Download Functional Utrastructure of Genlisea (Lentibulariaceae) Digestive

Annals of Botany 100: 195–203, 2007
doi:10.1093/aob/mcm109, available online at www.aob.oxfordjournals.org
Functional Utrastructure of Genlisea (Lentibulariaceae) Digestive Hairs
B AR TO S Z JA N P ŁACHN O 1 , * , M A ŁG O R Z ATA KOZ IE R A D Z K A - K I S Z K U R N O 2
and P IOT R ŚW I A˛T E K 3
1
Department of Plant Cytology and Embryology, Jagiellonian University, 52 Grodzka st., 31-044 Cracow, Poland,
Department of Genetics and Cytology, University of Gdańsk, Kładki 24 st., 80-822 Gdańsk, Poland and 3Department of
Animal Histology and Embryology, University of Silesia, 9 Bankowa st., 40-007 Katowice, Poland
2
Received: 19 March 2007 Returned for revision: 18 April 2007 Accepted: 23 April 2007 Published electronically: 4 June 2007
† Background and Aims Digestive structures of carnivorous plants produce external digestive enzymes, and play the
main role in absorption. In Lentibulariaceae, the ultrastructure of digestive hairs has been examined in some detail in
Pinguicula and Utricularia, but the sessile digestive hairs of Genlisea have received very little attention so far. The
aim of this study was to fill this gap by expanding their morphological, anatomical and histochemical
characterization.
† Methods Several imaging techniques were used, including light, confocal and electron microscopy, to reveal the
structure and function of the secretory hairs of Genlisea traps. This report demonstrates the application of cryo-SEM
for fast imaging of whole, physically fixed plant secretory structures.
† Key Results and Conclusion The concentration of digestive hairs along vascular bundles in subgenus Genlisea is a
primitive feature, indicating its basal position within the genus. Digestive hairs of Genlisea consist of three compartments with different ultrastructure and function. In subgenus Tayloria the terminal hair cells are transfer cells, but
not in species of subgenus Genlisea. A digestive pool of viscous fluid occurs in Genlisea traps. In spite of their
similar architecture, the digestive-absorptive hairs of Lentibulariaceae feature differences in morphology and
ultrastructure.
Key words: Genlisea, Lentibulariaceae, carnivorous syndrome, carnivorous plant, digestive hairs, ultrastructure, cryoscanning electron microscopy, morphology, cuticle, secretory glands, functional anatomy, digestive glands.
IN TROD UCT IO N
The Lentibulariaceae form the largest family of carnivorous
plants, with three genera (Pinguicula, Utricularia,
Genlisea) and about 300 species (Mabberley, 2000).
According to molecular studies, the genus Genlisea is
sister to Utricularia, and this pair is sister to the genus
Pinguicula (Jobson and Albert, 2002; Jobson et al., 2003;
Müller et al., 2004). Both Pinguicula and Utricularia
develop active traps, but both the physiology and functioning of the traps differ much between these genera.
Pinguicula are active ‘flypapers’ with slightly modified
leaves for carnivory, but Utricularia form suction bladders
(reviewed by Lloyd, 1942; Juniper et al., 1989; Legendre,
2000). In Genlisea a third special kind of trap has
evolved – eel (lobster-pot) traps (Lloyd, 1942;
Heslop-Harrison, 1975).
Genlisea species are small, rootless wetland plants which
produce corkscrew-shaped subsoil traps of foliar origin for
catching small soil organisms (Heslop-Harrison, 1975;
Juniper et al., 1989; Reut, 1993). Barthlott et al. (1998)
stated that Genlisea is highly specialized to trap protozoa;
however, later it was experimentally shown that the prey
caught depended on the kind of organisms available, and
that the plants trapped both protozoa and metazoa
(Płachno et al., 2005a; Studnička, 2003b). The basic structure of Genlisea traps is well known; each Genlisea trap
consists of a stalk, a vesicle and a tubular channel (neck)
*For correspondence. E-mail [email protected]
which divides into two helically twisted arms (Fig. 1A).
