Mutant Plants Deficient in Chloroplast Photorelocation Movement is Defective in Nuclear Photorelocation Movement.

We examined the nuclear avoidance response in Arabidopsis leaf adaxial pavement cells, which contain only a small number of tiny plastids (hereinafter the plastid in the pavement cells is referred to as simply “plastid”), because the multiple large chloroplasts in leaf mesophyll cells could interfere with the precise analysis of nuclear movement. Furthermore, the pavement plastids have some chloroplast-like characteristics, including chlorophyll autofluorescence and photorelocation movement. Fully developed pavement cells are interdigitating jigsaw puzzle-shaped cells (Fig. 1 A and D) in which spindle-shaped nuclei are associated with (or pulled by) thick longitudinal actin bundles (10) (Fig. 2 A and B). Most nuclei are positioned at the bottom of the cells in darkness (hereinafter referred to as the “dark position”), but are situated on the side walls under conditions of strong light (hereinafter the “light position”) (7, 10) (Fig. 1A).

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Fig. 2.

Cp-actin filament-dependent and -independent nuclear movement. (A) A nucleus along actin bundles (brackets) in a pavement cell. Note that some plastids (p) are attached to the nucleus (n). (B) Schematics of two types of nuclear movement, avoidance (arrow) and parallel (double-headed arrow), induced by side irradiation. Red lines indicate the actin bundles associated with nuclei. The area irradiated with blue light is indicated by a light-blue box. (C–F) Nuclear movement induced by side irradiation with blue light (dotted squares) in WT (C, parallel movement; D, avoidance movement), phot2-1 (E), and chup1-3 (F) cells. Photographs obtained at the beginning of blue light irradiation (i) and at the end of the observation period (ii) are shown. (Scale bars:10 μm). (G) Number of nuclei exhibiting avoidance (blue), parallel (red), or no movement (light green) in WT, phot2-1, and chup1-3 cells in darkness (dark), red light (RL), or blue light (BL) (n = 50). The effect of DCMU in WT plants under red or blue light is shown as well. (H–K) Enlarged view of the distribution of cp-actin filaments on plastids in WT nuclei showing parallel movement (H) and avoidance movement toward the upper part of the panel (I), phot2-1 (J), and chup1-3 (K) cells during blue light irradiation. (Scale bars: 2.5 μm.)

We performed a time-course analysis of the nuclear avoidance response (from the dark position to the light position) induced by continuous irradiation with strong blue light (50 μmol m⁻² s⁻¹) by counting the number of nuclei in the light position in leaves collected every 3 h (Fig. 1 B and C). Approximately 70% of the nuclei were in the dark position in dark-adapted WT cells, and the nuclear avoidance movement was completed within 6–9 h, resulting in ∼70% of the nuclei localized to the light position (Fig. 1 B and C). This response was completely impaired in phot2 mutant plants (Fig. 1B) (7), as was chloroplast movement (3).

Previous work has shown that the strong blue light-induced plastid movement from the pavement cell bottom to its anticlinal walls occurs in a phot2-dependent manner, similar to mesophyll chloroplasts (10). Indeed, photorelocation movement of plastids in pavement cells was induced by strong blue light in WT, but not in phot2 mutants (Movie S1), indicating that mesophyll chloroplasts and pavement cell plastids share the same mechanism of photorelocation movement. Thus, we examined the possible involvement of factors regulating chloroplast photorelocation movement in the nuclear avoidance response. Surprisingly, most of the mutants that were deficient in chloroplast movement also demonstrated impaired nuclear avoidance response in pavement cells (Fig. S1 A–D). Furthermore, the mutants were also deficient in the nuclear avoidance response in mesophyll cells (Fig. S1 E and F), indicating that nuclear movement is mediated by a common mechanism in both pavement and mesophyll cells. Most notably, a chloroplast unusual positioning 1 (chup1) mutant, which has defects in the actin-binding protein CHUP1 localized on both mesophyll chloroplasts and pavement plastids (Fig. S1G) and is completely impaired in chloroplast movement and positioning (11, 12) and plastid photorelocation movement in pavement cells (Movie S1), hardly showed a nuclear avoidance response (Fig. 1C).

