Name: Visualizing Stromule Frequency with Fluorescence Microscopy (Video) | JoVE Journal
Uploaded: 2016-11-23T14:00:00.000+00:00
Duration: 8 min 27 s
Description: Learn to visualize stromule frequency in plant cells using live cell confocal fluorescence microscopy with Nicotiana benthamiana. This protocol produces reproducible data on chloroplast dynamics.

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5 minutes

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 in N. benthamiana using Agrobacterium transformations13. This is less ideal than the transgenic lines, since Agrobacterium infiltrations induce some basal defense responses in N. benthamiana and interactions with Agrobacterium can alter stromule frequency in the leaf14, potentially complicating interpretation of results. Finally, to visualize stromule formation in vitro, chloroplasts may be extracted from any plant species, using either genetically encoded fluorophores or a fluorescent dye, as described in section 5 below.9,15
NOTE: Detailed methods for plant cultivation have been previously described.16 Briefly, grow N. benthamiana plants in 4&#34; pots filled with any professional soil mix that provides good drainage. Cover seedlings with a clear plastic dome for the first 10-14 days to provide a humid environment for germination. Add any standard fertilizer mix following manufacturer&#39;s instructions to 14-day-old plants. Grow plants under white light, using ~100 &#181;mol photons m-2 sec-1 light intensity. Water plants regularly.
1. Preparing Leaf Samples for Visualization
NOTE: Stromule dynamics are affected by wounding8, so tissue preparation should be conducted immediately before visualizing stromules by confocal fluorescence microscopy. Ideally, a sample should be visualized with 15 min after removal from the plant.
<ol>
	<li>Cut a small section of the leaf using a very sharp razor blade. Always use the same region of the leaf for consistency, and be certain to visualize the same surface (adaxial or abaxial) in all experiments.</li>
	<li>Immediately after cutting the leaf section, submerge the leaf section in a 5 ml needleless syringe filled with water, remove air from the syringe, and apply a vacuum by covering the syringe orifice with a finger and pulling the plunger.
	<ol>
		<li>Release the plunger gently to avoid damaging the leaf section. Repeat this two or three times, or until most of the air has been removed and the leaf appears to be deep green. 
		NOTE: This step removes air in the leaf that can interfere with accurate visualization of stromules.</li>
	</ol>
	</li>
	<li>Add a drop of water to a slide and place the leaf section on this drop. Then, add another drop of water to the top of the leaf, and place a cover slip over the leaf. If there are any air bubbles, gently tap the cover slip until they are removed. The slide is now ready for microscopy, and should be visualized immediately.</li>
</ol>
2. Visualizing Stromules with Confocal Fluorescence Microscopy
<ol>
	<li>With transmitted light, focus on a field of view near the center of the leaf section (away from the cells damaged by the razor blade). Use a 20X objective so that many chloroplasts can be visualized simultaneously. Save an image with transmitted light so that cell types can be distinguished later, if necessary.</li>
	<li>Switch the illumination source to a laser with appropriate excitation and emission filters for the fluorophore being used. For example, excite GFP with a 488 nm laser and collect emissions between 500 nm and 525 nm.
	<ol>
		<li>Using the microscope&#39;s software, select the GFP channel and click to adjust the pinhole aperture to 1 Airy unit. Select laser scanning, to start visualizing the sample, and then click the GFP channel to adjust the laser intensity and the detector gain to minimize the laser power while still clearly visualizing stromules. Once these settings have been determined, keep them as consistent as possible throughout all experiments.</li>
	</ol>
	</li>
	<li>Prepare a z-stack experiment that will collect a series of images through the leaf epidermis in the field of view.
	<ol>
		<li>For the z-stack, include all epidermal chloroplasts in the field of view to avoid bias (for example, if chloroplasts near the leaf surface have more stromules under conditions tested).</li>
		<li>Take images at the maximum interval possible that will include all stromules but minimize the time required for a z-stack. For example, if 1 Airy unit is equivalent to an in-focus confocal section that is 2 &#181;m deep, taking an image every 2 &#181;m is likely ideal. 
