Plant Nuclei Move to Escape Ultraviolet-Induced DNA Damage and Cell Death1[OPEN] Kosei Iwabuchi, Jun Hidema, Kentaro Tamura, Shingo Takagi, and Ikuko Hara-Nishimura* Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan (K.I., K.T., I.H.-N.); Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan (J.H.); and Graduate School of Science, Osaka University, Machikaneyama-cho 1-1, Toyonaka, Osaka 560-0043, Japan (S.T.) ORCID IDs: 0000-0002-3696-6806 (K.I.); 0000-0002-2289-2826 (S.T.); 0000-0001-8814-1593 (I.H.-N.).
A striking feature of plant nuclei is their light-dependent movement. In Arabidopsis (Arabidopsis thaliana) leaf mesophyll cells, the nuclei move to the side walls of cells within 1 to 3 h after blue-light reception, although the reason is unknown. Here, we show that the nuclear movement is a rapid and effective strategy to avoid ultraviolet B (UVB)-induced damages. Mesophyll nuclei were positioned on the cell bottom in the dark, but sudden exposure of these cells to UVB caused severe DNA damage and cell death. The damage was remarkably reduced in both blue-light-treated leaves and mutant leaves defective in the actin cytoskeleton. Intriguingly, in plants grown under high-light conditions, the mesophyll nuclei remained on the side walls even in the dark. These results suggest that plants have two strategies for reducing UVB exposure: rapid nuclear movement against acute exposure and nuclear anchoring against chronic exposure.
Being sessile, plants are constantly exposed to strong light. One of the mechanisms for coping with strong light is the relocation movement of organelles (Wada and Suetsugu, 2004; Takagi et al., 2011; Griffis et al., 2014). The nuclei move to the side walls of cells in response to strong light, a plant-specific phenomenon that is conserved in vascular plants such as the fern Adiantum capillus-veneris (Tsuboi et al., 2007) and the seed plant Arabidopsis (Arabidopsis thaliana; Iwabuchi et al., 2007). In leaves of Arabidopsis, nuclei of mesophyll and pavement cells are positioned at the center of the cell bottom in the dark and relocate to the side walls within 1 h of continuous irradiation with strong blue light (more than 50 mmol m22 s21; Iwabuchi et al., 2007; Iwabuchi et al., 2010). Analysis of Arabidopsis and Adiantum mutants indicated that the side-wall nuclear positioning is regulated by the blue-light receptor phototropin2 (Iwabuchi et al., 2007; Tsuboi et al., 2007; Iwabuchi et al., 2010).
1 This work was supported by a Specially Promoted Research Grant-in-Aid for Scientific Research to I.H.-N. (no. 22000014) and by Grants-in-Aid for Scientific Research to K.I. (no. 23-1024), I.H.-N. (no. 15H05776), J.H. (no. 23120502), S.T. (no. 20570037), and K.T. (no. 20570036) from the Japan Society for the Promotion of Science. * Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ikuko Hara-Nishimura ([email protected]). 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 article. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.15.01400
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Moreover, pharmacological analysis indicated that nuclear movement is dependent on the actin cytoskeleton (Iwabuchi et al., 2010). Recently, Higa et al. (2014) proposed a mechanism for moving nuclei to the side walls in pavement cells of Arabidopsis: plastids attach to the nuclei (which cannot move autonomously) and pull them toward the side walls. Plastids (chloroplasts) can autonomously move toward any direction within cells according to the direction or intensity of blue light (Tsuboi et al., 2009; Tsuboi and Wada, 2011; Wada, 2013). In Arabidopsis, chloroplasts are positioned on the cell bottoms in the dark and move to the side walls in strong light. Chloroplast movement is also regulated by phototropins, the actin cytoskeleton, and other proteins (Kong and Wada, 2014). The mechanism of the dark-induced cell-bottom positioning of nuclei is different from the mechanism of side-wall positioning. We reported that the cell-bottom positioning of nuclei is regulated by the plant-specific motor myosin XI-i (Tamura et al., 2013). In myosin XI-i mutants, the cell-bottom positioning is aberrant, but the side-wall positioning occurs normally (Tamura et al., 2013). The actin cytoskeleton is also required for the cell-bottom positioning (Iwabuchi et al., 2010). Thus, the dark-induced positioning of nuclei is regulated by both myosin XI-i and the actin cytoskeleton. The physiological roles of the nuclear movement remain unknown. Ultraviolet B (UVB) in sunlight (280– 320 nm) damages nuclear DNA by directly producing cyclobutane pyrimidine dimers (CPDs) and [6-4] photoproducts (Britt, 1996). These photoproducts inhibit transcription and replication (Batista et al., 2009), and if the damage cannot be repaired, cell death (apoptosis) occurs (Nawkar et al., 2013). Eventually, UVB causes carcinogenesis in animals (Pfeifer and Besaratinia,
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2012), and growth inhibition and reduced crop yields in plants (Hidema and Kumagai, 2006). To mitigate UV stress, plants have developed several protective mechanisms, such as DNA repair, pigmentation, and leaf thickening (Britt, 1996; Steyn et al., 2002). Here, we investigated whether nuclear movement is another UV-protection system. To this end, we examined (1) the effect of UVB on a dominant-negative mutant (actin8D, also called frizzy1) with a defect in actin polymerization (Kato et al., 2010), and (2) the effects of high light conditions and field conditions on the positioning of nuclei. Our results provide evidence for a new type of UV protection in plants. RESULTS AND DISCUSSION Actin Cytoskeleton Differently Regulates Nuclear Movement in Mesophyll and Pavement Cells
To confirm the involvement of the actin cytoskeleton in nuclear movement, we used actin8D, in which Glu272 in the hydrophobic loop of ACTIN8 is replaced with Lys, resulting in actin filament fragmentation (Kato et al., 2010). The dark-induced cell-bottom positioning of the nuclei was impaired in actin8D palisademesophyll cells (Fig. 1, A and B, Dark-adapted): 57% of the actin8D nuclei were aberrantly positioned on the
side walls even in the dark (Fig. 1C, Mesophyll cells). This result, together with the finding that myosin XI-i links the nuclear membrane and actin filaments to control dark-induced nuclear positioning in palisade-mesophyll cells (Tamura et al., 2013), indicates that the actin-myosin XI-i cytoskeleton drives nuclei to the cell bottom during darkening. However, in actin8D pavement cells, darkinduced cell-bottom positioning was not substantially impaired (Fig. 1, Pavement cells), suggesting involvement of the actin cytoskeleton in the dark-induced nuclear positioning depends on the cell type. On the other hand, in the presence of blue light, the nuclear relocation to the side walls was completely impaired in actin8D pavement cells (Fig. 1, Pavement cells), although the nuclear relocation was not able to be determined in actin8D palisade-mesophyll cells because 57% of the nuclei were positioned on the side walls before blue-light irradiation (Fig. 1, Mesophyll cells). These results indicate that nuclear movement is regulated differently in mesophyll cells and pavement cells (discussed below). Side-Wall Nuclear Positioning Protects Leaf Cells from UVB-Induced Cell Death
The question is what are the physiological meanings of switching the nuclear position within the cells. In spongy Figure 1. Nuclear positioning in mesophyll and pavement cells after dark adaptation and blue-light irradiation in a dominant-negative mutant of ACTIN8. A, Cross-sections of dark-adapted and 3-h blue-light-treated leaves of the wild type and actin8D (a dominant-negative mutant of ACTIN8). Blue, Cell walls stained with Calcofluor White; magenta, chloroplast autofluorescence; green (arrowheads), nuclei stained with Hoechst 33342. B, Pavement and mesophyll cells of wild-type and actin8D leaves after dark adaptation and 3-h blue-light treatment. Cells are outlined with yellow dotted lines. Nuclei stained with Hoechst 33342 are shown in blue. C, Side-wall nuclear-positioning rates of pavement and mesophyll cells of wildtype and actin8D leaves after blue-light irradiation. Data represent mean 6 SE (n = 5 leaves, *P , 0.05, **P , 0.01).
