Volume 4, Issue 5, Pages 886-895 (September 2011) Laser Stimulation of the Chloroplast/Endoplasmic Reticulum Nexus in Tobacco Transiently Produces Protein Aggregates (Boluses) within the Endoplasmic Reticulum and Stimulates Local ER Remodeling  Griffing Lawrence R.   Molecular Plant  Volume 4, Issue 5, Pages 886-895 (September 2011) DOI: 10.1093/mp/ssr072 Copyright © 2011 The Author. All rights reserved. Terms and Conditions

Figure 1 GFP–HDEL Fluorescence in the Same Leaf Epidermal Cell from an N. bethamiana Seedling Photobleached in Different Regions of Interest (ROI, White Rectangle). A 30-μm2 ROI was photobleached using a 405-nm laser set at 100% transmission and a 10-microsecond/pixel dwell time over two frames, as indicated by the presence of the ROI in the second and third frames. (A) Photobleaching time course of an ROI about 10 μm from the chloroplast. (B) Photobleaching time course of an ROI about 5 μm from the chloroplast. (C) Photobleaching time course of an ROI that includes the chloroplast. Upper left corner labels are in seconds. Scale bar = 10 μm. Representative of 10 experiments. (D, D’) 3-D reconstruction of the ER–chloroplast association prior to photobleaching. The ER is in green; the chloroplast is in red. (D) is from the perspective of inside the cell looking out, (D’) is outside the cell looking in. The ROI (black) is the same as that highlighted in (E). (E) Photobleaching experiment of another region showing prebleach (0 time, 3-D reconstruction in panel D), postbleach (0.428 s) and bolus formation persisting after 24.010 s. Scale bar = 2 μm. Molecular Plant 2011 4, 886-895DOI: (10.1093/mp/ssr072) Copyright © 2011 The Author. All rights reserved. Terms and Conditions

Figure 2 GFP–HDEL Fluorescence in a Leaf Epidermal Cell froman N. bethamiana Seedling Treated for 1 h with 25 μM Latrunculin b. (A) White-outlined ROI shows results of photobleaching this area for 100 milliseconds using 100% laser light transmission of the 405-nm laser (4 microseconds/pixel dwell time). Note that bleaching for this limited time does not completely bleach the ER. (B) Identical photobleaching conditions to (A) in white-outlined ROI that now includes the chloroplast. Bolus formation starts immediately and spreads through the network rapidly. Upper left-hand numbers are in seconds. Representative of three experiments. Scale bar = 10 μm. Molecular Plant 2011 4, 886-895DOI: (10.1093/mp/ssr072) Copyright © 2011 The Author. All rights reserved. Terms and Conditions

Figure 3 Bolus Recovery. (A) Dissipation of boluses 2 min after the time series shown in Figure 1C. The central region of the cell and region near the photostimulated chloroplast still retain the boluses, but they have dissipated completely in the upper right-hand region of the image. The image is a Z-axis ray-traced projection of an optical section series. Inset labeled shows the region used for the 3-D reconstruction in (B). (B) 3-D reconstruction of the region surrounding the initial photostimulation showing association of ER with photostimulated ER/chloroplast nexus, black arrows. (C) Further dissipation of the boluses after an additional 2 min following the optical sectioning in (A). The boluses have completely dissipated in the center of the cell. Photobleaching in the ROI (white rectangle) recovers normally. Upper left-hand numbers are in seconds. Representative of five experiments. Scale bar = 10 μm. Molecular Plant 2011 4, 886-895DOI: (10.1093/mp/ssr072) Copyright © 2011 The Author. All rights reserved. Terms and Conditions

