Creating Leaf Cell Suspensions for Characterization of Mesophyll and Bundle Sheath Cellular Features

Abstract

Imaging of mesophyll cell suspensions prepared from Arabidopsis has been pivotal for forming our current understanding of the molecular control of chloroplast division over the past 25 years. In this chapter, we provide a method for the preparation of leaf cell suspensions that improves upon a previous method by optimizing cellular preservation and cell separation. This technique is accessible to all researchers and amenable for use with all plant species. The leaf suspensions can be used for imaging chloroplast features within a cell that are important for photosynthesis such as size, number, and distribution. However, we also provide examples to illustrate how the cells in the suspensions can be easily stained to image other features, for example pit fields where plasmodesmata are located and organelles such as mitochondria, to improve our understanding of traits that are important for photosynthetic physiology.

Full text

Chapter 15
Creating Leaf Cell Suspensions for Characterization
of Mesophyll and Bundle Sheath Cellular Features
Roxana Khoshravesh and Tammy L. Sage
Abstract
Imaging of mesophyll cell suspensions prepared from Arabidopsis has been pivotal for forming our current
understanding of the molecular control of chloroplast division over the past 25 years. In this chapter, we
provide a method for the preparation of leaf cell suspensions that improves upon a previous method by
optimizing cellular preservation and cell separation. This technique is accessible to all researchers and
amenable for use with all plant species. The leaf suspensions can be used for imaging chloroplast features
within a cell that are important for photosynthesis such as size, number, and distribution. However, we also
provide examples to illustrate how the cells in the suspensions can be easily stained to image other features,
for example pit fields where plasmodesmata are located and organelles such as mitochondria, to improve our
understanding of traits that are important for photosynthetic physiology.
Key words Leaf cell suspensions, Light microscopy, Chloroplasts, Cellular architecture, Plasmodes-
mata, Callose, Live-cell fluorescence probes
1 Introduction
Microscopy-based imaging of cell architecture has been an essential
tool used to unravel the relationship between plant cellular form,
physiology, and evolution since the initial observations of plant cells
by Robert Hooke published in Micrographia in 1665. Current
techniques employed for characterization of plant cell structure
are quite sophisticated in comparison to those used during Hooke’s
time with, for example, the incorporation of confocal microscopy
for live-cell imaging to detect proteins labeled with fluorescent
probes such as GFP and YFP [1,2]. The integrated use of confocal
microscopy with one or more techniques such as light, scanning,
and transmission electron microscopy and immunohistochemistry
[3] has contributed substantially to our understanding of photo-
synthesis. As examples, these aforementioned techniques, used
either in combination or alone, have been indispensable for asses-
sing pyrenoid formation [4], thylakoid assembly [5,6], formation
Sarah Covshoff (ed.), Photosynthesis: Methods and Protocols, Methods in Molecular Biology, vol. 1770,
https://doi.org/10.1007/978-1-4939-7786-4_15,©Springer Science+Business Media, LLC, part of Springer Nature 2018
253

of dimorphic chloroplasts [7], plasmodesmata distribution between
the two photosynthetic cell types in C
4
species [8], and control of
plastid volume per cell [numbers and size; 9]. Many of the studies
characterizing plastid volume per cell have utilized a very easy,
rapid, and informative method to prepare mesophyll cell suspen-
sions that allows imaging of chloroplasts in intact cells using a
compound light microscope (Fig. 1a–c;[10]). This technique has
proven to be an essential tool for identification of plastid division
Fig. 1 Images of leaf cells in leaf cell suspension. (a) Spongy mesophyll cell from Arabidopsis (DIC). (b)
Palisade parenchyma cell from Heliotropium calcicola,aC
3
photosynthetic species (bright field). (c)Arabi-
dopsis mesophyll cells with 1–2 large plastids isolated from the mutant line pdv1pdv2 generated by
Miyagishima et al. [13]. DIC image by Dr. Siddhartha Dutta. (d) Bundle sheath cells of the C
4
photosynthetic
species Atriplex rosea stained with aniline blue illustrating callose at pit fields on a lateral wall between two
bundle-sheath cells (arrows). Imaged with fluorescence microscope. (e) Bundle-sheath cells of Oryza sativa
stained with IKI (brown). Imaged with DIC. Bars, 10 μm. bs bundle sheath; cchloroplast; mmesophyll; sstoma
254 Roxana Khoshravesh and Tammy L. Sage