		NOTE: The leaf epidermis is an uneven surface, so the total depth of the z-stack will vary depending on the sample; most z-stacks will require 10-20 images, but may include more.</li>
		<li>Adjust scanning speed and image resolution as necessary to make sure that the z-stack is collected quickly (typically within 5-10 min, if possible). Save the z-stack for later analysis.</li>
	</ol>
	</li>
</ol>
3. Image Processing
<ol>
	<li>Determine stromule frequency using any image analysis software. For this protocol, we recommend using ImageJ, which is publicly available from the National Institutes of Health (http://imagej.nih.gov/ij/), or the popular upgrade of ImageJ, Fiji (http://fiji.sc/Fiji).</li>
	<li>Merge the z-stack into a single image using the maximum intensity projection in the software.</li>
	<li>Identify and manually count all chloroplasts in the image.</li>
	<li>For each chloroplast, visually determine whether one or more stromules are seen extending from the chloroplast in the merged z-stack image. For examples, see Figure 1. 
	NOTE: Since the biological functions of stromules are not known, any thin, stroma-filled, tubular extension from the chloroplast can be considered a &#34;stromule&#34;, regardless of length. As a general rule, a stroma-filled extension from the chloroplast is a stromule if it is less than approximately 1 &#181;m in diameter along most of its length7. Ensure that one person analyzes all images to control for subjective differences in determining whether or not a chloroplast has a stromule. A second researcher should independently verify the stromule frequencies to further reduce subjective biases.</li>
</ol>
4. Experimental Design and Sampling
NOTE: Stromule frequency is highly variable between leaves, but several reports suggest that there is little variation in stromule frequency within an individual leaf9,17.
<ol>
	<li>Calculate the leaf stromule frequency from a single field of view of one leaf. Discard the leaf, and consider this an independent sample. Do not consider separate fields of view from one leaf as independent samples. 
	NOTE: There is no strong consensus on how to best measure changes in stromule activity, and until the function of stromules is more clearly defined, researchers should continue to be creative in quantifying stromule dynamics. For comparison across the literature, however, common standards of reporting should be used in addition to more creative approaches. At a minimum, always report the frequency of chloroplasts that have one or more stromules.</li>
	<li>Determine the frequency of chloroplasts that have stromules in a field of view. Use ImageJ to identify all chloroplasts in a field of view. Then, for each chloroplast, determine whether the chloroplast has formed at least one stromule. Report stromule frequency as the percentage of chloroplasts with a stromule. 
	NOTE: Some researchers transform the percentage, for example with the arcsine transformation, before reporting stromule frequencies; while this is an option for statistical analysis, the arcsine of stromule frequency is not an intuitive value, and does not need to be reported in the main text or figures of a study.
	<ol>
		<li>As an alternative approach, count the total number of stromules, and divide it by the total number of chloroplasts. 
		NOTE: Under most circumstances, this will yield similar results to approach 4.2; it may overestimate the stromule frequency, however, if a single chloroplast has formed many stromules. In the example shown under representative results, for example, several chloroplasts have two or more stromules, raising the number of stromules per chloroplast to 40/87 (versus a stromule frequency of 33/87).</li>
		<li>Alternatively, determine the stromule length instead of stromule frequency. Length can be measured in ImageJ by tracing the stromule with the freehand line tool. 
		NOTE: Since the biological role of stromules remains unknown, it is not clear that stromule length affects their function. Report significant differences in stromule length if they are observed, but also report the stromule frequencies (as described in 4.2). 
		NOTE: Stromule frequency can be highly variable among different leaves under the same growth conditions. Therefore, although determining stromule frequency can be time-consuming, experiments should be carefully designed to include large sample sizes. Using datasets from our lab, we determined that a sample size of at least 16 plants for each treatment is usually sufficiently powerful to determine if there is a statistically significant difference of at least a 1.5-fold change in stromule frequency between two conditions (with standard &#945; = 0.05 and &#946; = 0.80).</li>
	</ol>
	</li>
	<li>Statistical analysis of results.
	<ol>
		<li>To compare the mean stromule frequencies of two treatments, use the Welch&#39;s t test (also known as the heteroscedastic t test or t test assuming unequal variance). 
