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International Journal of Plant Sciences, May
1997 v158 n3 p249(10)
Peltate glandular trichomes of Leonotis
leonurus leaves: ultrastructure and histochemical characterization of
secretions. Lia Ascensao; Natalia Marques; M. Salome Pais.
Author's Abstract: COPYRIGHT 1997 University of
The histochemical characterization of the oleoresin produced by peltate
trichomes of Leonotis leonurus revealed
terpenoids and fiavonoid aglycones. At the onset of secretion, glandular cells
were more densely cytoplasmic than the other trichome cells. The lateral stalk
wall underwent cutinization, and the cuticle over the glandular cells became
thicker. During the active secretory stage, the most striking ultrastructural
features of glandular cells were the hypertrophy of the leucoplastidome and the
extensive proliferation of ER. The high development of these two cellular
compartments was related with the biosynthesis and transport of the secretory product.
A granulocrine secretion mechanism may operate alone or concurrently with an
eccrine process. In the glandular head, the loosening of the outer wall
fibrillar matrix, and the accumulation of secretion in the newly formed
interfibrillar spaces led to a secretory cavity development by detachment of
the cuticle and the outermost pectic layer of cell wall. Successive
accumulation of secretion in the secretory cavity conferred the spherical
shape, characteristic of a peltate gland, to the trichome. The interfibrillar
spaces, initially small and elongated, enlarged, became roundish, and appeared
as vesicles delimited by an electron-dense layer. These vesicle-like structures
are interpreted as lipophilic secretion globules in a hydrophilic phase. It is
suggested that pectic polysaccharide wall constituents may be the main
components of this hydrophilic matrix. The dense layer around the vesicles may
represent an interphase between lipophilic and hydrophilic compounds. The
secretion seemed to remain trapped in the secretory cavity, since no cuticular
disruption was observed.
Full Text: COPYRIGHT 1997 University of
Introduction
Many species of the Lamiaceae family have been used in folk medicine. The
genus Leonotis, frequently associated with Cannabis
through the term dagga, is often listed as a mild narcotic hallucinogen,
although toxicity and hallucinogenic properties seem rather insignificant (Duke
1985).
Leonotis leonurus R. Br. (lion's ear or
lion's tail), a perennial shrub widely distributed throughout South Africa and
tropical regions of America, is reputed to possess a great variety of medicinal
properties, being considered as emmenagogue, purgative, and vermifuge. Leaf
decoctions are used by Africans (Zulus, Hottentots, and Xhosas) to treat
asthma, fever, influenza, snakebite, epilepsy, skin diseases, and tapeworm. Dry
leaves, alone or mixed with tobacco, may lead to habituation when smoked
persistently (Watt and Breyer-Brandwigk 1962).
Phytochemical studies have revealed that L. leonurus produces labdane
diterpenoids (Kaplan and Rivett 1968; Purushothaman and Vasanth 1988) and an
essential oil rich in terpene hydrocarbons, mainly [Beta]-caryophyllene and
[Alpha]-pinene (Pedro et al. 1991).
The glandular trichomes that produce essential oils are a general feature of
the mint family. In L. leonurus the vegetative and reproductive organs bear
numerous glandular trichomes of two morphologically distinct types (peltate and
capitate) that also seem to have different secretion processes, as indicated by
SEM (Ascensao et al. 1995). It is, therefore, of interest to investigate the
ultrastructure of these trichomes to verify whether morphological differences
also are reflected in their cytology, their secretion process, and in the
chemical nature of the secreted material.
Although a considerable number of studies deal with the chemical composition
of the essential oils produced by Lamiaceae, the morphology and particularly
the ultrastructure of secreting glandular trichomes have been examined only in
a few species. Detailed studies were made on Mentha piperita (Amelunxen 1964),
Mentha spicata (Gershenzon et al. 1989), Salvia glutinosa and Salvia pratensis
(Schnepf 1972), Salvia officinalis (Venkatachalam et al. 1984), Monarda
fistulosa (Heinrich 1973; Heinrich et al. 1983), Origanum dictamnus
(Bosabalidis and Tsekos 1982), Teucrium scorodonia (Sevinate-Pinto and Antunes
1991), and Nepeta racemosa (Bourett et al. 1994).
