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Vesicular cells of the lodgepole pine dwarf
mistletoe (Arceuthobium americanum) fruit:
development, cytochemistry, and lipid analysis
Shannon K. Kelly, Cynthia M. Ross Friedman, and Ron G. Smith

Abstract: The lodgepole pine dwarf mistletoe, Arceuthobium americanum Nutt. ex Engelm., is a parasitic angiosperm that
infects conifers in western Canadian forests. A striking feature of the Arceuthobium life cycle is explosive seed dispersal,
triggered by hydrostatic build-up within the fruit, localized in a tissue called ‘‘viscin.’’ Viscin is composed of two cell
types: ‘‘viscin cells’’ that accrue pressure in their hydrated mucilage, and ‘‘vesicular cells’’ with a previously undetermined
function. The objective of this work was to investigate the development and composition of vesicular cells using microscopy, cytochemistry, and gas chromatography – mass spectroscopy. We found that vesicular cells are initiated from mesocarp cells lining the viscin cells. As development of the seed progresses, vesicular cells form a single layer, enlarge, and
become isodiametric. Later, the cells make up all of the remaining 3–4 outer mesocarp layers. Concomitant with enlargement, intracellular triacylglycerides (TAGs) largely composed of 16-carbon and 18-carbon fatty acids, accumulate. Immediately prior to discharge, vesicular cell boundaries become indistinct, and the cell contents become confluent, creating a
lipid mass between the viscin cells and the exocarp. Vesicular cells and their resulting lipid mass likely function in discharge by acting as a hydrophobic barrier to water loss and (or) as a repellant, resistant layer.
Key words: cytochemistry, dwarf mistletoe, gas chromatography – mass spectroscopy (GC-MS), triacylglycerides (TAGs),
viscin tissue.
Résumé : Le gui nain du pin lodgepole, Arceuthobium americanum, est une angiosperme parasite infectant les forêts de
l’Ouest canadien. On y observe une caractéristique bien particulière, soit la dispersion explosive des graines, déclenchée
par la formation d’une pression hydrostatique à l’intérieur du fruit, localisée dans un tissu ‘‘vicinal.’’ Ce tissu comporte
deux types de cellules: les ‘‘cellules vicinales’’ qui augmentent la pression dans leur mucilage hydraté et les "cellules vésiculaires", dont la fonction n’a encore jamais été définie. Les auteurs ont étudié le développement et la composition des
cellules vésiculaires à l’aide de la microscopie, de la cytochimie et de la chromatographie en phase gazeuse – spectroscopie de masse. Ils ont constaté que les cellules vésiculaires prennent naissance dans les cellules du mésocarpe bordant les
cellules vicinales. Au cours du développement de la graine, les cellules vésiculaires forment une seule couche, grossissent
et deviennent isodiamétriques. Plus tard, les cellules transforment toutes les cellules résiduelles des 3–4 couches du mésocarpe externe. De façon concomitante avec l’agrandissement, elles accumulent des triacylglycérides (TAGs) constitués surtout d’acides gras à 16 et 18 carbones. Immédiatement avant la décharge, le pourtour des cellules vésiculaires devient
indistinct, et les contenus des cellules deviennent confluents, créant une masse lipidique entre les cellules vicinales et
l’exocarpe. Les cellules vésiculaires et la masse lipidique qui en résulte interviennent vraisemblablement dans la décharge,
en agissant comme couche de résistance sous forme d’une barrière hydrophobe à la perte de l’eau ou comme répulsif.
Mots-clés : cytochimie, gui nain, chromatographie en phase gazeuse – spectrographie de masse (GC-MS), triacylglycérides
(TAGs), tissu vicinal.
[Traduit par la Rédaction]

Introduction
Mistletoes are flowering plants and aerial parasites belonging to the order Satantalales (Kuijt 1968; Cronquist
1981; Mathiasen et al. 2008). The mistletoe habit is believed
to have evolved five times independently in the Satantalales
(Der and Nickrent 2008; Mathiasen et al. 2008); while the
order is in a state of taxonomic revision, the classic mistleReceived 23 June 2009. Published on the NRC Research Press
Web site at botany.nrc.ca on 3 December 2009.
S.K. Kelly, C.M. Ross Friedman,1 and R.G. Smith.
Department of Biological Sciences, Thompson Rivers
University, P.O. Box 3010, Kamloops, BC V2C 5N3, Canada.
1Corresponding author (e-mail: cross@tru.ca).

