Plant Physiology Preview. Published on December 17, 2015, as DOI:10.1104/pp.15.01400
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Research area: Cell biology
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Article type: Research report
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Short title:
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UV avoidance behavior of the nucleus
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Corresponding author details:
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Prof. Ikuko Hara-Nishimura
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Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
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Tel.: +81 (75) 753-4142; Fax: +81 (75) 753-4142
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E-mail: [email protected]
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Plant nuclei move to escape ultraviolet-induced DNA
damage and cell death1[W]
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Kosei Iwabuchi, Jun Hidema, Kentaro Tamura, Shingo Takagi, and Ikuko
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Hara-Nishimura*
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Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan (K.I.,
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K.T, I.H-N.); Graduate School of Life Sciences, Tohoku University, Sendai 980-8577,
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Japan (J,H); Graduate School of Science, Osaka University, Machikaneyama-cho 1-1,
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Toyonaka, Osaka 560-0043, Japan (S.T.).
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One-sentence summary:
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In plant leaves exposed to ultraviolet B, nuclei are positioned to the side walls of cells in
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an actin-dependent manner to mitigate DNA damage and cell death.
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Footnote:
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This work was supported by a Specially Promoted Research Grant-in-Aid for
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Scientific Research to I.H-.N. (no. 22000014) and by Grants-in-Aid for Scientific
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Research to K.I. (no. 23-1024), I.H.-N. (no. 15H05776), to J.H. (no. 23120502), S.T.
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(no. 20570037), and K.T. (no. 20570036); from the Japan Society for the Promotion of
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Science (JSPS).
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* Address correspondence to [email protected].
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The author responsible for distribution of materials integral to the findings presented
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in this article in accordance with the policy described in the Instructions for Authors
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(www.plantphysiol.org) is: Ikuko Hara-Nishimura ([email protected]).
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[W] The online version of this article contains Web-only data.
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ABSTRACT
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A striking feature of plant nuclei is their light-dependent movement. In
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Arabidopsis thaliana leaf mesophyll cells, the nuclei move to the side walls of cells
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within 1–3 h after blue-light reception, although the reason is unknown. Here, we show
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that the nuclear movement is a rapid and effective strategy to avoid ultraviolet B
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(UVB)-induced damages. Mesophyll nuclei were positioned on the cell bottom in the
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dark, but sudden exposure of these cells to UVB caused severe DNA damage and cell
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death. The damage was remarkably reduced in both blue-light-treated leaves and mutant
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leaves defective in the actin cytoskeleton. Intriguingly, in plants grown under high-light
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conditions, the mesophyll nuclei remained on the side walls even in the dark. These
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results suggest that plants have two strategies for reducing UVB exposure: rapid nuclear
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movement against acute exposure and nuclear anchoring against chronic exposure.
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INTRODUCTION
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Being sessile, plants are constantly exposed to strong light. One of the
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mechanisms for coping with strong light is the relocation movement of organelles
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(Wada and Suetsugu, 2004; Takagi et al., 2011; Griffis et al., 2014). The nuclei move to
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the side walls of cells in response to strong light, a plant-specific phenomenon that is
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conserved in vascular plants such as the fern Adiantum capillus-veneris (Tsuboi et al.,
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2007) and the seed plant Arabidopsis thaliana (Iwabuchi et al., 2007). In leaves of
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Arabidopsis, nuclei of mesophyll and pavement cells are positioned at the center of the
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cell bottom in the dark and relocate to the side walls within 1 h of continuous irradiation
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with strong blue light (more than 50 µmol m-2 s-1) (Iwabuchi et al., 2007; Iwabuchi et al.,
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2010).
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Analysis of Arabidopsis and Adiantum mutants indicated that the side-wall
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nuclear positioning is regulated by the blue light receptor phototropin2 (Iwabuchi et al.,
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2007; Tsuboi et al., 2007; Iwabuchi et al., 2010). Moreover, pharmacological analysis
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indicated that nuclear movement is dependent on the actin cytoskeleton (Iwabuchi et al.,
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2010). Recently, Higa et al. (2014) proposed a mechanism for moving nuclei to the side
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walls in pavement cells of Arabidopsis: plastids attach to the nuclei (which cannot move
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autonomously) and pull them toward the side walls. Plastids (chloroplasts) can
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autonomously move toward any direction within cells according to the direction or
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intensity of blue light (Tsuboi et al., 2009; Tsuboi and Wada, 2011; Wada, 2013). In
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Arabidopsis, chloroplasts are positioned on the cell bottoms in the dark, and move to the
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side walls in strong light. Chloroplast movement is also regulated by phototropins, the
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actin cytoskeleton, and other proteins (Kong and Wada, 2014).