		NOTE: This test is not as powerful as the conventional Student&#39;s t test, but Welch&#39;s t test is advantageous because it does not assume that the variance in stromule frequency distribution is similar between treatments. 
		NOTE: Since stromule frequency is a &#34;proportion&#34;, its sampling distribution is predicted to be binomial rather than normal, which could theoretically skew statistical analysis if sample sizes are very low or if stromule frequency is very low (close to 0%) or very high (close to 100%). Some reports have used arcsine transformations before statistical analysis, which is intended to transform a binomial distribution to a normal distribution, and thus satisfy the standard requirements of a Student&#39;s t test or ANOVA. In practice, however, arcsine transformations have little or no effect on statistical analysis, and are therefore not recommended. Instead, repeat experiments to increase sample size, which will increase statistical power and reduce errors in interpretation of data.</li>
		<li>Whatever statistical approach is used, be sure to carefully report the sampling strategy, sample size, statistical test, and p value for each experiment. 
		NOTE: As with other fields of biology, a p value less than 0.05 may be considered &#34;statistically significant&#34;, although researchers should certainly report the precise p value obtained. If p is slightly above 0.05, increasing the sample size with two or three experiments may help to clarify whether or not the difference observed is reproducible and statistically significant.</li>
	</ol>
	</li>
</ol>
5. Extracting Intact Chloroplasts to Visualize Stromule Dynamics
NOTE: Several methods have been used to isolate chloroplasts from leaves, including a slightly different protocol in a recent study on stromule formation in vitro15. The protocol detailed below uses a relatively simple method that does not yield biochemically pure chloroplast samples, but does instead isolate a large quantity of intact, healthy chloroplasts9,18.
<ol>
	<li>Prepare cold extraction buffer: 50 mM Hepes NaOH, 330 mM sorbitol, 2 mM EDTA, 1.0 mM MgCl2, and 1.0 mM MnCl2. Adjust the pH to 6.9 with NaOH and HCl, and refrigerate before use. 
	NOTE: Although not necessary, using cold buffers and keeping samples on ice when possible will yield a higher proportion of intact chloroplasts.</li>
	<li>Prepare isolation buffer: 50 mM Hepes NaOH, 330 mM sorbitol, 2 mM EDTA, 1.0 mM MnCl2, 1.0 mM MgCl2, 10 mM KCl, and 1.0 mM NaCl. Adjust the pH to 7.6 with NaOH and HCl and refrigerate before use.</li>
	<li>If the leaves are not expressing a genetically encoded stromal fluorophore, prepare carboxyfluorescein diacetate (CFDA) solution: 50 mM CFDA stock solution in dimethyl sulfoxide (DMSO) (2.3 mg CFDA per 1.0 ml DMSO). 
	NOTE: The exact concentration is not critical. Keep CFDA stock in the dark at all times. Store small aliquots of 50 mM CFDA at -20 &#176;C.</li>
	<li>To isolate chloroplasts, remove ~5-10 g leaves from several plants and briefly rinse in cold water. Immediately transfer to 50 ml cold extraction buffer. Grind leaves using a blender with several short pulses. Filter through two or three layers of cheesecloth to remove leaf debris, divide the extracted chloroplasts into two 50 ml centrifuge tubes, and centrifuge for 1 min at 750 x g.</li>
	<li>Discard the supernatant and resuspend the green chloroplasts in 10 ml isolation buffer. Centrifuge again for 1 min at 750 x g, discard supernatant, and resuspend chloroplasts in isolation buffer to bring final volume to 5 ml.</li>
	<li>Transfer 20 &#181;l of the chloroplasts to a slide, cover with a coverslip, and begin microscopy. 
	NOTE: Chloroplasts from transgenic plants expressing plastid-targeted fluorescent proteins are now ready for visualization by confocal fluorescence microscopy.</li>
	<li>Stain chloroplasts from plants that are not expressing plastid-targeted fluorescent proteins to visualize stromules.
	<ol>
		<li>Add 5 &#181;l of 50 mM CFDA stock to the 5 ml chloroplasts-suspended in isolation buffer. Bring the final concentration to 50 &#181;M.</li>
		<li>Allow chloroplasts to incubate for 5 min, and then transfer a small aliquot (~50 &#181;l) to a slide, cover with a coverslip, and begin microscopy.</li>
		<li>Use a FITC or GFP filter set. Use a 20X objective to visualize many chloroplasts simultaneously, or a higher objective to visualize a single isolated chloroplast. 
