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Plant Cell Physiol. 45(2): 221–224 (2004)
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Short Communication
A Simple and Efficient Method for Isolating Trichomes for Downstream
Analyses
Xiaoguo Zhang and David G. Oppenheimer 1
Department of Botany and the University of Florida Genetics Institute, University of Florida, P.O. Box 118526, Gainesville, FL 32611-8526,
U.S.A.
;
and full-length cDNAs are available for a large percentage of
the genes (Seki et al. 2002a, Seki et al. 2002b).
Although a wealth of information has been obtained from
both genetic and molecular analyses of trichome development,
the biochemical analysis of Arabidopsis trichomes has lagged
behind. This is, in part, because trichomes make up only a
small fraction of the total number of cells of a leaf. Therefore,
if entire leaves are analyzed, trichome-specific biochemical
properties are overwhelmed by the contribution of the other
cells of the leaf. In addition, immunolocalization of trichome
proteins is impeded by the thick, secondary cell walls possessed by trichome cells. Current procedures for immunolocalization require a permeablization step to allow antibody access
to the intracellular milieu. Permeablization of mature trichomes using cell wall-degrading enzymes usually results in
loss of cell structure, and permeablization of mature trichomes
by freeze shattering usually results in broken trichomes. In
addition, measurement of the DNA content of trichomes is hindered by their attachment to the leaf. Staining of an intact Arabidopsis leaf with DAPI can result in unwanted fluorescence
contribution from the nuclei (and potentially cell walls) of the
non-trichome cells.
In this report, we describe a simple and efficient method
to isolate intact trichomes from Arabidopsis leaves that overcomes these limitations to trichome analysis. Our method is
based on the removal of Ca2+, which ionically cross-links the
pectins in the middle lamella that helps hold the trichome cells
to the leaf epidermis. The middle lamella lies between the primary walls of adjacent plant cells and originates from the cell
plate that forms during cytokinesis. The middle lamella is
important in abscission zones, and is responsible for holding
cells together. The major component of the middle lamella is
the anionic polysaccharide pectin.
In dicots, the primary pectin is rhamnogalacturonan I. The
backbone of rhamnogalacturonan I consists of long stretches of
α1,2-linked polygalacturonic acid alternating with α1,2-linked
rhamnose. Attached to the rhamnose are side chains of mostly
arabinose and galactose. The unsubstituted polygalacturonic
acid regions of the backbones of different pectin polymers are
held together by ionic bridges formed by divalent Ca2+. During
some cases of abscission, the ionic groups of the homogalac-
Arabidopsis trichomes are an excellent cell type to
address many questions in plant biology including the control of cell shape, endoreplication, and cell expansion.
Because trichomes comprise such a small percentage of the
cells of a leaf, biochemical analyses of trichomes are limited. To overcome this limitation, we developed a method
for removing trichomes from the leaf surface. Our method
allows the isolation of intact trichomes for use in downstream applications such as cell wall analysis, immunolocalization of trichome proteins, analysis of DNA content, and
proteomics. Also, this method will facilitate the isolation of
trichomes from practically any plant species.
Keywords: Arabidopsis — Cytoskeleton — Endoreplication
— Immunolocalization — Trichome.
Abbreviations: DAPI, 4′-6-diamidino-2-phenylindole; EGTA,
ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; FITC,
fluorescine isothiocyanate; PBST, phosphate-buffered saline with
Triton X-100; PIPES, 1,4-piperazine diethane sulfonic acid; SEM,
scanning electron microscopy.
Trichomes are useful as a model system to study many
processes central to plant development including acquisition
of cell fate, cell expansion, endoreplication, and cell polarity
(Hülskamp 2000, Qiu et al. 2002, Schellmann et al. 2002,
Schnittger and Hülskamp 2002, Schwab et al. 2000,
Szymanski et al. 2000, Walker et al. 2000, Wasteneys 2000).
