Development and Structure of Internal Glands and External Glandular Trichomes in Pogostemon cablin
Zhu J (2013) Development and Structure of Internal Glands and External Glandular Trichomes in Pogostemon cablin. PLoS
ONE 8(10): e77862. doi:10.1371/journal.pone.0077862
Development and Structure of Internal Glands and External Glandular Trichomes in Pogostemon cablin
Jiansheng Guo 0
Yongming Yuan 0
Zhixue Liu 0
Jian Zhu 0
Meng-xiang Sun, Wuhan University, China
0 1 Department of Molecular and Cell Biology, School of Life Science and Technology, Tongji University , Shanghai, China, 2 Firmenich Company, Shanghai , China
Pogostemon cablin possesses two morphologically and ontogenetically different types of glandular trichomes, one type of bristle hair on the surfaces of leaves and stems and one type of internal gland inside the leaves and stems. The internal gland originates from elementary meristem and is associated with the biosynthesis of oils present inside the leaves and stems. However, there is little information on mechanism for the oil biosynthesis and secretion inside the leaves and stems. In this study, we identified three kinds of glandular trichome types and two kinds of internal gland in the Pogostemon cablin. The oil secretions from internal glands of stems and leaves contained lipids, flavones and terpenes. Our results indicated that endoplasmic reticulum and plastids and vacuoles are likely involved in the biosynthesis of oils in the internal glands and the synthesized oils are transported from endoplasmic reticulum to the cell wall via connecting endoplasmic reticulum membranes to the plasma membrane. And the comparative analysis of the development, distribution, histochemistry and ultrastructures of the internal and external glands in Pogostemon cablin leads us to propose that the internal gland may be a novel secretory structure which is different from external glands.
The Lamiaceae comprise many commercially important species
because of their high content of essential oils, which are widely
used in pharmaceutical preparations, perfumery and cosmetics.
The development and histochemistry of glandular trichomes
occurring in plants of the Lamiaceae was well documented 
and they were recognized as the defense-related structures on the
aerial epidermis of leaves, stems and floral organs [7,8]. As to the
types of trichomes (capitate, non-glandular, peltate and different
versions and combinations of these), there was some variability in
Lamiaceae genera that can occur in a given species [9,10]. And
each type of trichomes had a different spatial arrangement and
function, secreting different combinations, or proportions, of
hydrophilic and lipophilic material.
Most published studies on the Lamiaceae had concentrated on
the ultrastructure of peltate trichomes or capitate trichomes in
relation to the secretory process . Basing upon the
remarkable ultrastructural transformations occurring at the onset
of secretion in the glandular trichomes, previous investigators
proposed a range of speculations concerning the possible sites of oil
synthesis and the possible mechanisms of oil secretion. There is
much distinction about the ultrastructure and secretory process in
different glandular trichomes of different species.
Pogostemon cablin is one of the tropical, aromatic crops in
Lamiaceae, and cultivated mainly in Southeast Asia, India and
Brazil. Previous studies on Pogostemon cablin have shown that there
are external or internal glands in leaves and stems [17,18]. These
structures are responsible for the production of sesquiterpenes
containing a large quantity of patchouli alcohol, of which more
than 30 percent of the essential oils [19,20]. However, little is
known about secretory process and the composition of the secreted
material in internal glands of leaves and stems. And the distinction
about the ultrastructure, secretory process and development
among the glands of Pogostemon cablin deserves to be studied for
elucidate the relationship between the external and internal
Development, Histochemistry and Secretion of Glands
The adaxial and abaxial surfaces of the investigated leaves and
stems of Pogostemon cablin showed numerous glandular trichomes
and bristle hairs (Figure S1). According to the morphology of
glandular trichomes, three types of trichomes were observed. Each
of the three external trichome types of Pogostemon cablin could be
easily characterized. Two of three external trichomes were
shortstalked capitate (type I) and peltate trichome (type II) and one was
long-stalked capitate trichome (type III) in Pogostemon cablin.
Internal glands were found in the palisade tissue of leaves (type
IV) (Figure 1A and B) and in the cortex parenchyma of stems (type
V) (Figure 2A and B). Every external trichome type started its
development with an epidermal cell, which first underwent a
periclinal cell division (Figure S3). Various colour reactions (Figure
S4) indicated that the secretion stored in the sub-cuticular space
(SCS) of mature external trichomes contained hydrophilic and
lipophilic components (Table 1). The head regions in secretory
external trichomes of Pogostemon cablin differed in size, structure,
composition of the metabolites and the secretory process.
Internal glands in leaves with a length of 40 mm (69) distributed
among palisade cells (Figure 1A, arrow). Mature internal glands
with a basal cell, a narrow stalk cell and one big cytoplasmically
dense head cell had a SCS which was often filled with secretory oil
(Figure 1B and Figure 4H).
The internal glands in leaves originated from a single
undifferentiated palisade cell-like which had a larger nucleus and
nucleolus (Figure 1C, arrow). The initial cell of internal glands
formed one big vacuolated basal cell and one small,
cytoplasmically dense apical cell through the unequal periclinal division
(Figure 1D, arrows). As the development processed, the apical cell
formed one narrow stalk cell and one big head cell with nucleus
and nucleolus (Figure 1E, arrows). Afterward, the head cell
enlarged to form the secretory cell which had a dense cytoplasm
and large nuclei with prominent nucleoli (Figure 1F). When the
internal glands came into secretory stage, the composition and the
localization of the secretory compounds in SCS could be detected.