		NOTE: CFDA will fluoresce inside the stroma of intact chloroplasts.</li>
		<li>If there is strong background fluorescence, wash the chloroplasts again in 10 ml isolation buffer after the 5 min incubation with CFDA, centrifuge for 1 min at 750 x g, discard supernatant, and resuspend in 5 ml isolation buffer.</li>
	</ol>
	</li>
</ol>

<ol><li>Koussevitzky, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signals+from+chloroplasts+converge+to+regulate+nuclear+gene+expression%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Signals from chloroplasts converge to regulate nuclear gene expression.</a> Science. 316 (5825), 715-719 (2007).</li>
<li>Burch-Smith, T. M., Brunkard, J. O., Choi, Y. G., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PNAS+Plus%3A+Organelle-nucleus+cross-talk+regulates+plant+intercellular+communication+via+plasmodesmata%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">PNAS Plus: Organelle-nucleus cross-talk regulates plant intercellular communication via plasmodesmata.</a> Proc. Natl. Acad. Sci. USA. 108 (51), E1451-E1460 (2011).</li>
<li>Stonebloom, S., Brunkard, J. O., Cheung, A. C., Jiang, K., Feldman, L., Zambryski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Redox+states+of+plastids+and+mitochondria+differentially+regulate+intercellular+transport+via+plasmodesmata%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Redox states of plastids and mitochondria differentially regulate intercellular transport via plasmodesmata.</a> Plant Physiol. 158 (1), 190-199 (2012).</li>
<li>Nomura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chloroplast-mediated+activation+of+plant+immune+signalling+in+Arabidopsis%5BTitle%5D)+AND+%22Nat.+Commun%22%5BJournal%5D)">Chloroplast-mediated activation of plant immune signalling in Arabidopsis.</a> Nat. Commun. 3, 926(2012).</li>
<li>Avendaño-Vázquez, A. -O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+uncharacterized+apocarotenoid-derived+signal+generated+in+%CE%B6-carotene+desaturase+mutants+regulates+leaf+development+and+the+expression+of+chloroplast+and+nuclear+genes+in+Arabidopsis%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">An uncharacterized apocarotenoid-derived signal generated in ζ-carotene desaturase mutants regulates leaf development and the expression of chloroplast and nuclear genes in Arabidopsis.</a> Plant Cell. 26 (June), 2524-2537 (2014).</li>
<li>Caplan, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chloroplast+stromules+runction+during+innate+immunity%5BTitle%5D)+AND+%22Dev.+Cell%22%5BJournal%5D)">Chloroplast stromules runction during innate immunity.</a> Dev. Cell. 34 (1), 45-57 (2015).</li>
<li>Hanson, M. R., Sattarzadeh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stromules%3A+recent+insights+into+a+long+neglected+feature+of+plastid+morphology+and+function%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Stromules: recent insights into a long neglected feature of plastid morphology and function.</a> Plant Physiol. 155 (4), 1486-1492 (2011).</li>
<li>Gray, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plastid+stromules+are+induced+by+stress+treatments+acting+through+abscisic+acid%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">Plastid stromules are induced by stress treatments acting through abscisic acid.</a> Plant J. 69 (3), 387-398 (2012).</li>
<li>Brunkard, J., Runkel, A., Zambryski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chloroplasts+extend+stromules+independently+and+in+response+to+internal+redox+signals%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Chloroplasts extend stromules independently and in response to internal redox signals.</a> Proc. Natl. Acad. Sci. USA. 112 (32), 10044-10049 (2015).</li>
<li>Dupree, P., Pwee, K. -H., Gray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+photosynthesis+gene-promoter+fusions+in+leaf+epidermal+cells+of+transgenic+tobacco+plants%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">Expression of photosynthesis gene-promoter fusions in leaf epidermal cells of transgenic tobacco plants.</a> Plant J. 1 (1), 115-120 (1991).</li>