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The mechanism of the dark-induced cell-bottom positioning of nuclei is different
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from the mechanism of side-wall positioning. We reported that the cell-bottom
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positioning of nuclei is regulated by the plant-specific motor myosin XI-i (Tamura et al.,
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2013). In myosin XI-i mutants, the cell-bottom positioning is aberrant but the side-wall
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positioning occurs normally (Tamura et al., 2013). The actin cytoskeleton is also
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required for the cell-bottom positioning (Iwabuchi et al., 2010). Thus, the dark-induced
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positioning of nuclei is regulated by both myosin XI-i and actin cytoskeleton.
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The physiological roles of the nuclear movement remain unknown. Ultraviolet B
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(UVB) in sunlight (280-320 nm) damages nuclear DNA by directly producing
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cyclobutane pyrimidine dimers (CPDs) and [6-4] photoproducts (Britt, 1996). These
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photoproducts inhibit transcription and replication (Batista et al., 2009), and if the
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damage cannot be repaired, cell death (apoptosis) occurs (Nawkar et al., 2013).
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Eventually, UVB causes carcinogenesis in animals (Pfeifer and Besaratinia, 2012) and
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growth inhibition and reduced crop yields in plants (Hidema and Kumagai, 2006). To
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mitigate UV stress, plants have developed several protective mechanisms such as DNA
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repair, pigmentation, and leaf thickening (Britt, 1996; Steyn et al., 2002). Here we
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investigated whether nuclear movement is another UV-protection system. To this end,
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we examined 1) the effect of UVB on a dominant-negative mutant (actin8D, also called
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frizzy1) with a defect in actin polymerization (Kato et al., 2010), and 2) the effects of
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high light conditions and field conditions on the positioning of nuclei. Our results
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provide evidence for a new type of UV-protection in plants.
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RESULTS AND DISCUSSION
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Actin Cytoskeleton Differently Regulates Nuclear Movement in Mesophyll and
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Pavement Cells
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To confirm the involvement of the actin cytoskeleton in nuclear movement, we
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used the actin8D, in which Glu-272 in the hydrophobic loop of ACTIN8 is replaced
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with lysine, resulting in actin filament fragmentation (Kato et al., 2010). The
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dark-induced-cell-bottom positioning of the nuclei was impaired in actin8D
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palisade-mesophyll cells (Fig. 1, A and B, Dark-adapted): 57% of the actin8D nuclei
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were aberrantly positioned on the side walls even in the dark (Fig. 1C, Mesophyll cells).
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This result, together with the finding that myosin XI-i links the nuclear membrane and
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actin filaments to control dark-induced nuclear positioning in palisade-mesophyll cells
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(Tamura et al., 2013), indicates that the actin-myosin XI-i cytoskeleton drives nuclei to
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the cell bottom during darkening. However, in actin8D pavement cells,
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dark-induced-cell-bottom positioning was not substantially impaired (Fig. 1, Pavement
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cells), suggesting involvement of actin cytoskeleton in the dark-induced nuclear
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positioning depends on the cell type. On the other hand, in the presence of blue light,
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the nuclear relocation to the side walls was completely impaired in actin8D pavement
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cells (Fig. 1, Pavement cells), although the nuclear relocation was not able to be
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determined in actin8D palisade-mesophyll cells because 57% of the nuclei were
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positioned on the side walls before blue-light irradiation (Fig. 1, Mesophyll cells).
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These results indicate that nuclear movement is regulated differently in mesophyll cells
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and pavement cells (discussed below).
