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Annals of Botany 84 : 459–466, 1999
Article No. anbo.1998.0944, available online at http:\\www.idealibrary.com on
Pathways for Nutrient Transport in the Pitchers of the Carnivorous Plant
Nepenthes alata
T. P A G E O W E N, Jr.*, K R I S T E N A. L E N N ON, M A T T H E W J. S A N T O and A M Y N. A N D E R S O N
Connecticut College, Department of Botany, 270 Mohegan AŠenue, New London, CT 06320–4196, USA
Received : 15 March 1999
Returned for revision : 25 March 1999
Accepted : 17 June 1999
Glands of the carnivorous pitcher plant Nepenthes alata are active in transport of materials into and out of the pitcher
lumen, indicating dual functions in both secretion and absorption. This study examined the potential for open
transport through these glands using the ultrastructural tracer lanthanum, which is restricted to the apoplast, and the
fluorescent symplastic tracer, 6(5)carboxyfluorescein. Glandular uptake of lanthanum from the pitcher fluid occurred
through the outer cell wall between irregularly spaced cutinized deposits, but was blocked from entering the
underlying mesophyll cell walls by thick endodermal-like regions. Similarly, lanthanum localization showed an open
apoplastic pathway from the petiole to the endodermal regions in the gland base. Thus, transport of materials into
or out of the gland must occur through the symplast. 6(5)Carboxyfluorescein showed that these glands transport
fluids directly from the pitcher fluid into vascular endings immediately beneath them via a symplastic route. When
applied to the petiolar vascular system, the fluorescent tracer freely entered immature pitchers, but was blocked from
entering the lumen of the mature pitcher by an endodermal zone. An ultrastructural survey showed infrequent pits
with plasmodesmatal connections to adjoining subepidermal cells. These results indicate that the function of the gland
is developmentally regulated. Prior to maturity, the primary function of the gland appears to be secretion. However,
at maturity, secretion is blocked by an endodermal layer, which limits the function of the gland to absorption. These
studies support the theory that the glands of Nepenthes alata are specialized for the bi-directional transport of
materials.
# 1999 Annals of Botany Company
Key words : Apoplastic transport, 6(5)carboxyfluorescein, carnivorous plants, digestive glands, endodermal layer,
Nepenthes alata Blanco, lanthanum, pitcher plants.
I N T R O D U C T I ON
The multicellular glands of carnivorous pitcher plants,
genus Nepenthes, are of considerable interest as model
systems for both plant development and membrane transport. Plants of this genus utilize a passive method of
attraction and entrapment to capture and digest their insect
quarry (for review see Slack, 1980 ; Juniper, Robins and
Joel, 1989). The lip of the pitcher, a ridged double edged
collar called the peristome, is characterized by the presence
of nectaries that attract insects to the pitcher opening
(Hooker, 1859 ; Lloyd, 1942 ; Owen and Lennon, 1999). A
lining of several layers of epicuticular wax (Lloyd, 1942) on
the upper region of the pitcher causes the insects to lose
their footing while foraging and to slip down the steep walls
of the pitcher into its base where they are trapped in a fluid.
At the base of the pitcher is a zone lined with multicellular
glands that have been shown to be uniform in morphology
(Adams and Smith, 1977 ; Owen and Lennon, 1999). These
glands are thought to be involved in the secretion of
digestive enzymes and in the absorption of insect-derived
nutrients (Fahn, 1979). In contrast, most plant glands have
the singular function of secretion (e.g. salt glands and many
nectaries ; Fahn, 1979). To our knowledge, no ultrastructural
observations of the glands of Nepenthes alata have been
* For correspondence.
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published. Of particular interest is the report of a cutinized,
or endodermal, cell wall layer in the basal region of the
gland (Juniper et al., 1989 ; Owen and Lennon, 1999). This
structure is common to glands in several carnivorous genera
(Heslop-Harrison, 1976) as well as to other secretory glands
such as salt glands (Lu$ ttge, 1971 ; Thomson, 1975). Like the
Casparian strip of roots, an endodermal, apoplastic barrier
at the base of the glands would require materials to flow
from the vasculature through the symplasm of the cells into
the secretory gland (Heslop-Harrison, 1976). Therefore, if
present, this barrier would limit apoplastic transport during
both secretion and absorption. The apoplastic pathways in
the digestive glands of Utricularia (Fineran and Gilbertson,
1980) and Brocchinia (Owen, Benzing and Thomson, 1988)
were traced at the ultrastructural level using the electrondense marker lanthanum. This marker was used to
investigate the presence and development of an endodermal
barrier in N. alata. In addition, the symplastic movement of
fluid through the gland was explored using the phloemspecific fluorescent tracer 6(5)carboxyfluorescein (CF ;
Grignon, Touraine and Durand, 1989).
