用相关光电子显微镜的方法确定拟南芥嫁接界面的超微结构单元

Clément Chambaud; Sarah J. Cookson; Nathalie  Ollat; Amélie  Bernard; Lysiane Brocard

doi:10.21769/BioProtoc.4590

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Targeting Ultrastructural Events at the Graft Interface of Arabidopsis thaliana by A Correlative Light Electron Microscopy Approach

用相关光电子显微镜的方法确定拟南芥嫁接界面的超微结构单元

LB Lysiane Brocard email

发布: 2023年01月20日第13卷第2期 DOI: 10.21769/BioProtoc.4590 浏览次数: 1793

评审: Wenrong HeYao XiaoAnonymous reviewer(s)

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Abstract

Combining two different plants together through grafting is one of the oldest horticultural techniques. In order to survive, both partners must communicate via the formation of de novo connections between the scion and the rootstock. Despite the importance of grafting, the ultrastructural processes occurring at the graft interface remain elusive due to the difficulty of locating the exact interface at the ultrastructural level. To date, only studies with interfamily grafts showing enough ultrastructural differences were able to reliably localize the grafting interface at the ultrastructural level under electron microscopy. Thanks to the implementation of correlative light electron microscopy (CLEM) approaches where the grafted partners were tagged with fluorescent proteins of different colors, the graft interface was successfully and reliably targeted. Here, we describe a protocol for CLEM for the model plant Arabidopsis thaliana, which unambiguously targets the graft interface at the ultrastructural level. Moreover, this protocol is compatible with immunolocalization and electron tomography acquisition to achieve a three-dimensional view of the ultrastructural events of interest in plant tissues.

Graphical abstract

Keywords: Plant grafting (植物嫁接)

Correlative microscopy (关联显微镜)

Arabidopsis thaliana (拟南芥)

Electron microscopy (电子显微镜)

Electron tomography (电子断层扫描法)

Background

Plant grafting is a process that has been used for centuries in agronomy to combine interesting agronomical traits together, such as resistance and fruit productivity (Mudge, 2009), or to study systemic biological processes, such as epigenetic regulations and long-distance signaling (Molnar et al., 2010; Turnbull, 2010; Turnbull et al., 2002; Wu et al., 2013; Bidadi et al., 2014; Zhang et al., 2014; Yang et al., 2015). Despite its widespread use, the cytologic events required for the establishment of the graft union remain poorly understood (Gautier and Chambaud et al., 2019). During graft union formation, tissues from both partners have to undergo a process of dedifferentiation to form callus cells and set up new pathways of communication such as phloem, xylem, and plasmodesmata. The movement of dyes or fluorescent probes between the scion and the rootstock have been used to resolve the kinetics of vascular connection at the graft interface, but no ultrastructural data are available for the establishment of cell-to-cell connection via plasmodesmata (Melnyk et al., 2015; Melnyk et al., 2018). Knowledge of the ultrastructural mechanisms underpinning graft union formation is currently very limited because locating precisely the site of contact between the two grafted partners under electron microscopy is challenging due to the difficulty to distinguish the origin/identity of each cell at the graft interface, i.e. to distinguish between cells from the scion and cells from the rootstock (Kollmann et al., 1985; Kollmann and Glockmann, 1985, 1991; Pina et al., 2017; Notaguchi et al., 2020). Attempts were successfully made by Kollmann and Glockmann (1985) and Notaguchi (2020), in which plants from different species showing noticeable ultrastructural differences in the callus cells produced at the graft interface were used.