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Side-wall Nuclear Positioning Protects Leaf Cells from UVB-induced Cell Death
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The question is what are the physiological meanings of switching the nuclear
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position within the cells. In spongy-mesophyll cells of dark-adapted leaves, the nuclei
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moved to the topside (Fig. 2A), which is the opposite direction to that in
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palisade-mesophyll cells. Similarly, the nuclear movements in pavement cells in
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dark-adapted leaves were directed downwards in adaxial (upper) side of a leaf and
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directed upwards in the abaxial (lower) side (Fig. 2A). Thus, plants in the dark tend to
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position the nuclei on the side toward the body centre as if to keep genetic materials
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farther from external environmental stresses. However, this nuclear positioning was
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fatal to mesophyll cells under certain conditions. Irradiating dark-adapted cotyledons
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with UVB at 2.5 W m−2 for 5 min (equivalent to midday sun) induced the death of
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mesophyll cells (Supplemental Fig. S1). By contrast, UV-induced cell death was
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noticeably suppressed in blue-light-treated cotyledons (Fig. 2B) and the dark-adapted
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actin8D cotyledons (Fig. 2C), both of which positioned most mesophyll nuclei on the
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side-walls of the cells (Fig. 1B). These results indicate that side-wall nuclear positioning
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protects leaf cells from UV-induced cell death.
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Side-wall Nuclear Positioning Mitigates DNA Damage to the Nuclei
To quantitatively determine whether the side-wall nuclear positioning reduces
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UV-induced DNA damage, blue-light-treated leaves and dark-adapted leaves were
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irradiated with UVB for 5 min. UVB-induced DNA damage of the leaves was assessed
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with an assay for CPDs, which were detected by immunostaining. In the
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blue-light-treated mesophyll cells, 76% of the nuclei were positioned on the side walls
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and their CPD levels were undetectable (Fig. 3A, right). By contrast, in the
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dark-adapted mesophyll cells, only 8% of the nuclei were positioned on the side walls
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and their CPD levels were high (Fig. 3A, left). Similar differences were observed in
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pavement cells (Fig. 3B), while little difference was observed in guard cells, in which
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the nuclei are less motile (Fig. 3C). To statistically analyze the correlation between
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side-wall nuclear positioning and the UV-induced DNA damage, we used the leaves
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treated with blue light for 0, 1, and 3 h, in which the side-wall nuclear-positioning rates
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increased during the course of the blue-light treatment (Fig. 1B; Supplemental Fig.
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S2A). The side-wall nuclear-positioning rates were negatively correlated with the CPD
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amounts in mesophyll cells and in pavement cells (Fig. 3D).
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Leaf nuclei are lens-shaped, so that the light-exposed surface area (the so-called
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projection area) depends on the angle of incident light. A statistical analysis revealed
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that the projection areas in pavement cells and mesophyll cells were negatively
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correlated with the side-wall nuclear-positioning rates (Fig. 3E; Supplemental Fig. S2C).
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These results suggest that the lens-shaped nuclei slide into the narrow space between the
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plasma membrane and vacuolar membrane, and thereby the nuclear movement of nuclei
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reduces DNA damage. Additionally, in mesophyll cells, nuclear movement causes them
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to be shielded by chloroplasts (Fig. 1C). The nuclei might move with chloroplasts in a
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way that reduces their light exposure because chloroplasts also exhibit a
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blue-light-dependent side-wall positioning, which avoids the strong-light induced
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stresses such as ROS generation (Kasahara et al., 2002; Wada et al., 2003). Pavement
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cell nuclei are also hauled by the blue-light-dependent plastid (chloroplast) movement
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(Higa et al., 2014).
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To minimize the effect of the blue-light-dependent chloroplast movement on the
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nuclear movement, we used the dark-adapted mesophyll cells of actin8D leaves, in
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which chloroplasts were positioned on the cell bottoms as in the wild type (Fig. 1C). In
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mesophyll cells, the side-wall nuclear-positioning rates were much higher in actin8D
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than in the wild type (Fig. 3F). In addition, the nuclear projection areas (Fig. 3G) and
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amounts of UVB-induced CPD (Fig. 3H) were lower in actin8D than in the wild-type.
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Taken together, these results show that the side-wall nuclear positioning reduces the
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amount of UVB-light that leaves receive, mitigating the DNA damage to the nuclei.
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Side-wall Nuclear Positioning Is Associated with Light Conditions During Plant
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Growth
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Next, we examined the effects of ambient light conditions on nuclear positioning.
