Name: visualizing stromule frequency with fluorescence microscopy
Uploaded: 2016-11-23T14:00:00
Description: Watch this Scientific Journal Video about Visualizing Stromule Frequency with Fluorescence Microscopy at JoVE.com

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

NOTE: Pour ce protocole, nous avons mis l&#39; accent sur le dosage de la fréquence stromule dans l&#39;épiderme de N. benthamiana laisse. Plusieurs lignées transgéniques stables ont été générés qui peuvent être utilisés à cette fin, y compris 35S PRO: FNRtp: EGFP 13 et NRIP1: Cerulean 6. Ces deux lignées montrent une expression robuste de fluorophores dans le stroma des chloroplastes des feuilles cultivées dans un large éventail de conditions. A...

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Lors de l&#39;instruction stromules, trois facteurs importants doivent être pris en considération tout au long de: (i) la manipulation du tissu végétal doit être maintenu à un minimum absolu, (ii) le système expérimental doit être maintenue constante, et (iii) les stratégies d&#39;échantillonnage doit être soigneusement planifiée pour assurer robuste, des données reproductibles sont analysées. Stromules sont remarquablement dynamiques: ils peuvent prolonger et se rétracter ra...

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;">EDTA</td>
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			<td height="42" style="height:42px;width:216px;">MgCl2</td>
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			<td height="42" style="height:42px;width:216px;">KCl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P3911</td>
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			<td height="42" style="height:42px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9625</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Laser Scanning Confocal Microscope&#160;</td>
			<td style="width:71px;">Carl Zeiss Inc</td>
			<td style="width:119px;">Model: LSM710</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Carboxyfluorescein diacetate (CFDA)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">21879&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">MX1458-6</td>
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			<td height="21" style="height:21px;width:216px;">Waring blender</td>
			<td style="width:71px;">Waring&#160;</td>
			<td style="width:119px;">Model: 31BL92</td>
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			<td height="21" style="height:21px;width:216px;">Fiji</td>
			<td style="width:71px;">fiji.sc</td>
			<td style="width:119px;"></td>
			<td>Open-source software for analyzing biological images</td>
		</tr>
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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.

Ce protocole a été utilisé pour visualiser la fréquence stromule à jour et la nuit dans les cotylédons de jeune N. plantules benthamiana. Les tranches d&#39;une pile z- ont été fusionnées en une seule image (figure 1A). Pour les besoins visuels, cette image était alors désaturée et inversée de telle sorte que stroma apparaît en noir (figure 1B). Les chloroplastes ont été marquées soit comme ayant aucun stromule...

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.

Visualisation Fréquence Stromule avec 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 ...

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