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

Agrobacterium mediated transient transformation of tobacco leaves
1. Culture Agrobacterium strain C58C1 carrying the target gene of interest in a 5 ml culture medium of Lurie broth overnight at 30 °C in an incubation shaker and spin down at 1,000 x g.
2. Transfer the bacterial cells into 5 ml of induction medium (see Recipes) and grow at 30 °C for 16 h with shaking.
3. Spin the cells down and re-suspended in 1 ml infiltration medium (see Recipes).
4. Measure the bacterial concentration to an optical density of 0.8 at 600 nm in a fresh 5 ml tube of infiltration medium. Infiltrate young leaves of about 4 weeks old tobacco plants, Nicotiana tabacum cv. xanthi using a needleless syringe. Visualize plant expression after 48 h.
Note: Protocol modified from Van den Ackerveken et al. (1996).
Preparation of Arabidopsis plant samples
1. GFP or YFP when viewed through the oculars with a broad emission filter coupled with a 488 nm LED will not only allow visualization of GFP or YFP (with less excitation efficiency) but also the plastids. This full spectrum visualization allows the user to differentiate damaged areas where plastids shift from red emission to orange. Furthermore in variegated tissues, ocular observation may be the only method of differentiating low GFP or YFP and damaged/stressed areas. Ocular observations have the benefit of much deeper optical sections, providing context for localization. In this case, 10x and 20x visualization provided clues to pursue vascular localization.
2. RFP excited with 543 nm light visualized with a broad emission filter will appear a deep orange color in a ruby red background from the plastids. With low RFP expression, it’s difficult to differentiate the two, and the CSLM should be used to separate the two.
3. The benefit of using LED over more common mercury vapor lamps is the reduction in photobleaching or degradation of the plastid pigments, which will shift emission causing erroneous analyses.
Preparation of enzymatically digested plant tissues and visualization
Partial enzymatic digestion of A. thaliana leaves for visualization of fluorescence in plant vasculature. Enzymatic digestion is done by modifying the protocol of Yoo et al. (2007) used for Protoplast Isolation Procedure.
1. Cut the leaves so most of the lamina is trimmed off from the midrib and veins and subjected to enzyme digestion.
  Note: A six to seven weeks old plant is normally used for this experiment.
2. Place the plant material to be digested in a Petri dish containing the enzyme solution and put it in a vacuum desiccator and vacuum infiltrate for 15 min.
  Note: A 5-10 ml cellulase/macerozyme solution (see Recipes) is enough for a flowering A. thaliana plant.
3. Take out the Petri dish from vacuum and continue digestion at RT for 2 h with gentle shaking on a platform shaker, between 30-50 RPM.
4. The proper digestion time is near when the epidermis is digested away, and mesophyll cells begin sloughing away. The slurry generated to visualize vascular parenchyma with intact leaf or is easily transferred with a transfer pipette or fine tip forceps as the samples are not allowed to fully digest to the protoplast stage. The sample can be transferred to a Petri dish containing 10 ml of the WI buffer. On a compound microscope, if the vasculature can be seen, the sample is ready for confocal analysis. Vasculature is easily identified by the helical thickenings in the xylem with transmitted light at 10x or 20x. Digestion time will vary with the hardiness of the plant material.
5. Mount samples on slides with coverslips for confocal microscopy. The incubation solution is used to mount the sample, as it will support the cells during imaging.

Data analysis

Factors that affect sample observations:

Transgene construct features influence microscopy outcomes
Promoter selection can influence signal strength significantly in our experiments. Consequently, we have combined both transient, high level expression experiments with native promoter, stable transformation experiments for any constructs to be tested (Figure 4). For proteins targeted to subcellular organelles, it appears that truncation of the full-length gene can sometimes produce unreliable results. Figure 5 shows that the MSH1 protein, which targets to the nucleoid subcompartment of the plastid, can be found localized to the nucleoid only when at least domains I–III of the protein coding region are included in the analysis (Virdi et al., 2016).

Figure 4. Expression pattern in leaves and level of expression vary depending on the promoter used. A. pCAMBIA1302C::35S::MSH1::GFP transient expression in tobacco. There are more punctate GFP expression signals when the gene construct is driven by the CaMV 35S promoter. B. pCAMBIA1302C (-35S)::NativePro::MSH1::GFP stable expression in Arabidopsis cells. The native promoter resulted in fewer punctate GFP signals (as seen by number of green dots per plastid). All scale bars are 25 µm. GFP pseudocolored - green, chloroplast autofluorescence pseudocolored - red, maximum intensity projections.

Figure 5. Protein localization pattern can vary depending on truncations in Arabidopsis thaliana Col-0 leaf tissue. A. Native promoter::MSH1(ATG to Domain I)::GFP; domain I alone gives a diffused signal in the plastids. Single frame image. B. Native promoter::MSH1(ATG to Domain III)::GFP. Single frame image. C. Native promoter::MSH1(ATG to Domain V)::GFP. MSH1 ATG start to domain III is necessary to give the punctate localization. Maximum intensity projection. GFP pseudocolored green, chloroplast autofluorescence pseudocolored red for all images. All scale bars are 25 µm. D. Schematic diagram of the different domains of MSH1 from Arabidopsis thaliana used for the truncation studies (Virdi et al., 2016).
Tissue preparation can also influence resolution in a confocal microscopy study
Plants stably transformed with a full-length gene construct under control of its native promoter offer the opportunity to document spatial and temporal expression behaviors. Figure 6 shows the result of partial tissue digestion in revealing the localization of the MSH1 protein within specialized plastids of the vascular parenchyma tissues. These observations are confirmed, in Figure 7, by stem cross-sections that show MSH1-GFP signal within the vascular bundle tissues. However, it is essential that the tissues assayed are not derived from a stressed plant. Figure 8 shows that artifactual fluorescence in a plant under stress can be confused with RFP signal.