The digestive hairs have the same simple architecture consisting of three functional compartments: basal cell, middle
cell, and a large head formed of four to eight terminal
secretory cells (Goebel, 1891; Lloyd, 1942; Reut, 1993;
Płachno, 2006). The details of its trap physiology and ultrastructure are still poorly recognized. So far only general
observations of hair structure have been reported (Lloyd,
1942; Juniper et al., 1989; Reut, 1993; Müller et al.,
2002). Juniper et al. (1989) found that these hairs resemble
the sessile hairs of Byblis and Pinguicula, and suggested
that their function and mechanism were also similar. For
a proper understanding of the functions of the trap, detailed
knowledge of the ultrastructure of these hairs is required.
On that basis, comparison of digestive hair ultrastructure
in Pinguicula, Utricularia and Genlisea, revealing structural and topographical differences and similarities, can
shed light on the evolution of the carnivorous syndrome
in Lentibulariaceae.
It is well known that chemical fixation of biological
material often fails to rapidly stabilize all cell compartments, and may produce many artefacts (Mersey and
McCully, 1978). To avoid these artefacts, several techniques have been employed to investigate nearly intact
plant and animal tissues: various fluorophores, cryo-fixation, UV microscopy and environmental SEM (Lichtscheidl
et al., 1990; Craig and Beaton, 1996; Pauluzzi et al.,
1996; Hepler and Gunning, 1998; Walther and Müller,
1999). Recently, some of them have been used to study
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196
Płachno et al. — Functional Ultrastructure of Genlisea Digestive Hairs
F I G . 1. (A) Traps of very young hybrid Genlisea lobata Genlisea violacea plant, the trap arms are very short (b, vesicle; n, neck; a, arm); (B) vesicle of
Genlisea violacea; (C) vesicle of Genlisea sp. ‘Itacambira Beauty’; (D) vesicle of Genlisea margaretae, hairs concentrate along the vascular bundle.
plant secretory structures (Turner et al., 2000, Kolb and
Müller, 2004; Adlassnig et al., 2005; Sakamoto et al.,
2006). In this paper the use of several imaging techniques,
including light, confocal and electron microscopy, are
described to distinguish the functional structure of the
secretory hairs of Genlisea traps. The application of
cryo-SEM for fast imaging of intact plant secretory structures without chemical fixation is highlighted.
M AT E R I A L S A N D M E T H O D S
Plants of subgenus Genlisea (G. hispidula, G. aurea,
G. pygmaea, G. repens, G. margaretae) and subgenus
Tayloria (G. lobata, G. violacea f. Giant, G. uncinata, a
species not yet described Genlisea sp. ‘Itacambira
Beauty’, hybrid G. lobata G. violacea f. Giant) were cultivated in the Department of Plant Cytology and
Embryology, Jagiellonian University in Cracow. They
were grown under a 16-h photoperiod in pots containing a
mixture of wet peat and sand. The peat contained naturally
occurring small organisms.
Cryo-scanning electron microscopy
For cryo-scanning microscopy, fresh trap parts of
Genlisea hispidula were frozen in a mixture of solid and
liquid nitrogen (‘slush’, 2210 8C) and transferred to a cryotransfer system (Gatan) connected to the FE-SEM. Then the
samples were fractured and the ice sublimated at 295 8C
for 5 min. The freshly prepared surfaces were observed in
a Hitachi S-4800 scanning electron microscope at 3 kV.
Finally, the same samples were sputter-coated with gold
(approx. 5 nm thick) and observed again.
Light and transmission electron microscopy
Traps [mainly Genlisea hispidula (immature and mature
traps), Genlisea lobata Genlisea violacea f. Giant, but
also Genlisea repens and Genlisea violacea f. Giant] were
hand-sectioned with a razor blade and fixed in 2.5 % formaldehyde and 2.5 % glutaraldehyde in 0.05 M cacodylate
buffer ( pH 7.0) for 4 h at room temperature. The material
was post-fixed in 1 % OsO4 in cacodylate buffer for 24 h
at 48C, rinsed in the same buffer, treated with 1 % uranyl
acetate in distilled water for 1 h, dehydrated with acetone
and embedded in Epon 812 (Fullam, Latham, NY, USA)
or Spurr’s resin. Semi-thin sections were stained with
methylene blue/toluidine blue O and examined with an
Olympus BX60 microscope. Ultrathin sections were cut
on Leica Ultracut UCT and Sorvall MT 2B ultramicrotomes. After contrasting with uranyl acetate and lead
citrate, the sections were examined in a Hitachi H500 or
Philips CM 100 electron microscope.