The histochemical tests for detecting lipids compounds have given
the positive results in this internal gland. Gold-yellow staining of
secretion in the head and neck-cell area with Neutral red under
UV light indicated the presence of total lipids (Figure 1G),
although staining with Sudan black B for total lipids was slightly
positive (Figure 1I). Staining with Sudan III showed up faintly in
the head cell, but the oil droplet contained in the SCS showed an
intense jacinth staining (Figure 1H). These results were also
confirmed using OsO4 for unsaturated lipids (Figure 1J). The Nadi
reaction showed up an intense violet or blue-violet staining of the
oil droplet contained in the SCS (Figure 1K). The head cell of
mature internal gland became stained fluorescent yellow-orange
with the Naturstoffreagent A which indicated flavones (Figure 1L).
The histochemical tests carried out to detect polysaccharides
compound, using ruthenium Red and PAS, showed up very weak
reactions (not shown).
Another internal glands type with a length of 120 mm (625)
distributed among cortex cells of stems (Figure 2A and B). The
internal glands, differing from external glandular trichomes or
internal glands in leaves, originated from a signal meristematic cell
close to phloem with nucleus and nucleolus (Figure 2C). Through
a series of anticlinal division, a uniseriate row of three cells was
formed (Figure 2D and E). The lower cell of the row corresponded
to the basal cell of glands, the upper to the glands head and the
intermediate to the stalk.
In the young internal gland of stems, the head cell did not have
cuticle outer cell wall and was much smaller than that of mature
internal glands in size (Figure 2E). With a further thickening of
stem, the head cell lengthened and was covered with a thick
cuticle. Fully-developed internal glands consisted of one long,
cytoplasmically dense and cuticle-covered head cell, one narrow
stalk cell and one vacuolated basal cell (Figure 2G). When
subjected to UV light, the secretory material staining with Neutral
red contained in the SCS revealed an intense gold-yellow
secondary fluorescence, whereas slight chloroplasts fluorescing
red (chlorophyll autofluorescence) were seen in stalk region
(Figure 2H). The material secreted into the SCS contained
lipophilic substances as tested with Sudan III (Figure 2I) and
Sudan black B (Figure 2J). Staining with OsO4 (Figure 2K), for
unsaturated lipids, showed positive results which confirmed the
results of the tests using Neutral red, Sudan III and Sudan black B.
The Nadi reaction resulting in an intense violet or blue-violet
staining of the secretion contained in the SCS indicated the
presence of terpenoids (Figure 2L). The fluorochrome for
flavonoid detection, Naturstoffreagenz A induced a yellow-green
secondary fluorescence in head cell and secreted exudate
(Figure 2M). Similar to internal glands of leaves, the glands in
stems gave negative or weak reactions with the tests used for
The release of secreted exudate in SCS of the two internal gland
types to the environment through cuticle was not observed.
However, each of the three external trichome types of Pogostemon
cablin showed intense secretion release in different ways under
cryo-SEM (Figure S2). The secreted material was possibly released
via the random fibrillar network on the reticulate cuticle of
shortstalked capitate glandular trichomes or the gaps between the
spherical accumulations of cutin at the apex of peltate glandular
trichomes (Figure S5). The cuticle of external and internal glands
showed different thicknesses, whereas the cuticle of internal glands
of leaves had similar morphology and thicknesses with that of
internal glands of stems (Figure S5).
Ultrastructure of External Glands
In the secretory stage, the head cells of three external trichome
types of Pogostemon cablin showed different ultrastructural features,
although they all had dense cytoplasm with nucleus and nucleolus,
numerous plastids and small vacuole. In the two secretory cells of
short-stalked capitate glandular trichomes, long and narrow
cisternae of rough endoplasmic reticulum, forming stacks, were
found in the parietal cytoplasm, lying parallel to each other and to
the plasma membrane (Figure 3A and B). And numerous Golgi
vesicles were found in head cell and close to plasma membrane
(Figure 3C). Unlike short-stalked capitate trichomes, the secretory
cell of peltate glandular trichomes contained numerous short
segments of SER which surrounded Golgi and plastids (Figure 3D
and E). And many vesicles between plasma membrane and cell
wall were observed (Figure 3F, arrows). In contrast to short-stalked
capitate and peltate glandular trichomes, the long-stalked capitate
glandular trichomes contained fewer long SER and sparse Golgi
(Figure 3G and J). And the apical cell was strongly polarized.
Numerous small vacuoles occupied the middle and basal parts of
the cell and surrounded the nucleus (Figure 3G and H). The
plastidome consisted of amoeboid plastids that often contained
moderately large plastoglobuli and lipid-like material and
occupied the apical region of the secretory cell (Figure 3I and J).
Similar to peltate glandular trichomes, one vesicle between cell
wall and plasma membrane was present in several specimens
Ultrastructure of Internal Glands
The internal glands located in mesophyll parenchyma
ultrastructurally resembled peltate trichomes (Figure 4A). In early
secretory stage, the head cell of internal glands was characterized
by enlarged plastids, numerous small vacuoles, an extensive short
rough endoplasmic reticulum (RER) and the detachment of the
thick cuticle from the outer cell walls to form an extensive
extracellular SCS (Figure 4B and C). In SCS, a smooth-textured,
lipid-like material occurred within the fibrillar substance as thin,
sheet-like layers (Figure 4B). The mature plastids containing small
plastoglobuli were surrounded by short RER segments (Figure 4C).
