlinal wall into concave, convex, and intermediate regions indicated that, in strong blue light, nuclei became relocated preferably to a concave region of the anticlinal wall, nearest the center of the cell. Mutant analyses verified that light-dependent nuclear positioning was regulated by phototropin2, while dark positioning of nuclei was independent of phototropin. Nuclear movement was inhibited by an actin-depolymerizing reagent, latrunculin B, but not by a microtubule-disrupting reagent, propyzamide. Imaging actin organization by immunofluorescence microscopy revealed that thick actin bundles, periclinally arranged parallel to the longest axis of the epidermal cell, were associated with the nucleus in darkness, whereas under strong blue light, the actin bundles, especially in the vicinity of the nucleus, became arranged close to the anticlinal walls. Light-dependent changes in the actin organization were clear in phot1 mutant but not in phot2 and phot1phot2 mutants. We propose that, in Arabidopsis, blue-light-dependent nuclear positioning is regulated by phototropin2-dependent reorganization of the actin cytoskeleton.Positioning organelles is essential for cellular activities. The nucleus changes its position in a programmatic way during development and the cell cycle (Britz, 1979; Nagai, 1993; Chytilova et al., 2000). For example, before asymmetrical divisions that give rise to the formation of root hair cells or guard mother cells, the nucleus migrates to the future division plane (Britz, 1979). In elongating root hair cells of Arabidopsis (Arabidopsis thaliana), the nucleus is maintained at a fixed distance from the apex (Ketelaar et al., 2002).While the nuclear migrations before mitosis and in root hairs are developmental, nuclear positioning is also regulated environmentally. In the fern, Adiantum capillus-veneris, nuclei in prothallial cells change their intracellular positions in response to light (Kagawa and Wada, 1993, 1995). The nuclei are located along the anticlinal walls in darkness and move toward the outer periclinal walls in weak light and to the anticlinal walls in strong light (Kagawa and Wada, 1993, 1995; Tsuboi et al., 2007). This response is called light-dependent nuclear positioning. Since the response is induced in cells that exhibit neither cell division nor expansion, it is believed to have a physiological role, distinct from the nuclear positioning associated with development.Recently, light-dependent nuclear positioning was reported in the spermatophyte Arabidopsis (Iwabuchi et al., 2007). In epidermal and mesophyll cells of dark-treated leaves, nuclei are distributed along the inner periclinal wall. Under strong light, they become located along the anticlinal walls. In mesophyll cells, nuclear movement from inner periclinal to anticlinal walls is induced repeatedly and specifically by blue light of high-fluence rate (more than 50 μ mol m⁻² s⁻¹) and is regulated by the blue light receptor phototropin2. Interestingly, mesophyll cells of the phot2 mutant have aberrantly positioned nuclei even in darkness. By contrast, the involvement of phototropins in nuclear positioning has not yet been examined for epidermal cells.Phototropin is a blue light receptor containing two light oxygen voltage domains at the N terminus, which bind an FMN chromophore, and a Ser/Thr kinase domain at the C terminus, which undergoes blue-light-dependent autophosphorylation (Briggs et al., 2001a; Christie, 2007). Arabidopsis possesses phototropins1 and 2 (Huala et al., 1997; Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). Phototropins are shown microscopically and biochemically to localize to the plasma membrane region (Briggs et al., 2001b; Sakamoto and Briggs, 2002; Kong et al., 2006) and mediate several responses, including phototropism (Liscum and Briggs, 1995; Sakai et al., 2001), stomatal opening (Kinoshita et al., 2001), and chloroplast movements (Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). In general, phototropin1 is more sensitive to light than its paralog and mediates low-fluence-rate light responses, whereas phototropin2 functions predominantly under higher fluence rates (Sakai et al., 2001).While the photoreceptor eliciting these nuclear movements has been revealed, the motile system responsible for moving the nuclei is still unknown. In general, organelle movements depend on the cytoskeleton, with the specific roles for actin and microtubules dependent on the organelle and species (Wada and Suetsugu, 2004). In land plants, the actin cytoskeleton plays a pivotal role in positioning organelles, including nuclei, chloroplasts, mitochondria, and peroxisomes (Wada and Suetsugu, 2004; Takagi et al., 2009).The role of the cytoskeleton in developmental nuclear movements has been investigated. In growing root hairs of Arabidopsis, the nuclear movements are driven along actin filaments (Ketelaar et al., 2002), whereas, in tobacco (Nicotiana tabacum) BY-2 cells, the cell-cycle-based nuclear migration before mitosis is found to depend on microtubules (Katsuta et al., 1990). In interphase Spirogyra crassa cells, centering of nuclei is regulated by both actin filaments and microtubules, but in distinct ways (Grolig, 1998). To the best of our knowledge, the cytoskeletal basis of environmentally induced nuclear movements in land plants has not been elucidated.The best-characterized organelle movements are the light-induced orientation movements of chloroplasts, and although exceptions have been reported, this movement depends on actin (Britz, 1979; Takagi, 2003; Wada et al., 2003). Under weak light, chloroplasts gather at the periclinal walls, perpendicular to the direction of light (accumulation response), whereas under strong light, they become positioned along the anticlinal walls, parallel to the direction of light (avoidance response). Recently, for Arabidopsis, Kadota et al. (2009) characterized the nature of the actin filaments probably involved in these movements. With the onset of either accumulation or avoidance response, short actin filaments appear at the leading edge of each chloroplast.In Arabidopsis, light-dependent nuclear positioning shows similarities to the chloroplast avoidance response, with regard to the direction of movement, relevant photoreceptor (phototropin2), and effective fluence rate (Iwabuchi and Takagi, 2008). On the other hand, nuclei are larger than chloroplasts and might require thicker, more rigid actin bundles for effective motility. Here, we investigate the involvement of the actin cytoskeleton as well as phototropin in regulatory system for nuclear positioning in epidermal cells of Arabidopsis leaves.