拟南芥表皮、叶肉和血管实质细胞的激光扫描共聚焦显微镜检查

Christian  Elowsky; Yashitola  Wamboldt; Sally  Mackenzie

doi:10.21769/BioProtoc.2150

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Peer-reviewed

Laser Scanning Confocal Microcopy for Arabidopsis Epidermal, Mesophyll, and Vascular Parenchyma Cells

拟南芥表皮、叶肉和血管实质细胞的激光扫描共聚焦显微镜检查

CE Christian Elowsky ^*

YW Yashitola Wamboldt ^*

SM Sally Mackenzie email

(*contributed equally to this work) 发布: 2017年03月05日第7卷第5期 DOI: 10.21769/BioProtoc.2150 浏览次数: 13764

评审: Arsalan DaudiAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Molecular Plant, featuring study using the protocol.

Feb 2016

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Abstract

Investigation of protein targeting to plastids in plants by confocal laser scanning microscopy (CLSM) can be complicated by numerous sources of artifact, ranging from misinterpretations from in vivo protein over-expression, false fluorescence in cells under stress, and organellar mis-identification. Our studies have focused on the plant-specific gene MSH1, which encodes a dual targeting protein that is regulated in its expression and resides within the nucleoid of a specialized plastid type (Virdi et al., 2016). Therefore, our methods have been optimized to study protein dual targeting to mitochondria and plastids, spatial and temporal regulation of protein expression, and sub-organellar localization, producing a protocol and set of experimental standards that others may find useful for such studies.

Keywords: Confocal (共聚焦)

Autofluorescence (自发荧光)

Organellar (细胞器)

Background

Protein targeting behavior in plants is influenced by amino-terminal presequences as well as internal sequence features that can influence suborganellar localization behaviors (Baginsky and Gruissem, 2004). Combined with promoter-driven spatial and temporal regulation in expression, a protein’s activity can be extremely precise and specialized by virtue of timing and location. In the case of MSH1, this nuclear-encoded, plant-specific protein is dual targeted to mitochondria and plastids (Xu et al., 2011). Promoter features direct its expression to reproductive, epidermal and vascular parenchyma cells (Virdi et al., 2016). Internal protein features direct its localization to the mitochondrial and plastid nucleoid, as well as to the plastid thylakoid membrane. Discovery of these unusual protein features was greatly facilitated by laser scanning confocal microscopy using methodologies described here. Much of this detail would have been overlooked using more traditional organellar subfractionation methodologies.

Materials and Reagents

5 ml tube
Needleless syringe
Double edge razor blades (Electron Microscopy Sciences, Personna^TM, catalog number: 72000 )
Glass cover slips, No. 1.5 (VWR, catalog number: 16004-302 )
Dissecting needle or probe pin
Petri dish (VWR, catalog number: 25384-302 )
Centrifuge tube (VWR, catalog number: 89039-668 )
0.45 μm filter (VWR, catalog number: 28145-481 )
Disposable transfer pipette (Fisher Scientific, catalog number: 13-7117M )
Glass slides (VWR, catalog number: 48300-048 )
Arabidopsis leaf/flower/stem tissue (Ecotype Col-0, 6 weeks old plants)
Lurie broth
Tween 20 (Sigma-Aldrich, catalog number: P1379 )
Incubation solution
Potassium phosphate dibasic (K₂HPO₄) (Sigma-Aldrich, catalog number: P3786 )
Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P5655 )
Ammonium sulfate, (NH₄)₂SO₄ (Sigma-Aldrich, catalog number: A4418 )
Sodium citrate dihydrate (Sigma-Aldrich, catalog number: C8532 )
Magnesium sulfate (MgSO₄) (1 M stock solution) (Sigma-Aldrich, catalog number: M2643 )
Glucose (Sigma-Aldrich, catalog number: G8270 )
Glycerol (Sigma-Aldrich, catalog number: G5516 )
MES (Sigma-Aldrich, catalog number: M2933 )
KOH or NaOH
Acetosyringone (3’,5’-dimethoxy-4’-hydroxyacetophenone) (Sigma-Aldrich, catalog number: D134406 )
MS medium basal salts (Sigma-Aldrich, catalog number: M5519 )
Cellulase ‘onozuka’ R-10 (Yakult Honsha, Tokyo, Japan)
Macerozyme R-10 (Yakult Honsha, Tokyo, Japan)
Mannitol (Sigma-Aldrich, catalog number: M1902 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C7902 )
BSA (optional) (Sigma-Aldrich, catalog number: A7906 )
Induction medium (see Recipes)
Infiltration medium (see Recipes)
Cellulase/macerozyme solution (see Recipes)
Washing and incubation solution (WI) (see Recipes)

Equipment

387/478/555 nm triple-bandpass filter excitation with a broad emission filter ~400-700 nm
LED illumination, 488 nm, 543 nm (Lumencor AURA light engine)
Incubator shaker
Platform shaker
Forceps (VWR, catalog number: 82027-440 ) or (Cole-Parmer Instrument, catalog number: EW-07287-09 )
90i upright compound microscope (Nikon Instruments)
10x Plan Apo 0.45NA (Nikon Instruments, model: OFN25 )
20x Plan Apo 0.75NA (Nikon Instruments, model: lambda )
60x Plan Apo VC water immersion lens 1.2NA (Nikon Instruments, model: MRD07602 )
Confocal laser scanning microscope (CLSM) (Nikon Instruments, model: A1⁺ )
Autoclave
Water bath
Vacuum desiccator

Software

NIS-Elements software

Procedure

English

中文翻译

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如何引用

Elowsky, C., Wamboldt, Y. and Mackenzie, S. (2017). Laser Scanning Confocal Microcopy for Arabidopsis Epidermal, Mesophyll, and Vascular Parenchyma Cells. Bio-protocol 7(5): e2150. DOI: 10.21769/BioProtoc.2150.