Scanning electron microscopy
The procedures for preparing samples (G. hispidula,
G. lobata, G. violacea f. Giant, G. uncinata, G. pygmaea,
G. repens, Genlisea sp. ‘Itacambira Beauty’, G. margaretae, G. lobata G. violacea f. Giant) for conventional
Płachno et al. — Functional Ultrastructure of Genlisea Digestive Hairs
SEM were as described earlier (Płachno et al., 2005b, c).
Briefly, traps were hand-sectioned with a razor blade and
fixed as for TEM. The material was later dehydrated in
an ethanol and acetone series, and critical-point dried
using liquid CO2. The dried tissues were sputter-coated
with gold and viewed in a Hitachi S-4700 microscope.
Cytochemical observations and confocal microscopy
For cytochemical observations, both fresh and fixed sectioned hairs were used. The PAS reaction was used for
detection of water-insoluble polysaccharides with
1,2-glycol groups (We˛dzony, 1996). Auramine 0 was used
for cutin and lipid localization (We˛dzony, 1996). Hairs
were incubated with the membrane-permeable fluorophore
DIOC6(3) (dihexaoxacarbocyanine iodide) to label
mitochondria and endoplasmic reticulum (Terasaki, 1994).
Living hairs in DIOC6(3) solution were observed with a
confocal microscope (ICS Leica Microsystem Heidelberg,
Mannheim, Germany).
R E S U LT S
Distribution and general morphology of hairs
Three patterns of digestive hair distribution were observed
in Genlisea vesicles (Fig. 1B – D and Table 1). In subgenus
Genlisea the hairs are densely clustered; thus the heads are
flattened to a more cube-like shape. Genlisea vesicle hairs
TA B L E 1. Comparison of some fine-structural features of
digestive hairs in subgenera Genlisea and Tayloria
Subgenus Genlisea
Distribution of
digestive hairs in
vesicle
Hairs concentrated along
vascular bundles
Middle cell
Endodermal
Terminal cells
Wall labyrinth
Vacuole
ER
Dictyosomes
Plastids
Mitochondria
Cuticle
Absent
Large, with one or few
spherical inclusions
Well developed; rER
membrane seems to be
anastomosed with
plasmalemma
With small
electron-translucent
vesicles, in abudance near
outer and radial walls also
some near nucleus
Small, with small starch
grains and plastoglobules
Numerous
Well developed, with true
pores well visible in TEM
Subgenus Tayloria
Not concentrated
along vascular
bundles; in distal part
vesicle of Genlisea sp.
‘Itacambira Beauty’
vesicle hairs form
rows
As in subgenus
Genlisea
Well developed
As in subgenus
Genlisea
Well developed, rER
in close proximity to
invaginated plasma
membrane near wall
ingrowths
A few, poorly
developed near wall
ingrowths, sometimes
near outer walls
As in subgenus
Genlisea
Numerous, especially
near wall ingrowths
As in subgenus
Genlisea
197
are mushroom-shaped, with a short stalk and a globular
head (Fig. 2A). The terminal cells usually have a radial
arrangement (Fig. 2B), but this pattern is disrupted in
some hairs.
Structure of hairs
The basic structure of the digestive hairs is uniform
throughout the genus. Some fine structural differences of
the subgenera Genlisea and Tayloria are compared in
Table 1.