Botany 87: 1177–1185 (2009)

toe families Loranthaceae and Viscaceae resolve as two of
the five mistletoe clades. Dwarf mistletoes (genus Arceuthobium, family Viscaceae) are obligate aerial parasites on
members of Pinaceae and Cupressaceae. These parasites
seriously compromise the health of the host trees, many of
which are important timber species, but also influence numerous ecological processes (Hawksworth and Wiens
1996). Arceuthobium americanum Nutt. ex Engelm. infects
jack pine (Pinus banksiana Lamb.) in western Canada and
lodgepole pine (Pinus contorta var. latifolia Engelm.) in
western North America (Godfree et al. 2002).
As Arceuthobium has economic and ecological importance, a good understanding of its life cycle, in which the
fruit plays a pivotal role, is required. In many Arceuthobium
species, including A. americanum, the fruit and seed take

doi:10.1139/B09-079

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two growing seasons to fully develop (Hawksworth and
Wiens 1996). The Arceuthobium fruit, with its single, integumentless seed, has several intriguing characteristics. Arguably though, the most noteworthy of these features is its
mode of seed dispersal: the fruit’s ategmic seed is explosively discharged from the mature fruit of essentially all
members of the genus aside from A. verticilliflorum Englm.
Ballistic discharge is a hallmark of this genus; birds typically disperse the seeds of other mistletoes.
Forcible seed discharge in Arceuthobium is thought to be
accomplished by way of a hydrostatic pressure that builds
up in a mucilaginous tissue called ‘‘viscin’’ (Hawksworth
and Wiens 1996). Ross Friedman and Sumner (2009) confirmed that the viscin tissue of A. americanum represents
the mesocarp, and is a complex tissue consisting of two cell
types: viscin cells and vesicular cells. Viscin cells, which
are elongated and secrete a hydrophilic mucilage, comprise
the bulk of this tissue in A. americanum. The pressure required for discharge accumulates within the extracellular
mucilage of the viscin cells, which are found attached to
the modified endocarp that envelops the ategmic seed following discharge. Viscin tissue, including both viscin cells
and vesicular cells, are found in other mistletoes (see Sallé
1983; Gedalovich and Kuijt 1987).
In A. americanum, mature vesicular cells are isodiametric,
containing a large hydrophobic component, and are found
external and adjacent to the viscin cells (Ross Friedman and
Sumner 2009). Relative to the cells of the exocarp, vesicular
cells seem enlarged and swollen. Vesicular cells remain with
the fruit exocarp following discharge, and their function is
obscure. Sallé (1983) described ‘‘vacuolated’’ cells in Viscum album L. (Viscaceae), which are found at the outer periphery of the viscin cell layer, and have the same general
appearance as the vesicular cells in A. americanum. Ross
Friedman and Sumner (2009) suggested that these vacuolated cells are synonymous with the vesicular cells described
for A. americanum. Gedalovich and Kuijt (1987), who
coined the term ‘‘vesicular cells’’, observed them in the Loranthaceous mistletoe Phthirusa pyrifolia (H.B.K.) Eichler.
However, vesicular cells in this species are slightly more
elongated than those in A. americanum and are interspersed
with the viscin cells rather than being located on the periphery of the viscin cell layer. Studies on vesicular cells are
limited. Ross Friedman and Sumner (2009) noted the point
in development when the vesicular cells became more prominent, but there have been no published investigations into
the origin and development of the vesicular cells in any mistletoe.
In addition, the vesicular cells in Viscum album L. (Sallé
1983) and P. pyrifolia (Gedalovich and Kuijt 1987) do not
show evidence of being hydrophobic like those of
A. americanum (Ross Friedman and Sumner 2009). As Arceuthobium species are dispersed explosively, perhaps the
hydrophobic nature of the vesicular cells in the dwarf mistletoes when compared to the bird dispersed mistletoes is
important. Furthermore, no previous study has determined
the identity of the hydrophobic material.
The objectives of this work are (i) to carefully describe
the origin and development of the vesicular cells of
A. americanum using modern techniques of microscopy;

Botany Vol. 87, 2009

and (ii) to employ cytochemical and biochemical techniques
to elucidate the types(s) of hydrophobic material that is
found in the vesicular cells. In turn, this information may
further our knowledge of the life cycle of A. americanum
and may offer an insight into the mechanism of seed dispersal in these important forest pathogens.