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mesophyll cells of dark-adapted leaves, the nuclei moved to the top side (Fig. 2A), which is the opposite direction to that in palisade-mesophyll cells. Similarly, the nuclear movements in pavement cells in dark-adapted leaves were directed downward in the adaxial (upper) side of a leaf and directed upward in the abaxial (lower) side (Fig. 2A). Thus, plants in the dark tend to position the nuclei on the side toward the body center as if to keep genetic materials farther from external environmental stresses. However, this nuclear positioning was fatal to mesophyll cells under certain conditions. Irradiating dark-adapted cotyledons with UVB at 2.5 W m22 for 5 min (equivalent to midday sun) induced death of mesophyll cells (Supplemental Fig. S1). By contrast, UV-induced cell death was noticeably suppressed in blue-light-treated cotyledons (Fig. 2B) and the dark-adapted actin8D cotyledons (Fig. 2C), both of which positioned most mesophyll nuclei
on the side walls of the cells (Fig. 1B). These results indicate that side-wall nuclear positioning protects leaf cells from UV-induced cell death. Side-Wall Nuclear Positioning Mitigates DNA Damage to the Nuclei
To quantitatively determine whether the side-wall nuclear positioning reduces UV-induced DNA damage, blue-light-treated leaves and dark-adapted leaves were irradiated with UVB for 5 min. UVB-induced DNA damage of the leaves was assessed with an assay for CPDs, which were detected by immunostaining. In the blue-light-treated mesophyll cells, 76% of the nuclei were positioned on the side walls and their CPD levels were undetectable (Fig. 3A, right). By contrast, in the dark-adapted mesophyll cells, only 8% of the nuclei
Figure 2. Significant reduction of UVB-induced cell death in blue-light-treated cotyledons and actin8D cotyledons. A, Cross-section of a darkadapted leaf of a 3-week-old plant. Blue, Cell walls stained with Calcofluor White; magenta, chloroplast autofluorescence; green (arrowheads), nuclei stained with Hoechst 33342. B, A set of the darkadapted and 3-h blue-light-treated cotyledons were irradiated with UVB for 5 min (+ UVB) and unirradiated (2 UVB). Dead cells were stained with trypan blue. Bars = 1 mm. Data of dead cells represent mean 6 SE (n = 5–7 leaves, **P , 0.01). C, A set of the dark-adapted wild-type and actin8D cotyledons were irradiated with UVB for 5 min (+ UVB) and unirradiated (2 UVB). Dead cells were stained with trypan blue. Bars = 1 mm. Data of dead cells represent mean 6 SE (n = 5–6 leaves, **P , 0.01).
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were positioned on the side walls and their CPD levels were high (Fig. 3A, left). Similar differences were observed in pavement cells (Fig. 3B), while little difference was observed in guard cells, in which the nuclei are less motile (Fig. 3C). To statistically analyze the correlation between side-wall nuclear positioning and the UVinduced DNA damage, we used the leaves treated with blue light for 0, 1, and 3 h, in which the side-wall nuclear-positioning rates increased during the course of
the blue-light treatment (Fig. 1B; Supplemental Fig. S2A). The side-wall nuclear-positioning rates were negatively correlated with the CPD amounts in mesophyll cells and pavement cells (Fig. 3D). Leaf nuclei are lens shaped, so that the light-exposed surface area (the so-called projection area) depends on the angle of incident light. A statistical analysis revealed that the projection areas in pavement cells and mesophyll cells were negatively correlated with the side-wall
Figure 3. UVB-induced DNA damage is negatively correlated with nuclear positioning on the side walls. A to C, Mesophyll (A), pavement (B), and guard cells (C) after dark adaptation and 3-h blue-light treatment are shown together with nuclei stained with Hoechst 33342 (blue) and the side-wall nuclear-positioning rates (%, mean 6 SE). See Supplemental Figure S2 for original data. Shown are a nucleus stained with Hoechst 33342 (Nucleus) and a heat-map visualization of the UVB-induced CPD amounts (CPD). See “Materials and Methods” for details. Bars = 20 mm. D, Negative correlations between UVB-induced CPD amounts and side-wall nuclear-positioning rates in mesophyll and pavement cells. See Supplemental Figure S2, A and B, for original data. Linear regressions: y = 20.5583x + 58.759 and R2 = 0.8014 for mesophyll cells; y = 21.3465x + 128.42 and R2 = 0.9612 for pavement cells; y = 2.9351x + 59.02 and R2 = 0.98489 for guard cells. E, Negative correlations between nuclear projection areas and side-wall nuclear-positioning rates in mesophyll and pavement cells. See Supplemental Figure S2, A and C, for original data. Linear regressions: y = 20.4514x + 67.242 and R2 = 0.99615 for mesophyll cells; y = 20.4353x + 52.71 and R2 = 0.993 for pavement cells; y = 20.1148x + 9.5488 and R2 = 0.0248 for guard cells. F to H, Side-wall nuclear-positioning rates (F), nuclear projection area (G), and CPD amount (H) of the dark-adapted mesophyll cells of wild-type and actin8D leaves. See “Materials and Methods” and Supplemental Figure S6 for details. Data represent mean 6 SE (top, n = 5 leaves, **P , 0.01; middle, n = 5 leaves, **P , 0.01; bottom, n = 5 leaves, **P , 0.01). Plant Physiol. Vol. 170, 2016
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Figure 4. Positioning of nuclei on the side walls is associated with light conditions during plant growth. A to D, Effects of light conditions on nuclear positioning in mesophyll and pavement cells of dark-adapted leaves. Plants were grown for 3 weeks under the light conditions indicated (A and B) and grown in the sun (C and D). Nuclei (arrowheads), chloroplasts (magenta), and cell walls (blue) are shown in the left panels. Cells outlined with yellow dotted lines and nuclei stained with Hoechst (blue) are shown in the right panels. Bars = 20 mm. Side-wall nuclear-positioning rates in the dark-adapted 682