Basal cell and middle cell
The cylindrical basal cell is laterally linked with
epidermal cells, and below with parenchyma cells. In
some hairs the basal cell is in direct contact with a tracheary
element. It is highly vacuolated; the cytoplasm with
organelles is concentrated towards the middle cell. The
middle cell is more or less cylindrical, with broadened
terminal parts. The part of this cell that supports terminal
cells is especially large and more or less conical. The
lateral wall is impregnated with electron-translucent
material – cutin (Casparian strip). This wall is thickened
especially at the base of the cell, where the middle cell is
linked with the basal cell. The lateral wall apparently is
brittle, because it fractures easily during material processing. The cell is strongly polarized. The vacuole occupies
the middle and basal parts of the cell and contains highly
osmiophilic material. In TEM, the vacuole consists of
both electron-translucent and solidly electron-dense osmiophilic compartments. In cryo-SEM, homogenous material
fills the whole vacuole (Fig. 2A). During preparation for
TEM, probably some of this material was washed out
from the vacuole. The hairs readily absorb Auramine 0,
which is accumulated in the middle cell. The vacuole is
polymorphic, consisting of Auramine-positive and
Auramine-negative parts. Most of the cytoplasm with the
nucleus, mitochondria, ER and plastids with osmiophilic
inclusions lies near the terminal cells (Fig. 2C). All
mitochondria within the hairs exhibit well-developed
cristae. The transverse wall between the stalk and the
basal cell is thin and penetrated by numerous plasmodesmata. The transverse wall between the stalk and the
terminal cell is also thin, but the wall surface is larger
than that between the stalk and the basal cell.
Secretory cells
The cytoplasm of the terminal cell is concentrated mostly
towards its base and radial walls (Fig. 2A). Here the prominent nucleus is localized, as well as dictyosomes, numerous
mitochondria (Fig. 3A), and plastids with small starch
grains and plastoglobules (Fig. 3B). A large vacuole
occupies most of the cell, and is surrounded by a thin
peripheral layer of cytoplasm in which there are ribosomes,
mitochondria, and dictyosomes (not hypertrophied) with
small electron-translucent vesicles (Fig. 3C) and ER. In
the vacuole is a large spherical inclusion visible by light
microscopy and cryo-SEM (Fig. 2A) but not by TEM. By
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Płachno et al. — Functional Ultrastructure of Genlisea Digestive Hairs
F I G . 2. (A) Fractured hair of Genlisea hispidula (cryo-SEM) (BC, basal cell; MC, middle cell; TC, terminal cell; IN, vacuolar inclusion; V, vacuole; N,
nucleus); (B) radial arrangement of terminal cells in Genlisea uncinata hair; (C) part of section through middle cell and terminal cells (MC, middle cell;
TC, terminal cell; WL, wall labyrinth; V, vacuole; N, nucleus; M, mitochondrion, Genlisea lobata Genlisea violacea. Scale bar ¼ 2 mm.
cryo-SEM this inclusion appears homogenous. In hairs
from immature traps of G. hispidula, rough endoplasmic
reticulum (rER) profiles are situated mainly along the
outer walls of the terminal cells. Portions of rER elements
(Fig. 3D) and electron-translucent vesicles are in close
association with the plasmalemma. Sometimes the rER
membrane seems to be anastomosed with the plasmalemma
(Fig. 3D). Multivesicular and paramural bodies are also
observed (Fig. 3E). The latter seem to be in close contact
with rER membranes (Fig. 3F). The surfaces of both the
outer and radial walls are irregular; however, a wall labyrinth sensu stricto is not formed. Similar observations were
made in mature hairs of this species (Fig. 3G). In contrast
to Genlisea hispidula (Fig. 2A) and G. repens,
G. violacea G. lobata and G. violacea feature radial
and outer walls ingrowths (the terminal cells are
transfer cells). The wall ingrowths are of reticular type
and consist of two different structural regions: a dense
core, and peripheral electron-translucent substance surrounding it. The wall ingrowths are PAS-positive; they
branch and anastomose to form the wall labyrinth. There
are many mitochondria (Fig. 3H), rER and some microbodies in close proximity to the invaginated plasma membrane. Unlike in the walls between terminal and stalk
cells (Fig. 4A), plasmodesmata were not observed in the
radial walls of the secretory cells (Fig. 4B), indicating the
absence of a symplastic connection between these cells.