Materials and methods
Site description, collection, and initial sample
preparation
The research site, dominated by lodgepole pine trees
heavily infected with A. americanum, was located adjacent
to Stake Lake (50831’N, 120828’W), 30 km south of Kamloops, British Columbia. Pistillate aerial shoots of
A. americanum representing a range of developmental stages
were photographed at the site with a Nikon E995 3.34 mp
camera (Nikon, Tokyo, Japan) and collected twice weekly
over the growing season (1 May 2008 through 30 August
2008). All of the samples had fruit that were in their second
year of development. On average, about 2 shoots were collected each time, for a total of about 60 shoots obtained over
the entire collection period; each shoot contained at least 5
fruits, and thus 300 fruits were obtained. All of the samples
were taken from the host trees at about 1.5 m above the
ground, as close to the trunk or branch as possible.
At the site, most shoots were placed in a fixative consisting of 2% (w/v) paraformaldehyde + 2% (w/v) glutaraldehyde in 0.1 mol/L phosphate buffer (pH 6.8). The material
was kept at 4 8C overnight, then rinsed in the same buffer.
For transmission electron microscopy (TEM), 1 of the 2
fixed shoots collected per sample date was postfixed with
2% (w/v) osmium tetroxide in the same buffer for 4 h. All
fixed material was then dehydrated in an ethanol series and
embedded in Spurr’s resin (Spurr 1969). Some of the samples taken from 16 June 2008 and 16 August 2008 were not
fixed at the site but were instead stored at –80 8C.
Staining of sectioned fruit for brightfield light
microscopy
About 30 sections from each non-osmicated fruit (1 median; 29 nearly median  5 fruit for a total of 5 median
and 145 nearly median sections per date) 2 mm thick were
obtained with a Sorvall Porter-Blum JB-4 microtome (Sorvall, Newtown, Connecticut, USA) and affixed to gelatincoated glass slides (Jensen 1962). The slides were subjected
to the following brightfield light-level cytochemical staining
procedures. Stained sections were viewed with a Nikon
Eclipse E400 light microscope and photos taken using a Nikon E995 3.34 mp camera (Nikon, Tokyo, Japan).
Toluidine Blue O
For routine staining to highlight general morphology, as
well as to localize phenolics and pectic acids, at least 5 sections per date (including median sections for determining dimension) were stained with 0.05% (w/v) Toluidine Blue O
(C.I. 52040, Sigma) in a 0.05 mol/L benzoate buffer
(pH 4.4). Under these acidic conditions, phenolics such as
tannins stain blue-green, while pectins stain pink (Jensen
1962; O’Brien et al. 1964; Sumner 1988).
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Kelly et al.

Periodic acid – Schiff’s treatment
A periodic acid – Schiff’s (PAS) treatment was used to
localize insoluble carbohydrates, which stain pink under this
regime (O’Brien and McCully 1981). Slides containing at
least 5 sections per date were incubated in saturated 2,4 dinitrophenyl hydrazine in 15% (v/v) acetic acid for 30 min to
block endogenous aldehyde, immersed in 1% (w/v) periodic
acid for 30 min to oxidize vicinal 1,2 glycol groups to aldehydes, subjected to Schiff’s reagent for 45 min, and rinsed
with distilled water (distilled water was substituted for periodic acid in the controls).
Aniline blue black stain
Aniline blue black (ABB) stain was used to localize protein following the methods outlined by O’Brien and
McCully (1981). Slides containing at least 5 sections per
date were placed into 1% (w/v) ABB (C.I. 20470, Polysciences, Warrington, Pennsylvania, USA) in 7% (v/v) acetic acid
for 15 min at 65 8C. The slides with sections were rinsed in
7% (v/v) acetic acid then mounted in glycerol containing 5%
(v/v) acetic acid. A positive reaction results in a bright to
dark blue stain, whereas a negative reaction results in no
colour to a dull grey appearance. The ABB procedure was
also used to counter stain PAS-treated sections (at least five
per date).
Sudan Black B stain
Sudan Black B (SBB) stain was prepared using the
method outlined in Ross (2002) as modified from Bronner
(1975). The stain, 0.3% (w/v) of SBB (C.I. 26150, Sigma;
sigmaaldrich.com) in 70% (v/v) ethanol, was heated to
37 8C for 14 h, and filtered twice. Prior to staining, slides
were incubated in 70% (v/v) ethanol for 2 min, and then
placed into stain at 55 8C for 1 h, rinsed in 70% (v/v) ethanol for 1 min followed by 15 min in 80% (v/v) ethanol at
room temperature. When viewed, hydrophobic materials
such as lipids stain black (Bronner 1975; Kiernan 2007).
Luxol Fast Blue stain
A solution of 0.1% (w/v) Luxol Fast Blue (LFB) was prepared using 95% (v/v) ethanol (Kiernan 2007). The slides
were heated to 56 8C for 16 h, rinsed with 95% ethanol,
and then distilled water. The slides were placed in a 0.05%
(w/v) lithium carbonate solution for 30 s followed by 30 s in
a 70% (v/v) ethanol solution, rinsed with distilled water,
counter stained with a 0.1% (w/v) crystal violet solution,
and rinsed with 95% ethanol. The LFB treatment stains
phospholipids blue (Kiernan 2007).
Preparation of vesicular cell smears from unfixed
samples and subsequent staining with Nile Red for
fluorescence microscopy
Whole fruit stored at –80 8C (about five fruit per two
sampling dates) were brought to room temperature, placed
on gelatin-coated slides, and manipulated under a Kyowa
Optical SDZ-AL dissecting microscope (Kyowa Optical, Tokyo, Japan). The seed and viscin cell coating was squeezed
out and discarded. The remaining outer fruit shell was cut
open to reveal the vesicular cells layers, which were re-