<li>Charuvi, D., Kiss, V., Nevo, R., Shimoni, E., Adam, Z., Reich, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gain+and+loss+of+photosynthetic+membranes+during+plastid+differentiation+in+the+shoot+apex+of+Arabidopsis%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Gain and loss of photosynthetic membranes during plastid differentiation in the shoot apex of Arabidopsis.</a> Plant Cell. 24 (3), 1143-1157 (2012).</li>
<li>Chiang, Y. -H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+characterization+of+the+GATA+transcription+factors+GNC+and+CGA1+reveals+their+key+role+in+chloroplast+development%2C+growth%2C+and+division+in+Arabidopsis%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Functional characterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development, growth, and division in Arabidopsis.</a> Plant Physiol. 160 (1), 332-348 (2012).</li>
<li>Schattat, M., Barton, K., Baudisch, B., Klösgen, R. B., Mathur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plastid+stromule+branching+coincides+with+contiguous+endoplasmic+reticulum+dynamics%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Plastid stromule branching coincides with contiguous endoplasmic reticulum dynamics.</a> Plant Physiol. 155 (4), 1667-1677 (2011).</li>
<li>Erickson, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agrobacterium-derived+cytokinin+influences+plastid+morphology+and+starch+accumulation+in+Nicotiana+benthamiana+during+transient+assays%5BTitle%5D)+AND+%22BMC+Plant+Biol%22%5BJournal%5D)">Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays.</a> BMC Plant Biol. 14 (1), 127(2014).</li>
<li>Ho, J., Theg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+formation+of+stromules+in+vitro+from+chloroplasts+isolated+from+Nicotiana+benthamiana%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The formation of stromules in vitro from chloroplasts isolated from Nicotiana benthamiana.</a> PLoS One. 11 (2), e0146489(2016).</li>
<li>Brunkard, J. O., Burch-Smith, T. M., Runkel, A. M., Zambryski, P. Investigating plasmodesmata genetics with virus-induced gene silencing and an Agrobacterium-mediated GFP movement assay. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBrunkard%2C+JO+%22Method.+Mol.+Biol%22">Method. Mol. Biol</a>. , 185-198 (2015).</li>
<li>Schattat, M. H., Klösgen, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+stromule+formation+by+extracellular+sucrose+and+glucose+in+epidermal+leaf+tissue+of+Arabidopsis+thaliana%5BTitle%5D)+AND+%22BMC+Plant+Biol%22%5BJournal%5D)">Induction of stromule formation by extracellular sucrose and glucose in epidermal leaf tissue of Arabidopsis thaliana.</a> BMC Plant Biol. 11, 115(2011).</li>
<li>Joly, D., Carpentier, R. Rapid isolation of intact chloroplasts from spinach leaves. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aJoly%2C+D+%22Method.+Mol.+Biol%22">Method. Mol. Biol</a>. 684, 321-325 (2011).</li>
<li>Waters, M. T., Fray, R. G., Pyke, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stromule+formation+is+dependent+upon+plastid+size%2C+plastid+differentiation+status+and+the+density+of+plastids+within+the+cell%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell.</a> Plant J. 39 (4), 655-667 (2004).</li>
</ol>

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.

<table><tbody><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>NaOH</td><td>Fischer-Scientific</td><td>S320-1</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma-Aldrich</td><td>S1876</td><td></td></tr><tr><td>EDTA</td><td>Fischer-Biotech</td><td>BP121</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>221279</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M0250</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9625</td><td></td></tr><tr><td>Laser Scanning Confocal Microscope&amp;nbsp;</td><td>Carl Zeiss Inc</td><td>Model: LSM710</td><td></td></tr><tr><td>Carboxyfluorescein diacetate (CFDA)</td><td>Sigma-Aldrich</td><td>21879&amp;nbsp;</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>EMD</td><td>MX1458-6</td><td></td></tr><tr><td>Waring blender</td><td>Waring&amp;nbsp;</td><td>Model: 31BL92</td><td></td></tr><tr><td>Fiji</td><td>fiji.sc</td><td></td><td>Open-source software for analyzing biological images</td></tr></tbody></table>

visualizing stromule frequency with fluorescence microscopy

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...

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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