J.O.B. and A.M.R. were supported by predoctoral fellowships from the National Science Foundation.

NOTE: For this protocol, we have focused on assaying stromule frequency in the epidermis of N. benthamiana leaves. Several stable transgenic lines have been generated that can be used for this purpose, including 35SPRO:FNRtp:EGFP13 and NRIP1:Cerulean6. Both of these lines show robust expression of fluorophores in the chloroplast stroma of leaves grown under a wide range of conditions. Alternatively, chloroplast-targeted fluorophores may be transiently expressed ...

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The authors have nothing to disclose.

When investigating stromules, three important factors must be considered throughout: (i) manipulation of the plant tissue must be kept to an absolute minimum, (ii) the experimental system must be kept consistent, and (iii) sampling strategies must be carefully planned to ensure robust, reproducible data are analyzed.
Stromules are remarkably dynamic: they can extend and retract rapidly before an observer&#39;s eyes under the microscope. Moreover, stromule frequency varies significantly in resp...

Chloroplasts are dynamic organelles in plant cells responsible for photosynthesis and a host of other metabolic processes. Signaling pathways from the chloroplast also exert significant influence on plant physiology and development, coordinating plant responses to environmental stress, pathogens, and even leaf shape1-6. Recently, biologists have gained interest in a poorly understood aspect of chloroplast structure: stromules, very thin stroma-filled tubules that extend from the surface of the chloroplast7.
The biological functions of stromules remain unknown, although stromule frequency is known to vary in response to environmental stimuli7-9, and stromules may be capable of transmitting signaling molecules between organelles6. All types of plastids (not only the green, photosynthetic chloroplasts, but also clear leucoplasts, starch-filled amyloplasts, and pigmented chromoplasts, to name a few types of plastids) make stromules, and stromules are found in all land plant species that have been examined to date. Stromules can extend and retract dynamically, appearing or disappearing within seconds, or they can remain relatively stationary for long times. One of the major hurdles facing stromule biologists is that stromules are often studied using dramatically different methods, tissues, and species, making comparisons across the stromule biology literature difficult. Going forward, standard practices and thorough descriptions of the experimental systems used to study stromules will be critical to discovering the function of these ubiquitous features of chloroplast morphology.
Here we describe methods for visualizing stromule formation in the epidermal chloroplasts of Nicotiana benthamiana leaves. In the mesophyll, chloroplasts are densely packed into large, three-dimensional cells, which makes it difficult to accurately and rapidly visualize stromules by confocal microscopy. By contrast, epidermal cells are relatively flat, contain fewer chloroplasts, and are at the surface of the leaf, allowing for easy and rapid visualization of stromules. N. benthamiana is an ideal model system for these experiments because, unlike many plant species, all cells in the epidermis of N. benthamiana make chloroplasts10. In the epidermis of most plants, including Arabidopsis thaliana, only the stomatal guard cells have chloroplasts, while other epidermal cells have "leucoplasts", plastids that are clear, relatively amorphous, and nonphotosynthetic9,11,12. Thus, whereas a single field of view of an A. thaliana epidermis might show only a handful of chloroplasts in a pair of guard cells, a field of view of an N. benthamiana epidermis will include dozens or even hundreds of chloroplasts. All of the methods described here, however, can be modified to investigate other questions in stromule biology; for example, we have used the same approach to study leucoplast stromules of A. thaliana9.

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			<td height="42" style="height:42px;width:216px;">Laser Scanning Confocal Microscope&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Carboxyfluorescein diacetate (CFDA)</td>
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			<td height="21" style="height:21px;width:216px;">Dimethyl sulfoxide (DMSO)</td>
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			<td>Open-source software for analyzing biological images</td>
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visualizing stromule frequency with fluorescence microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

This protocol was used to visualize stromule frequency at day and at night in the cotyledons of young N. benthamiana seedlings. Slices from a z-stack were merged into a single image (Figure 1A). For visual purposes, that image was then desaturated and inverted so that stroma appears black (Figure 1B). The chloroplasts were labeled either as having no stromules (green asterisk) or having at least one stromule (magenta asterisk). Of the 87...

Watch this Scientific Journal Video about Visualizing Stromule Frequency with Fluorescence Microscopy at JoVE.com

Visualizing Stromule Frequency with Fluorescence Microscopy

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

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.

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8.0K Views.  USDA. 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 chlorop...