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moved with a scalpel and smeared onto the slides and left
to air-dry.
A staining solution of Nile Red was made by adding 2–
10 mL of a 500 mg/mL stock solution (oxidized from Nile
Blue A, C.I. 51180, Sigma) to 1 mL of 75% (v/v) glycerol
(Greenspan et al. 1985). A drop of the stain was placed on
the slides, which were left to incubate for 5 min. Under
450–500 nm band excitation, neutral lipids, primarily triacyleglycerides (TAGs), appear yellow-gold in color (528 nm
band emission in acetone). Under 520–580 nm band excitation, phospholipids, amphipathic lipids, and strongly hydrophobic proteins appear red (570–620 nm emission). All
samples were viewed with a Nikon Eclipse E400 light microscope fitted with a Nikon episcopic fluorescence attachment. Brightfield light microscopy was also used to ensure
that the vesicular cells had been correctly identified.
Transmission electron microscopy
For TEM, about 5 ultrathin sections 50–90 nm in thickness (grey to gold interference colours) were obtained from
each osmicated fruit (5 sections  5 fruit per date) with a
Reichert-Jung Ultracut ultramicrotome (Reichert-Jung, Buffalo, New York, USA) and adhered to the shiny side of uncoated copper hex grids (200 mesh). The sections were
stained with saturated uranyl acetate in 50% methanol, and
counterstained with 0.1% aqueous lead citrate. All sections
were observed and photographed with a Hitachi Model H7000 TEM (Hitachi, Tokyo, Japan) at an operating voltage
of 75 kV.
Gas chromatograph-mass spectroscopy analysis of
vesicular cell contents
The seeds and viscin cell coating of about 10 previouslyfrozen A. americanum fruit collected 16 August 2008 were
removed and discarded, and the remaining outer fruit shells,
which contained the vesicular cell layers, were weighed and
transferred to a microfuge tube containing 1 mL of hexanes.
The fruit shells were macerated to separate the intracellular
lipids from the membrane bound lipids, and centrifuged for
45 s at 5500 r/min (7500g). The supernatant, which contained the vesicular cell extract, was removed and transferred to a new microfuge tube and dried. The residual
nonvesicular fruit tissue in the pellet was retained in the first
tube.
To saponify and methylate the fatty acid (FA) acyl
groups, 1 mL of Meth-Prep II1 (Alltech, Deerfield, Illinois,
USA) was added to both tubes. Then, 0.5 mL of hexanes
was added to each tube, followed by centrifugation for 30 s.
The hexane portion was removed, and the derivatized samples were analyzed using a Saturn 2200 GC-MS (Varian,
Palo Alto, California, USA). The samples were injected
(1 mL; split ratio 1/200) into a 30 m  0.25 mm internal diameter (VF-5MS) column with a starting temperature of
150 8C, increasing at 5 degrees/min (helium carrier). Fragments with a m/z ratio of 50 to 500 were monitored. For
comparison, standard fatty acid methyl esters (FAMEs), derived from FAs ranging from 14 to 18 carbons (GLC-80;
Supelco, Bellefonte, PA, USA), were run, and the National
Institute of Standards and Technology (NIST) mass spectral
search program (version 1.7) was searched (NIST 1999).
The GC-MS experiment was conducted in duplicate.
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Botany Vol. 87, 2009

Figs. 1–3. Development of Arceuthobium americanum fruit (arrowheads). Fig. 1. Observed near the beginning of the second growing season
(15 May 2008). Scale bar = 15 mm. Fig. 2. Observed 16 June 2008. Scale bar = 8 mm. Fig. 3. Observed near the end of the growing season
(22 August 2008). Scale bar = 6 mm.