The cuticle of the terminal cells is clearly seen in TEM
as an electron-dense layer having discontinuities manifest
as bright areas (Fig. 4C). The discontinuities are developed
as pores, and are especially visible above a thick pectic
layer continuous with the common middle lamella of
Płachno et al. — Functional Ultrastructure of Genlisea Digestive Hairs
199
F I G . 3. (A) In vivo labelling of mitochondria, endoplasmic reticulum and nuclear envelope, using DIOC6(3), in terminal cells of Genlisea violacea
f. Giant, Scale bar ¼ 10 mm. (B) Part of terminal cell cytoplasm with mitochondria (M), plastid (P) and rough endoplasmic reticulum (rER);
Genlisea lobata Genlisea violacea; scale bar ¼ 500 nm. (C) Peripheral layer of cytoplasm between vacuole and outer cell wall in terminal cell of
Genlisea hispidula: CW, cell wall; D, dictyosome; scale bar ¼ 500 nm. (D) Part of the section through terminal cells of young hair of Genlisea hispidula
showing rough endoplasmic reticulum in close contact with plasma membrane: rER, rough endoplasmic reticulum; RCW, radial walls; scale bar ¼
500 nm. (E) Paramural body in terminal cell of young hair of Genlisea hispidula: PB, paramural body; rER, rough endoplasmic reticulum; RCW,
radial walls; scale bar ¼ 500 nm. (F) Close contact of rER profile with paramural body (PB) in terminal cell of young hair of Genlisea hispidula: cell
wall, CW; scale bar ¼ 250 nm. (G) Part of the section through terminal cell of a mature hair of Genlisea hispidula: D, dictyosom; Pb, paramural
body; RCW, radial walls; scale bar ¼ 500 nm. (H) Part of section through terminal cells of mature hair of Genlisea lobata Genlisea violacea: In,
wall in-growth; M, mitochondrion; Mb, microbody; rER, rough endoplasmic reticulum; RCW, radial walls; scale bar ¼ 500 nm.
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Płachno et al. — Functional Ultrastructure of Genlisea Digestive Hairs
F I G . 4. (A) Part of transverse wall between stalk and terminal cell penetrated by numerous plasmodesmata, Genlisea lobata Genlisea violacea: MC,
middle cell; TC, terminal cell; In, wall ingrowth; scale bar ¼ 500 nm. (B) Part of the radial wall between terminal cells of Genlisea hispidula, showing
absence of plasmodesmata: M, mitochondrion; RCW, radial walls; D, dictyosome; scale bar ¼ 500 nm. (C) Cuticular pore (asterisk) in cuticle of terminal
cells of mature Genlisea lobata Genlisea violacea hair: M, mitochondrion; scale bar ¼ 830 nm.
anticlinal cell walls. In unimpregnated wall material, separate cutin cystoliths also occur. Cuticular pores were
detected by various techniques, both in chemically fixed
and in frozen material (for more details, see Płachno
et al., 2005b).
DISCUSSION
Hair architecture in Lentibulariaceae
Besides having a similar architecture (basal cell, middle cell
and secretory cells), the digestive-absorptive hairs of the
Lentibulariaceae present morphological and ultrastructural
differences (Table 2). For example, the middle cell in all
three genera has a Casparian strip-like lateral wall, which
is an apoplastic barrier, but this cell has a highly developed
wall labyrinth only in Utricularia. This is associated with
rapid water transport during removal of water from
Utricularia bladders (Fineran and Lee, 1975; Fineran,
1985). The terminal cells of Genlisea share similarities
with those of both Utricularia and Pinguicula. For
example, the large vacuole, which is the dominant organelle, is also characteristic of the arms – the distal parts
of quadrifid and bifid terminal cells of Utricularia
(Fineran and Lee, 1975). One of the main functions of
TA B L E 2. Comparison of some fine-structural features of digestive hairs in Lentibulariaceae (after Vintéjoux, 1974; Beltz,
1975; Fineran and Lee, 1975; Fineran, 1985; Heslop-Harrison and Heslop-Harrison,1981; Juniper et al., 1989; Płachno,
2006; and the present results)
Pinguicula
Middle cell
Shape
Wall labyrinth
Lateral wall
Vacuole
Protein crystal in
nucleus
Terminal cells
Shape
Wall labyrinth
Vacuole
Protein crystal in
nucleus
Plastids
Mitochondria
Cuticle
Plano-convex or concavo-convex
Genlisea
Utricularia
Discoid
Absent
Casparian strip-like