Figure 6. Partial enzymatic digestion of A. thaliana leaf midrib and side veins to study sub-cellular localization of fluorescence. Native Pro::MSH1::GFP stable transformed A. thaliana Col-0 leave digested with cellulose and macerozyme as described above. MSH1::GFP expression in specialized plastids can be seen concentrated around phloem and xylem but not in the mesophylls. All scale bars are 25 µm. A. Minor leaf vein. GFP pseudocolored - green, chloroplast autofluorescence pseudocolored - red, xylem autofluorescence pseudocolored - blue. Maximum intensity projection. B. Midrib of leaf. GFP pseudocolored - green, chloroplast autofluorescence pseudocolored - red.

Figure 7. Hand sectioning technique to visualize tissue specificity of proteins. A. Cross section of native promoter::MSH1::GFP stable transformed floral stems hand sectioned as described in Materials and Methods. MSH1::GFP expression localizes to the vascular system. GFP pseudocolored green, chloroplast autofluorescence pseudocolored red, xylem autofluorescence pseudocolored blue with crosstalk in the GFP channel. B. Cross section of a dually transformed A. thaliana with MSH1::GFP and PPD3::RFP (AT1G76450); co-localization of two proteins in the same tissue and plastid type. GFP pseudocolored - green, RFP pseudocolored - red, chloroplast autofluorescence pseudocolored - blue. Single frame images and all scale bars are 25 µm.

Figure 8. Artifacts that users need to be aware of in Confocal Microscopy. A stressed A. thaliana Col-0 leaf vacuoles. Scale bar is 25 µm. Chloroplast autofluorescence pseudocolored - green, vacuole autofluorescence pseudocolored - red. Maximum intensity projection.

Recipes

Induction medium (1 L)
10.5 g K₂HPO₄
4.5 g KH₂PO₄
1 g (NH₄)₂SO₄
0.5 g sodium citrate dihydrate
1 ml MgSO₄ (1 M stock solution)
2 g glucose
5 ml glycerol
10 mM MES
Adjust pH to 5.6 with KOH or NaOH and autoclave at 115 °C for 20 min
Cool down medium and add antibiotics and 50 μg/ml acetosyringone
Infiltration medium
Half strength MS medium basal salts
10 mM MES
Adjust pH to 5.6 with KOH
Add 150 μg/ml acetosyringone before use
Cellulase/macerozyme solution
1.5% cellulase ‘onozuka’ R-10 (0.15 g/10 ml)
0.4% macerozyme R-10 (Yakult Honsha, Tokyo, Japan) (0.04 g/10 ml)
0.4 M mannitol (0.8 M mannitol stock)
20 mM KCl (2 M KCl stock)
20 mM MES, pH 5.7 (0.2 M MES stock)
Heat the enzyme solution in a 55 °C water bath for 10 min in 15 ml centrifuge tube (stirring not needed)
Cool it to room temperature
Add 10 mM CaCl₂ (1 M CaCl₂ stock); 0.1% BSA (optional) (10% stock, sterile)
Pass the enzyme through a 0.45 μm filter
Washing and incubation solution (WI)
0.5 M mannitol
4 mM MES, pH 5.7
20 mM KCl

Acknowledgments

We wish to thank Dr. Kamaldeep Virdi and Sunil Kumar for supplying images from their research in Arabidopsis for demonstration purposes. We also thank Cecil Renfro for her assistance with photography. We gratefully acknowledge support from the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of DOE (DE-FG02-10ER16189) to S.M. for this work.

References

Baginsky, S. and Gruissem, W. (2004). Chloroplast proteomics: potentials and challenges. J Exp Bot 55(400): 1213–1220.
Van den Ackerveken, G., Marois, E. and Bonas, U. (1996). Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell. Cell 87(7):1307–1316.
Virdi, K. S, Wamboldt, Y., Yu, J., Laurie, J. D., Kumar, S., Kundariya, H., Xu, Y., Elowsky, C., Basset, G., Bricker, T., Leubker, S., Keren, I. and Mackenzie, S. A. (2016). MSH1 is a plant organellar DNA binding and thylakoid protein under precise spatial regulation to alter development. Mol Plant 9: 245–60.
Xu, Y. Z., Arrieta-Montiel, M. P., Virdi, K. S., de Paula, W. B., Widhalm, J. R., Basset, G. J., Davila, J. I., Elthon, T. E., Elowsky, C. G., Sato, S. J., Clemente, T. E. and Mackenzie, S. A. (2011). MutS HOMOLOG1 is a nucleoid protein that alters mitochondrial and plastid properties and plant response to high light. Plant Cell 23: 3428–3441.
Yoo, S. D., Cho, Y. H. and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7): 1565-1572.