mutants, such as those with one or two chloroplasts per cell
(Fig. 1c), thereby aiding in transforming our understanding of
the molecular control of plastid division over the past 25 years
[10–14]. More recently the leaf cell suspension method has been
employed to study shifts in chloroplast numbers, size, and position-
ing in mesophyll and bundle-sheath cells during the evolution from
C
3
to C
4
photosynthesis in over 12 lineages [15,16] and identifi-
cation of candidate genes involved in the evolutionary transition of
chloroplast traits between those two cell types [16]. The latter two
studies [15,16] underscore the applicability of this technique for
use in a broad number of plant species.
Here, we describe the method for preparation of cell suspen-
sions from leaves for imaging of chloroplasts and other cellular
features that are important for photosynthetic physiology. We pro-
vide slight modifications to the original technique to enhance the
ease of cell separation and quality of cell preservation through the
use of a lower concentration of tissue fixative and a pectinase
treatment. The pectinase treatment enables leaf cell suspensions
to be made from a wide range of species that have tightly adherent
cells. Enhanced cell preservation provides a more accurate assess-
ment of planar chloroplast size and chloroplast distribution within
the cell, a trait important for understanding light perception, CO
2
diffusion, and refixation of photorespired CO
2
[15–17]. Chloro-
plasts in the isolated cells are easily viewed in the absence of staining
with regular bright-field microscopy (Fig. 1b) as well as by using
differential interference contrast imaging (DIC; Fig. 1a, c, e). Thus,
this technique is accessible to all researchers and amenable for use
with all plant species.
Although this quick and easy procedure can be used for rapid
screening of plastid traits in all plant species, the leaf cell suspen-
sions are also valuable for characterizing other cellular features
important for photosynthetic physiology. Plasmodesmata reside in
pit fields and C
4
species have been demonstrated to have a greater
pit field area at the mesophyll-bundle sheath interface than C
3
species [8]. Leaf cell suspensions can be utilized to image pit fields
using the fluorochrome aniline blue, which stains plasmodesmata
associated β-1,3 glucan (callose, Fig. 1d;[18]). This use of the leaf
cell suspensions provides either an alternative to time- and labor-
intensive techniques for immunolocalization of callose on cleared
leaf tissue [8] or an initial rapid screening for pit field/plasmodes-
mata phenotypes prior to the use of more time- and labor-intensive
techniques. As a second example, leaf suspensions can be employed
to image mitochondria and peroxisomes to test hypotheses addres-
sing the role(s) of these organelles in C
4
evolution; unfixed leaf
sections can be used for live-cell labeling with fixable fluorescent
probes that stain mitochondria and peroxisomes. These labeled
cells can subsequently be prepared for leaf cell suspensions. The
combined use of leaf cell suspensions and fixable fluorescent probes
Leaf Cell Suspensions 255