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In plants grown under high-light conditions (200–220 µmol m−2 s−1) for 3 weeks, most
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mesophyll nuclei remained on the side walls even in the dark, although in plants grown
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under low-light condition (30-50 µmol m−2 s−1), most mesophyll nuclei remained on the
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cell bottom in the dark (Fig. 4A). The side-wall nuclear-positioning rates of
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high-light-grown plants remarkably increased in an incubation-time-dependent manner
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(Fig. 4B). This was not the case with pavement nuclei (Fig. 4, A and B). The
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high-light-grown plants exhibited blue-light-induced nuclear positioning in both cell
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types (Supplemental Fig. S3, A and B). As expected, in Arabidopsis plants grown at a
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sunny spot in the field, most mesophyll nuclei remained on the side walls in the dark
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(Fig. 4, C and D). Hence, in sun leaves, the nuclei do not relocate from the side walls to
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the cell bottoms during darkening in order to prepare for sunlight the next day, while in
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shade leaves the nuclei relocate to the side walls to reduce their exposure to UVB light
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(Fig. 4E).
The present study provides two modes of UVB avoidance behavior of plant
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nuclei: Mode I is for plants grown in the low light and Mode II is for plants grown in
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the sun (Fig. 4E). In Mode I, pavement nuclei are located on the cell bottom in the dark
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(Iwabuchi et al., 2007), relocated rapidly to the side walls during light irradiation
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depending on actin (this study; Iwabuchi et al., 2010) and plastid movements (Higa et
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al., 2014), and then move back to the cell bottom during dark adaptation depending on
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actin (Iwabuchi et al., 2010) and an actin-myosin XI-i cytoskeleton (Tamura et al.,
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2013). On the other hand, mesophyll nuclei are anchored on the center of the cell
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bottom depending on actin (this study) and an actin-myosin XI-i cytoskeleton (Tamura
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et al., 2013). Nuclear movements during both light irradiation and dark adaptation
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depend on actin (Iwabuchi et al., 2010), but not on myosin XI-i (Tamura et al., 2013). A
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significant difference between Mode I and Mode II is the mesophyll nuclear positioning
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in the dark: nuclei are anchored on the cell bottom in Mode I, while nuclei locate on the
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side walls in Mode II. This result suggests that the actin-and-myosin XI-i system for the
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cell-bottom nuclear anchoring is not functional under the high-light conditions.
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Swichtching nuclear positions through the acin-and-myosin-XI-i system could be
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important for adaptation to environments in plants.
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Other methods, also induced by blue light, were reported to reduce the amount of
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UVB light received by leaves: accumulation of the UVB-absorbing pigment
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anthocyanin (Ahmad et al., 1995) and thickening of leaves (Lopez-Juez et al., 2007).
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However, accumulating sufficient amounts of anthocyanin required more than 12 h of
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continuous blue-light irradiation (Supplemental Fig. S4A) and leaf thickening required
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more than 50 h (Supplemental Fig. S4B). Therefore, these two responses are too slow to
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avoid UVB injury. In contrast, nuclear relocation to the side wall requires only 1-3 h of
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blue-light irradiation (Fig. 1B). Nuclear relocation is an effective and rapid strategy to
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avoid UVB-induced damage and cell death. However, UVB had no ability to induce
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nuclear relocation, although UVA with a longer wavelength (320–400 nm) induced it
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within 3-h irradiation (Supplemental Fig. S6). Thus, plants might use blue/UVA light as
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an indicator of the presence of UVB. This is consistent with the result that the
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side-wall-nuclear-positioning is regulated by the blue/UVA photoreceptor phototropin 2
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(Iwabuchi et al., 2007; Iwabuchi et al., 2010). Sessile plants might have developed such
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nuclear positioning strategies to overcome their inability to move away from excess
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light and to survive fluctuating environmental conditions.
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MATERIALS AND METHODS
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Plants and Growth Conditions
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Arabidopsis thaliana ecotype Columbia was used as the wild-type plant and the
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actin8D mutant (Kato et al., 2010) was in the Columbia background. Seeds were sown
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on compost and grown for 1–5 weeks at 22°C under conditions of 16 h white light (30–
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50 µmol m-2 s-1 or 200–220 µmol m-2 s-1) and 8 h dark. Unless otherwise stated, 4–
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5-week-old plants or 7-day-old seedlings grown under 30–50 µmol m-2 s-1 light were
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used. The wild Arabidopsis thaliana (ecotype unknown) was harvested from
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Kamogawa river in Japan.