Arabidopsis trichomes afford many advantages for their use in
developmental studies. First, trichomes are relatively large, single cells (up to 500 µm long) and can be easily observed using
a dissecting microscope. Trichomes differentiate from the plant
epidermis so invasive procedures are not necessary to observe
their development. Due to the ease of genetic analysis in Arabidopsis, there is a wealth of genetic data on trichome development; mutations affecting each stage of trichome development
have been isolated (Hülskamp et al. 1994, Larkin et al. 1994,
Luo and Oppenheimer 1999, Oppenheimer 1998, Schellmann
et al. 2002). Finally, the Arabidopsis genome has been completely sequenced (The Arabidopsis Genome Initiative 2000),
1
Corresponding author: E-mail, [email protected]; Fax, +1-352-392-3993.
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A method for isolating trichomes
Fig. 1 Isolation of trichomes from Arabidopsis leaves. (A) Light micrograph of a sample of isolated wild-type Arabidopsis trichomes stained
with 0.05% Toluidine Blue. (B) Scanning electron micrographs of Arabidopsis leaves before and (C) after trichome removal. Fully expanded
leaves were fixed and dehydrated in 100% methanol, critical point dried, coated with gold/palladium and viewed at 10 kV. Scale bars are: (B)
1.5 mm, and (C) 1.2 mm.
turonan regions are methylated. The methylation decreases the
negative charges to which the Ca2+ binds, thus decreasing the
number of ionic bridges. This, in turn, decreases the ability of
the adjacent cells in the abscission zone to hold together. We
exploited this property of pectins to dissociate upon Ca2+
removal from the middle lamella to develop a method to
remove trichomes from leaves of Arabidopsis plants.
Generally, trichome removal was carried out as follows.
Leaves from plants grown in soil or on plates were collected,
fixed and treated with EGTA. Individual leaves or leaf pieces
were placed in a Petri dish. Trichomes were removed by gentle
rubbing using a small paintbrush. The denuded leaves were discarded and the trichomes were transferred by pipette to a
microcentrifuge tube. We found that it was important to keep a
wetting agent (such as 0.05% Triton X-100) present at all times
to prevent the trichomes from floating and sticking to the sides
of the microcentrifuge tube. Low speed centrifugation was used
to collect the trichomes and they were washed several times to
remove the EGTA solution. Once washed, the trichomes were
ready for further analyses. Fig. 1A shows a Toluidine Bluestained sample of isolated Arabidopsis trichomes demonstrating that the trichome preparation was free of other cell types,
and that the trichomes retained their shapes after removal from
the leaves.
To determine the efficiency of trichome removal, we
examined leaves by SEM before (Fig. 1B) and after (Fig. 1C)
trichomes were removed. We found that we routinely removed
more than 90% of the trichomes on the leaves. This result
establishes that not only is this procedure an efficient means to
remove and collect Arabidopsis trichomes, but also that the col-
lected trichomes are a representative sample of the trichomes
present on the leaves.
One of our motivations for developing this procedure was
to use the trichomes for immunolocalization studies. Microscopic observations of trichomes in our lab indicated that the
base of the trichome has a thinner cell wall than the stalk and
branches. We reasoned that it would be possible to use mild
enzymatic digestion to create an entry point for the antibodies
in the trichome base without destroying the trichome structure.
To test this, we used anti-actin antibodies to label the actin
cytoskeleton in mature trichomes following a mild pectinase
treatment. Immunolabeling of the actin cytoskeleton of the trichome was strong and uniform throughout the volume of the
trichome cell (Fig. 2A).
We also examined whether the isolated trichomes would
be suitable for nuclear DNA content measurements by staining
isolated mature trichomes with DAPI. We found that staining
of mature trichome nuclei was strong and consistent (Fig. 2B).
Because the trichomes are stained and viewed after their
removal from the leaves, background fluorescence from other
leaf cell nuclei or cell walls is eliminated.
Here we describe a method for easily isolating mature trichome cells from the epidermis of Arabidopsis plants. This
method is straightforward and requires no specialized skills or
equipment. Furthermore, in principle, this method should be
applicable to any plant species that has large, single-celled trichomes. There are numerous applications for which a pure
population of a particular cell type is advantageous including
biochemical, microarray, and proteomic analyses. This procedure opens the door for comparative studies of trichomes
between wild-type Arabidopsis and the myriad trichome
A method for isolating trichomes
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Fig. 2 Immunolocalization and DAPI staining of isolated Arabidopsis trichomes. (A) Immunolocalization of the actin cytoskeleton in an isolated
trichome. Scale bar is 200 µm. (B) Isolated trichome stained with DAPI. Trichome nucleus is visible as the bright spot in the center of the image.