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nuclear-positioning rates (Fig. 3E; Supplemental Fig. S2C). These results suggest that the lens-shaped nuclei slide into the narrow space between the plasma membrane and vacuolar membrane, and thereby the nuclear movement of nuclei reduces DNA damage. Additionally, in mesophyll cells, nuclear movement causes them to be shielded by chloroplasts (Fig. 1C). The nuclei might move with chloroplasts in a way that reduces their light exposure because chloroplasts also exhibit a blue-lightdependent side-wall positioning that avoids the stronglight-induced stresses such as ROS generation (Kasahara et al., 2002; Wada et al., 2003). Pavement cell nuclei are also hauled by the blue-light-dependent plastid (chloroplast) movement (Higa et al., 2014). To minimize the effect of the blue-light-dependent chloroplast movement on the nuclear movement, we used the dark-adapted mesophyll cells of actin8D leaves, in which chloroplasts were positioned on the cell bottoms as in the wild type (Fig. 1C). In mesophyll cells, the side-wall nuclear-positioning rates were much higher in actin8D than in the wild type (Fig. 3F). In addition, the nuclear projection areas (Fig. 3G) and amounts of UVB-induced CPD (Fig. 3H) were lower in actin8D than in the wild-type. Taken together, these results show that the side-wall nuclear positioning reduces the amount of UVB light that leaves receive, mitigating the DNA damage to the nuclei. Side-Wall Nuclear Positioning Is Associated with Light Conditions During Plant Growth
Next, we examined the effects of ambient light conditions on nuclear positioning. In plants grown under high-light conditions (200–220 mmol m22 s21) for 3 weeks, most mesophyll nuclei remained on the side walls even in the dark, although in plants grown under low-light conditions (30–50 mmol m22 s21), most mesophyll nuclei remained on the cell bottom in the dark (Fig. 4A). The side-wall nuclear-positioning rates of high-light-grown plants remarkably increased in an incubation-time-dependent manner (Fig. 4B). This was not the case with pavement nuclei (Fig. 4, A and B). The high-light-grown plants exhibited blue-light-induced nuclear positioning in both cell types (Supplemental Fig. S3, A and B). As expected, in Arabidopsis plants grown at a sunny spot in the field, most mesophyll nuclei remained on the side walls in the dark (Fig. 4, C and D). Hence, in sun leaves, the nuclei do not relocate from the side walls to the cell bottoms during darkening in order to prepare for sunlight the next day, while in shade leaves the nuclei relocate to the side walls to reduce their exposure to UVB light (Fig. 4E).
This study provides two modes of UVB avoidance behavior of plant nuclei: Mode I is for plants grown in the low light, and Mode II is for plants grown in the sun (Fig. 4E). In Mode I, pavement nuclei are located on the cell bottom in the dark (Iwabuchi et al., 2007), relocated rapidly to the side walls during light irradiation depending on actin (this study; Iwabuchi et al., 2010) and plastid movements (Higa et al., 2014), and then moved back to the cell bottom during dark adaptation depending on actin (Iwabuchi et al., 2010) and an actinmyosin XI-i cytoskeleton (Tamura et al., 2013). On the other hand, mesophyll nuclei are anchored on the center of the cell bottom depending on actin (this study) and an actin-myosin XI-i cytoskeleton (Tamura et al., 2013). Nuclear movements during both light irradiation and dark adaptation depend on actin (Iwabuchi et al., 2010) but not on myosin XI-i (Tamura et al., 2013). A significant difference between Mode I and Mode II is the mesophyll nuclear positioning in the dark: nuclei are anchored on the cell bottom in Mode I, while nuclei locate on the side walls in Mode II. This result suggests that the actin-and-myosin XI-i system for the cellbottom nuclear anchoring is not functional under high-light conditions. Switching nuclear positions through the actin-and-myosin-XI-i system could be important for adaptation to environments in plants. Other methods, also induced by blue light, were reported to reduce the amount of UVB light received by leaves: accumulation of the UVB-absorbing pigment anthocyanin (Ahmad et al., 1995) and thickening of leaves (López-Juez et al., 2007). However, accumulating sufficient amounts of anthocyanin required more than 12 h of continuous blue-light irradiation (Supplemental Fig. S4A), and leaf thickening required more than 50 h (Supplemental Fig. S4B). Therefore, these two responses are too slow to avoid UVB injury. By contrast, nuclear relocation to the side wall requires only 1 to 3 h of blue-light irradiation (Fig. 1B). Nuclear relocation is an effective and rapid strategy to avoid UVB-induced damage and cell death. However, UVB had no ability to induce nuclear relocation, although ultraviolet A (UVA) with a longer wavelength (320–400 nm) induced it within 3 h of irradiation (Supplemental Fig. S5). Thus, plants might use blue/UVA light as an indicator of the presence of UVB. This is consistent with the result that the side-wall nuclear positioning is regulated by the blue/UVA photoreceptor phototropin2 (Iwabuchi et al., 2007; Iwabuchi et al., 2010). Sessile plants might have developed such nuclear positioning strategies to overcome their inability to move away from excess light and to survive fluctuating environmental conditions.
Figure 4. (Continued.) leaves are shown for plants grown for 1 to 3 weeks under the light conditions indicated (mean 6 SE, n = 5 leaves, *P , 0.05, **P , 0.01; B) and are shown for four independent plants grown in the sun (D). E, Two modes of UVB avoidance behavior of plant nuclei. Mode I is for low-light-acclimated plants in shade, and Mode II is for high-light-acclimated plants in the sun. Shown are involvements of actin and myosin XI-i in each step of nuclear anchoring on the cell bottom in the dark, side-wall nuclear positioning during light irradiation, and nuclear movement to the cell bottom during dark adaptation. See the text for explanations. Plant Physiol. Vol. 170, 2016
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MATERIALS AND METHODS Plants and Growth Conditions Arabidopsis (Arabidopsis thaliana) ecotype Columbia was used as the wildtype plant, and the actin8D mutant (Kato et al., 2010) was in the Columbia background. Seeds were sown on compost and grown for 1 to 5 weeks at 22°C under conditions of 16 h white light (30–50 mmol m22 s21 or 200–220 mmol m22 s21) and 8 h dark. Unless otherwise stated, 4- to 5-week-old plants or 7-d-old seedlings grown under 30 to 50 mmol m22 s21 light were used. The wild Arabidopsis (ecotype unknown) was harvested from Kamogawa River in Japan.
Dark and Light Treatments For dark treatment, detached leaves placed on germination medium plates (half-strength Murashige and Skoog salts, 0.025% MES-KOH, pH 5.7, and 0.5% gellan gum) or seedlings on soil were placed in the dark for 16 to 24 h. For light treatment, samples were irradiated with 100 mmol m22 s21 blue light (470 nm) or 30 mmol m22 s21 red light (660 nm) using an LED light source system (IS-mini; CCS). For UVB irradiation, 15 W m22 UVA and 2.5 W m22 UVB were applied for 5 min or 3 h using UVA and UVB sources (FL20SBLB, FL20SE; Toshiba). Light intensity was measured using a quantum sensor (LI-190SA; LI-COR) or a UVB sensor (SD204cos; LI-COR).