Giving sequence to our work on L. leonurus, we describe in this article the
ultrastructural development of glandular cells of peltate trichomes and their
histochemistry. Special emphasis is given to the subcuticular space of these
glandular trichomes where the essential oils accumulate.
Material and methods
TRANSMISSION ELECTRON MICROSCOPY (TEM)
Leaves of Leonotis leonurus R. Br., grown
at the Lisbon Botanic Garden, were fixed at different stages of development
with 3% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, at 4 [degrees]
C for 4-6 h, and postfixed for 2 h in 2% unbuffered osmium tetroxide
(OS[O.sub.4]). After dehydration in a graded acetone series, the material was
embedded in Epon-Araldite resin. Thin sections were conventionally stained with
uranyl and lead citrate and examined with a JEOL 100C electron transmission
microscope at 80 kV.
For polysaccharide detection, ultrathin sections were collected on
Formvar-coated gold grids and submitted to the PATAg test (Thiery 1967).
LIGHT MICROSCOPY (LM)
The majority of the histochemical tests used in this study have been
referred to in a previous paper (Ascensao and Pais 1987). Sections of fresh
material cut on a Reichert cryostat microtome were stained with Sudan black B,
Sudan IV, and Nile blue A for total lipids; Nadi reagent and anthimonium
trichloride for terpenoids; Wagner and Dirtmar reagents for alkaloids; periodic
acid-Schiff (PAS) reagent for polysaccharides with vicinal glycol groups; and
mercuric bromophenol blue for proteins. The presence of phenolic compounds was
tested with ferric trichloride (Johansen 1940), vanillin-hydrochloric acid (
Semithin sections of material prepared for TEM were also stained for lipids
with
SCANNING ELECTRON MICROSCOPY (SEM)
Leaves were fixed for 2 h at 4 [degrees] C with 2% glutaraldehyde in 0.1 M
sodium cacodylate buffer at pH 7.2. After washing in the same buffer and
dehydrating in acetone, the material was critical-point dried, sputter-coated
with gold, and observed in a Jeol JSM T220 scanning electron microscope at 15
kV.
Results
Peltate trichomes are quite common on Leonotis
leonurus leaves and flowers (Ascensao et al. 1995). They occur on both surfaces
of leaf, calyx, and corolla, mixed with capitate trichomes, while on the
stamens and gynoecium they are the only constituents of the indumentum. Fully
developed peltate trichomes are about 60 [[micro]meter] ([+ or -]15) in height
and 50 [[micro]meter] ([+ or -]10) in diameter at the spherical head
[ILLUSTRATION FOR FIGURE 1 OMITTED]. They consist of a short stalk cell and a
large head with eight secretory cells arranged in a circle [ILLUSTRATION FOR
FIGURES 2, 5 OMITTED].
The secretion contained in the subcuticular space stained red with Sudan IV
([ILLUSTRATION FOR FIGURE 3 OMITTED], asterisk) and dark blue with
Secretion showed a blue autofluorescence under ultraviolet light, indicating
phenolic compounds. Fluorochromes for flavonoids, such as aluminum chloride and
The trichome cell walls stained strongly with PAS. However, the outer layer
of glandular cell walls stained lightly with PAS and intensely with Ruthenium
red, whereas in the inner layer, the staining intensity observed was the
reverse. The thick cuticle on the stalk lateral walls stained heavily with
Sudan IV.
The ultrastructure of the glandular cells underwent dramatic changes during
peltate trichome development, going through three different stages: a
presecretory, a secretory, and a postsecretory phase.
PRESECRETORY PHASE. In the presecretory phase, which corresponds to the
differentiation of the trichome, the ultrastructure of the glandular cells was
similar to the other meristematic cells of the leaf and flower. They contained
a large nucleus with a prominent nucleolus and a dense cytosol, rich in
ribosomes [ILLUSTRATION FOR FIGURES 6, 8 OMITTED]. The multishaped plastids had
dense stromas, scarce internal membranes, and few starch grains [ILLUSTRATION
FOR FIGURES 8, 9, 10 OMITTED]. Occasional dictyosomes and a few cisternae of
endoplasmic reticulum, dispersed throughout the cell, were observed [ILLUSTRATION
FOR FIGURES 8, 9 OMITTED]. Plasmodesmata were frequent on the periclinal stalk
walls and on the anticlinal glandular cell walls. At this stage glandular cells
had a thin cuticle ([ILLUSTRATION FOR FIGURE 9 OMITTED], arrow), comparable to
the one found in epidermal cells.