to detect mitochondria, peroxisomes, as well as other cellular fea-
tures is tractable for use with plants that are not easily transformed
with organelle-specific probes such as GFP or YFP. Finally, Pyke
and Leech [10] used potassium iodide (IKI), which stains starch, to
enhance chloroplast detection as illustrated in Fig. 1e. IKI staining
can also be used in combination with leaf cell suspensions when an
investigator wishes to follow the course of starch accumulation in
specific leaf cell types during a given time period. Once cells have
been imaged, cellular parameters can be quantified to test hypoth-
eses of interest as previously described [3,10–18].
2 Materials
2.1 Tissue Fixation 1. EM-grade glutaraldehyde aqueous solution, 25%.
2. EM-grade paraformaldehyde aqueous solution, 20%.
3. 0.1 M Sodium cacodylate buffer, titrate to the pH 6.9 with
2 M HCl.
4. Fixative: 0.5% Glutaraldehyde in 0.1 M sodium cacodylate
buffer, or 1% paraformaldehyde in sodium cacodylate buffer
(see Note 1)
5. Double-edged razor blades (see Note 2).
6. Dissecting microscope.
7. Borosilicate glass vials, 1 dram volume.
8. High precision fine tip tweezers.
9. Transfer pipettes, 1 mL.
2.2 Preparation
of Leaf Cell
Suspensions
1. NaOH pellets.
2. 0.2 M Disodium EDTA, pH 9.0: Add 37.22 g disodium EDTA
and bring to approximately 450 mL volume. Stir to produce a
milky solution. Add NaOH pellets slowly, while stirring, to
clear the solution. Bring to a total volume of 500 mL by adding
water, pH to 9.0.
3. Digestion buffer, pH 5.3: 0.15 M Sodium hydrogen phos-
phate, 0.04 M citric acid.
4. Pectinase from Aspergillus niger (polygalacturonase, 1 U/mg).
5. Water bath.
6. Sonicating bath.
7. Glass microscope slides, 25 75 1 mm.
8. Glass coverslips, No. 1., 22 50 mm.
9. Wooden applicator stick.
256 Roxana Khoshravesh and Tammy L. Sage

10. Light microscope adjusted for imaging with bright field
and/or differential interference contrast microscopy (DIC)
with digital image capture capability or confocal microscope.
11. Fluorescence microscope with appropriate filters for aniline
blue excitation (excitation filter 390 nm; dichroic mirror
420 nm; emission filter 460 nm).
2.3 Detection
of Callose, Cellular
Feature of Interest
with Fluorescent Probe
or Starch
1. 0.01% Aniline blue in 150 mM K
2
HPO
4
(see Note 3) for
callose detection.
2. Appropriate live-cell labeling fixable fluorescent probe for
imaging cell structure of interest (see Note 4).
3. 1% IKI solution in dH
2
O for starch detection.
3 Methods
3.1 Tissue Fixation 1. Prepare all materials required for fixation.
2. Place a drop of fixative on the dissecting scope-stage plate and
immerse the leaf tissue into the drop (Fig. 2a). Add more
fixative if required to cover the leaf tissue.
3. Use the single-edged razor to cut the tissue into 1–2 mm
2
pieces.
Use tweezers to hold the leaf sample (Fig. 2a;see Note 5).
4. Fill the vial with fixative.
5. Using tweezers or a 1 mL plastic transfer pipette with the tip
cut off, transfer the pieces of tissue into the vial. Overfill the vial
with fixative and quickly place the lid on the vial ensuring that
no air is introduced into the vial (Fig. 2b). Most tissue pieces
will immediately sink to the bottom of the vial (Fig. 2c;see
Note 6).
6. Leave fixed tissue at room temperature in the dark for 1 h.
Fig. 2 The process of tissue fixation. (a) A razor blade cutting immersed tissue into 1–2 mm wide strips to
enhance fixative penetration. Note the presence of tape on the razor to protect fingers. (b) Tissue pieces
floating on fixative in an overfilled vial. (c) Tissue pieces should immediately sink to the bottom of the vial
following rapid placement of the lid onto the overfilled vial. Samples sinking immediately after placement of lid
on the vial indicates that fixative is penetrating the tissue
Leaf Cell Suspensions 257

