The overall goal of this procedure is to visualize and determine stromule frequency using live cell confocal fluorescence microscopy within leaves, and for visualizing stromules in vitro using chloroplast extracted from leaves. These experimental methods can help answer key questions in plant cell biology and chloroplast research by discovering how stromules function. The main advantage of these techniques is that they produce reproducible and robust data, even of these highly dynamic chloroplast structures.
We had the idea for the chloroplast isolation study because some research suggested that stromule formation required cytosolic structures, like the cytoskeleton, to form. So we decided to blend up the cell, and see if stromules could still form. To prepare leaf samples, begin by using a very sharp razor blade to cut a small section of an Nicotiana benthamiana leaf.
Immediately after cutting the leaf section, submerge it in a five milliliter syringe filled with water. Then, remove the air from the syringe, and apply a vacuum by using a finger to cover the syringe orifice, before pulling the plunger. Gently release the plunger to avoid damaging the leaf section.
Then repeat the air removal two or three times, or until the air has been removed, and the leaf appears to be deep green. Add a drop of water to a slide, and place the leaf section on the drop. Then add another drop of water to the top of the leaf, and add a cover slip.
If there are any air bubbles, gently tap the cover slip until they are removed. With transmitted light, and a 20x objective, focus on a field of view near the center of the leaf section, visualizing multiple chloroplasts simultaneously. Save an image with transmitted light, so that cell types can be distinguished later if necessary.
Then switch to laser illumination with the appropriate excitation and emission filters for the fluorophore being used, and save another image. Using the microscope software, select the GFP channel, and click to adjust the pinhole aperture to one airy unit. Select laser scanning to start visualizing the sample, and then click the GFP channel and detector gain to minimize the laser power while still clearly visualizing stromules.
Next, prepare a Z stack experiment that will collect the series of images through the leaf epidermis in the field of view. Adjust the scanning speed and image resolution as necessary to make sure that the Z stack is collected quickly. Then save the Z stack for later analysis.
Using image J, merge the Z stack into a single image using the maximum intensity in the software. Then identify and manually count all chloroplasts in the image. For each chloroplast, visually determine whether one or more stromules are seen extending from the chloroplast in the merged Z stack image.
To extract intact chloroplasts, prepare cold extraction buffer using the following reagents. Then, with NaOH and HCl, adjust the pH to 6.9, and refrigerate before use. Prepare isolation buffer, and adjust the pH to 7.6.
If the leaves are not expressing a genetically encoded stromofluorophore, prepare Carboxyfluorescein diacetate, or CFDA solution. To isolate chloroplasts, remove approximately 5 to 10 grams of leaves from several plants and briefly rinse in cold water. Then immediately transfer the leaves to 50 milliliters of cold extraction buffer.
Using a blender with several short pulses, grind the leaves, then filter the mixture through two to three layers of cheese cloth to remove leaf debris. Divide the extracted chloroplast into two 50 milliliter centrifuge tubes, and centrifuge for one minute at 750 x g. Discard the supernatant, and use 10 milliters of isolation buffer to re-suspend the green chloroplasts.
Then centrifuge again for one minute at 750 x g, discard the supernatant, and use isolation buffer to re-suspend the chloroplasts up to a final volume of five milliliters. If the chloroplasts are from transgenic plants expressing plastid-targeted fluorescent proteins, transfer 20 microliters of the chloroplasts to a slide and add a cover slip. Then carry out microscopy.
To stain chloroplasts from plants that are not expressing plastid-targeted fluorescent proteins, add five microliters of 50 millimolar CFDA stock to the five milliliters of chloroplasts in isolation buffer. After allowing the chloroplasts to incubate for five minutes transfer a small aliquot to a slide, add a cover slip and carry out microscopy. Finally, use a FITC or GFP filter set and a 20x objective to visualize multiple chloroplasts simultaneously.
Or a higher objective to visualize a single isolated chloroplast. A merged stack showing GFP stomule frequency in the codoledence of young N.Benthamiana seedlings is shown here. In this panel, the image was de-saturated and inverted so that the stroma appears black.
The chloroplasts were labelled either as having no stromules, or having at least one stromule. Of the 87 epidermal chloroplasts visualized, 33 have stromules, for a frequency of 37.9 percent in this leaf. This graph represents the analysis of over 23, 000 chloroplasts, and several hundred cells from a single leaf from 21 different plants.
The average stromule frequency during the day was 20.8 plus or minus 1.8 percent, and the average nighttime stromule frequency was 12.8 plus or minus 0.9 percent, indicating a significantly higher daytime frequency, as determined by the Welch's T test. In this figure, chloroplast stromules were observed in vitro after isolation from N.benthamiana expressing plastid-targeted GFP, or after using CFDA to stain chloroplasts from N.benthamiana, or Spinachia oleracea. While conducting live cell imaging of stromules, it is important to work quickly, and to manipulate the plant tissue as little as possible.
Following this procedure, other methods like gene silencing or chemical treatments can be used to discover additional molecular players or pathways involved in stromule formation and function. This technique has been used by researchers in plant cell biology and plant immunity to study the role of stromules in cellular signalling pathways and plant responses to pathogens. After watching this video, you should have a good understanding of how to calculate stromule frequency in a leaf, and how to observe stromules in extracted chloroplasts.
And remember that the lasers and the electrical voltage supplying a scanning electron confocal microscope can be extremely hazardous, and it's important to understand the system requirements for using this equipment.