To overcome the requirement of cytological differences to accurately identify cell origin and properly localize the graft interface, we propose a protocol for correlative light and electron microscopy (CLEM) to unambiguously target the graft interface at the ultrastructural level. At present, the use of CLEM approaches is still scarce for the analyses of plant tissues (Bell et al., 2013; Marion et al., 2017). Two out of the three CLEM protocols developed for plants are unfortunately not easily compatible with electron tomography due to either the weakness of the embedded resin used (glycol methacrylate) or the absence of resin (Tokuyasu method) (Marion et al., 2017). The protocol described by Bell et al. (2013) presents the advantage of being easily accessible but, because it is based on a chemical fixation, only permits a coarse ultrastructural preservation. Here, in order to combine fluorescence and high ultrastructural resolution in the same sample, we adapted the protocol described by Kukulski et al. (2012a and 2012b), an in-resin fluorescence CLEM method for plant tissues (Kukulski et al., 2012a and 2012b). This protocol allows us to preserve fluorescence and maintain the ultrastructure; further, in combination with electron tomography, this method allows to reach a level of resolution enabling the visualization of the bilayer of the plasma membrane. Moreover, this protocol is compatible with immunogold labeling and is adapted for hypocotyls and roots of plantlets of Arabidopsis thaliana. For our study, the hypocotyl of a scion and a rootstock expressing a yellow fluorescent protein (YFP) and monomeric red fluorescent protein (mRFP), respectively, fused to the endoplasmic reticulum–retention motif HDEL, were grafted (Chambaud et al., 2022). By optimizing (1) the grafting procedure, (2) the duration of the freeze-substitution (FS) steps, (3) the thickness of the ultrathin sections, (4) the sample mounting for light microscopy (LM) observation, and (5) the LM acquisition, we were able to observe fluorescence signals and precisely target the graft interface. The compatibility of our CLEM protocol with our previously described electron tomography protocol (Nicolas et al., 2018), allowed to access the three-dimensional (3D) view of ultrastructural elements specifically at the graft interface (Chambaud et al., 2022). Our CLEM protocol has the potential to unravel multiple plant cell processes at the ultrastructural level and was in fact already applied successfully to study autophagy (Gomez et al., 2022) and phase-to-phase separation processes (Dragwidge et al., 2022).