CHUP1 plays an important role in the generation of short actin filaments on chloroplasts (i.e., cp-actin filaments), which are essential for chloroplast photorelocation movement and positioning in mesophyll cells (9). Importantly, we detected cp-actin filaments on pavement cell plastids (SI Results and Discussion and Movies S1 and S2). These results indicate that, in addition to using the same photoreceptor, nuclear movement is mediated by a similar mechanism as that of chloroplast movement.

Using transgenic plants expressing nuclear-targeted GFP, we analyzed nuclear dynamics at 10-min intervals for 12 h under strong light conditions (Fig. 1D). When the light was switched on, the nuclei of dark-adapted WT plants, which in darkness had been stationary at the bottom of the cell, began to show random movement (Fig. 1D). This movement differed from the chloroplast avoidance response, in which chloroplasts move directly to anticlinal walls using the shortest route (13). In addition, although the nuclei arrived at the side walls, they often returned to the cell bottom (Fig. 1D). Unexpectedly, the phot2 and chup1 mutants, which had been deficient in the nuclear avoidance response in the fixed-cell experiments described above (Fig. 1 B and C), did not have stationary nuclei on the cell bottom, but exhibited random movements that were either similar to (phot2) or more vigorous than (chup1) those of WT plants (Fig. 1D).

A comparison of the maximum speed and total distance traveled over 12 h of light-induced nuclear movement found that the chup1 nuclei showed more rapid movement and traveled a longer distance than the WT and phot2 nuclei (Fig. 1 E and F). This finding is similar to what was observed for chloroplasts in chup1 cells, which were dissociated from the plasma membrane and thus moved rapidly (9). However, the phot2 and chup1 nuclei remained at the side walls for a much shorter time than in WT cells (Fig. 1G), indicating that the approach and anchorage of nuclei to the side walls are important mechanisms in the blue light-induced nuclear avoidance response.

The Nuclear Periphery Lacks Detectable Actin Filament Structures, in Contrast to Cp-Actin Filaments Present on Chloroplasts.

Mutant analyses suggested that nuclear movement and positioning could be mediated by an actin-based mechanism similar to the cp-actin filament-based mechanism used by chloroplasts (9). Although cp-actin filaments can be observed at the front of moving chloroplasts and around the entire periphery of stationary chloroplasts (9), we did not detect any actin filament structure similar to cp-actin filaments around the nuclei in the cells that we examined (SI Results and Discussion and Movie S3). Moreover, we did not observe any components involved in chloroplast and nuclear photorelocation movements on the nuclear envelope (Fig. S1 G and H). Therefore, it is unlikely that cp-actin filament-like short actin filaments along the nuclear periphery mediate nuclear photorelocation movement.

We further analyzed nuclear photorelocation movement in lines expressing GFP-tagged actin-binding proteins by partial cell irradiation with strong blue light provided by a blue laser and examination using a time-lapse confocal laser scanning microscope (14). We observed two types of movements when strong light was applied to one side of the nucleus along the long axis (“side irradiation”; Fig. 2B): movement parallel to the actin bundles, termed “parallel movement” (Fig. 2 B and C and Movie S4) and movement in which the nuclei always escaped from the irradiated side toward the nonirradiated side, termed “avoidance movement” (Fig. 2 B and D and Movie S2). Thus, using a combination of both movements, nuclei can move in any direction (similar to chloroplast photorelocation movement) regardless of the direction of the attached actin bundles.

Nuclear Avoidance Movement Is Dependent on Cp-Actin Filament-Mediated Movement of Plastids.

Over the course of microscopic evaluations, we found that several plastids were always attached to nuclei and accompanied the nuclei during nuclear movements (Fig. 2A, Fig. S2A, and SI Results and Discussion). Thus, we hypothesized that the attached plastids carried the nuclei in a cp-actin filament-dependent manner. We precisely examined nuclear movement in WT and mutant pavement cells exposed to nuclear side irradiation with blue light (Fig. 2 C–G). In the WT cells, ∼40% of the photorelocated nuclei exhibited avoidance movement, and the remainder exhibited parallel movement, regardless of whether they were abaxial or adaxial epidermal cells (Fig. 2G and Fig. S2B). Conversely, the avoidance movement was rarely induced in the phot2 mutant plants, and ∼60% of the nuclei showed no movement, although the proportion of parallel movement was comparable to that in the WT plants (∼40%) (Fig. 2G, Fig. S2C, and SI Results and Discussion). Furthermore, the cp-actin filaments on the plastids were not reorganized in response to strong light in the phot2 mutant (Fig. 2 E and J and Movie S5). These results indicate that phot2 mediates avoidance movement via cp-actin filament regulation.