RER appeared to closely approach or contact plastids and
osmiophilic deposits were consistently present along RER
segments near the plastids (Figure 4C and D). In addition, RER
seemed to be in close contact with plasma membrane, indicating
the continuity between RER and plasma membrance (Figure 4E).
In the late secretory stage, SCS filled with numerous lipid
spherosomes surrounded by a lot of fibrillar substance became
very big (Figure 4J). Plastids often contained large plastoglobuli
and oil droplets (Figure 4I). Unlike capitate trichomes, the internal
glands lacked Golgi bodies and large vesicles but, numerous
mitochondria were found. The lateral cell wall of the narrow stalk
cell thickened and had become densely staining (Figure 4A,
arrow). The narrow stalk cell with dense cytoplasm contained
larger nucleus and elliptical chloroplasts with starch grains.
Abandent small mitochondria and a few of small vacuoles were
also observed in the narrow stalk cell (Figure 4F). The basal cell
with natural chloroplasts remained vacuolate, and its peripheral
cytoplasm appeared to contain fewer organelles than the stalk or
secretory cell (Figure 4A). The periclinal cell wall, bordering the
stalk cell, usually contained branched plasmodesmata (Figure 4G).
Compared with internal glands in leaves, the internal glands of
stems had one longer, cytoplasmically dense and cuticle-covered
head cell, one narrow stalk cell and one vacuolated basal cell
(Figure 5A). In secretory stage, the SCS formed by the detachment
of the thick cuticle from the outer cell walls was filled with
numerous spherosomes (Figure 5B, asterisks). The secreted
material, stored in the periplasmic space (Figure 5C, asterisks),
passing through the wall, accumulated temporarily in the SCS.
The oil droplets surrounded by membrane-like structure (arrow) in
SCS contained lipid droplets and electron-opaque material outside
the oil droplets (Figure 5D). After the head cell reached the
characteristic structure of a fully-developed gland, numerous
mature plastids with small plastoglobuli, the marked proliferation
of the endomembrane system, an increase in the number of
mitochondria and a lot of small vacuoles were the remarkable
feature of the secretory cell (Figure 5E and I). In this stage, the
secretory cell contained two nuclei with clear nucleolus (Figure 5A).
Plastids were found only in secretory cells, usually in close contact
with short cisternae of smooth endoplasmic reticulum (SER)
(Figure 5I, arrows). These plastids were variable in form and
lacked chloroplasts and starch grains (Figure 5F and I). The small
vacuoles near plastids and short cisternae of SER often contained
larger highly osmiophilic spherosomes (Figure 5F). At the apical
region of the secretory cell, vacuoles variable in form seemed to be
exhausted and in close contact with electron-opaque material
(Figure 5H and L). In addition, vacuoles with electron-opaque
material were often observed near the plasma membrane
(Figure 5L). Short segments of SER filled with osmiophilic droplets
appeared closely approach or contacted the plasma membrane
and plastids (Figure 5G and I). Numerous mitochondria were
extensively distributed within the cytoplasm near other organelles
(Figure 5E). The Golgi was sparse and not well-developed. The
stalk cell had two big vacuoles and cellulosic cell walls that were
stained black. The cytoplasm was dense and had several
mitochondria. The stalk cell contained one triangular nucleus
with clear nucleolus and many plastids with big stanch grains
(Figure 5J). The basal cell was highly vacuolated and its peripheral
cytoplasm appeared to contain fewer organelles than the stalk or
secretory cells. Plasmodesmata occurred frequently between the
head cell and the stalk cell, and also occurred between the basal
cell and the stalk cell, but were less numerous (Figure 5K). There
was not obvious connection between the internal gland and
parenchymal cells around.
Development and Histochemistry
Observations on transverse section of leaves showed internal
glands of Pogostemon cablin distributed among palisade cells, which
(B) portion of head cell showing Golgi (G) and plastids (P) in close contact with rough endoplasmic reticulum (RER) near cell wall (CW); (C) numerous
Golgi (G) with many vesicles near the plasma membrane (PM). (DF) Peltate glandular trichomes in secretory stage: (D) longitudinal section through a
secretory peltate glandular trichome with plentiful electron-light lipid deposits (*) in the sub-cuticular space; (E) the higher magnification of (D)
showing Golgi (G) and plastids (P) in close contact with the short segments of smooth endoplasmic reticulum (SER); (F) vesicles (arrows) are found
between cell wall (CW) and plasma membrane (PM). (GK) Ultrastructural aspects of the long-stalked capitate trichomes: (G) the mature glandular
trichomes with a cytoplasmically dense apical cell, a narrow stalk cell and an elongated vacuolated stalk cell; (H) the higher magnification of (G)
showing the secretory cell with the sub-cuticular space (SCS), many small vacuoles (V), abandent mitochondria (M) and numerous small plastids (P); (I)
the details of the cytoplasm of the secretory cell showing prevalence of mitochondria (M) and plastids (P) with plastoglobuli in the apical region; (J)
the sparse smooth endoplasmic reticulum (SER) close to a lipid-filled plastid (P) (arrow); (K) a bigger vesicle (arrow) between cell wall (CW) and plasma
membrane. (D) Oil droplets in SCS contain lipid droplets (L) and membrane-like structure (arrow) outside the oil droplet. (E) Portion of head cell
showing numerous mitochondria (M), small vacuoles (V) and plastids (P). (F) Vacuoles with electron-opaque material (arrows) are in close to plastid (P)
and mitochondria (M). (G) The smooth endoplasmic reticulum (arrows) is observed to be in close to plasma membrane (PM). (H) Portion of mature
internal glands showing electron-opaque material (arrows) in close contact with vacuoles. (I) The smooth endoplasmic reticulum (arrows) in close to
plastids (P). (J) The stalk cell with thickened lateral wall (arrow) contains big vacuoles (V), the nucleus (N) and numerous plastids (P). (K) The details of
cell wall showing plasmodesmata (arrows) that connect the narrow stalk cell and head cell. (L) The details of head cell showing that vesicles (V) near
plasma membrane (PM) are in close connect with electron-opaque material (arrows).