Results
Fruit development and overview of vesicular cells in the
nearly-mature fruit
The fruit of A. americanum develops over the second season post-fertilization, and enlarges from May (Fig. 1)
through June (Fig. 2) into July, becoming recurved as the
end of the growing season approaches (Fig. 3). Mature vesicular cells examined about one month prior to explosive
discharge (early August) are isodiametric, have a diameter
of approximately 40 mm, and are large relative to the exocarp cells (Fig. 4). Vesicular cells are found in about 3–4
conspicuous cell layers in the upper three-quarters of the
fruit, immediately outside of the viscin cells. Vesicular cell
contents do not stain with Toluidine Blue O.
Origin, development,and cytochemistry of the vesicular
cells
The mesocarp is about 4–5 cell layers thick at the beginning of the second growing season (early- to mid-May). At
this time, the innermost uniseriate layer of mesocarp begins
to differentiate into the viscin cells (Figs. 5 and 6). The
outer 3–4 layers of mesocarp have prominent nuclei
(Fig. 6), and are somewhat flattened radially; these are the
‘‘vesicular initials’’ — cells destined to develop into the vesicular cells. Aside from the nuclei, which react positively to
the ABB stain for protein, the contents of vesicular initials
do not show a positive staining reaction when subjected to
a combined PAS (insoluble carbohydrate) – ABB (proteins)
procedure (Fig. 6), nor do they stain positively for the identification of hydrophobic materials when treated with SBB
(not shown).
Vesicular initials retain their appearance and staining reactivity until the end of May – early June: at this time, they
still do not stain positively when treated with either PAS

(Fig. 7) or ABB (Fig. 8), but the innermost layer of vesicular initials in the upper quarter of the fruit now reacted positively for hydrophobic materials when stained with SBB
(Fig. 9), indicating that differentiation into the enlarged vesicular cells is imminent. Within the course of a few days, a
wave of SBB-positive staining proceeds basipetally in the
innermost layer of vesicular initials, signaling that this inner
layer of vesicular cell initials in the upper three-quarters of
the fruit is poised to initiate differentiation. All remaining
outer mesocarp layers/vesicular initials rapidly become
SBBpositive as well.
By early- to mid- June, the inner layer of vesicular initials
in the upper quarter of the fruit — the first that displayed a
positive reaction to SBB — have become vesicular cells, as
they have enlarged to an average diameter of about 30 mm
(Fig. 10). The single layer of vesicular cells remained PAS
negative, but strongly SBB positive (Fig. 11). Aside from
their cellular boundaries, they were also negative for LFB,
which suggests that their contents are not composed of phospholipids (Fig. 12). When treated with Nile Red and viewed
with the excitation/emission filter pairing of 450–500/
528 nm, the vesicular cells in whole mount smears fluoresced yellow-gold, which is a positive identification of
TAGs (Fig. 13). Conversely, when treated with Nile Red
and viewed with the excitation/emission pairing for phospholipids, amphipathic lipids, and strongly hydrophobic proteins, no fluorescence was evident (not shown). A few viscin
cells and associated mucilage was evident when viewed
under brightfield LM (Fig. 14), where it is clear that only
the vesicular cells were fluorescing when stained with Nile
Red and observed with the filter pairing for TAGs (compare
Fig. 14 with Fig. 13). When viewed with TEM, vesicular
cells show a large osmiophilic deposit, as well as several
smaller peripheral osmiophilic bodies, and the cell wall is a
typical primary wall (Fig. 15).
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Figs. 4–9. Organization of vesicular cells and initiation of their development in Arceuthobium americanum; ed, endocarp (crushed); en,
endosperm; ex, exocarp; ey, embryo; m, mesocarp; ve, vesicular cells; vi, viscin cells. Fig. 4. Near-median longitudinal section through a
nearly mature fruit sampled 1 August 2008 and treated with Toluidine Blue O. Vesicular cells are found in about 3–4 conspicuous cell
layers in the upper three-quarters of the fruit, just outside of the viscin cell layer. Scale bar = 120 mm. Fig. 5. Near-median longitudinal
section of fruit sampled near the beginning of the second growing season (15 May 2008); whole fruit at this stage seen in Fig. 1. The
mesocarp is about 4–5 cell layers thick, and its innermost uniseriate layer is differentiating into viscin cells. Scale bar = 200 mm. Fig. 6.
Serial section to the region of differentiating viscin cells in the inset in Fig. 5 and subjected to periodic acid – Schiff’s (PAS) treatment
followed by aniline blue black (ABB) staining. The outer 3–4 layers of mesocarp have prominent nuclei (arrowheads); these mesocarp cells,
also referred to as ‘‘vesicular initials’’, will develop into vesicular cells. Scale bar = 30 mm. Figs. 7, 8, and 9. Serial sections of the developing mesocarp in the upper quarter of a fruit sampled early June (1 June 2008). The viscin cells are continuing to develop; the outer 3–4
layers of mesocarp/viscin initials have a negative reaction to PAS (Fig. 7) and ABB (Fig. 8), but are now positive (arrowheads) for Sudan
black B (Fig. 9). Scale bars in Figs. 7, 8, and 9 = 30 mm.