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Dark and Light Treatments
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For dark treatment, detached leaves placed on GM plates (half-strength MS salts,
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0.025% MES-KOH, pH 5.7, and 0.5% Gellan gum) or seedlings on soil were placed in
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the dark for 16-24 h. For light treatment, samples were irradiated with 100 µmol m-2 s-1
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blue light (470 nm) or 30 µmol m-2 s-1 red light (660 nm) using an LED light source
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system (IS-mini; CCS). For UVB irradiation, 15 W m-2 UVA and 2.5 W m-2 UVB were
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applied for 5 min or 3 h using UVA and UVB sources (FL20SBLB, FL20SE; Toshiba).
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Light intensity was measured using a quantum sensor (LI-190SA; LI-COR) or a UVB
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sensor (SD204cos; LI-COR).
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Nuclear Staining
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Samples were fixed in fixation buffer (50 mM PIPES, 10 mM EGTA, and 5 mM
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MgSO4, pH 7.0) containing 2% formaldehyde and 0.3% glutaraldehyde for 1 h. Fixed
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samples were stained with 5 µg/mL Hoechst 33342 (CalBiochem), diluted in fixation
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buffer supplemented with 0.03% Triton X-100 for 1.5 h.
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Nuclear Area Measurement
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After nuclear staining, cells on the adaxial side were imaged using a fluorescence
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microscope (Axioskop 2 plus; Zeiss) equipped with a charge-coupled device (CCD)
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camera (VB-7010; Keyence). To determine the nuclear projection area, Hoechst-stained
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images, which had been converted to 32-bit grayscale images, were binarized, their
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nuclei were outlined and their surface area was determined using Image J
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(http://rsb.info.nih.gov/ij).
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Leaf Thickness Measurement
Fixed leaves were cut into approximately 3 × 5 mm pieces and samples were
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embedded in 5% agar. Transverse 200 µm-thick sections were prepared using a
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vibrating blade microtome (VT1000; Leica). Sections were stained with Hoechst as
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described for nuclear staining and then with 0.2 µg/mL Calcofluor White
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(Sigma-Aldrich) for 10 min. Sections were observed using a fluorescence microscope
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(Axioskop 2 plus) or a confocal laser scanning microscope (LSM780 META; Zeiss).
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The thickness of three regions in each section was determined using Image J and the
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mean of these three measurements was calculated as the thickness of the leaf.
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Anthocyanin Content Measurement
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The leaf anthocyanin content was determined as reported previously (Zhang et al.,
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2011). The fresh weight of each leaf was measured using an electronic balance
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(XS105DU; Mettler Toled). The absorbance at 530 nm of sample solutions was
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measured using a plate reader (Infinite 200 PRO; Tecan). The amount of anthocyanin
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was expressed as absorbance at 530 nm per gram of leaf fresh weight.
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Immunofluorescence Microscopy
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After UVB irradiation, leaves were fixed as described for nuclear staining. Before
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fixation, leaves were placed in the dark for 28 h so that all nuclei were positioned at the
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bottom. This was important because the immunofluorescence signal might be
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influenced by the position of the nuclei. For actin8D mutant analysis, leaves were
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centrifuged at 13,000 × g for 1 min to artificially relocate nuclei to the bottom
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(Supplemental Fig. S5). After fixation, leaves were cut into two pieces of approximately
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5 × 10 mm and fixed to a cover glass with the adaxial side facing upwards using
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cyanoacrylate glue (Konishi). Samples were further cut into pieces of approximately 1 ×
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1 mm on the cover glass and digested with fixation buffer containing 1% Cellulase
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“Onozuka” RS (Yakult) and 0.1% Pectolyase Y-23 (Kyowa Chemical Products) for 5
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min at 37°C. The adaxial layer was detached from the cover glass and further digested
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for 1 min. Samples were permeabilized with fixation buffer containing 0.5% Triton
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X-100 for 1 h and blocked in fixation buffer containing 20% fetal bovine serum
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(Thermo Scientific) for 1 h.
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To label CPDs, samples were immunostained with the mouse monoclonal primary
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TDM-2 antibody (Cosmo Bio; diluted 1:500) at 37°C overnight and with
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Alexa-488-conjugated anti-mouse IgG (Invitrogen; diluted 1:500) for 3 h. Antibodies
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were diluted in fixation buffer supplemented with 5% fetal bovine serum. Nuclei were
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stained with fixation buffer containing 5 µg/mL Hoechst 33342 for 15 min in the dark.