was similar to the intact sub-dermal secretory cavities of Eucalyptus
 and elaiophores of Oncidium trulliferum . In contrast to the
elaiophores of Oncidium trulliferum which secreted and stored oils
under a distinct subepithelial layer, the oils of the internal glands
were secreted and stored in the SCS like the external glandular
trichomes. Compared to the internal glands in leaves, numerous
internal glands with long head cell among cortex cells of stems had
bigger SCS filled with oil droplets. Observations on the
development of the two internal glands types showed that the
glands of stems originated from a single meristematic cell close to
phloem and the internal glands in leaves originated from one
undifferentiated palisade cell. These results indicated that the two
internal gland types both originated from similar ground
meristem. The absence of the two internal gland types in
primordial leaves and apical dome of the stem apex suggested
cellular differentiation at a later stage during the maturation of
stems and leaves. However, cell differentiation terminates in early
leaf development, which is described by Cutter . These
observations may not provide further evidence for this hypothesis.
An abundance of terpenoids was reported in the leaves and
stems of Pogostemon cablin . Monoterpenes dominate the
secretory material of Rutaceae species  and the Lamiaceae
; Sesquiterpenes and other terpenes are the main components
of the essential oil of the Asteraceae . Terpenoids have many
different functions in plants such as attracting pollinating insects to
flowers or protecting the plant from destruction by herbivores and
other pathogens [27,28]. The result of the Nadi reagent test
revealed the presence of the terpenoids in the secretory material
stored in SCS of all mature gland types of Pogostemon cablin. The
abundant terpenoids in the external and internal glands of
Pogostemon cablin may protect the plant from destruction by
herbivores through influencing their alimentary system.
Histochemical test on the two fully developed internal glands indicated
that the secretion contained lipophilic components. These results
are similar to the phytochemical data available for other species of
the Lamiaceae . The presence of lipid compounds in palisade
tissue has also been reported by Maria et al.  in Lantana camara.
Only the internal glands and short-stalked capitate glandular
trichomes contained flavones. The flavones had a diverse array of
physiological functions, not only acting as antioxidants and/or
sunscreen pigments to protect plants from oxidative and UV light
damage but also as modulators of auxin transport [31,32]. These
functions of flavones had been accentuated by the presence of
flavonoids in internal glands. And polysaccharides were found only
in the two capitate glandular trichome types. The different
composition of the secretory material in the five secretory gland
types may suggest that the function of these epidermal appendages
and internal glands differed from each other.
Interestingly, the composition of the secretory material in
internal glands which distributed among the cortical cells of stems
was similar with the internal glands in leaves. And the two internal
gland types with similar internal cuticle both originated from
ground meristem. Taking the similarities in development,
morphology and histochemistry between the two internal gland types
into consideration, it is possible that these two secretory gland
types have close evolutionary relationship, although their locations
and size are different. And the similar structures of two internal
gland types with peltate glandular trichomes, including a
subcuticular space filled with secretory material and three
components: a basal region, a stalk region and a head region, lead us to
purpose that the internal glands of Pogostemon cablin may be the
similar secretory structure type with peltate glandular trichomes.
However, the internal glands had different origin compared with
external glandular trichomes. Possible differences within the
secretory glands, as observed through developmental differences
between internal glands and external glandular trichomes, were
accentuated by the absence of polysaccharides and the presence of
flavonoids in internal glands. In addition, the distribution of glands
in Pogostemon cablin and the absence of secretion release in internal
glands also indicated the difference between internal glands and
The stalk cells of the mature internal glands observed in this
study all appeared to contain densely-stained cytoplasm, unusual
plastids with starch grains, and an intact nucleus, occasionally with
a moderately large nucleolus. These observations, together with
the presence of obvious plasmodesmata on the periclinal walls of
2, negative; +, positive.
stalk cells and the lack of chloroplasts in the head cells indicate that
it is likely that these cells are related to their essential role in the
supply of carbon substrates to the non-photosynthetic head cells.
In addition, the lateral wall of the stalk cells of external glandular
trichomes forms a boundary to the exterior and is heavily
suberized. It is interesting that similar suberization of lateral wall
was observed in internal glands. The thickened lateral wall is
deemed to contribute to support the head cell, as supposed by
previous work. As the internal glands are among the palisade cells
or cortex cells, it is unlikely that stalk cells are specialized for
supporting the head cell. The result of histochemistry tests of
mature internal glands showed that there was weak reaction in
stalk cells. It seems that at least some of the specializations of stalk
cells of internal glands are related to essential oil biosynthesis, not
only supply of carbon substrates.