At the later presecretory phase, the stalk cell and the basal cells
developed central vacuoles. In addition, the gland cuticles began to thicken,
while cuticles of epidermal cells remained thin. This cuticle-thickening
process was first detected on the stalk lateral walls [ILLUSTRATION FOR FIGURE
8 OMITTED].
SECRETORY PHASE. In the beginning of the secretory phase, the increase in
thickness of gland cuticles was evident [ILLUSTRATION FOR FIGURES 11, 12
OMITTED]. Cuticles appeared reticulate due to a fibrillae network that
permeated through an apparently amorphous matrix. The fibrillae seemed to have
some continuity with the structural elements of the pectocellulosic layer
beneath ([ILLUSTRATION FOR FIGURE 11 OMITTED], arrows). During this stage,
several ultrastructural changes occurred in glandular cells, e.g.,
densification of cytoplasm, increase in ribosome number, and an extensive
development of the plastid compartment that was accompanied by ER
proliferation. Most of the cell volume was occupied by numerous large and
amoeboid plastids. They were closely stacked and were separated by only a thin
layer of cytoplasm, containing ER cisternae associated with the outer membrane
of the plastidial envelope ([ILLUSTRATION FOR FIGURES 13 OMITTED], 14, arrows).
These polymorphic plastids, frequently bell-shaped, were typically leucoplasts
characterized by a very dense stroma and a poorly developed system of internal
membranes. Leucoplasts did not contain thylakoids; they had only short membrane
tubules connected with the envelope inner membrane. The tubules had dilated
portions that very often were filled with an electron-dense content
([ILLUSTRATION FOR FIGURES 13, 14 OMITTED], arrowheads).
Following the hypertrophy of the leucoplastidome, a high proliferation of
smooth endoplasmic reticulum (SER) occurred. A large number of long and sinuous
cisternae were parallel or around the cell organelles [ILLUSTRATION FOR FIGURE
15 OMITTED]. Although neither direct connections between ER membranes and the plasmalemma
nor extrusion vesicles containing the secretory product were detected, a very
sinuous plasmalemma was observed ([ILLUSTRATION FOR FIGURE 16 OMITTED],
arrows).
Concurrently with the production of the secretory material, an extracellular
space developed over the glandular cells. It began in the outer cell wall by a
loosening of the fibrillar matrix ([ILLUSTRATION FOR FIGURE 17 OMITTED],
arrows). The gaps between the fibrils began as small and elongated
electron-translucent areas, often in a tandem arrangement ([ILLUSTRATION FOR
FIGURES 18, 19 OMITTED], arrows), indicating their aggregation and conferring
to the outer cell wall the appearance of a loose fibrillar mesh [ILLUSTRATION
FOR FIGURES 21, 22 OMITTED].
As a result of the wall loosening and of secretion accumulation, a
subcuticular space was formed by the detachment of the cuticle and the
outermost pectic layer of cell wall [ILLUSTRATION FOR FIGURES 3, 7, 20
OMITTED]. Vesicle-like structures of irregular surface, surrounded by a
fibrillar-granular matrix, could be seen within the subcuticular space
([ILLUSTRATION FOR FIGURES 21, 22 OMITTED], stars). The accumulation of
secretion in the subcuticular space gave a spherical shape to the trichome,
characteristic of a mature peltate gland.
At this stage the secretory cavity had numerous round vesicle-like
structures of different sizes. These vesicles, with an electron-light content,
appeared delimited by an electron-dense layer and apparently increased in size
by coalescence [ILLUSTRATION FOR FIGURE 23 OMITTED]. The inner layer of
glandular cell wall stained heavily by PATAg test, while the residual matrix of
outer wall and the bounding vesicle layer stained lightly [ILLUSTRATION FOR
FIGURE 24 OMITTED].