MATERIALS AND METHODS
Pitcher plants (Nepenthes alata Blanco) were grown in a
greenhouse (22–28 mC) of Connecticut College in sphagnum
moss without fertilizer. Tissue for electron microscopy was
collected from mature pitchers, fixed in 1n25 or 2n5 % (v\v)
# 1999 Annals of Botany Company
460
Owen et al.—Transport Pathways in Nepenthes Pitchers
F. 1. Low magnification transmission electron micrograph showing a near median section of the digestive gland. Uptake of fluid from the pitcher
lumen occurs through cell layer one (Fig. 18). Note the continuous epidermis (E) under the gland (E*) and the proximity of the vascular tissues
(X). A segment of the small epidermal cell (R) that characteristically arches over the upper gland surface is shown. Bar l 10 µm. F. 2. Electron
micrograph of a region of the thick outer wall of two gland cells. A thin cuticle is present on the outer surface (arrow). The wall matrix has
Owen et al.—Transport Pathways in Nepenthes Pitchers
glutaraldehyde in 0n05  phosphate buffer (pH 7n0) for
4–5 h at room temperature, washed in buffer, and post-fixed
with 1 % (v\v) OsO in buffer overnight at 4 mC. The tissue
%
was washed in buffer, dehydrated through an acetone series
and embedded in epoxy resin (Spurr, 1969). Ultrathin
sections were cut on a Sorvall MT-2 ultramicrotome with a
glass or diamond knife, then stained with 2 % (w\v)
aqueous uranyl acetate and lead citrate (Reynolds, 1963).
Samples were viewed with a Zeiss EM-109 transmission
electron microscope.
The apoplastic pathway was examined using lanthanum
as an electron-dense tracer. To study the glandular pathway,
the lumen of an intact pitcher was rinsed in tap water and
the basal region filled with a 2 % (w\v) lanthanum nitrate
solution, pH 7n0 (Revel and Karnovsky, 1967). Alternatively, the lanthanum solution was layered within a 1-cm
diameter ring of grease applied to an excised pitcher surface
and placed in a humid chamber made from a watersaturated filter paper lining an inverted beaker. The vascularsupplied pathway was examined by placing the subtending
petiole (cut to 6-cm length underwater), with an entire
attached pitcher, in a vial of the tracer. Care was taken not
to let the pitcher interior come in direct contact with the
lanthanum solution. Pitchers were incubated for 4–24 h in
lanthanum or in tap water, then fixed and stained for
electron microscopy as above, except that the fixatives were
buffered to pH 7n2. Material was incubated in distilled water
as a control.
6(5) Carboxyfluorescein (CF ; Sigma) was dissolved in a
minimum amount of 0n3  KOH, then brought to a final
concentration of 1 m in distilled water after adjusting the
pH to 6n3 (Grignon et al., 1989). Existing fluid was removed
from unopened pitchers using a 1 cc insulin syringe inserted
into the base of the pitcher, then replaced with tracer
injected through the pitcher lid, or from open traps by
inverting the pitcher and replacing pitcher fluid with enough
tracer to fully cover the basal glandular region. Alternatively, tracer was fed into the expanded petiole of the
pitchers via the ‘ reverse-flap ’ technique (Salisbury and
Ross, 1992) in mature, open pitchers, or starting at the time
coinciding with the appearance of fluid within closed,
immature pitchers. A small section of the expanded petiole
was cut to expose vascular tissue, and the cut end of a 400 µl
centrifuge tube (USA\Scientific Plastics, Ocala, FL, USA)
was inverted and placed around the section. Tracer was then
inserted into the well of the tube. In all cases, the traps were
incubated with the tracer for 4–72 h. Each pitcher was cut
from its petiole and immediately rinsed several times with
distilled water to remove any unabsorbed tracer. CF was
detected in free-hand sections or whole tissue using an
461
Olympus BH-2 epifluorescence microscope equipped with
an ultraviolet cube, UG1 exciter filter and 420 nm barrier
filter. Distilled water was used as a control. As an additional
control, tracer was applied to the outer epidermis of the
pitcher and examined as above.
