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 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.
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.
stromules을 조사 할 때, 세 가지 중요한 요인 전반에 걸쳐 고려되어야한다 : 절대 최소로 유지해야 식물 조직의 (ⅰ) 조작, (ⅱ) 실험 시스템은 일관성을 유지해야하며, (ⅲ) 샘플링 전략은 신중을 계획해야합니다 강력한 보장, 재생 가능한 데이터 분석된다.
Stromules는 매우 동적 : 그들은 확장하고 현미경 관찰자의 눈 앞에 빠르게 철회 할 수 있습니다. 또한 stromule 주파수 (예 : 잎…
The authors have nothing to disclose.
J.O.B. and A.M.R. were supported by predoctoral fellowships from the National Science Foundation.
Hepes | Sigma-Aldrich | H3375 | |
NaOH | Fischer-Scientific | S320-1 | |
Sorbitol | Sigma-Aldrich | S1876 | |
EDTA | Fischer-Biotech | BP121 | |
MnCl2 | Sigma-Aldrich | 221279 | |
MgCl2 | Sigma-Aldrich | M0250 | |
KCl | Sigma-Aldrich | P3911 | |
NaCl | Sigma-Aldrich | S9625 | |
Laser Scanning Confocal Microscope | Carl Zeiss Inc | Model: LSM710 | |
Carboxyfluorescein diacetate (CFDA) | Sigma-Aldrich | 21879 | |
Dimethyl sulfoxide (DMSO) | EMD | MX1458-6 | |
Waring blender | Waring | Model: 31BL92 | |
Fiji | fiji.sc | Open-source software for analyzing biological images |