Figure 4 2-D and 3-D Analysis of ER Dense Cisternal and Tubular Network. Same cell as that in Figure 1, 10 min after photostimulation. (A) ER cluster is seen as the bright region is associated with the photobleached chloroplast. Note that the white-outlined ROI is photobleached and recovers normally. Upper left-hand numbers are in seconds. (B) The image is a Z-axis ray-traced projection of an optical section series. Scale bar = 10 μm. (C, C’) 3-D reconstruction using isosurfaces of region containing photostimulated nexus in (B). (D, D’) 3-D reconstruction using isosurfaces of non-photostimulated region in (B). The intimate association of the chloroplast with the cortical ER can be seen from both the outside-in and inside-out views. For (C) and (D), the chloroplast is red and the ER is green. (C) and (D) are shown from the perspective of outside looking into the cell, while (C’) and (D’) are shown from the perspective of being inside the cell looking out. Representative of 10 experiments. Molecular Plant 2011 4, 886-895DOI: (10.1093/mp/ssr072) Copyright © 2011 The Author. All rights reserved. Terms and Conditions

Figure 5 A Different Cell from that in Figures 1 and 4 Shows the Same Response, but Does Not Re-Initiate Bolus Formation upon Further Stimulation. (A) Note that bolus formation develops around the white ROI after initial photobleaching. (B) Five minutes later, there is an ER cluster surrounding the chloroplast. When an ROI containing the chloroplast is photobleached using the same settings that caused the initial response, the fluorescence recovers normally and there is no additional bolus formation. Upper left-hand numbers are in seconds. Representative of 10 experiments. Scale bar = 10 μm. Molecular Plant 2011 4, 886-895DOI: (10.1093/mp/ssr072) Copyright © 2011 The Author. All rights reserved. Terms and Conditions

Figure 6 Epidermal Cell in Mature N. tabacum Leaf Transiently Expressing YFP–RHD3. (A) Z-axis ray-traced projection of optical sections containing the lower cortical region of the cell. Chloroplasts (autofluorescence, magenta) lie adjacent to the perinuclear ER and nuclear envelope labeled with YFP–RHD3 (green). The white rectangular ROI is the site of the photostimulation of a perinuclear chloroplast for 90 milliseconds, 4 microseconds/pixel dwell time at 100% laser light transmission. The inset is a 3-D reconstruction of the region directly below it, showing the nucleus (n) and the photostimulation ROI (black rectangle). (B) Z-axis ray-traced projection of optical sections containing the same region as (A) after 5 min. Boluses now decorate the nucleus (n) and the ER is clustering (white arrows) around the photostimulated chloroplast. The inset is a 3-D reconstruction of the region directly below it, showing the punctate nature of the YFP–RHD3 in the perinuclear region and the clusters of ER around the photostimulated chloroplasts (black arrows). (C) Z-axis ray-traced projection of optical sections containing the outer cortical region of the same cell as (A), showing a typical tubular ER network labeled with YFP–RHD3 (green) and the infrequent occurrence of chloroplasts in this region (autofluorescence, magenta). (D) Z-axis ray-traced projection of the same cell, same region, 11 min after photostimulating the ER/chloroplast nexus shown by the ROI in (A). Representative of five experiments. Scale bar = 10 μm. Molecular Plant 2011 4, 886-895DOI: (10.1093/mp/ssr072) Copyright © 2011 The Author. All rights reserved. Terms and Conditions

Figure 7 Heat-Stressed Cells (40–45°C, 30 min) Do Not Show Bolus Formation, but Do Produce an ER Cluster in the Vicinity of the ER/Chloroplast Nexus upon Photostimulation. (A) Photobleached ER/Chloroplast nexus (white-outlined ROI) for 130 milliseconds, 4 microsecond/pixel dwell time, at 100% laser light transmission. The chloroplast and ER become photobleached, but there is no bolus formation. (B) Separate experiment with cells from a heat-stressed (40–45°C, 30 min) seedling photobleaching an ER/chloroplast nexus, where the chloroplast edge is very close to the plane of section. Photobleaching for 40 milliseconds, 4 microsecond/pixel dwell time, at 100% laser transmission, does not produce boluses. Numbers are in seconds. (C) Z-axis ray-traced projection of the same cell as in (B) 7 min after photostimulation. Representative of three experiments. Scale bar = 10 μm. Molecular Plant 2011 4, 886-895DOI: (10.1093/mp/ssr072) Copyright © 2011 The Author. All rights reserved. Terms and Conditions