Site of Oil Biosynthesis
In the previous studies about secretory cells of external glands
from taxonomically distant plants, similar ultrastructural features,
including dense cytoplasm, extensive endoplasmic reticulum,
amoeboid leucoplasts, relatively few Golgi, and abundant
mitochondria, have been noted [13,16,3334]. Basing largely upon
observations of the apparent sites of lipid accumulation, previous
investigators supposed the possible sites of oil biosynthesis such as
SER , vacuoles , cytoplasm , and the combination of
plastids and SER . However, there is a lack of information on
the site of monoterpene and lipids biosynthesis of the internal
glands in Pogostemon cablin.
The observations obtained by TEM showed that the internal
glands of leaves in early secretory stage contained abundant small
plastids which were reported to be found only in external
glandular trchomes. Following the accumulation of secretory oil
in the SCS, the plastids became mature and contained internal
membranes and small plastoglobuli, similar to that noted in other
plants by Glenn et al.  and Franceschi and Giaquinta .
The oil droplets in mature plastids strongly indicated that the
plastids may play an important role in oil biosynthesis. The
osmiophilic inclusions-containing RER and the very close
association with plastids suggests an important role for RER in
lipids biosynthesis, as suggested in Peppermint by Glenn and Rodney
Similar to the internal glands of leaves, the main ultrastructural
characteristics of the fully secreting internal glands in stems,
including numerous plastids in close contact with a well-developed
tubular membrane system filled with osmiophilic inclusions,
abundant mitochondria, small vacuoles containing
electronopaque material, poorly developed Golgi bodies and lipid-like
material in close contact with small vacuole, were first observed in
the present work. These observations supply evidence that internal
glands in stems are typical lipid and monoterpenes secreting
glands. Although the lipids-containing plastids were not observed
in internal glands of stems, the extensive development of the
plastid compartment with profuse tubular osmiophilic structures,
together with the high proliferation of the SER filled with
osmiophilic inclusions, indicated that the plastids and SER may
play an important role in lipids and monoterpenes biosynthesis, as
suggested in other plants [11,16,37]. Amelunxen  has
supposed that vacuoles are the possible sites of terpenoid synthesis.
In the long secretory cell of internal glands in stems of Pogostemon
cablin, small vacuoles containing electron-opaque material and
lipid-like material in close contact with small vacuoles strongly
suggested that the vacuoles may have an important role in lipids
biosynthesis. In addition, during the excretion of the lipid-like
material out of small vacuoles, the vacuoles seemed to be
exhausted anomalous and the extraplasmatic space was much
enlarged. Thus, the possible role of vacuoles in lipids biosynthesis
has been accentuated by these observations. However, no futher
evidence has been ovserved to support the hypothesis. As supposed
by previous researchers [6,34,40], vacuoles may not produce, but
only process the secretory material.
Histochemical staining has shown that, in addition to lipids and
terpenoids, the internal glands in stems and leaves of Pogostemon
cablin also contained abundant flavones. And it is possible that
some of the ultrastructural features described here may represent
specializations for the biosynthesis of the flavones. The remarkable
changes of SER during the development of internal glands may
indicate the role of SER in flavones biosynthesis, which has been
suggested by the location of flavonoid and phenylpropanoid
biosynthetic enzymes in ER [41,42].
Similar to internal glands, plastids in three external glandular
trichome types of Pogostemon cablin were the organelles that showed
the most striking changes during the development of the
trichomes. Although the secretory cells of peltate trichomes and
short-stalked capitate trichomes in this work did not contain the
lipid-containing plastids which could indicate the important role of
plastids in the oil biosynthesis, the correlation between the striking
changes of plastids and the secretory process may suggest that the
plastids had a role in lipids and monoterpene biosynthesis. And the
lipid-containing plastids in long-stalked capitate trichomes strongly
indicated the important role of plastids in the lipids biosynthesis.
However, the difference in the number of ER and Golgi among all
the glands types of Pogostemon cablin may suggest their different roles
in the oil biosynthesis.
Possible Secretory Mechanisms
Basing upon the very close association of SER with plastids and
the plasma membrane, and the presence of lipid deposits within
the SER, previous investigators postulated a secretion mechanism
for secretory material mediated by direct SER-plasma membrane
connections . Multivesicular bodies, as noted by Tanchak
and Fowke  and Tse et al. , connect with both endocytosis
and secretion. And Paramural bodies, as reported in Claceolaria
trichomes  and Genlisea Digestive Hairs , may also play an
important role in the transport of synthesized material.
In the internal glands of leaves and stems of Pogostemon cablin, we
suggest that the secretory material may also be transported directly
from ER to the cell wall via connecting ER membranes with the
plasmalemma. The abundent secretory oil in the peripheral
cytoplasm and periplasmic space, as described in Zeyheria Montana
by Silvia et al. , indicated the accumulation of oil not through
multivesicular bodies or paramural bodies. Many previous
investigators have noted similar ultrastructural features of the
secretory oil in the SCS of mature external glands. Unlike the
electron-light lipid deposits in other secretory glands of Pogostemon
cablin, the secretory material in the SCS of mature internal glands
in stems appeared many spherosomes with membrane-like
structure and electron-light lipid droplets among electron-opaque
substance. We suppose that the electron-opaque substance in SCS
is as same as the electron-opaque material in close contact with
small vacuoles. And the small vacuole with electron-opaque
material close to plasma membrane suggested that the export of
this material from the secretory cells to the storage space may be
similar to the lanthanum transport mechanisms in Atriplex halimus
, although a series of oil-containing small vacuole connecting
the big vacuole and the plasma membrane were not observed. And
the SCS of internal glands in leaves filled with numerous lipid
spherosomes surrounded by a lot of fibrillar substance whose
function was not clear.