Throughout June and July, the vesicular cells in this single layer increased in size to their maximum diameter of
about 40 mm, but the results for all cytochemical staining reactions remained unchanged. The cell walls did not appear
to change in appearance or dye reactivity at either the light
or electron level of microscopy, staining pink with Toluidine

Blue O (not shown) and PAS (Fig. 10), blue with ABB (not
shown), and not staining with either SBB (Fig. 11) or LFB
(Fig. 12). In early August, the remaining outer 2–3 layers of
mesocarp (the remaining vesicular initials) rapidly enlarged,
following the same basipetal wave, so that all of the outer
3–4 cell layers of the mesocarp (the vesicular initials) in the
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Botany Vol. 87, 2009

Figs. 10–17. Maturation of vesicular cells and results of cytochemical staining; d, SBB-positive deposit; ex, exocarp; ve, vesicular cell; vi,
viscin cells. Figs. 10–12. Serial sections of the developing viscin cells and vesicular cells in the upper quarter of a fruit sampled in mid-June
(16 June 2008); whole fruit at this stage seen in Fig. 2. Vesicular cells are still only found in one cell layer, and are periodic acid – Schiff’s
negative (Fig. 10), strongly positive to Sudan black B (SBB) (Fig. 11), and negative to Luxol fast blue (Fig. 12) aside from a positive reaction (arrowheads) at the vesicular cell boundaries. Scale bars in Figs. 10–12 = 30 mm. Figs. 13 and 14. Whole mounts of vesicular cells at
the same stage of development as seen in Figs. 10–12. In Fig. 13, vesicular cells stained with Nile Red and observed under 450–500 nm
band excitation, 528 nm band emission fluoresce yellow-gold. Fig. 14 shows whole vesicular cells with their characteristic enlarged appearance when viewed with brightfield light microscopy. Viscin cell materials (arrow) and other debris (arrowhead), obvious under light microscopy (Fig. 13), do not fluoresce (Fig. 14). Scale bars in Figs. 13 and 14 = 30 mm. Fig. 15. A vesicular cell viewed with TEM possesses a
large osmiophilic deposit as well as several smaller peripheral osmiophilic bodies (arrowheads). The cell wall is a typical primary wall
(arrows). Scale bar = 10 mm. Fig. 16. Section through vesicular cells in the upper quarter of a fruit sampled 1 August 2008 and stained with
Toluidine Blue O. The remaining outer 2–3 layers of mesocarp (remaining vesicular initials) have now differentiated into vesicular cells.
Scale bar = 80 mm. Fig. 17. Section through the upper quarter of a fruit sampled 22 August 2008 and stained with SBB; whole fruit at this
stage seen in Fig. 3. The cell walls amongst adjacent vesicular cells become indistinct, leaving cell wall remnants (arrowheads) and a large
SBB-positive deposit between the viscin cells and the cells of the exocarp. Scale bar = 80 mm.

upper three-quarters of the fruit had differentiated into vesicular cells (Fig. 16). These cells were all filled with a highly
hydrophobic component.
By mid- to late August, immediately prior to explosive
discharge, an intriguing phenomenon occurred: the cell walls
amongst adjacent vesicular cells apparently broke down or
otherwise become indistinct, leaving a large SBB-positive
deposit between the viscin cells and the cells of the exocarp
(Fig. 17). Remnants of broken wall provide evidence for the
breakdown.
Gas chromatography-mass spectroscopy analysis of
vesicular cell contents
The residue from the vesicular cell hexanes extract had
the consistency of an oily liquid. The GC-MS analysis of
the derivatized vesicular residue yielded several peaks that
had retention times within the range of the FAME standards.
The largest peak eluted had a similar retention time and the
same mass spectra profile as hexadecanoic acid methyl ester, derived from hexadecanoic acid (16:0; see Table 1).
Other compounds present in the chromatograph had retention times and possessed mass spectra consistent with them
being derived from octadecanoic acid (18:0), octadecenoic
acid (18:1), and octadecadienoic acid (18:2) in the vesicular
cell extract. The GS-MS analysis of the nonvesicular fruit
tissue had more components, indicated by the multiple
peaks, most of which did not correspond to FAMEs (data
not shown).