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Each specimen was mounted on a glass slide with 0.1% p-phenylenediamine diluted in
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13 mM NaCl, 0.51 mM Na2HPO4, 0.16 mM KH2PO4 (pH 9.0–9.5 with KOH), and 50%
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glycerol, and observed with a fluorescence microscope (Axioskop 2 plus). The exposure
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time was 0.04 s. Images of Hoechst staining and CPD staining were acquired for each
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nucleus. The mean signal intensity of each nucleus was determined using Image J.
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Hoechst-stained images that had been converted to 32-bit grayscale were binarized and
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nuclei were outlined. The extracted outlines were overlaid onto the corresponding
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CPD-stained images, and the mean signal intensity within each outline was measured.
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The CPD signal intensity of each UVB-irradiated nucleus was subtracted from the
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average intensity of non-UVB-irradiated nuclei. The net intensity of the CPD signal in
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the nucleus of each cell type was expressed relative to the fluorescent intensity of CPDs
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in the nucleus of a pavement cell that had undergone dark treatment followed by UVB
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irradiation. Heat-maps of CDP levels were created using the Image J plug-in HeatMap
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Histogram (http://www.samuelpean.com/heatmap-histogram/).
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Cell Death Measurement
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Seedlings were irradiated with 2.5 W m-2 UVB for 5 min and then with 30 µmol m-2
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s-1 red light for 5 days. Emitted light with a wavelength of 491–552 nm (488 nm
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excitation) was defined as autofluorescence of dead cells. Staining with trypan blue,
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which has intrinsic fluorescence in the far-red region of the spectra(Mosiman et al.,
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1997), was performed as described previously (Kim et al., 2008). To quantify dead cells,
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each trypan-blue-stained cotyledon was scanned from the upper to lower surface with a
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confocal microscope (LSM780; 610–758 nm emission, 488 nm excitation). The
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maximum intensity projection image of each cotyledon was binarized, and the total
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surface area of dead cells per leaf area was determined using Image J.
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Statistics
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All data with error bars are represented as mean ± s.e. by using StatPlus. The
P-values were determined with unpaired Student’s t-test.
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Sequence data from this article can be found in the GenBank/EMBL data libraries under
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accession number ACTIN8, At1g49240.
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Supplemental Data
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The following data are available in the online version of this article.
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Supplemental Figure S1. Detection of UVB-induced cell death in mesophyll cells.
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Supplemental Figure S2. Nuclear positioning on the side walls, nuclear projection
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area, and UVB-induced DNA damage in nuclei after blue-light irradiation.
Supplemental Figure S3. Distribution of nuclei in leaves of plants grown under
various light conditions.
Supplemental Figure S4. Changes in leaf thickness and accumulation of the
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UV-absorbing pigment anthocyanin during blue light treatment for UV protection.
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Supplemental Figure S5. Distribution of nuclei in mesophyll, pavement, and guard
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cells before and after centrifugation of leaves.
Supplemental Figure S6. Different irradiation effects of UVA and UVB on nuclear
positioning.
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ACKNOWLEDGMENTS
We are grateful to Masao Tasaka (Nara Institute of Science and Technology) for his
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donation of actin8D and to Tobias Baskin (University of Massachusetts) and James
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Raymond (Eigoken) for critical readings of this manuscript.
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AUTHOR CONTRIBUTIONS
K.I. and I.H.-N. designed the project. K.I. performed all experiments. K.T., J.H., and
S.T. discussed the project and data. K.I. and I.H.-N. wrote the paper.
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FIGURE LEGENDS
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Figure 1. Nuclear positioning in mesophyll and pavement cells after dark
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adaptation and blue-light irradiation in a dominant-negative mutant of ACTIN8.
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A, Cross sections of dark-adapted and 3-h-blue-light treated leaves of wild type and
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actin8D (a dominant-negative mutant of ACTIN8). Blue, cell walls stained with
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Calcofluor White; magenta, chloroplast autofluorescence; green (arrowheads), nuclei
352
stained with Hoechst 33342. B, Pavement and mesophyll cells of wild-type and actin8D
353
leaves after dark adaptation and 3-h-blue-light treatment. Cells are outlined with yellow
354
dotted lines. Nuclei stained with Hoechst 33342 are shown in blue. C, Side-wall
355
nuclear-positioning rates of pavement and mesophyll cells of wild-type and actin8D
356
leaves after blue-light irradiation. Data represent mean ± SEM (n = 5 leaves, *P < 0.05,