However, not only RER in close contact with plasma
membrane was observed, but also abundant vesicles which were
similar to paramural bodies were observed in the secretory cells of
peltate glandular trichomes. In addition, abundant Golgi stacks
with numerous bodies of the short-stalked capitate trichomes
during the secretory phase were observed to have close approach
with RER and plasma membrane. In the secretory cell of
longstalked capitate trichomes, the vesicle was observed between cell
wall and plasma membrane. Thus, it is possible that there are
several ways in which secretory material is transported in external
glands. We suggest a similar secretion mechanism to internal
glands for short-stalked capitate trichomes by direct SER-plasma
membrane connections. And abundant vesicles between cell wall
and plasma membrane in peltate trichomes and long-stalked
capitate trichomes may suggest the export of secretory oil by the
paramural bodies process, as postulated in Claceolaria trichomes
 and Genlisea Digestive Hairs . The Golgi bodies in close
contact with plasma membrane suggest that the final secretion
product may be transported to the cell surface via Golgi vesicles,
and released into the periplasmic space through the fusion of Golgi
vesicles with plasma membrane. A similar mechanism of secretion
has been reported in other trichomes [13,51]. The way in which
the secretory oil is transported from secretory cells to SCS may
provide new evidence to point out the difference between the
external and internal glands. And the different way of essential oil
secretion in different glands may indicate the diversity of the
export of substance.
In summary, the data on developmental stage, histochemistry
and ultrastructures about the site of oil biosynthesis and secretory
mechanism of internal glands in the study indicated that the
internal glands may be a novel secretory structure distributed
within the plant which was different from external glands on
leaves, although they all had similar structures. The internal glands
appeared inside stems and leaves and occurred later than external
glands during the development that may reveal the adaptability of
secretory glands to plant diseases and insect pests stress in long
The identical composition of the secretory material and the
similar ultrastructure between the two types of internal glands,
including plastids with similar shape, poorly developed Golgi
bodies, numerous short cisternaes of rough endoplasmic reticulum
and thin cuticle, indicated that there may be close evolutionary
relationship between the two internal gland types. And the
distribution of internal glands from leaves to stems may also
further reflect its biological function and adaptability to
environment in different plant organs. Each of these lipid secreting glands
had its specific ultrastructure which may reveal the site of lipid
biosynthesis and may have different function. They demonstrated
a different way of secretory processing and releasing. These results
indicated the diversity of the site of oil biosynthesis and secretory
mechanisms of secretory glands in a given species. For the question
as to why and for what Pogostemon cablin leaves and stems need all
these different gland types, further clarification of the functions
and ecophysiological and evolutionary roles of these secretory
glands is required.
Materials and Methods
To establish the experiments, Pogostemon cablin was grown in
growth chambers under defined climatic conditions with a
photoperiod of 16 h. Day and night temperatures were
respectively 22uC and 18uC. And the relative humidity was from 50% to
70%. Fresh leaves at various developmental stages of maturity
were selected for the investigation of glandular trichomes and
internal glands. Young stem and mature stem were harvested for
the study of internal glands.
Scanning (SEM) and Transmission (TEM) Electron
For conventional scanning electron microscopy (CSEM), leaves
and stems at various developmental stages were fixed in
glutaraldehyde (2.5% with 0.1 M phosphate buffer, at pH 7.3,
overnight at 4uC). Sectional material was washed in the phosphate
buffer (pH 7.3). Dehydration was done in an acetone dilution
series (30%, 50%, 70%, 90%, followed by 36100%). After critical
drying with Leica EM CPD300 automated critical point dryer, the
samples were mounted on double-sided carbon tape on stubs.
They were then plasma coated with 10 nm gold and viewed with a
Hitachi S-3400N scanning electron microscope. For cryo-SEM,
samples were fixed in liquid nitrogen, sublimated and gold-coated
in Quorum PP2000T Cryo-SEM system. For transmission
electron microscopy, samples were fixed in glutaraldehyde (2.5%
with 0.1 M phosphate buffer, at pH 7.3), post-fixed in osmium
tetroxide (1%, in 0.1 M phosphate buffer, pH 7.3), dehydrated in
an acetone dilution series and infiltrated with Eponate 12 resin.
Sections for transmission electron microscopy were cut to a
thickness of 70 nm with diamond knives and a Leica EM UC6
ultramicrotome. The sections were stained with either 1% (w/v)
aqueous uranyl acetate and 1% (w/v) lead citrate. Specimens were
viewed with a JEM-1230 (JEOL, Tokyo) transmission electron
The development and histochemistry of external trichomes and
internal glands were studied with light microscopy. Sections for
light microscopy were cut to a thickness of 0.5 to 1 mm with glass
knives and stained with toluidine blue. The main classes of
metabolites in secreted material of glandular trichomes were
observed in fresh and fixed hand-sections, using following different
histochemical tests. Neutral red and Sudan black B were used to
localize total lipids, osmium tetroxide for unsaturated lipids,
Naturstoffreagent A for detection of flavonoids (under UV 365
emission LP 397), periodic acid-Schiff (PAS) reagent for
polysaccharides, Sudan III for lipids, NADI reagent for terpenes,
ruthenium red for pectins. The observations were made under
an Olympus microscope.
