Supplementary data for the chapter:
“Fluorescent protein applications in plants”

By: R. Howard Berg and Roger N. Beachy, Danforth Plant Science Center
In: Fluorescent Proteins, 2nd Edition (2008), Kevin Sullivan, editor Method in Cell Biology,
Volume 85, Elsevier Inc. Chapter 8, pp 153-177

ER bodies in Arabidopsis: a case for correlative analysis using electron microscopy

This time-lapse movie is of cells in Arabidopsis flowers, filament cells of the stamen. The transgenic plant expresses ER-targeted GFP, which accumulates in spindle-shaped bodies that are highly motile (in this movie each frame was acquired in 24 seconds, no time elapsed between frames, total time = 46 minutes). These images from confocal light microscopy suggest that the signal accumulates in a “new”, spindle-shaped organelle. However, examining these more closely using transmission electron microscopy (TEM, figures on the right) allows a more simple interpretation. In Arabidopsis and other members of the Brassicaceae some proteins accumulate in the ER, forming crystalline bodies (asterisk) that produce local enlargements in this organelle (note the ribosomes on the membrane surface in the higher magnification micrograph, confirming that it is ER). These are what are seen in the time-lapse movie, showing that they are highly motile. The GFP signal molecule is distributed throughout the ER, in its lumen, and are particularly bright in the larger volume formed by the crystals. Mag marker = 1 µm. For a more detailed look at this story, see: B.E.S. Gunning (1998), “The identity of mystery organelles in Arabidopsis plants expressing GFP, Trends in Plant Science, 3:417.

Time lapse technique: balancing temporal and spatial resolution

Highly dynamic events in time-lapse imaging require high temporal resolution, i.e., more frequent imaging per unit time. This might be accomplished in confocal imaging by using fewer slices and a larger pinhole (i.e., sacrificing Z axis resolution) and keeping the field of view relatively small (to reduce the number of pixels to be scanned). These two movies, Agg1-tl1.mov and Agg1-tl3.mov, illustrate these concepts. Made using the same region of leaf tissue of Arabidopsis, the signal is a YFP fusion with Agg1, the gamma subunit of heterotrimeric G protein (Q. Zeng, X. Wang, and M.P. Running, Danforth Plant Science Center, 2008, Plant Physiology, 143: 1119-1131). Agg1-tl1 was made using relatively high Z-axis spatial resolution and low temporal resolution, and Agg1-tl3 vice versa. The high spatial resolution image, covering an area of 900 x 587 pixels, was constructed using a smaller pinhole and eight optical sections, compresses time (74X) too much to see details of signal movement and exhibits significant photobleaching over the 40 minute acquisition period, due to the increased excitation light dosage resulting from the higher number of optical sections. The high temporal resolution image, sampling a smaller field of view (512 x 512 pixels) and using a larger pinhole and three optical sections, has eight times better temporal resolution (9X compression) that clearly shows the nature of the signal dynamics and no signal fade over the five minute acquisition period (same excitation dose for both acquisitions). By cropping the field of view and reducing the number of slices, temporal resolution was improved. Pixel size is similar in both images, voxel size is larger in Agg1-tl3.

4D imaging of viral replication complexes

As shown in our 3D imaging page, TMV proteins interact with plant components in forming replication factories (viral replication complexes). Critical to the replication and spread of the virus, these complexes form bodies that are motile, facilitating cell-to-cell spread of the virus. 4D confocal imaging of fluorescent protein-tagged molecules in this movie show that the bodies move on actin filaments. Details: Arabidopsis leaf epidermal cell infected with CG-TMV; actin filaments were labeled using a GFP fusion with the actin-binding domain of talin, viral replication complexes were labeled using a GFP fusion with TMV movement protein; this is viewable in stereo with red/cyan glasses; confocal imaging with one frame every 10 seconds, 250 frames, 42 minutes total.

Vacuole dynamics in soybean root tips

3Dvac.jpg  Vacuole biogenesis in soybean root tip cells, 3D projection of multiphoton optical sections.  The green color is due to autofluorescence of isoflavonoids contained within the vacuoles.  Developing vacuoles form a cage of polygonal tubes surrounding the cell nucleus.