Fig 1.
In vitro stromule formation requires plant extract.
A-C: A concentrated cell extract fraction was added to fixed chloroplasts and observed under a microscope. D-F: Isolated chloroplasts treated with a solution of 0.5% BSA, 2 mM ATP and 200 μM GTP. G-I: A zoomed in image of a stimulated chloroplast shows multiple stromules with nodules emanating from a single plastid. Scale bars in panels A–D correspond to 30 μm; those in G–I, 10 μm. All images were organized by showing the cerulean (grey), autofluorescence (red), and merged images from left to right. White arrows indicate stromules. The total elapsed time of the recording was 10 minutes and 23 seconds.
Fig 2.
Stromules are dynamic structures that also move throughout the z planeA-L: An image sequence through the z axis of two chloroplasts that have been stimulated to form stromules.
The scale bar corresponds to 10 μm; the 12 panels cover 19.8 μm in the z direction. The white arrows mark stromule branches.
Fig 3.
Time-lapse image series of stromule formation in vitro.
Time (min) is shown in the lower left of each panel. A+B: Image of chloroplasts before the addition of the cell extract. C+D: After 1 minute, the addition of cell extract stimulates stromule formation within ~10 seconds. E+H: Stromules remain present for the remainder of the time course. White arrows mark stromules. The scale bar corresponds to 15 μm.
Fig 4.
Stromules are stimulated by a 100 kDa or greater sized protein or protein complex.
A-B: Images showing isolated chloroplasts before and after the addition of the heat-inactivated cell extract respectively. C-D: Pre and post images of isolated chloroplasts treated with the pellet fraction after a 100,000 x g ultracentrifugation of the whole-cell extract, respectively. E-F: Isolated chloroplasts before and after the addition of the supernatant fraction after a 100,000 x g ultracentrifugation of the whole-cell extract. G-H: Images of isolated chloroplasts before the addition and after a 15 minute incubation with a cell extract fraction containing proteins smaller than 100 kDa but greater than 10 kDa respectively. Stromules are labeled with white arrows. The scale bar corresponds to 15 μm.
Fig 5.
Stromule formation does not require exogenous ATP.
A-H: Time-lapse images showing isolated chloroplasts incubated with apyrase-treated cell extract, with duration of the reaction shown in the bottom center of each panel. The apyrase-treated cell extract was added after 1 minute. Stromules are labeled with white arrows. The scale bar corresponds to 15 μm.