Discussion
Origin, development, and fate of the vesicular cells
The vesicular cells of A. americanum begin developing
about 2 weeks after the onset of viscin-cell development.
Their development is characterized by cell enlargement,
which is primarily achieved by the prolific accumulation of
SBB positive, osmiophilic, intracellular material, as well as
an increase in the number of cell layers from one to three
or four. While many TEM sections were obtained, the large
lipid component of the vesicular cells obscured the ultrastructural characteristics.
Immediately prior to explosive discharge, the vesicular
cell contents become confluent, creating an SBB-positive
deposit between the viscin cells and the cells of the exocarp.

The fate of the vesicular cell walls, plasma membranes, and
other cellular contents has not been determined; as we could
not detect visible changes in the cell walls, the breakdown
event must have been rapid. The formation of this hydrophobic SBB-positive mass is highly reminiscent of the late
developmental stages of a typical amoeboid (syncytial)
anther wall tapetum. Over the course of pollen development,
the cells of the tapetum undergo apoptosis to form a large
syncytial (or coenocytic) ‘‘lipid body,’’ which is largely
composed of TAGs (Shukla et al. 1998; Murphy and Vance
1999).
Nature of the hydrophobic component of the vesicular
cells
Based on our results, and on the resemblance of vesicular
cell development to the development in an amoeboid tapetum, we believe the hydrophobic component of the vesicular
cells is largely composed of TAGs. Potential candidates for
hydrophobic components of plant cells include TAGs, terpenes (volatile oils), phospholipids, and suberins, although
typically only TAGs are found as large intracellular bodies
(Murphy 1993; Fahn 2000; Zweytick et al. 2000). Our cytochemical evidence suggests that the SBB positive hydrophobic material within vesicular cells is largely composed of
TAGs, as the Nile Red staining with the filter pairing for
neutral lipid (essentially TAG) identification (Greenspan et
al. 1985) showed a positive result, while the material did
not stain with LFB, a phospholipid stain (Kiernan 2007).
The cytochemical results are supported by our GC-MS
data, which indicate that FAs, making up the acyl groups of
TAGs, are a key component of the vesicular cell extract.
However, as isomers can exist for FAs, particularly regarding double-bond positioning in the unsaturated FAs, we cannot conclusively identify the FAs present in the vesicular
cell extract, although we suspect that we have recognized
likely candidates. The GC-MS results indicate that hexadecanoic acid (16:0) is apparently the predominant FA of the
vesicular extract, with octadecanoic acid (18:0), octadecadienoic acid (18:2), and octadecenoic acid (18:1) also being
present. These FAs are most likely incorporated into TAGs.
This is not surprising, as FAs composing TAGs in plants
and animals are most commonly 16, 18, and 20 carbons in
length (Harwood 1997). Furthermore, hexadecanoic acid
and octadecenoic acid are the most common FAs of plants:
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Kelly et al.

being the most common saturated unsaturated FAs, respectively (Cseke et al. 2006). It has also been reported that octadecadienoic acids are the most common polyunsaturated

1183

FA in plants, and octadecenoic acid is the FA with the highest molecular weight most common to plants (Harwood
1997).
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263, 150, 67

264, 222, 180, 135, 97, 55

255, 199, 143, 74

263, 150, 67

264, 222, 180, 55

255, 199, 143, 74
298

296

294

298
13.86
12 500

13.79

296
—
4 700

13.36

294
13.26
13.31
6 300

Note: The vesicular cells were separated from the seeds, extracted in hexane, dried, methylated with Meth-Prep II1 (Alltech), and repartitioned in hexane; the identity of the FAMEs as analyzed by GCMS was determined by comparing the retention times and mass spectra of the sample output with that of the standards and the NIST Library (NIST 1999).

Sample
227, 185, 143, 74
Sample
270
Standard
Standard
10.43

FAME
Hexadecanoic acid
methyl ester
Octadecadienoic acid
methyl ester
Octadecenoic acid
methyl ester
Octadecanoic acid
methyl ester

Total ion count
17 200

Sample
10.38

270

Parent ion (m/z)
Retention time (min)

Table 1. Fatty acid methyl esters (FAMEs) present in the vesicular extract of Arceuthobium americanum fruit as determined by GC-MS.