Large
Present
Cylindrical with broadened terminal parts,
almost covered by the glandular head
Absent
Casparian strip-like
Large, with osmiophilic deposits
Absent
Forming head
Forming head
Developed
Developed in species of subgenus Tayloria
Large, with osmiophilic content
Present or absent
Large, with one or few spherical inclusions
Absent
Consists of stalk terminated by arm
projecting into trap lumen
Absent; only small wall ingrowths
occur
In the arm, large with crystal
Absent
Very large, ramifying leucoplast
invested by endoplasmic reticulum
Numerous, with well-developed cristae
Discontinuous, lacking well-formed
pores
Small, with small starch grains
Not very conspicous
As in Pinguicula
Well developed, with true pores visible in
both TEM and SEM
As in Pinguicula
The open structure, lacking
well-formed pores
Well developed
Casparian strip-like
Inconspicous, bigger only in old hair
Absent
Płachno et al. — Functional Ultrastructure of Genlisea Digestive Hairs
this vacuole in both genera is enlargement of the cell
(contact between the cell surface and external environment), which is important for secretion and absorption
processes.
Taking together previously published work on Lentibulariaceae hairs (Fineran and Lee, 1975; Heslop-Harrison
and Heslop-Harrison, 1981; Fineran, 1985) and the present
results, it is clear that the most complicated terminal cell
in the digestive hairs of this family has evolved in Utricularia. In contrast to the sessile hairs in Pinguicula and
Genlisea, in Utricularia the quadrifid and bifid terminal
cells not only play a role in secretion and absorption but
also partially take over the function of the middle cell.
According to Heslop-Harrison and Heslop-Harrison
(1981), in Pinguicula digestive hairs the radial walls of
terminal cells have wall ingrowths. This is similar to the
present observation in the Genlisea hybrid studied and
G. violacea. In both genera they consist mainly of polysaccharides with 1,2-glycol groups ( pectins). It should be
added that Heslop-Harrison (1976) reported finding transfer cells in G. africana but did not describe the ultrastructure of the hairs. In the present study it was found that the
wall ingrowths of Genlisea are similar to the reticular wall
ingrowths described in other plants (Gunning and Pate,
1969). Transfer cells are developed for intensive shortdistance transport between the symplast and apoplast
(Gunning and Pate, 1974; Offler et al., 2003). In Genlisea
the wall labyrinth is well developed and transport should
also be very intensive and active, because the complexity
of wall ingrowths is directly correlated with the intensity
of transport by the transfer cell (Gunning and Pate,
1969). In interpreting the present results and the findings
from a molecular study by Jobson et al. (2004), it
should be borne in mind that the concentration of hairs
along vascular bundles in species of subgenus Genlisea
may be inherited from an ancestor (subgenus Genlisea is
most probably the basal lineage; Jobson et al. 2004;
present results). According to Jobson et al. (2004),
active water pumping might have occurred in the Genlisea
ancestor trap. In recent Genlisea species, active transport
of water with organisms into traps has not been observed;
prey entered the traps unaided and later moved inside
(Adamec, 2003; Płachno et al., 2005a). In summary, the
functional specialization and degree of complexity of Lentibulariaceae traps and digestive hairs are strongly correlated with differences in the rates of their molecular
evolution (Jobson and Albert, 2002; Jobson et al., 2004;
Müller et al., 2004).
Mode of secretion
The presence of well-developed rER and numerous mitochondria in terminal cells of Genlisea showed that these cells
are active and have the essential apparatus for enzyme biosynthesis, as in digestive glands of other carnivorous plant
(reviewed by Juniper et al., 1989). In the closely related
Pinguicula, it has been suggested that the main synthesis
of digestive enzymes occurs on membrane-bound ribosomes
of the rER (Schnepf, 1961; Vassilyev and Muravnik, 1988).
The present observations may suggest the mode of secretion.