Materials and Reagents

Plant culture
1. 1.5 mL Eppendorf tubes (Sarstedt, catalog number: 72.708)
2. 12 N or 37% hydrochloric acid (Carlo Erba, catalog number: 524526)
3. 1.5 mL Eppendorf tube rack (Ratiolab cardboard grid, inserts for cryobox, catalog number: 5020147)
4. Square plastic culture plates for A. thaliana seedlings, vertical culture (VWR, catalog number: 391-0444)
5. Bleach (Orapi Europe, catalog number: OR0011)
6. Murashige and Skoog (MS) medium + vitamins (Duchefa Biochemie, catalog number: M0222.0050) for seedlings
7. Plant agar for seedlings culture and grafting (Duchefa Biochemie, catalog number: P1001.1000)
8. Anapore tape (Euromedis, catalog number: 135320) to seal culture plates
9. KOH
10. Bleach solution for seeds sterilization (Recipe 1)
11. Culture medium for seedling and grafting (Recipe 2)
  1. Half-strength MS medium for seedlings
  2. Water medium for grafting
Transgenic lines
For grafting experiments, we used 35S::HDEL_mRFP and 35S::HDEL_YFP lines, which have strong fluorescent expression (Nelson et al., 2007;Lee et al., 2013). Both lines showed a fluorescence signal that was easily visible through oculars with a zoom microscope with the medium pre-set level for the excitation power and the adapted set filter.
Plant grafting
1. Round Petri dishes, 90 × 14.2 mm for grafting (generally used for bacterial culture) (VWR, catalog number 391-0439)
2. Neutral nylon membrane hybond-N (GE Healthcare, catalog number: RPN 203 N)
3. Paraffin film (Bemis, Parafilm ‘M’) cut into strips of approximately 1 cm wide to seal the Petri dishes
High-pressure freezing (HPF)
1. Screw-top 2 mL tubes (Sarstedt, catalog number: 72.693)
2. Toothpicks
3. Bovine serum albumin (BSA) fraction V (Sigma-Aldrich, catalog number: 05482)
4. Methyl cyclohexane (MCH) (Merck Schuchardt OHG 85662 Hohenbrunn, Germany)
5. Distilled water to cut sample
6. Membrane carriers (100 μm deep with 1.5 mm diameter) (Leica Microsystems, catalog numbers: 16707898)
7. 20% BSA solution for cryoprotection during HPF (see Recipe 3)
Freeze-substitution (FS)
1. Disposable regular pipettes, 1, 2, and 3 mL (VWR, catalog numbers: 612-2850, 612-2851, and 612-1681) and FS-specific 1 mL pipettes with thin tips (Ratiolab, catalog number: 2600155)
2. Wheaton glass sample vials with snap-cap for cryomix preparation (DWK Life Sciences, WHEATON, catalog number: 225536)
3. Personal cartridge half mask 6100 (Honeywell International, catalog number: 1029471)
4. Aluminum foil
5. Uranyl acetate powder (Merck, catalog number: 8473)
6. Pure methanol (Fisher Scientific, catalog number: M/4062/17)
7. Ultrapure 100% ethanol and acetone (VWR, catalog numbers: 83813.440 and 20066.558, respectively)
8. Colored nail polish (color does not matter)
9. 12.5 mL reagent containers with screw caps (Leica Microsystems, catalog number: 16707158)
10. HM20 resin (Electron Microscopy Sciences, catalog number 14345)
11. Reagent bath (Leica Microsystems, catalog number: 16707154)
12. Plastic mold flow-through rings (Leica Microsystems: 16707157)
13. Cryosubstitution uranyl-acetate stock solution (20%) (see Recipe 4)
14. Cryosubstitution mix (see Recipe 5)
15. HM20 solutions (see Recipe 6)
Ultramicrotomy
1. Crystallizing dish (Fisher Scientific, catalog number: 11766582)
2. Fast absorbent paper filters (Whatman, Filter papers 41) (GE Healthcare, catalog number: 1441-070)
3. Glass rods (Fisher Scientific, catalog number: 12441627)
4. Gilder standard hexagonal 200 mesh copper grids (Electron Microscopy Sciences, catalog number: G200HS-Cu) to coat with parlodion or commercial grids with formvar (Electron Microscopy Sciences, catalog number: FCFT200-CU)
5. Razor blades for coarse block preparation (Electron Microscopy Sciences, catalog number: 71990)
6. Pasteur pipet (VWR, catalog number: 612-1720)
7. 0.2 μm syringe filter (VWR, catalog number: 514-0061)
8. 10 mL syringe (Terumo, catalog number: SS+10ES1)
9. Solid parlodion (Electron Microscopy Sciences, catalog number: 19220)
10. Isoamyl-acetate (Sigma-Aldrich, catalog number: W205532)
11. Toluidine blue powder (Sigma-Aldrich, catalog number: T3260)
12. Sodium borate powder
13. Ultrapure (Milli-Q) water
14. 2% parlodion solution for grid filming (see Recipe 7)
15. Toluidine blue solution (see Recipe 8) for screening block sections on tabletop microscope during resin block milling
Electron microscopy
1. Gold colloid 5 nm (BBI Solution, catalog number: EMGC5)
2. Gold fiducials solution (see Recipe 9)