In the chup1 mutant, which lacks cp-actin filaments (Fig. 2 F and K), parallel movement of nuclei was predominant (SI Results and Discussion and Fig. S2 D–F) and avoidance movement was rarely induced (Fig. 2G and Movie S6). Thus, the nuclear avoidance movement induced by side irradiation is dependent on the plastid movement regulated by cp-actin filaments. Importantly, parallel movement along the actin bundles, but not avoidance movement, was induced by side irradiation with red light that was unable to induce nuclear photorelocation movement (7). Red light-induced parallel movement of nuclei was strongly suppressed by the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), indicating that this movement is mostly photosynthesis-dependent (Fig. 2G and SI Results and Discussion). Furthermore, the blue light-induced avoidance response was still induced in leaf cells treated with DCMU (SI Results and Discussion and Fig. S2G). Thus, these results strongly suggest that the blue light-induced nuclear avoidance response of nuclei is dependent on plastid movement regulated by cp-actin filaments.

Nuclear Avoidance Movement Is Dependent on Plastids Attached to the Nucleus.

To convincingly demonstrate that plastids carry nuclei during photomovement, we analyzed the light-induced movement of nuclei without plastid attachment. Although we could not identify nuclei without attached plastids in WT plants or mutants deficient in chloroplast movement, we found that mutants deficient in plastid division, such as the plastid division 1 (pdv1)/pdv2 double mutant (15) and paralog of arc6 (parc6) (16), had pavement cells in which nuclei were not associated with plastids (Fig. 3A, Fig. S3, and SI Results and Discussion). In the plastid division mutants, the nuclei without attached plastids exhibited predominantly parallel movement and very infrequent avoidance movements, similar to findings in the chup1 mutant (Fig. 3 C and D). In contrast, mutant cells with nuclei attached to plastids showed both avoidance and parallel movements at a ratio similar to that seen in WT cells (Fig. 3 B and D).

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Fig. 3.

Plastids are essential for the nuclear avoidance response. (A) Nuclei associated with or without plastids (p). Nuclei in WT plants are always associated with plastids (i). In the plastid division mutant parc6, nuclei associated with (+) large plastids (ii) or without (−) large plastids (iii) can be observed. (B and C) Side irradiation-induced movement of nuclei associated with plastids (B) or not associated with plastids (C) in parc6. Other details are the same as those in Fig. 2. (D) Number of nuclei exhibiting avoidance (blue), parallel (red), or no movement (light green) in WT and parc6 plants (n = 50). WT data from Fig. 2G are shown for comparison. (E) Number of plastids associated with nuclei on the side walls or at the bottom in the parc6 pavement cells before or after blue light irradiation for 12 h (n = 60). (Scale bars: 10 μm.)

If plastid-free nuclei cannot move in response to blue light, then the ratio of plastid-free nuclei at the bottom of the cell must increase as a result of the movement of plastid-bearing nuclei to the side wall. Indeed, after 12 h of blue light irradiation, the ratio of nuclei without plastids on the cell bottom increased sharply, and most of the nuclei on the side walls had one or two attached plastids. In dark-adapted cells, the nuclei on both the cell bottom and the side walls were associated with two plastids in most cases (Fig. 3E, Fig. S3, and SI Results and Discussion). Furthermore, when blue light-adapted plants were transferred to the dark and the movement of nuclei from the side wall to the bottom was induced (i.e., dark positioning), the ratio of plastid-bearing nuclei (often two plastids) at the bottom of the cell increased as a result of the movement of plastid-bearing nuclei from the side wall to the bottom (Fig. S3E). These results clearly demonstrate that only the nuclei with plastids were capable of moving in response to ambient light conditions.