POSTSECRETORY PHASE. During this phase, an increase in vacuolation was
observed. Vacuoles containing a flocculent material and membrane debris
occupied the greatest volume in the glandular cells and limited the cytoplasm
to a thin parietal layer. Secretions that accumulated in the subcuticular space
compressed the glandular disk cells, inducing their anticlinal walls to fold
up. There was no indication of spontaneous cuticle rupture. A progressive
degradation of the cellular constituents was observed.
Discussion
In spite of the poor specificity of the histochemical tests, they can be
useful to localize in situ the main chemical classes of metabolites present in
plant secretions. Our histochemical results indicate that the secretion of Leonotis leonurus peltate trichomes is an
oleoresin containing terpenoids (essential oils and resiniferous acids) and
flavonoid aglycones as its main constituents. Proteins, polysaccharides,
alkaloids, and tannins were not detected in the secretion. These results are
consistent with those obtained by GC and GC/MS. Mono- and sesquiterpenes were
identified in the essential oil of L. leonurus (Pedro et al. 1991), and the
presence of labdane diterpenoids was also reported (Kaplan and Rivett 1968;
Purushothaman and Vasanth 1988). Tannins appear to be absent in Lamiaceae, and only
a few minor alkaloids seem to occur in this family (Richardson 1992). Small
amounts of polysaccharides were histochemically detected in the secretion of
the peltate trichomes of some Lamiaceae species (Werker et al. 1985). After our
TEM studies of the secrettry cavity development, we are convinced that the
polysaccharides detected may be cell wall components, corresponding probably to
the loose fibrillar mesh of the outer cell wall, where the secrettry products
accumulated.
At the ultrastructural level, the trichome stalk cell of L. leonurus showed
little structural specialization besides the cutinization of lateral cell
walls, the lack of chloroplasts, and numerous plasmodesmata in the periclinal
walls. Similar features were reported by several authors for trichomes of other
Lamiaceae (Amelunxen 1964; Schnepf 1974; Bosabalidis and Tsekos 1982; Fahn
1988; Bourett et al. 1994).
Cutinization of the side walls of stalk cell was frequently observed in
glandular trichomes. It is generally assumed that cutinized walls may block the
back flow of secretions stored in the subcuticular space, preventing the
intoxication of mesophyll cells. The lack of chloroplasts in the trichome cells
together with the presence of a barrier to apoplastic flow in the stalk indicate
that precursors of secretion components might come from the mesophyll.
Plasmodesmata in the periclinal walls of stalk cells may provide transport of
photosynthates into the glandular cells, contributing to secretion rate
regulation.
At the secretion stage, the most striking ultrastructural features of the
peltate trichome glandular cells were the extensive development of the
leucoplastidial compartment and the high proliferation of SER. The
hypertrophied leucoplastidome in close association with ER is characteristic of
various glandular structures secreting essential oils and resins (Dell and
McComb 1978; Charon et al. 1987; Fahn 1988; Kleinig 1989; Wagner 1991; Duke and
Paul 1993).
The involvement of leucoplasts and ER in terpene secretion has been biochemically
demonstrated. For monoterpenes, plastids are clearly implicated as the
exclusive site of synthesis. In fact, the biosynthesis of monoterpene
hydrocarbons in vitro was achieved by a leucoplast-enriched fraction (Gleizes
et al. 1983; Pauly et al. 1986), and the biosynthesis of geranyl pyrophosphate
was recently localized in plastids (Soler et al. 1992). The synthesis of
farnesyl pyrophosphate and its derived sesquiterpenes occurred in the
cytosol/SER (Gleizes et al. 1980; Belingheri et al. 1988; Hugueney and Camara
1990) as the synthesis of triterpenes (Goodwin 1979). On the other hand,
correlation studies, performed in a large number of species, indicated a
constant relationship between the expansion of leucoplastidome and the ratio of
monoterpenes in the oil and the extension of SER and the rate of sesquiterpene
or oxygenated compounds (Cheniclet and Carde 1985).
The essential oil of L. leonurus, analyzed by GC and GC/MS (Pedro et al.
1991), showed that the sesquiterpenes were the major fraction of the oil
(59.4%), followed by monoterpenes (30.4%). In this species, apparently, the
ultrastructural features and the results of the essential oil analysis of L.
leonurus did not confirm the correlation found by Cheniclet and Carde (1985);
however, it is necessary to consider that on L. leonurus organs two kinds of
glandular trichomes occur and that probably both are involved in essential oil
production.