Equipment

Plant culture
1. Desiccator used as hermetic chamber
2. Fume hood
3. Glass crystallizing dish
4. Fridge for vernalization
5. -80°C freezer for sterilization
Plant grafting
1. Dissecting microscope with a minimal working distance of 5 cm (Leica Microsystems, model: Leica MZFLIII)
2. Glass beaker for sterilization of tools with ethanol
3. Spray for ethanol
4. Dissecting micro-knives 15° 5 mm (Fine Science Tools, catalog number: 72-1551)
5. Precision tweezers [EMS style 5X, catalog number: 78320-5X (soft) and 78520-5X (hard)]
Fluorescence screening
1. Zoom microscope (Zeiss Axiozoom.V16) equipped with a 0.5× objective with a numerical aperture of 0.125. The fluorescent part is composed by the fluorescence source Optoprim sola SE II and filter sets (GFP filter set: excitation window between 460 and 480 nm and emission window between 500 and 548 nm; mRFP filter set: excitation window between 540 and 580 nm and emission window up to 593 nm).
High-pressure freezing (HPF)
1. Liquid nitrogen, liquid nitrogen container, and adapted personal protection gear
2. Air compressor (JUN-AIR)
3. Leica EM-PACT1 machine (Leica Microsystems)
4. Leica loading system
5. Pod holders (Leica, catalog number: 16706832)
6. Pods (Leica, catalog number: 16706838)
7. Regular biomolecular pipettes, ranging from 2 to 1,000 µL
8. Binocular with transmission illumination for root dissection (Nikon, model: SMZ-10A)
9. Heating surface for quick drying of pods and pod holders during the session
10. Insulated tweezers for manipulation in liquid nitrogen (VOMM Germany, 22 SA ESD)
11. Metal containers for frozen sample transfer and associated screwdriver for lifting procedures (provided with EMPACT1)
12. Torque screwdriver (TOHNICHI, Torque driver RTD60CN) set to 30 N.cm
Freeze-substitution (FS)
1. Leica automatic freeze-substitution AFS2 (Leica Microsystems, model: Leica AFS2)
2. Leica freeze-substitution processor FSP (Leica Microsystems, model: Leica FSP)
3. Metal socket for mold containers
4. Microneedles for separating the membrane carriers from frozen samples [microneedle 0.025 mm (Electron Microscopy Science, catalog number: 62091-01) and 5 µm (Ted Pella, catalog number: 13625)]
5. Ventilated fume hood
Ultramicrotomy
1. Block holders
2. Diamond trimming knife for first milling steps to reach the sample (Drukker 8 mm Histoknife Dry, discontinued)
3. Diamond knives Trim20 dry (Diatome, catalog number: DTB20), Ultra wet 45° (Diatome, catalog number: DU4530), and Histo wet (Diatome, catalog number: DH4560)
4. ELMO glow discharger (optional) (CORDOUAN Technologies)
5. Grid carriers for grid storage
6. Leica Ultracut UC7 (Leica Microsystems, model: Leica Ultracut UC7)
7. Tabletop microscope (Olympus, model: CX-41)
8. Precision weight scale for parlodion weighting (Sartorius, model: TE124S)
Confocal microscopy
1. Sensitive confocal microscope (Zeiss Microscopy, model: Zeiss LSM 880) and 63× NA 1.4 oil immersion objective
Electron microscopy
1. FEI Tecnai G2 Spirit 120 kV equipped with Eagle 4K × 4K high sensibility CCD camera
2. Advanced tomography holder (Fischione instruments, model: 2020)

Software

Zeiss Zen Dark version 2.3 software (https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html) used for confocal acquisition
Tecnai imaging and analysis (TIA) version 4.5 software was used in conjunction with transmission electron microscopy (TEM) User Interface software version 4.5 to control and acquire micrograph with the electron microscopy (https://www.fei.com/software/)
Xplore3D version 4.5 (FEI) was used for automated tilt series acquisitions (https://www.fei.com/software/)
ImageJ version 1.53n (https://imagej.nih.gov/ij/download.html) with the Bio-Format version 6.9 plugin (https://www.openmicroscopy.org/bio-formats/) to open and process the Zeiss .czi files and to open the .mrc tilt series files and tomograms.
IMOD suite version 4.11.6 (http://bio3d.colorado.edu/imod/). All processing of tomograms was done using the IMOD suite (Kremer et al., 1996), from the alignment of the tilt series to tomogram reconstruction
For visualization purposes only, Adobe Photoshop version 23.0.1 is used for TEM mosaic assembling using photomerge function.
ICY version 2.4.2.0 and its plugin ec-CLEM version 1.0.1.5 (https://icy.bioimageanalysis.org/plugin/ec-clem/) were used for LM and TEM correlation

Procedure

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Chambaud, C., Cookson, S. J., Ollat, N., Bernard, A. and Brocard, L. (2023). Targeting Ultrastructural Events at the Graft Interface of Arabidopsis thaliana by A Correlative Light Electron Microscopy Approach. Bio-protocol 13(2): e4590. DOI: 10.21769/BioProtoc.4590.