Nuclear Staining Samples were fixed in fixation buffer (50 mM PIPES, 10 mM EGTA, and 5 mM MgSO4, pH 7.0) containing 2% formaldehyde and 0.3% glutaraldehyde for 1 h. Fixed samples were stained with 5 mg/mL Hoechst 33342 (CalBiochem), diluted in fixation buffer supplemented with 0.03% Triton X-100 for 1.5 h.
Nuclear Area Measurement After nuclear staining, cells on the adaxial side were imaged using a fluorescence microscope (Axioskop 2 plus; Zeiss) equipped with a CCD camera (VB7010; Keyence). To determine the nuclear projection area, Hoechst-stained images, which had been converted to 32-bit grayscale images, were binarized, their nuclei outlined, and their surface area determined using Image J (http:// rsb.info.nih.gov/ij).
Leaf Thickness Measurement Fixed leaves were cut into approximately 3- 3 5-mm pieces, and samples were embedded in 5% agar. Transverse 200-mm-thick sections were prepared using a vibrating blade microtome (VT1000; Leica). Sections were stained with Hoechst as described for nuclear staining and then with 0.2 mg/mL Calcofluor White (Sigma-Aldrich) for 10 min. Sections were observed using a fluorescence microscope (Axioskop 2 plus) or a confocal laser scanning microscope (LSM780 META; Zeiss). The thickness of three regions in each section was determined using Image J, and the mean of these three measurements was calculated as the thickness of the leaf.
Anthocyanin Content Measurement The leaf anthocyanin content was determined as reported previously (Zhang et al., 2011). The fresh weight of each leaf was measured using an electronic balance (XS105DU; Mettler Toledo). The A530 of sample solutions was measured using a plate reader (Infinite 200 PRO; Tecan). The amount of anthocyanin was expressed as A530 per gram of leaf fresh weight.
Immunofluorescence Microscopy After UVB irradiation, leaves were fixed as described for nuclear staining. Before fixation, leaves were placed in the dark for 28 h so that all nuclei were positioned at the bottom. This was important because the immunofluorescence signal might be influenced by the position of the nuclei. For actin8D mutant analysis, leaves were centrifuged at 13,000g for 1 min to artificially relocate nuclei to the bottom (Supplemental Fig. S6). After fixation, leaves were cut into two pieces of approximately 5 3 10 mm and fixed to a cover glass with the adaxial side facing upward using cyanoacrylate glue (Konishi). Samples were 684
further cut into pieces of approximately 1 3 1 mm on the cover glass and digested with fixation buffer containing 1% Cellulase “Onozuka” RS (Yakult) and 0.1% Pectolyase Y-23 (Kyowa Chemical Products) for 5 min at 37°C. The adaxial layer was detached from the cover glass and further digested for 1 min. Samples were permeabilized with fixation buffer containing 0.5% Triton X-100 for 1 h and blocked in fixation buffer containing 20% fetal bovine serum (Thermo Scientific) for 1 h. To label CPDs, samples were immunostained with the mouse monoclonal primary TDM-2 antibody (Cosmo Bio; diluted 1:500) at 37°C overnight and with Alexa-488-conjugated antimouse IgG (Invitrogen; diluted 1:500) for 3 h. Antibodies were diluted in fixation buffer supplemented with 5% fetal bovine serum. Nuclei were stained with fixation buffer containing 5 mg/mL Hoechst 33342 for 15 min in the dark. Each specimen was mounted on a glass slide with 0.1% p-phenylenediamine diluted in 13 mM NaCl, 0.51 mM Na2HPO4, 0.16 mM KH2PO4 (pH 9.0–9.5 with KOH), and 50% glycerol, and observed with a fluorescence microscope (Axioskop 2 plus). The exposure time was 0.04 s. Images of Hoechst staining and CPD staining were acquired for each nucleus. The mean signal intensity of each nucleus was determined using Image J. Hoechst-stained images that had been converted to 32-bit grayscale were binarized and nuclei were outlined. The extracted outlines were overlaid onto the corresponding CPD-stained images, and the mean signal intensity within each outline was measured. The CPD signal intensity of each UVB-irradiated nucleus was subtracted from the average intensity of non-UVB-irradiated nuclei. The net intensity of the CPD signal in the nucleus of each cell type was expressed relative to the fluorescent intensity of CPDs in the nucleus of a pavement cell that had undergone dark treatment followed by UVB irradiation. Heat maps of CPD levels were created using the Image J plug-in HeatMap Histogram (http://www.samuelpean.com/heatmap-histogram/).
Cell Death Measurement Seedlings were irradiated with 2.5 W m22 UVB for 5 min and then with 30 mmol m22 s21 red light for 5 d. Emitted light with a wavelength of 491 to 552 nm (488 nm excitation) was defined as autofluorescence of dead cells. Staining with trypan blue, which has intrinsic fluorescence in the far-red region of the spectra (Mosiman et al., 1997), was performed as described previously (Kim et al., 2008). To quantify dead cells, each trypan-blue-stained cotyledon was scanned from the upper to lower surface with a confocal microscope (LSM780; 610–758 nm emission, 488 nm excitation). The maximum intensity projection image of each cotyledon was binarized, and the total surface area of dead cells per leaf area was determined using Image J.
Statistics All data with error bars are represented as mean 6 SE using StatPlus. The P values were determined with unpaired Student’s t test. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number At1g49240 (ACTIN8).
Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Detection of UVB-induced cell death in mesophyll cells. Supplemental Figure S2. Nuclear positioning on the side walls, nuclear projection area, and UVB-induced DNA damage in nuclei after bluelight 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 UV-absorbing pigment anthocyanin during blue-light treatment for UV protection. Supplemental Figure S5. Different irradiation effects of UVA and UVB on nuclear positioning. Supplemental Figure S6. Distribution of nuclei in mesophyll, pavement, and guard cells before and after centrifugation of leaves. Plant Physiol. Vol. 170, 2016
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ACKNOWLEDGMENTS We are grateful to Masao Tasaka (Nara Institute of Science and Technology) for his donation of actin8D and to Tobias Baskin (University of Massachusetts) and James Raymond (Eigoken) for critical readings of this article. Received September 9, 2015; accepted December 12, 2015; published December 17, 2015.