Figure S1 Morphology and distribution of glandular
trichomes on the surface of Pogostemon cablin. (AF)
(SEM): Adaxial surface (A) and abaxial surface (B) view with
peltate glandular trichomes and short-stalked capitate glandular
trichomes in secretory phase. (C) The surface view of stems
showing long-stalked capitate trichomes (arrows). (D) Long-stalked
capitate trichomes (arrows) among glandular trichomes and
nonglandular trichomes on the leaf veins. (E) Distribution of glandular
trichomes on stem apex of Pogostemon cablin. (F) Higher
magnification of (E) showing fully developmental peltate glandular
trichomes and short-stalked capitate glandular trichomes and the
tuberculate epidermal cells depicted by arrows.
Figure S2 SEM micrographs showing the morphology
and the secretion of three glandular trichome types in
Pogostemon cablin. (AC) The secretion of short-stalked
capitate glandular trichomes: (A) Pre-secretory stage with the
secretory centre (arrow); (B) beginning of the secretory release
(arrow); (C) the secretory droplets are getting bigger and the
number increase to two (arrows). (DF) The secretion of
longstalked capitate glandular trichomes: (D) beginning of the secretory
release and the oil droplet is small (arrow); (E) the oil droplet is
getting bigger (arrow); (F) the small oil droplet (arrow) outside the
apical cell. (GI) The secretion of peltate glandular trichomes: (G)
Pre-secretory stage without thickened cuticle; (H) secretory stage
with protruding cuticle; (I) the collapse of the sub-cuticular space
Figure S3 Semithin sections of three glandular
trichome types in different developmental phases showing
the process of development. (AE) The development of
shortstalked capitate glandular trichomes: (A) protruding epidermal cell
with an asymmetrical cytoplasmic distribution containing
vacuolate basal portions and cytoplasmically dense apical portions; (B)
two-celled stage with one cytoplasmically dense apical cell and one
vacuolate cell; (C) three-celled stage with one big cytoplasmically
dense apical cell; (D) four-celled stage with two cytoplasmically
dense apical cells without cuticle; (E) mature short-stalked capitate
glandular trichomes with one sub-cuticular space containing
essential oil (arrow). (FJ) The developmental process of
longstalked capitate glandular trichomes: (F) protruding epidermal cell
with a vacuolate basal region and an apical region containing the
nucleus; (G) glandular trichome initial after periclinal cell divisions
with a vacuolate basal cell and a apical cell containing the nucleus
in the apical region; (H) three-celled stage showing a vacuolate
basal cell,a vacuolate stalk cell, and an apical region containing the
nucleus; (I) glandular trichomes in pre-secretory stage with a
cytoplasmically dense apical cell, a narrow stalk cell and an
elongated stalk cell; (J) mature long-stalked glandular trichomes
with one sub-cuticular space containing essential oil (arrow). (K
O) The developmental process of peltate glandular trichomes: (K)
protruding epidermal cell with a vacuolate basal region and an
apical region containing the nucleus; (L) two-celled stage with one
cytoplasmically dense apical cell and one vacuolate basal cell; (M)
three-celled stage with one cytoplasmically dense apical cell
containing two nucleus, one narrow stalk cell and one vacuolate
basal cell; (N) mature peltate glandular trichomes with one
subcuticular space (arrow); (O) post-secretory glandular trichomes
with the collapse of the sub-cuticular space.
Figure S4 Bright field and fluorescence micrographs of
three glandular trichome types showing histochemical
characterization of secretory products. (AH)
Histochemistry of the short-stalked capitate glandular trichomes: (A)
Ruthenium Red test showing the apical cells stained red; (B)
gold-yellow secondary fluorescence observed with Neutral Red
under UV light; (C) positive staining reaction with Sudan III in the
apical cells and weak reaction in the stalk cell; (D) the apical cells
stained blue with Sudan Black B and the stalk cell stained black;
(E) OsO4 test showing the apical cells and the droplet (arrow)
stained black; (F) NADI staining for terpenes is positive in the
apical cells; (G) the apical cells react positively with
Naturstoffreagent A; (H) PAS test for polysaccharides in apical cells. (IO)
Histochemistry of the long-stalked capitate glandular trichomes: (I)
ruthenium Red test showing the apical cells stained red; (J) yellow
staining of secretion in sub-cuticular space with Neutral red; (K)
secretory material stained with Sudan III; (L) positive staining
reaction with Sudan Black B; (M) black staining of secretion with
OsO4, secretory process is visible; (N) secretory material reacts
positively for terpenes with NADI; (O) mature trichome reacts
positively in PAS test for polysaccharides in the head cell and stalk
cell. (PT) Histochemistry of the peltate glandular trichomes: (P)
gold-yellow secondary fluorescence observed with Neutral Red
under UV light; (Q) secretory material in the sub-cuticular space
positive stained with Sudan III; (R) staining for total lipids with
Sudan Black B; (S) positive staining reaction with OsO4 in the
head cells and weak reaction in the narrow stalk cell; (T) NADI
staining for terpenes is positive in the sub-cuticular space.