RESULTS
Gland ultrastructure
The cells of the digestive glands were arranged in discrete
layers, with a top layer of elongate, thick-walled cells
subtended by two to three layers of rounded to flattened
cells (Figs 1 and 18). The fourth cell layer appeared
continuous with the sunken epidermis of the pitcher lumen.
A thin cuticle layer was present on the surface of the outer
gland cells (Fig. 2). The cells of this layer were characterized
by greatly thickened exterior cell walls that, at the
ultrastructural level, were composed of electron-dense
material interspersed with small, lighter-staining regions
(Fig. 2). This staining pattern was observed in the outer
two-thirds of the wall. The inner portion of the cell wall was
uniformly light staining (Fig. 2).
In contrast, the exterior cell wall of the adjacent epidermal
cells contained a thick, uniformly staining cuticular material
(Fig. 3) that extended into the outer wall regions of all cell
layers of the glands (Figs 3 and 4). Additionally, the walls of
cell layers two, three and four (Fig. 18) were thickened and
uniformly cutinized (Fig. 5). The cuticle material penetrated
into the branching lateral walls from these main tangential
gland walls.
The walls of the gland cells lacked ingrowths or
protrusions. In addition to the thicker regions of the
cutinized walls, the only other distinctive wall shapes were
pit-fields with plasmodesmata which were often observed
between cell layers three and four (Figs 6 and 18) and less
frequently throughout the rest of the gland.
The base of the gland was subtended by two layers of
highly vacuolate cells adjacent to the ends of tracheary
elements (Fig. 7). They generally contained large osmiophilic
deposits. Based on cell shape, position and gland development, the cell layers immediately beneath the gland are
significant not only through their ontogeny (Owen and
Lennon, 1999), but because they form the structural and
functional interface between the gland and the vasculature.
All gland cells had prominent nuclei, rudimentary plastids,
and numerous mitochondria (Fig. 8). Endoplasmic
reticulum (ER) and Golgi were present and appeared evenly
distributed. However, these elements of the endomembrane
system were not present in notably large amounts. Cell
irregular, darkly staining deposits of cutin within a lighter-staining background. These deposits are lacking at the inner margins of the wall (*).
Tissue of mature pitcher was treated with lanthanum from the petiole. Bar l 1 µm. F. 3. Epidermis (E)–gland cell (GC) junction. The adjoining
walls are thickened and heavily cutinized (arrowheads) as compared to cellulosic walls (*). Lanthanum (La) is restricted by this endodermal
barrier. From mature pitcher incubated with lanthanum. Bar l 0.5 µm. F. 4. Higher magnification of the endodermal barrier at the epidermis
(E)—gland (GC) interface. Note the proximity of the plasma membrane to the completely cutinized walls (arrowheads) and the accumulation of
lanthanum deposits at the wall—endodermis junction (arrows). Bar l 0.5 µm. F. 5. Electron micrograph showing the intersection between cell
layers two and three of the gland (GC ; see Fig. 18) and the sunken epidermal (E*) and sub-epidermal (SE) cells in a region near the middle of
the gland. The cutinized walls (C) form an effective endodermal barrier to apoplastic transport. Lanthanum (arrows) applied from the pitcher.
Bar l 2 µm. F. 6. Pit-fields (arrows) with plasmodesmata at the epidermis (E)–gland cell (GC) junction. Lanthanum was applied to the pitcher
fluid ; note the lack of lanthanum tracer in the cell walls. N, Nucleus ; C, cutinized wall. Bar l 2 µm.
462
Owen et al.—Transport Pathways in Nepenthes Pitchers
F. 7. Electron micrograph showing a tracheary element (X) adjacent to two subepidermal cells (SE) below the gland. The secondary cell wall
thickenings of the tracheary element lack lanthanum deposits in this tissue. Lanthanum was applied to the pitcher fluid. Bar l 1 µm. F. 8. The
general ultrastructure of the cytoplasm of a cell in layer one. The plastids (P) lack elaborate granal membranes. Also present are several
mitochondria (M) and a dictyosome (D). Bar l 0.25 µm. F. 9. Electron micrograph of pitcher tissue incubated with lanthanum supplied
Owen et al.—Transport Pathways in Nepenthes Pitchers
layers one and two (refer to Fig. 18) contained numerous
small vacuoles frequently containing abundant osmiophilic
material.