3.2 Live-Cell
Labeling with Fixable
Fluorescent Probe
1. Place a drop of fluorescent probe solution on the dissecting
scope-stage plate.
2. Place live tissue directly in the fluorescent probe solution and
cut the tissue into 1–2 mm
2
pieces with a razor as described in
Subheading 3.1,step 3.
3. Transfer the tissue into a vial filled with the fluorescent probe
solution. Overfill the vial with the probe solution and quickly
place the lid on the vial ensuring that no air is introduced into
the vial as described above.
4. Incubate in the dark at room temperature for time recom-
mended by probe supplier.
5. Remove the fluorescent probe solution from the vial with a
transfer pipette and replace with phosphate-buffered saline
(PBS) solution (1X PBS, 3 x 10 min).
6. Remove the PBS and fix in 1% paraformaldehyde in sodium
cacodylate buffer.
7. Proceed as described in Subheading 3.3.
3.3 Preparation
of Leaf Cell
Suspensions
1. Remove the fixative from the vial using a transfer pipette and
add 2 mL of 0.2 M disodium EDTA to the vial.
2. Heat the vials in a water bath for 2–3 h at 55 C on a floating
rack in a water bath. Either store at 4C(see Note 7)and
proceed as described in Subheading 3.4 or if cells do not separate
during squashing for imaging, proceed to the next step.
3. Remove EDTA from the vials and rinse the samples with diges-
tion buffer for 15–20 min at room temperature. Add enough
buffer to cover the tissue.
4. Incubate in 2% pectinase dissolved in digestion buffer for 1 h at
45 C in a water bath.
5. Rinse in digestion buffer (2 30 min) to remove pectinase at
room temperature.
6. Store samples at 4 C until ready for viewing (see Note 8).
7. If using the samples for aniline blue detection of callose,
remove the digestion buffer, rinse in tap water (2 30 min),
and replace with 1 mL of aniline blue solution, room tempera-
ture (see Note 9).
3.4 Microscopic
Observation
3.4.1 Imaging Unstained
Tissue
1. Use a pipette to place a drop of digestion buffer onto a glass
microscope slide.
2. Use tweezers to transfer a piece of digested leaf tissue from the
vial directly into the digestion buffer on the microscope slide.
3. Cover tissue with a coverslip and gently squash the tissue with a
wooden applicator stick until the cells are separated as visua-
lized under the dissecting scope (see Note 10).
258 Roxana Khoshravesh and Tammy L. Sage

4. View the cell suspension with bright-field microscopy or DIC.
3.4.2 Imaging Tissue
Stained with Fluorescent
Probe
1. Mount and squash digested leaf tissue on a microscope slide as
directed in Subheading 3.4.1.
2. View the cell suspension with a conventional fluorescence
microscope or confocal microscope as recommended by fluo-
rescent probe manufacturer.
3.4.3 Imaging Tissue
Stained with IKI
1. Mount digested leaf tissue on microscope slide as directed in
Subheading 3.4.1.
2. Use a pipette or dropper to place a drop of IKI directly on the
tissue.
3. Allow the stain to penetrate for 5 min.
4. Cover tissue with a coverslip and gently squash as directed in
Subheading 3.4.1.
5. View the cell suspension with bright-field microscopy or DIC.
4 Notes
1. Paraformaldehyde is recommended for confocal microscopy
and if fluorescent probes are being employed to detect cellular
features of interest.
2. Break double-edged razor blade in half before removing the
packaging paper and then clean with 70% EtOH to remove oil
and packaging residue. Cover broken edge of the razor blade
with tape to protect fingers (Fig. 2a).
3. To make aniline blue solution, add aniline blue to K
3
PO
4
,
cover with foil, and place in room temperature overnight.
The initially blue solution will turn a clear light yellow and is
ready to use. Store indefinitely at 4 C.
4. Choose a fluorescent probe that is retained after fixation. Zhu
et al. [19] describes the use of fluorescent probes for sensing
and imaging organelles.
5. Very small tissue size allows the digestion solutions to penetrate
into the cell walls and facilitates the process of digestion. If
fixing grass leaves, longitudinal thin sections parallel to the
veins increase the efficiency of the middle lamella digestion.
6. This method of tissue fixation eliminates the need for vacuum
infiltration of tissues that can lead to cellular artifact. If tissue
does not sink, shake the vial. Use only tissue samples that sink
Leaf Cell Suspensions 259

to the bottom. If no tissue sinks, open the lid, add more
fixative, dry the lid, and try again.
7. Tissue can be stored in EDTA for up to 6 months at 4 C.
8. Tissue can be preserved in the digestion buffer for 2–4 weeks
at 4 C.
9. Tissue can be preserved in the aniline blue for 4 months at 4 C.
10. If you have trouble separating the cells, then place the vial
containing the digested leaf tissue in a sonicating bath for
1–2 min to facilitate cell separation.
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