Other prominent fragment sizes (m/z)

Botany Vol. 87, 2009

Standard or NIST Library
227, 185, 171, 143, 74

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Plant TAGs are almost invariably liquid at room temperature and thus considered ‘‘oils’’ (Lersten et al. 2006). The
liquid consistency of the extruded vesicular cell extract
coupled with the presence of the unsaturated FAs in the vesicular cell TAGs suggests that the vesicular TAGs are oils.
The positive staining reaction of the vesicular cells with osmium tetroxide corroborates this suggestion, as osmium tetroxide reacts with unsaturated lipids (Lison 1960). We plan
future work on the cellular origin of the lipids, as the TEM
work here was not able to capture the dynamic aspects of
lipid formation during vesicular cell development.
Relevance of vesicular cell structure and development: a
discussion of function
In Arceuthobium, the seeds are explosively discharged,
and there is no evidence of endozoochory (Hawksworth and
Wiens 1996). Furthermore, while vesicular cells have been
observed in the bird dispersed Viscum album (Sallé 1983)
and Phthirusa pyrifolia (Gedalovich and Kuijt 1987), these
vesicular cells do not have a hydrophobic component; in
fact, Gedalovich and Kuijt (1987) stated that the vesicular
cells may also serve as a specialized storage area for (water)
soluble nutrients for the disseminator. Therefore, even if the
vesicular cells of A. americanum shared an evolutionary history with those of other mistletoes, the function in bird dispersal has long been lost. While the formation of a
contiguous vesicular cell lipid mass follows a similar pattern
of development to that of an amoeboid tapetum, which
serves to provide nutrition to developing pollen grains, such
a deposit in the explosive fruit of A. americanum likely has
function in roles other than as an energy source.
The vesicular cells, then, must have a key role in explosive discharge. As the hydrophobic vesicular cells essentially line the hydrophilic viscin cells, the developing
vesicular cells could act as a seal to ensure that water accumulating in the viscin mucilage does not leak out, thus allowing for an increase in hydrostatic pressure. The increase
in vesicular cell size and number of layers throughout development might be required to counter the force building in
the region of the viscin cells. The final breakdown of the vesicular cell boundaries leading to the formation of a large
TAG deposit, which occurs as discharge becomes imminent,
may be needed to provide resistance to the impending explosion, and the contiguous TAG deposit, being oily, may
contribute a hydraulic component to the building pressure.
Furthermore, the degenerate vesicular cell layers and TAG
deposit remains with the exocarp following discharge, which
suggests that the degenerate layers and deposit might have
functioned as an abscission layer or perhaps even as a hydrophobic repulsion with the hydrophilic viscin cell layer.
Such repulsion would be based upon the polar energy of cohesion among water molecules (viscin cell mucilage) and
hydrophobic attraction (TAG deposit) due to the hydrophobic effect (Van Oss 2006), and while not necessarily contributing to the explosive force within the fruit, would enable a
particularly clean severance.
We plan to investigate the fruit and viscin tissue of two
nonconformist mistletoes: Korthalsella sp. (Viscaceae),
which is said to have mildly explosive fruit, and
A. verticilliflorum, which is the only Arceuthobium species
that does not explosively discharge its seeds (Hawksworth
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Kelly et al.

and Wiens 1996). We expect to find vesicular cells in both
mistletoes, but we expect those of Korthalsella to have features similar to those of A. americanum. Conversely, we suspect A. verticilliflorum vesicular cells may be divergent
from those of A. americanum. Future work will also include
further study of the viscin cells, the other component of the
viscin tissue in all mistletoes. The uniqueness of the development, location, and oily content of the vesicular cells considered as a whole suggests that these cells are required for
the process of explosive seed discharge in A. americanum:
vesicular cells and their resulting lipid mass may act as a
hydrophobic barrier to water loss and (or) as a repellant, resistant layer.

Acknowledgements
The research was funded by a Natural Sciences and Engineering Research Council of Canada Discovery Grant provided to C.M.R.F. The authors thank Trent Hammer (Dept.
of Physical Sciences, Chemistry) at Thompson Rivers University (TRU) for his assistance and excellent advice with
GC-MS, as well as Carolynne Fardy (Dept. of Biological
Sciences at TRU) for her support with laboratory work. The
authors also thank Kelly Anne Ross (Pacific Agri-Food Research Centre) and Jonathan Van Hamme (Dept. of Biological Sciences at TRU) for helpful comments on the
manuscript. The authors also greatly appreciate the advice
on mistletoe taxonomy provided by Bruce Ford (Dept. of
Biological Sciences, University of Manitoba) and Daniel
Nickrent (Dept. of Plant Biology, Southern Illinois University – Carbondale).

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