Lanthanum tracer
A lanthanum solution was applied to the pitchers in two
ways to examine the open apoplastic transport routes
present in the tissue, from vasculature to the glands, and
from the pitcher fluid into the glands directly. In the TEM,
lanthanum appears as black, irregularly shaped deposits
(Thomson, Platt and Campbell, 1973), and is easy to
distinguish compared to the water-treated controls. Pitchers
in which the subtending petiole had been immersed in a
lanthanum solution had electron-dense deposits throughout
the cell walls of the pitcher mesophyll (not shown). In
addition, the xylem underlying the glands was labeled, with
deposits around the wall thickenings (Fig. 9). Lanthanum
also penetrated into the wall spaces of the adjacent sunken
epidermal cells, or basal cells, under the glands (Fig. 9)
including the unusual epidermal ridge that overhangs each
gland (Figs 10 and 18). However, lanthanum was not
present in the cell wall spaces of the glands at the basal
region of the large cutinized zone (shown in Fig. 5).
Similarly, the tracer was absent from the apoplastic space of
the gland cells adjacent to the epidermis (not shown).
When lanthanum was applied to the pitcher surface and
allowed to penetrate for up to 24 h, deposits localized within
the walls of the glands but were limited to the outer two cell
layers of the gland (Figs 3–5, 11–13). The tracer permeated
the wall of the outer cells but was restricted to the electrontransparent spaces interspersed among the apparent
cutinized occlusions within the outer wall or to the inner
uniform regions (Fig. 11). Also, lanthanum was deposited
throughout the inner cell walls of cell layer one (Figs 12 and
18). The tracer appeared to be restricted from entering the
walls of cell layer four (see Fig. 18) by the cutinized zones in
the gland-epidermis interface (Figs 3 and 4) and by the thick
epidermal cuticle (Fig. 3).
6(5) Carboxyfluorescein tracer
Bi-directional symplastic transport through the glands
and supporting pitcher tissues was examined by following
the movement of a fluorescent tracer. To monitor fluid
uptake, the pitcher fluid of closed, developing pitchers
(immature) or of open, mature pitchers was removed and
replaced with an equal volume of 6(5) carboxyfluorescein
tracer (CF). Alternately, tracer was fed into the mid-vein of
the expanded petiole of the trap (Salisbury and Ross, 1992).
Each set was incubated for 4–24 h before examination by
fluorescence microscopy.
463
The cutinized regions of the glands, and to a lesser extent
the underlying mesophyll and lignified vascular tissues,
autofluoresced weakly compared to the fluorescence of the
tracer (not shown). In immature and mature pitchers in
which CF replaced the pitcher fluid, the fluorescent tracer
was observed to move into the glands (Fig. 14) as well as
through the glands into the interconnected vascular bundles
directly underlying the glands (Figs 15 and 16). When
applied to vascular bundles in the petiole of immature
pitchers, CF permeated the glands and entered the pitcher
fluid (not shown). In mature, open pitchers, in contrast, CF
was transported from the petiole to the base of the glands
but did not appear to enter the glands or the pitcher fluid
(Fig. 17). In all cases, the tracer was confined to the vascular
bundles ; no tracer migrated to the surrounding pitcher
tissues. In control tissue in which CF had been applied to
the outer epidermal surface of the pitcher, there was no
evidence of absorption of the tracer.
DISCUSSION
This study examined the possible dual functions of the
digestive glands of N. alata in absorption and secretion,
using both ultrastructural and fluorescent tracers.
Reportedly, the glands secrete degradative enzymes, including ribonucleases and phosphatases (Matthews, 1960),
proteases (Amagase, 1972 a ; Jentsch, 1972) and possibly
chitinases (Amagase, 1972 b), as well as ions, including
chloride (Lu$ ttge, 1971) and calcium (Massa, 1998). However, the origin of these enzymes in pitcher plants has been
recently brought into question (Luciano et al., 1998 ; Santo,
Massa and Owen, 1998). Either simultaneously, or as a
result of these secretions, the glands absorb small byproducts from digested insects (Juniper et al., 1989). Thus,
the physiological activities of these glands share characteristics with specialized secretory glands such as salt glands
(Thomson, 1975) and with the absorptive trichomes of
bromeliads (Benzing et al., 1976).