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Kong SG, Wada M (2014) Recent advances in understanding the molecular mechanism of chloroplast photorelocation movement. Biochim Biophys Acta 1837: 522–530 López-Juez E, Bowyer JR, Sakai T (2007) Distinct leaf developmental and gene expression responses to light quantity depend on bluephotoreceptor or plastid-derived signals, and can occur in the absence of phototropins. Planta 227: 113–123 Mosiman VL, Patterson BK, Canterero L, Goolsby CL (1997) Reducing cellular autofluorescence in flow cytometry: an in situ method. Cytometry 30: 151–156 Nawkar GM, Maibam P, Park JH, Sahi VP, Lee SY, Kang CH (2013) UVInduced cell death in plants. Int J Mol Sci 14: 1608–1628 Pfeifer GP, Besaratinia A (2012) UV wavelength-dependent DNA damage and human non-melanoma and melanoma skin cancer. Photochem Photobiol Sci 11: 90–97 Steyn WJ, Wand SJE, Holcroft DM, Jacobs G (2002) Anthocyanins in vegetative tissues: a proposed unified function in photoprotection. New Phytol 155: 349–361 Takagi S, Islam MS, Iwabuchi K (2011) Dynamic behavior of doublemembrane-bounded organelles in plant cells. Int Rev Cell Mol Biol 286: 181–222 Tamura K, Iwabuchi K, Fukao Y, Kondo M, Okamoto K, Ueda H, Nishimura M, Hara-Nishimura I (2013) Myosin XI-i links the nuclear membrane to the cytoskeleton to control nuclear movement and shape in Arabidopsis. Curr Biol 23: 1776–1781 Tsuboi H, Suetsugu N, Kawai-Toyooka H, Wada M (2007) Phototropins and neochrome1 mediate nuclear movement in the fern Adiantum capillus-veneris. Plant Cell Physiol 48: 892–896 Tsuboi H, Wada M (2011) Chloroplasts can move in any direction to avoid strong light. J Plant Res 124: 201–210 Tsuboi H, Yamashita H, Wada M (2009) Chloroplasts do not have a polarity for light-induced accumulation movement. J Plant Res 122: 131– 140 Wada M (2013) Chloroplast movement. Plant Sci 210: 177–182 Wada M, Kagawa T, Sato Y (2003) Chloroplast movement. Annu Rev Plant Biol 54: 455–468 Wada M, Suetsugu N (2004) Plant organelle positioning. Curr Opin Plant Biol 7: 626–631 Zhang Y, Zheng S, Liu Z, Wang L, Bi Y (2011) Both HY5 and HYH are necessary regulators for low temperature-induced anthocyanin accumulation in Arabidopsis seedlings. J Plant Physiol 168: 367–374
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A striking feature of plant nuclei is their light-dependent movement. In Arabidopsis (Arabidopsis thaliana) leaf mesophyll cells, the nuclei move to the side walls of cells within 1 to 3 h after blue-light reception, although the reason is unknown. Here, we show that the nuclear movement is a rapid and effective strategy to avoid ultraviolet B (UVB)-induced damages. Mesophyll nuclei were positioned on the cell bottom in the dark, but sudden exposure of these cells to UVB caused severe DNA damage and cell death. The damage was remarkably reduced in both blue-light-treated leaves and mutant leaves defective in the actin cytoskeleton. Intriguingly, in plants grown under high-light conditions, the mesophyll nuclei remained on the side walls even in the dark. These results suggest that plants have two strategies for reducing UVB exposure: rapid nuclear movement against acute exposure and nuclear anchoring against chronic exposure.
Being sessile, plants are constantly exposed to strong light. One of the mechanisms for coping with strong light is the relocation movement of organelles (Wada and Suetsugu, 2004; Takagi et al., 2011; Griffis et al., 2014). The nuclei move to the side walls of cells in response to strong light, a plant-specific phenomenon that is conserved in vascular plants such as the fern Adiantum capillus-veneris (Tsuboi et al., 2007) and the seed plant Arabidopsis (Arabidopsis thaliana; Iwabuchi et al., 2007). In leaves of Arabidopsis, nuclei of mesophyll and pavement cells are positioned at the center of the cell bottom in the dark and relocate to the side walls within 1 h of continuous irradiation with strong blue light (more than 50 mmol m22 s21; Iwabuchi et al., 2007; Iwabuchi et al., 2010). Analysis of Arabidopsis and Adiantum mutants indicated that the side-wall nuclear positioning is regulated by the blue-light receptor phototropin2 (Iwabuchi et al., 2007; Tsuboi et al., 2007; Iwabuchi et al., 2010).
1 This work was supported by a Specially Promoted Research Grant-in-Aid for Scientific Research to I.H.-N. (no. 22000014) and by Grants-in-Aid for Scientific Research to K.I. (no. 23-1024), I.H.-N. (no. 15H05776), J.H. (no. 23120502), S.T. (no. 20570037), and K.T. (no. 20570036) from the Japan Society for the Promotion of Science. * Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ikuko Hara-Nishimura ([email protected]). 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 article. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.15.01400
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Moreover, pharmacological analysis indicated that nuclear movement is dependent on the actin cytoskeleton (Iwabuchi et al., 2010). Recently, Higa et al. (2014) proposed a mechanism for moving nuclei to the side walls in pavement cells of Arabidopsis: plastids attach to the nuclei (which cannot move autonomously) and pull them toward the side walls. Plastids (chloroplasts) can autonomously move toward any direction within cells according to the direction or intensity of blue light (Tsuboi et al., 2009; Tsuboi and Wada, 2011; Wada, 2013). In Arabidopsis, chloroplasts are positioned on the cell bottoms in the dark and move to the side walls in strong light. Chloroplast movement is also regulated by phototropins, the actin cytoskeleton, and other proteins (Kong and Wada, 2014). The mechanism of the dark-induced cell-bottom positioning of nuclei is different from the mechanism of side-wall positioning. We reported that the cell-bottom positioning of nuclei is regulated by the plant-specific motor myosin XI-i (Tamura et al., 2013). In myosin XI-i mutants, the cell-bottom positioning is aberrant, but the side-wall positioning occurs normally (Tamura et al., 2013). The actin cytoskeleton is also required for the cell-bottom positioning (Iwabuchi et al., 2010). Thus, the dark-induced positioning of nuclei is regulated by both myosin XI-i and the actin cytoskeleton. The physiological roles of the nuclear movement remain unknown. Ultraviolet B (UVB) in sunlight (280– 320 nm) damages nuclear DNA by directly producing cyclobutane pyrimidine dimers (CPDs) and [6-4] photoproducts (Britt, 1996). These photoproducts inhibit transcription and replication (Batista et al., 2009), and if the damage cannot be repaired, cell death (apoptosis) occurs (Nawkar et al., 2013). Eventually, UVB causes carcinogenesis in animals (Pfeifer and Besaratinia,
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2012), and growth inhibition and reduced crop yields in plants (Hidema and Kumagai, 2006). To mitigate UV stress, plants have developed several protective mechanisms, such as DNA repair, pigmentation, and leaf thickening (Britt, 1996; Steyn et al., 2002). Here, we investigated whether nuclear movement is another UV-protection system. To this end, we examined (1) the effect of UVB on a dominant-negative mutant (actin8D, also called frizzy1) with a defect in actin polymerization (Kato et al., 2010), and (2) the effects of high light conditions and field conditions on the positioning of nuclei. Our results provide evidence for a new type of UV protection in plants. RESULTS AND DISCUSSION Actin Cytoskeleton Differently Regulates Nuclear Movement in Mesophyll and Pavement Cells
To confirm the involvement of the actin cytoskeleton in nuclear movement, we used actin8D, in which Glu272 in the hydrophobic loop of ACTIN8 is replaced with Lys, resulting in actin filament fragmentation (Kato et al., 2010). The dark-induced cell-bottom positioning of the nuclei was impaired in actin8D palisademesophyll cells (Fig. 1, A and B, Dark-adapted): 57% of the actin8D nuclei were aberrantly positioned on the
side walls even in the dark (Fig. 1C, Mesophyll cells). This result, together with the finding that myosin XI-i links the nuclear membrane and actin filaments to control dark-induced nuclear positioning in palisade-mesophyll cells (Tamura et al., 2013), indicates that the actin-myosin XI-i cytoskeleton drives nuclei to the cell bottom during darkening. However, in actin8D pavement cells, darkinduced cell-bottom positioning was not substantially impaired (Fig. 1, Pavement cells), suggesting involvement of the actin cytoskeleton in the dark-induced nuclear positioning depends on the cell type. On the other hand, in the presence of blue light, the nuclear relocation to the side walls was completely impaired in actin8D pavement cells (Fig. 1, Pavement cells), although the nuclear relocation was not able to be determined in actin8D palisade-mesophyll cells because 57% of the nuclei were positioned on the side walls before blue-light irradiation (Fig. 1, Mesophyll cells). These results indicate that nuclear movement is regulated differently in mesophyll cells and pavement cells (discussed below). Side-Wall Nuclear Positioning Protects Leaf Cells from UVB-Induced Cell Death
The question is what are the physiological meanings of switching the nuclear position within the cells. In spongy Figure 1. Nuclear positioning in mesophyll and pavement cells after dark adaptation and blue-light irradiation in a dominant-negative mutant of ACTIN8. A, Cross-sections of dark-adapted and 3-h blue-light-treated leaves of the wild type and actin8D (a dominant-negative mutant of ACTIN8). Blue, Cell walls stained with Calcofluor White; magenta, chloroplast autofluorescence; green (arrowheads), nuclei stained with Hoechst 33342. B, Pavement and mesophyll cells of wild-type and actin8D leaves after dark adaptation and 3-h blue-light treatment. Cells are outlined with yellow dotted lines. Nuclei stained with Hoechst 33342 are shown in blue. C, Side-wall nuclear-positioning rates of pavement and mesophyll cells of wildtype and actin8D leaves after blue-light irradiation. Data represent mean 6 SE (n = 5 leaves, *P , 0.05, **P , 0.01).