357
**P < 0.01).
358
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Figure 2. Significant reduction of UVB-induced cell death in blue-light treated
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cotyledons and actin8D cotyledons. A, Cross-section of a dark-adapted leaf of a
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3-week-old plant. Blue, cell walls stained with Calcofluor White; magenta, chloroplast
362
autofluorescence; green (arrowheads), nuclei stained with Hoechst 33342. B, A set of
363
the dark-adapted and 3-h-blue-light-treated cotyledons were irradiated with UVB for 5
364
min (+ UVB) and unirradiated (- UVB). Dead cells were stained with trypan blue. Bars,
365
1 mm. Data of dead cells represent mean ± SEM (n = 5–7 leaves, **P < 0.01). C, A set
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of the dark-adapted wild-type and actin8D cotyledons were irradiated with UVB for 5
367
min (+ UVB) and unirradiated (- UVB). Dead cells were stained with trypan blue. Bars,
368
1 mm. Data of dead cells represent mean ± SEM (n = 5–6 leaves, **P < 0.01).
369
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Figure 3. UVB-induced DNA damage is negatively correlated with nuclear
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positioning on the side walls. A-C, Mesophyll (A), pavement (B), and guard cells (C)
372
after dark adaptation and 3-h-blue-light treatment are shown together with nuclei
373
stained with Hoechst 33342 (blue) and the side-wall nuclear-positioning rates (%, mean
374
± SEM). See Supplemental Fig. S2 for original data. A nucleus stained with Hoechst
375
33342 (nucleus) and a heat-map visualization of the UVB-induced CPD amounts (CPD).
376
See METHOD SUMMARY for details. Bars, 20 µm. D, Negative correlations between
17
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
377
UVB-induced CPD amounts and side-wall nuclear-positioning rates in mesophyll and
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pavement cells. See Supplemental Fig. S2A and 2B for original data. Linear
379
regressions: y = -0.5583x + 58.759 and R² = 0.8014 for mesophyll cells; y = -1.3465x +
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128.42 and R² = 0.9612 for pavement cells; y = 2.9351x + 59.02 and R² = 0.98489 for
381
guard cells. E, Negative correlations between nuclear projection areas and side-wall
382
nuclear-positioning rates in mesophyll and pavement cells. See Supplemental Fig. S2A
383
and 2C for original data. Linear regressions: y = -0.4514x + 67.242 and R² = 0.99615
384
for mesophyll cells; y = -0.4353x + 52.71 and R² = 0.993 for pavement cells; y =
385
-0.1148x + 9.5488 and R² = 0.0248 for guard cells. F-H, side-wall nuclear-positioning
386
rates (F), nuclear projection area (G), and CPD amount (H) of the dark-adapted
387
mesophyll cells of wild-type and actin8D leaves. See MATERIALS AND METHODS
388
and Supplemental Fig. S5 for details. Data represent mean ± SEM (upper, n = 5 leaves,
389
**P < 0.01; middle, n = 5 leaves, **P < 0.01; bottom, n = 5 leaves, *P < 0.05, **P <
390
0.01).
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Figure 4. Positioning of nuclei on the side walls is associated with light conditions
393
during plant growth. A-D, Effects of light conditions on nuclear positioning in
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mesophyll and pavement cells of dark-adapted leaves. Plants were grown for 3 weeks
395
under the light conditions indicated (A, B) and grown in the sun (C, D). Nuclei
396
(arrowheads), chloroplasts (magenta), and cell walls (blue) are shown in left panels.
397
Cells outlined with yellow dotted lines and nuclei stained with Hoechst (blue) are
398
shown in right panels. Bars, 20 µm. Side-wall nuclear-positioning rates in the
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dark-adapted leaves are shown for plants grown for 1–3 weeks under the light
400
conditions indicated (mean ± SEM, n = 5 leaves, *P < 0.05, **P < 0.01) (B), and are
401
shown for four independent plants grown in the sun (D). E, Two modes of UVB
402
avoidance behavior of plant nuclei. Mode I is for low-light-acclimated plants in shade
403
and Mode II is for high-light-acclimated plants in the sun. Shown are involvements of
404
actin and myosin XI-i in each step of nuclear anchoring on the cell bottom in the dark,
405
side-wall nuclear-positioning during light irradiation, and nuclear movement to the cell
406
bottom during dark adaptation. See the text for explanations.
407
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