Numerous mitochondria and rudimentary plastids were
noted in the gland cells. This indicates that energy dependent
transport processes may rely on mitochondrial respiration
rather than photosynthesis (Faraday and Thomson, 1986).
ER and Golgi were present, but were not strikingly abundant
nor present at levels sufficient to encourage the suggestion
that they were involved in the synthesis and\or secretion of
enzymes. However, it is possible these actions occur
primarily in immature pitchers or under special stimulation
in mature pitchers and thus the assumed correlation of
proliferation of ER and Golgi with these functions was not
observed in these studies.
The outer cell wall of the digestive glands in N. alata was
unusual in appearance, with a matrix of irregularly shaped
through the petiole. Lanthanum deposits (arrows) surround the tracheary element (X) wall thickenings and heavily infiltrate the subepidermal
(SE) cell walls. Bar l 2 µm. F. 10. Lanthanum (arrow) freely penetrated the open cell wall spaces from the petiole to the pitcher. Electron
micrograph showing the epidermal ridge (R) that is characteristic of these glands. Note the thick cuticle (*). Bar l 2 µm. F. 11. Electron
micrograph of the outer cell wall of cell layer one (see Fig. 18) incubated with lanthanum. The tracer was excluded from the electron-dense
interstices but penetrated the wall matrix especially in the region adjacent to the plasma membrane (arrow). Bar l 1 µm. F. 12. Electron
micrograph of the outer cell wall of a gland cell in layer one (see Fig. 18) from a region below the irregular matrix. The tracer is primarily located
adjacent to the membrane. Bar l 1 µm. F. 13. Electron micrograph of a region of cell layer two (see Fig. 18) showing the apoplastic continuity
from the pitcher fluid through the upper gland. Note apparent density of the cytoplasm of these cells. D l dictyosome. Bar l 0.5 µm.
464
Owen et al.—Transport Pathways in Nepenthes Pitchers
darkly stained regions interspersed with smaller, lighter
stained areas. The density of the darker wall regions was
similar to that of the intact cuticle of the epidermis,
suggesting the wall was filled with cutinized deposits. This is
consistent with a model for the genus proposed by Juniper
et al. (1989) but based on unpublished work. Interestingly,
glands from other carnivorous genera, including Utricularia
(Fineran, 1985) and Brocchinia reducta (Owen et al., 1988),
have similar striated cutin structures in the outer wall of the
gland cells that contact the digestive fluid. In B. reducta,
which relies on rain water to fill the leaf rosette that traps
insects, this wall compresses when desiccated, forming a
barrier that presumably limits evaporative water loss (Owen
and Thomson, 1991).
Movement of the lanthanum tracer delineates open
apoplastic spaces at the ultrastructural level, as it cannot
cross membranes or cutinized cell walls (Thomson et al.,
1973 ; Nagahashi, Thomson and Leonard, 1974 ; Fineran
and Gilbertson, 1980 ; Peterson, Swanson and Hull, 1986).
The movement of lanthanum from the pitcher lumen into
the outer walls of cell layer one (Fig. 18) and not into the
walls of the neighbouring epidermal cells demonstrates that
the outer walls of the gland are permeable and therefore
provide the only sites for the movement of solutes into or
out of the lower portion of the pitcher.
In almost all varieties of glands investigated in higher
plants, special wall incrustations are located at strategic
positions to separate the secretory cells from the apoplast of
the plant (Lu$ ttge, 1971). In most cases, glandular cells
surmount an endodermoid layer with one or more basal
cells underneath (Fahn, 1988 ; Juniper et al., 1989).
Restriction of lanthanum by cutin-impregnated walls at the
gland-epidermis junction and to the base of cell layer two
(Fig. 18) within the gland of Nepenthes indicated that the
wall is impermeable in these locations. This cutinized zone
must serve to limit unregulated apoplastic transport from
the gland into the underlying tissues, a function similar to
that of the endodermis in roots. In a complementary
experiment, lanthanum supplied to the pitcher via the
petiole penetrated the open apoplasm, including the vascular
system, up to the gland base, but was prevented from being
transported further into the gland by a cutinized layer.
Thus, transport through the gland must be symplastic.