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mesophyll cells of dark-adapted leaves, the nuclei moved to the top side (Fig. 2A), which is the opposite direction to that in palisade-mesophyll cells. Similarly, the nuclear movements in pavement cells in dark-adapted leaves were directed downward in the adaxial (upper) side of a leaf and directed upward in the abaxial (lower) side (Fig. 2A). Thus, plants in the dark tend to position the nuclei on the side toward the body center as if to keep genetic materials farther from external environmental stresses. However, this nuclear positioning was fatal to mesophyll cells under certain conditions. Irradiating dark-adapted cotyledons with UVB at 2.5 W m22 for 5 min (equivalent to midday sun) induced death of mesophyll cells (Supplemental Fig. S1). By contrast, UV-induced cell death was noticeably suppressed in blue-light-treated cotyledons (Fig. 2B) and the dark-adapted actin8D cotyledons (Fig. 2C), both of which positioned most mesophyll nuclei
on the side walls of the cells (Fig. 1B). These results indicate that side-wall nuclear positioning protects leaf cells from UV-induced cell death. Side-Wall Nuclear Positioning Mitigates DNA Damage to the Nuclei
To quantitatively determine whether the side-wall nuclear positioning reduces UV-induced DNA damage, blue-light-treated leaves and dark-adapted leaves were irradiated with UVB for 5 min. UVB-induced DNA damage of the leaves was assessed with an assay for CPDs, which were detected by immunostaining. In the blue-light-treated mesophyll cells, 76% of the nuclei were positioned on the side walls and their CPD levels were undetectable (Fig. 3A, right). By contrast, in the dark-adapted mesophyll cells, only 8% of the nuclei
Figure 2. Significant reduction of UVB-induced cell death in blue-light-treated cotyledons and actin8D cotyledons. A, Cross-section of a darkadapted leaf of a 3-week-old plant. Blue, Cell walls stained with Calcofluor White; magenta, chloroplast autofluorescence; green (arrowheads), nuclei stained with Hoechst 33342. B, A set of the darkadapted and 3-h blue-light-treated cotyledons were irradiated with UVB for 5 min (+ UVB) and unirradiated (2 UVB). Dead cells were stained with trypan blue. Bars = 1 mm. Data of dead cells represent mean 6 SE (n = 5–7 leaves, **P , 0.01). C, A set of the dark-adapted wild-type and actin8D cotyledons were irradiated with UVB for 5 min (+ UVB) and unirradiated (2 UVB). Dead cells were stained with trypan blue. Bars = 1 mm. Data of dead cells represent mean 6 SE (n = 5–6 leaves, **P , 0.01).
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were positioned on the side walls and their CPD levels were high (Fig. 3A, left). Similar differences were observed in pavement cells (Fig. 3B), while little difference was observed in guard cells, in which the nuclei are less motile (Fig. 3C). To statistically analyze the correlation between side-wall nuclear positioning and the UVinduced DNA damage, we used the leaves treated with blue light for 0, 1, and 3 h, in which the side-wall nuclear-positioning rates increased during the course of
the blue-light treatment (Fig. 1B; Supplemental Fig. S2A). The side-wall nuclear-positioning rates were negatively correlated with the CPD amounts in mesophyll cells and pavement cells (Fig. 3D). Leaf nuclei are lens shaped, so that the light-exposed surface area (the so-called projection area) depends on the angle of incident light. A statistical analysis revealed that the projection areas in pavement cells and mesophyll cells were negatively correlated with the side-wall
Figure 3. UVB-induced DNA damage is negatively correlated with nuclear positioning on the side walls. A to C, Mesophyll (A), pavement (B), and guard cells (C) after dark adaptation and 3-h blue-light treatment are shown together with nuclei stained with Hoechst 33342 (blue) and the side-wall nuclear-positioning rates (%, mean 6 SE). See Supplemental Figure S2 for original data. Shown are a nucleus stained with Hoechst 33342 (Nucleus) and a heat-map visualization of the UVB-induced CPD amounts (CPD). See “Materials and Methods” for details. Bars = 20 mm. D, Negative correlations between UVB-induced CPD amounts and side-wall nuclear-positioning rates in mesophyll and pavement cells. See Supplemental Figure S2, A and B, for original data. Linear regressions: y = 20.5583x + 58.759 and R2 = 0.8014 for mesophyll cells; y = 21.3465x + 128.42 and R2 = 0.9612 for pavement cells; y = 2.9351x + 59.02 and R2 = 0.98489 for guard cells. E, Negative correlations between nuclear projection areas and side-wall nuclear-positioning rates in mesophyll and pavement cells. See Supplemental Figure S2, A and C, for original data. Linear regressions: y = 20.4514x + 67.242 and R2 = 0.99615 for mesophyll cells; y = 20.4353x + 52.71 and R2 = 0.993 for pavement cells; y = 20.1148x + 9.5488 and R2 = 0.0248 for guard cells. F to H, Side-wall nuclear-positioning rates (F), nuclear projection area (G), and CPD amount (H) of the dark-adapted mesophyll cells of wild-type and actin8D leaves. See “Materials and Methods” and Supplemental Figure S6 for details. Data represent mean 6 SE (top, n = 5 leaves, **P , 0.01; middle, n = 5 leaves, **P , 0.01; bottom, n = 5 leaves, **P , 0.01). Plant Physiol. Vol. 170, 2016
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Figure 4. Positioning of nuclei on the side walls is associated with light conditions during plant growth. A to D, Effects of light conditions on nuclear positioning in mesophyll and pavement cells of dark-adapted leaves. Plants were grown for 3 weeks under the light conditions indicated (A and B) and grown in the sun (C and D). Nuclei (arrowheads), chloroplasts (magenta), and cell walls (blue) are shown in the left panels. Cells outlined with yellow dotted lines and nuclei stained with Hoechst (blue) are shown in the right panels. Bars = 20 mm. Side-wall nuclear-positioning rates in the dark-adapted 682