In L. leonurus peltate trichomes, the transport of terpenes to the cellular
surface may be via ER, since dictyosomes were not common in the glandular cells
and, therefore, the participation of the Golgi apparatus in the synthesis and
transport of essential oils seemed to be negligible; moreover, proteins and
polysaccharides were absent in the secretion, as shown by histochemical tests.
The elimination of secretory product to the subcuticular space is not fully
understood. Although extrusion vesicles were not detected, the sinuous aspect
of the plasmalemma suggests fusion of vesicles. One may speculate that a
granulocrine secretion occurs via ER vesicles or through transient
ER-plasmalemma fusion, which implies the existence of an alternative pathway of
membrane flow. Some components of the oleoresin, such as [Beta]-pinene, must be
sequestered within a membrane compartment. It was proved that this compound
inhibits the mitochondrial and chloroplastidial electron transport (Douce et
al. 1978; Pauly et al. 1981). Otherwise, Gleizes et al. (1980) have assumed
that sesquiterpenes as steroids, for instance, may act as integral components
of the membrane structure. The difficulties showing anastomosis between the
exocytotic vesicles and the plasmalemma by TEM may be explained by the massive
secretion of terpenoids in a very short period of time. Nevertheless, an eccrine
pattern of secretion cannot be excluded for some compounds. This view is
supported by Stern et al. (1987), who suggested that volatile terpenoids
apparently cross the plasmalemma as single molecules.
Our observations on the peltate trichomes of L. leonurus indicate that the
secretion released in the cellular surface passes the inner cell wall, does not
accumulate in the periplasmic space, and fills up the interfibrillar spaces of
the outer cell wall. The thickening of the apical glandular cell wall seems to
result largely from the loosening of the outer wall fibrils rather than by
deposition of wall material, since only scarce dictyosomes are found.
The wall loosening, presumably the result of enzymatic action of
endoglycanases, and the accumulation of secretory products may contribute to
the subcuticular space development. Secretory cavities are common in many types
of exotropic glands that secrete lipophilic compounds (Schnepf 1974; Fahn
1988).
In most of the secretory cavities described, accumulation of the secretory
product occurs between the cuticle and the wall of the glandular cells. On the
contrary, in L. leonurus peltate trichomes, the bounding dermal sheath of the
secretory cavity consists of cuticle and a portion of the pectic outermost cell
wall layer. The presence of a cuticular sheath in some Lamiaceae peltate
trichomes was reported by Werker (1993). This wall reinforcement along the
cuticle may give resistance to the secretory cavity when large amounts of
secretion are stored. To the best of our knowledge, a similar secretory cavity
was described by TEM and an analogous formation mechanism was suggested (Kim
and Mahlberg 1991, 1995) only in the glandular trichomes of Cannabis sativa.
It seems likely that the round vesicle-like structures that fill up the
large secretory cavity may correspond to the secretion, dispersed as lipophilic
globules in a hydrophilic phase. The histochemical reactions and the Thiery
test indicate that pectic polysaccharide wall components must be the main
constituents of this hydrophilic residual matrix and the single dense layer
around the vesicles may represent an interface between lipophilic and
hydrophilic compounds. Vesicle-like structures within secretory cavities were
also reported in Humulus lupulus (Oliveira and Pais 1990) and in C. sativa
(Mahlberg and Kim 1992). According to these authors, the dense layer delimiting
the secretory vesicles may contain proteinaceous components such as those that
occur at the oil bodies in plant cells or at low density lipoprotein bodies in
animal cells (Kim and Mahlberg 1995).
In L. leonurus peltate trichomes, the secretion remains trapped in the
intact subcuticular space unless external factors, such as extreme climatic
conditions or grazing, cause its disruption. However, we still believe that
highly volatile secretory products can escape through microchannels at the
reticulate cuticle.
Acknowledgments
We are grateful to Instituto de Biotecnologia e Quimica Fina - Centro de
Biotecnologia Vegetal da Junta Nacional de Investigacao Cientifica e
Tecnologica (JNICT) for the financial support of this work.
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