When placed in the basal region of the pitchers, the
fluorescent tracer rapidly moved though the glands into the
vascular system. Grignon et al. (1989) demonstrated that
6(5)carboxyfluorescein is a reliable tracer of the phloem
symplast, and transportable over long distances without
leakage into the apoplast. Since the lanthanum experiments
F 14–17. Fluorescence micrographs of whole tissues and hand
sections showing the movement of the fluorescent symplastic tracer
6(5)carboxyfluorescein (CF) through the pitchers (Figs 14–16). F. 14.
CF moves exclusively through the glands (G) and into the vascular
bundles (arrows) below. There is a weak autofluorescence of chlorophyll
in the background. Tracer was applied to mature pitchers via the
pitcher fluid. Bar l 0.1 mm. F. 15. Hand section through a gland and
adjacent tissues. The tracer lines the epidermal cuticle (arrow),
penetrates the gland (G) and enters the vascular system (arrowheads)
below the gland. Tracer was applied to mature pitchers via the pitcher
fluid. Bar l 0.1 mm. F. 16. Vascular uptake of the tracer showing
interconnectedness of glands (arrowheads) and underlying vascular
strands which appear as a fluorescent network. Tracer was applied via
the ‘ reverse-flap ’ technique to mature pitchers. Bar l 0.25 mm. F.
17. Transport of the tracer is restricted by the endodermal layer at the
gland (G)—epidermis interface. Tracer applied to the petiolar vascular
system of a mature pitcher via the ‘ reverse-flap ’ technique was
restricted to the vascular tissue (V) at the base of the gland. Bar l
0.05 mm.
Owen et al.—Transport Pathways in Nepenthes Pitchers
465
F. 18. Model of the gland of Nepenthes alata, and of the observed transport pathways through it. The gland is made up of three cell layers.
Cell layer one is characterized by a thick outer wall, which is in contact with the pitcher fluid. The walls of cell layers two and three are impregnated
with what appears to be cutin, forming an endodermis-like barrier to apoplastic transport. The gland is subtended by a sunken epidermal layer,
which is continuous with the epidermis of the pitcher lumen, and a subepidermis ; cell layers four and five respectively. Vascular bundles are seen
in close proximity to the base of the gland. Secretion, which occurs only in immature pitchers, is apoplastic. Absorption from the pitcher fluid
is symplastic through cell layer one, then must occur apoplastically due to the presence of the endodermis (black walls). Each gland is
characterized by the presence of a modified epidermal cell, called the epidermal ridge, which overhangs the gland.
showed that the glands are apoplastically isolated, the CF
must have crossed the plasmalemma to enter the symplast
for plasmodesmatal transport. This indicates that the
movement of fluids, presumably containing insect components under normal circumstances, from the pitcher
lumen, through the gland, to the underlying vascular system
occurs symplastically.
Over the course of its development, the pitcher of
Nepenthes alata elongates, expands, and fills with several
millilitres of fluid before the flap-like lid opens (Owen and
Lennon, 1999). During this study, we examined the pathway
of solute movement into the developing pitcher using CF as
a tracer. This was done by applying CF to vascular tissue of
the expanded petiole via the ‘ reverse-flap ’ technique
(Salisbury and Ross, 1992). The fluorescent probe travelled
freely from a major petiole vein, through the immature
pitcher, and into the pitcher fluid. In contrast, CF applied to
the petiole of mature pitchers did not enter the gland or the
pitcher lumen. This suggests that the endodermal barrier
develops more closely to the time of pitcher opening. A
more precise study of the development of this endodermal
barrier is underway.
Clearly, the digestive glands of N. alata are specialized
glands that share many ultrastructural features with those
of other carnivorous genera and other secretory glands. The
alignment of the glands directly over the vascular system is
efficient for the conduction of solutes into or out of the
pitcher. Further, the endodermal layer within the gland
provides a mechanism to regulate the content of the
transported materials. The different pathways and apoplastic barriers are summarized in Fig. 18. These characteristics make the glands of N. alata an interesting model
system in which to further examine rates and specificity of
transport in plant cells.
A C K N O W L E D G E M E N TS
We thank Dr Kathy Platt for her helpful suggestions during
the preparation of this manuscript. This work was supported
in part by an NSF Instrument and Laboratory Improvement
grant (DUE-9552109) and the Keck Undergraduate Science
Program.
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