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nuclear-positioning rates (Fig. 3E; Supplemental Fig. S2C). These results suggest that the lens-shaped nuclei slide into the narrow space between the plasma membrane and vacuolar membrane, and thereby the nuclear movement of nuclei reduces DNA damage. Additionally, in mesophyll cells, nuclear movement causes them to be shielded by chloroplasts (Fig. 1C). The nuclei might move with chloroplasts in a way that reduces their light exposure because chloroplasts also exhibit a blue-lightdependent side-wall positioning that avoids the stronglight-induced stresses such as ROS generation (Kasahara et al., 2002; Wada et al., 2003). Pavement cell nuclei are also hauled by the blue-light-dependent plastid (chloroplast) movement (Higa et al., 2014). To minimize the effect of the blue-light-dependent chloroplast movement on the nuclear movement, we used the dark-adapted mesophyll cells of actin8D leaves, in which chloroplasts were positioned on the cell bottoms as in the wild type (Fig. 1C). In mesophyll cells, the side-wall nuclear-positioning rates were much higher in actin8D than in the wild type (Fig. 3F). In addition, the nuclear projection areas (Fig. 3G) and amounts of UVB-induced CPD (Fig. 3H) were lower in actin8D than in the wild-type. Taken together, these results show that the side-wall nuclear positioning reduces the amount of UVB light that leaves receive, mitigating the DNA damage to the nuclei. Side-Wall Nuclear Positioning Is Associated with Light Conditions During Plant Growth
Next, we examined the effects of ambient light conditions on nuclear positioning. In plants grown under high-light conditions (200–220 mmol m22 s21) for 3 weeks, most mesophyll nuclei remained on the side walls even in the dark, although in plants grown under low-light conditions (30–50 mmol m22 s21), most mesophyll nuclei remained on the cell bottom in the dark (Fig. 4A). The side-wall nuclear-positioning rates of high-light-grown plants remarkably increased in an incubation-time-dependent manner (Fig. 4B). This was not the case with pavement nuclei (Fig. 4, A and B). The high-light-grown plants exhibited blue-light-induced nuclear positioning in both cell types (Supplemental Fig. S3, A and B). As expected, in Arabidopsis plants grown at a sunny spot in the field, most mesophyll nuclei remained on the side walls in the dark (Fig. 4, C and D). Hence, in sun leaves, the nuclei do not relocate from the side walls to the cell bottoms during darkening in order to prepare for sunlight the next day, while in shade leaves the nuclei relocate to the side walls to reduce their exposure to UVB light (Fig. 4E).
This study provides two modes of UVB avoidance behavior of plant nuclei: Mode I is for plants grown in the low light, and Mode II is for plants grown in the sun (Fig. 4E). In Mode I, pavement nuclei are located on the cell bottom in the dark (Iwabuchi et al., 2007), relocated rapidly to the side walls during light irradiation depending on actin (this study; Iwabuchi et al., 2010) and plastid movements (Higa et al., 2014), and then moved back to the cell bottom during dark adaptation depending on actin (Iwabuchi et al., 2010) and an actinmyosin XI-i cytoskeleton (Tamura et al., 2013). On the other hand, mesophyll nuclei are anchored on the center of the cell bottom depending on actin (this study) and an actin-myosin XI-i cytoskeleton (Tamura et al., 2013). Nuclear movements during both light irradiation and dark adaptation depend on actin (Iwabuchi et al., 2010) but not on myosin XI-i (Tamura et al., 2013). A significant difference between Mode I and Mode II is the mesophyll nuclear positioning in the dark: nuclei are anchored on the cell bottom in Mode I, while nuclei locate on the side walls in Mode II. This result suggests that the actin-and-myosin XI-i system for the cellbottom nuclear anchoring is not functional under high-light conditions. Switching nuclear positions through the actin-and-myosin-XI-i system could be important for adaptation to environments in plants. Other methods, also induced by blue light, were reported to reduce the amount of UVB light received by leaves: accumulation of the UVB-absorbing pigment anthocyanin (Ahmad et al., 1995) and thickening of leaves (López-Juez et al., 2007). However, accumulating sufficient amounts of anthocyanin required more than 12 h of continuous blue-light irradiation (Supplemental Fig. S4A), and leaf thickening required more than 50 h (Supplemental Fig. S4B). Therefore, these two responses are too slow to avoid UVB injury. By contrast, nuclear relocation to the side wall requires only 1 to 3 h of blue-light irradiation (Fig. 1B). Nuclear relocation is an effective and rapid strategy to avoid UVB-induced damage and cell death. However, UVB had no ability to induce nuclear relocation, although ultraviolet A (UVA) with a longer wavelength (320–400 nm) induced it within 3 h of irradiation (Supplemental Fig. S5). Thus, plants might use blue/UVA light as an indicator of the presence of UVB. This is consistent with the result that the side-wall nuclear positioning is regulated by the blue/UVA photoreceptor phototropin2 (Iwabuchi et al., 2007; Iwabuchi et al., 2010). Sessile plants might have developed such nuclear positioning strategies to overcome their inability to move away from excess light and to survive fluctuating environmental conditions.
Figure 4. (Continued.) leaves are shown for plants grown for 1 to 3 weeks under the light conditions indicated (mean 6 SE, n = 5 leaves, *P , 0.05, **P , 0.01; B) and are shown for four independent plants grown in the sun (D). E, Two modes of UVB avoidance behavior of plant nuclei. Mode I is for low-light-acclimated plants in shade, and Mode II is for high-light-acclimated plants in the sun. Shown are involvements of actin and myosin XI-i in each step of nuclear anchoring on the cell bottom in the dark, side-wall nuclear positioning during light irradiation, and nuclear movement to the cell bottom during dark adaptation. See the text for explanations. Plant Physiol. Vol. 170, 2016
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MATERIALS AND METHODS Plants and Growth Conditions Arabidopsis (Arabidopsis thaliana) ecotype Columbia was used as the wildtype plant, and the actin8D mutant (Kato et al., 2010) was in the Columbia background. Seeds were sown on compost and grown for 1 to 5 weeks at 22°C under conditions of 16 h white light (30–50 mmol m22 s21 or 200–220 mmol m22 s21) and 8 h dark. Unless otherwise stated, 4- to 5-week-old plants or 7-d-old seedlings grown under 30 to 50 mmol m22 s21 light were used. The wild Arabidopsis (ecotype unknown) was harvested from Kamogawa River in Japan.