(C) DIC image of the same trichome as shown in (B).
mutants that are available. In addition, this method will facilitate comparative studies of trichomes between species.
Wild-type Arabidopsis plants (Columbia and RLD ecotypes) plants were grown in soilless potting mixture (Metro
Mix 360) supplemented with PGP (Pollock and Oppenheimer
1999) under continuous fluorescent illumination at 25°C. For
immunolabeling, plants were grown under aseptic conditions
on MS medium solidified with 0.8% (w/v) agar. Plates were
incubated in a growth chamber under a 16 h : 8 h light : dark
cycle at 22°C.
Trichomes were removed from leaves as follows. Fully expanded leaves with mature trichomes were fixed in 1.5% (v/v)
formaldehyde, 0.5% (v/v) glutaraldehyde in PEMT (25 mM
PIPES, 2.5 mM EGTA, 0.5 mM MgSO4, 0.05% [v/v] Triton X100, pH 7.2) or PBST (phosphate-buffered saline containing
0.05% [v/v] Triton X-100, pH 7.2) for 40 min to 3 h at room
temperature. Alternative fixatives such as FAE (3.7% [v/v],
50% [v/v] ethanol, 5% [v/v] acetic acid) or 50% (v/v) ethanol,
5% (v/v) acetic acid also have been used successfully. Fixation
may be omitted, if needed, depending on particular downstream applications. Following fixation, leaves were washed
three times in PEMT (or PBST) for 10 min to remove fixative.
Leaves were transferred to 20 ml glass scintillation vials containing approximately 10 ml of PEMT supplemented with
50 mM EGTA, vacuum infiltrated for 10–15 min, and incubated 16–24 h at 4°C. Concentrations of EGTA as high as
250 mM and times as short as 1 h (at 50°C) have been used
successfully. Leaves were transferred to Petri dishes and
trichomes were removed by gentle rubbing using an artist’s
paintbrush (4 mm wide, flat nylon bristles). Trichomes were
transferred by pipette (with the aid of a dissecting microscope)
to 2 ml microcentrifuge tubes. Trichomes were washed three
times with PBST by gently centrifuging (1,600–5,000×g for
4 min) and removal of the supernatant taking care not to disturb
the loose trichome pellet. Trichomes were gently resuspended
in the desired volume of PBST. Alternatively, trichomes were
transferred to 70 µm nylon cell strainers (Falcon, 2350) and
further manipulations were carried out by placing the cell
strainers in Petri dishes containing the desired solutions.
To facilitate viewing for trichome branch length, etc.
measurements, trichomes were stained with 0.05% (w/v) Toluidine Blue supplemented with 0.05% (v/v) Triton X-100 (as a
wetting agent) for 10–60 min. and rinsed once or twice in
PBST. To visualize the trichome nucleus, isolated trichomes
were stained with 0.2 µg ml–1 DAPI in PBST for 1–3 h followed by destaining in PBST without DAPI for 0.5–1 h.
Immunolocalization of the trichome cytoskeleton was performed essentially as described by Sugimoto et al. (2000).
Monoclonal anti-actin antibody clone C4 (ICN Biochemicals,
Inc.) was used at a 1 : 400 dilution to localize the actin
cytoskeleton. FITC-conjugated goat anti-mouse IgG secondary
antibody (Sigma) was used at a 1 : 100 dilution.
For SEM analysis, pubescent or denuded leaves were
transferred to anhydrous 100% methanol for at least 1 h. The
methanol was changed twice with at least 30 min between
changes. Samples were critical point dried, coated with gold/
palladium, and viewed at 10 kV.
Acknowledgments
The authors gratefully acknowledge the assistance of Jolanta
Nunley (University of Alabama) with the SEMs, and Guy and Kim
Caldwell (University of Alabama) for the use of their epifluorescent/
DIC microscope. The authors also thank Josh Vacik for his initial work
on the development of this procedure. The authors thank Paris Grey for
critical reading of the manuscript. This work was supported by NSF
(Award # 0352916).
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A method for isolating trichomes
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(Received October 12, 2003; Accepted November 17, 2003)