201
Multivesicular bodies are connected with both secretion and
endocytosis (e.g. Tanchak and Fowke, 1987; Tse et al.,
2004). Paramural bodies probably are also connected with
transport of synthesized material, as in Claceolaria trichomes
(Sacchetti et al., 1999). In Genlisea, we suggest that digestive enzymes may also be transported directly from ER to
the cell wall via connecting ER membranes with the plasmalemma. This mechanism was previously suggested for
Dionaea (Robins and Juniper, 1980) and Pinguicula
(Vassilyev and Muravnik, 1988). Using a high-pressure
freezing complex of actin and ER, which is associated with
organelles and the cell wall, stalk cells of Drosera capensis
were observed in emergences (Lichtscheidl et al., 1990).
Cortical ER may play a key role in anchoring the cytoskeleton, in facilitating seretion, and also in the communication of
signals between cytoplasm and the exterior of the cell
(reviewed by Lichtscheidl and Hepler, 1996). Phosphatase
activity is connected with the outer and radial walls of
mature Genlisea digestive hairs (Płachno, et al., 2006;
Fig. 1). In other carnivorous plants (Nepenthes, Dionaea,
Pinguicula), synthesis of digestive enzymes in the prematuration stage of gland development has been suggested
(Vassilyev, 1977; Robins and Juniper, 1980; HeslopHarrison and Heslop-Harrison, 1981; Vassilyev and
Muravnik, 1988; Owen et al., 1999). In Drosophyllum lusitanicum there is continuous enzyme (acid hydrolase) secretion
in both immature and mature unstimulated digestive glands
(Vassilyev, 2005). In mature traps of Genlisea, digestive
hairs are stimulated continuously, because prey enter
opened traps all the time. Enzyme secretion occurs in the
absence of prey in plants from in vitro sterile culture
(Płachno, 2006; Płachno et al., 2006). For these reasons,
we suggest that digestive enzymes are continuously secreted
to the trap interior (continuous digestive activity). Mucilage
has been observed inside Genlisea traps (Studnička, 2003a;
Płachno et al., 2005b). Thus, digestive pools of viscous
fluid occur in Genlisea. However, unlike in the secretory
cells of mucilage glands of Drosophyllum, Drosera,
Utricularia and Pinguicula (Schnepf, 1961, 1963;
Vintéjoux and Shoar-Ghafari, 1997, 2005) hypertrophy of
dictyosomes and large vesicles containing mucilage were
not observed in Genlisea.
Mode of absorption
The pores in the cuticle are involved in both secretion of
enzymes and absorption of the products of digestion. The
present ultrastructural observations confirm earlier reports
on cuticular discontinuities in Genlisea hairs seen by
SEM and vital staining (Płachno et al., 2005b). In carnivorous plants, true cuticular pores have been detected only in
Drosera (Williams and Pickard, 1969, 1974 after Juniper
et al., 1989; Ragetli et al., 1972; Heslop-Harrison, 1975)
and recently in Roridula (Anderson, 2005).
In the present study, cryo-SEM was found to be a very
useful method for visualizing not only the morphology
but also the cytomorphology of whole plant secretory
structures.
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Płachno et al. — Functional Ultrastructure of Genlisea Digestive Hairs
ACK N OW L E D G E M E N T S
The first author is grateful to Dr Elena Gorb and
Dr Stanislav Gorb (Evolutionary Biomaterials Group, MPI
for Metals Research, Stuttgart, FRG) for their hospitality
and for providing the opportunity to use the cryo-SEM,
and to Prof. Irene K. Lichtscheidl and her group
(Department of Cell Physiology and Scientific Films,
University of Vienna, Austria) for the use of confocal
microscopy facilities and for hosting the author’s stay at
the University of Vienna. We thank Kamil Pásek (http://
www.bestcarnivorousplants.com), Dr Gerfield Deutsch,
Andreas Fleischman and Dr Lubomir Adamec for providing
plants for this study. This work was supported in part by a
grant from the Jagiellonian University, Cracow (DBN-414/
CRBW/18/2006).
L I T E R AT U R E CI T E D
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