Dark and Light Treatments For dark treatment, detached leaves placed on germination medium plates (half-strength Murashige and Skoog salts, 0.025% MES-KOH, pH 5.7, and 0.5% gellan gum) or seedlings on soil were placed in the dark for 16 to 24 h. For light treatment, samples were irradiated with 100 mmol m22 s21 blue light (470 nm) or 30 mmol m22 s21 red light (660 nm) using an LED light source system (IS-mini; CCS). For UVB irradiation, 15 W m22 UVA and 2.5 W m22 UVB were applied for 5 min or 3 h using UVA and UVB sources (FL20SBLB, FL20SE; Toshiba). Light intensity was measured using a quantum sensor (LI-190SA; LI-COR) or a UVB sensor (SD204cos; LI-COR).
Nuclear Staining Samples were fixed in fixation buffer (50 mM PIPES, 10 mM EGTA, and 5 mM MgSO4, pH 7.0) containing 2% formaldehyde and 0.3% glutaraldehyde for 1 h. Fixed samples were stained with 5 mg/mL Hoechst 33342 (CalBiochem), diluted in fixation buffer supplemented with 0.03% Triton X-100 for 1.5 h.
Nuclear Area Measurement After nuclear staining, cells on the adaxial side were imaged using a fluorescence microscope (Axioskop 2 plus; Zeiss) equipped with a CCD camera (VB7010; Keyence). To determine the nuclear projection area, Hoechst-stained images, which had been converted to 32-bit grayscale images, were binarized, their nuclei outlined, and their surface area determined using Image J (http:// rsb.info.nih.gov/ij).
Leaf Thickness Measurement Fixed leaves were cut into approximately 3- 3 5-mm pieces, and samples were embedded in 5% agar. Transverse 200-mm-thick sections were prepared using a vibrating blade microtome (VT1000; Leica). Sections were stained with Hoechst as described for nuclear staining and then with 0.2 mg/mL Calcofluor White (Sigma-Aldrich) for 10 min. Sections were observed using a fluorescence microscope (Axioskop 2 plus) or a confocal laser scanning microscope (LSM780 META; Zeiss). The thickness of three regions in each section was determined using Image J, and the mean of these three measurements was calculated as the thickness of the leaf.
Anthocyanin Content Measurement The leaf anthocyanin content was determined as reported previously (Zhang et al., 2011). The fresh weight of each leaf was measured using an electronic balance (XS105DU; Mettler Toledo). The A530 of sample solutions was measured using a plate reader (Infinite 200 PRO; Tecan). The amount of anthocyanin was expressed as A530 per gram of leaf fresh weight.
Immunofluorescence Microscopy After UVB irradiation, leaves were fixed as described for nuclear staining. Before fixation, leaves were placed in the dark for 28 h so that all nuclei were positioned at the bottom. This was important because the immunofluorescence signal might be influenced by the position of the nuclei. For actin8D mutant analysis, leaves were centrifuged at 13,000g for 1 min to artificially relocate nuclei to the bottom (Supplemental Fig. S6). After fixation, leaves were cut into two pieces of approximately 5 3 10 mm and fixed to a cover glass with the adaxial side facing upward using cyanoacrylate glue (Konishi). Samples were 684
further cut into pieces of approximately 1 3 1 mm on the cover glass and digested with fixation buffer containing 1% Cellulase “Onozuka” RS (Yakult) and 0.1% Pectolyase Y-23 (Kyowa Chemical Products) for 5 min at 37°C. The adaxial layer was detached from the cover glass and further digested for 1 min. Samples were permeabilized with fixation buffer containing 0.5% Triton X-100 for 1 h and blocked in fixation buffer containing 20% fetal bovine serum (Thermo Scientific) for 1 h. To label CPDs, samples were immunostained with the mouse monoclonal primary TDM-2 antibody (Cosmo Bio; diluted 1:500) at 37°C overnight and with Alexa-488-conjugated antimouse IgG (Invitrogen; diluted 1:500) for 3 h. Antibodies were diluted in fixation buffer supplemented with 5% fetal bovine serum. Nuclei were stained with fixation buffer containing 5 mg/mL Hoechst 33342 for 15 min in the dark. Each specimen was mounted on a glass slide with 0.1% p-phenylenediamine diluted in 13 mM NaCl, 0.51 mM Na2HPO4, 0.16 mM KH2PO4 (pH 9.0–9.5 with KOH), and 50% glycerol, and observed with a fluorescence microscope (Axioskop 2 plus). The exposure time was 0.04 s. Images of Hoechst staining and CPD staining were acquired for each nucleus. The mean signal intensity of each nucleus was determined using Image J. Hoechst-stained images that had been converted to 32-bit grayscale were binarized and nuclei were outlined. The extracted outlines were overlaid onto the corresponding CPD-stained images, and the mean signal intensity within each outline was measured. The CPD signal intensity of each UVB-irradiated nucleus was subtracted from the average intensity of non-UVB-irradiated nuclei. The net intensity of the CPD signal in the nucleus of each cell type was expressed relative to the fluorescent intensity of CPDs in the nucleus of a pavement cell that had undergone dark treatment followed by UVB irradiation. Heat maps of CPD levels were created using the Image J plug-in HeatMap Histogram (http://www.samuelpean.com/heatmap-histogram/).
Cell Death Measurement Seedlings were irradiated with 2.5 W m22 UVB for 5 min and then with 30 mmol m22 s21 red light for 5 d. Emitted light with a wavelength of 491 to 552 nm (488 nm excitation) was defined as autofluorescence of dead cells. Staining with trypan blue, which has intrinsic fluorescence in the far-red region of the spectra (Mosiman et al., 1997), was performed as described previously (Kim et al., 2008). To quantify dead cells, each trypan-blue-stained cotyledon was scanned from the upper to lower surface with a confocal microscope (LSM780; 610–758 nm emission, 488 nm excitation). The maximum intensity projection image of each cotyledon was binarized, and the total surface area of dead cells per leaf area was determined using Image J.
Statistics All data with error bars are represented as mean 6 SE using StatPlus. The P values were determined with unpaired Student’s t test. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number At1g49240 (ACTIN8).
Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Detection of UVB-induced cell death in mesophyll cells. Supplemental Figure S2. Nuclear positioning on the side walls, nuclear projection area, and UVB-induced DNA damage in nuclei after bluelight 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 UV-absorbing pigment anthocyanin during blue-light treatment for UV protection. Supplemental Figure S5. Different irradiation effects of UVA and UVB on nuclear positioning. Supplemental Figure S6. Distribution of nuclei in mesophyll, pavement, and guard cells before and after centrifugation of leaves. Plant Physiol. Vol. 170, 2016
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ACKNOWLEDGMENTS We are grateful to Masao Tasaka (Nara Institute of Science and Technology) for his donation of actin8D and to Tobias Baskin (University of Massachusetts) and James Raymond (Eigoken) for critical readings of this article. Received September 9, 2015; accepted December 12, 2015; published December 17, 2015.
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