Figure S5 EM micrographs showing the cuticle of
mature external and internal glands in Pogostemon
cablin. (A) A random fibrillar network (arrow) on the reticulate
cuticle of short-stalked capitate glandular trichomes is evident; (B)
the thin cuticle of long-stalked capitate glandular trichomes; (C)
TEM and (D) SEM micrographs showing the gaps (arrow)
between the spherical accumulations of cutin at the apex of
peltate glandular trichomes; (E) the cuticle of internal glands in
leaves with high electron density; (F) the cuticle of internal glands
Conceived and designed the experiments: JG YY JZ. Performed the
experiments: JG. Analyzed the data: ZL JZ. Contributed reagents/
materials/analysis tools: YY JZ. Wrote the paper: JG.
1. Werker E , Putievsky E , Ravid U , Dudai N , Katzir I ( 1993 ) Glandular hairs and essential oil in developing leaves of Ocimum basilicum L. (Lamiaceae) . Annals of Botany 71 : 43 - 50 .
2. Werker E , Ravid U , Putievsky E ( 1985a ) Structure of glandular hairs and identification of the main components of their secreted material in some species of the Labiatae . Israel Journal of Botany 34 : 31 - 45 .
3. Fahn A ( 1988 ) Secretory tissues in vascular plants . New Phytologist 108 : 229 - 257 .
4. Fahn A ( 2000 ) Structure and function of secretory cells . In: Hallahan DL , Gray JC , Callow JA, eds. Advances in botanical research incorporating advances in plant pathology , volume 31 : plant trichomes . London: Academic Press.
5. Ascensao L , Marques N , Pais MS ( 1995 ) Glandular trichomes on vegetative and reproductive organs of Leonotis leonurus (Lamiaceae) . Annals of Botany 75 : 619 - 626 .
6. Huang SS , Bruce K Kirchoff , Liao JP ( 2008 ) The capitate and peltate glandular trichomes of Lavandula pinnataL . (Lamiaceae): histochemistry, ultrastructure, and secretion. Journal of the Torrey Botanical Society 135 ( 2 ): 155 - 167 .
7. Rodriguez E , Healy PL , Mehta I ( 1984 ) Biology and Chemistry of Plant Trichomes . New York : Plenum Press.
8. Wagner GJ ( 1991 ) Secreting Glandular Trichomes: More than Just Hairs . Plant Physiology 96 : 675 - 679 .
9. Venkatachalam KV , Kjonaas R , Croteau R ( 1984 ) Development and essential oil content of secretory glands of sage (Salvia officinalis) . Plant Physiology 76 : 148 - 150 .
10. Werker E ( 2000 ) Trichome diversity and development . In Advances in Botanical Research Incorporating Advances in Plant Pathology , Vol. 31 : Plant Trichomes (Hallahan, D.L. and Gray , J.C., eds). San Diego/Boston/London: Academic Press. 1 - 35 .
11. Turner GW , Gershenzon J , Croteau RB ( 2000 ) Development of Peltate Glandular Trichomes of Peppermint . Plant Physiology 124 : 665 - 679 .
12. Heinrich G , Schultze W ( 1985 ) Composition and site of biosynthesis of the essential oil in fruits of Phellodendron amurense Rupr . (Rutaceae). Israel Journal of Botany 34 : 205 - 217 .
13. Ascensao L , Pais MS ( 1998 ) The leaf capitate trichomes of Leonotis leonurus: Histochemistry, ultrastructure and secretion . Annals of Botany 81 : 263 - 271 .
14. Ascensao L , Marques N , Pais MS ( 1997 ) Peltate glandular trichomes of Leonotis leonurus leaves: ultrastructure and histochemical characterization of secretions . International Journal of Plant Sciences 158 : 249 - 258 .
15. Gersbach PV ( 2002 ) The essential oil secretory structures of Prostanthera ovalifolia (Lamiaceae) . Annals of Botany 89 : 255 - 260 .
16. Machado SR , Gregorio EA , Guimaraes E ( 2006 ) Ovary Peltate Trichomes of Zeyheria montana (Bignoniaceae): Developmental Ultrastructure and Secretion in Relation to Function . Annals of Botany 97 : 357 - 369 .
17. Maeda E , Miyake H ( 1997 ) Leaf Anatomy of Patchouli (Pogostemon patchouli) with Reference to the Disposition of Mesophyll Glands . Japanese Journal of Crop Science 66 ( 2 ): 307 - 317 .
18. Henderson W , Hart JW , How P , Judge J ( 1970 ) Chemical and morphological studies on sites of sesquiterpene accumulation in Pogostemon cablin (Patchouli) . Phytochemistry 9: 1219 - 1228 .
19. Nabeta K , Kawachi J , Sakurai M ( 1993 ) Volatile production in cultured cells and plantlet regeneration from protoplast of patchouli, Pogostemon cablin . Developments in food science 32: 577 - 589 .
20. Sugimura Y , Toi N ( 1991 ) Yield and quality of essentia1 oils from aroma crops . Japanese Journal of Crop Science 60 : 324 - 331 .
21. Goodger JQD , Heskes AM , Mitchell MC , King DJ , Neilson EH , etc. ( 2010 ) Isolation of intact sub-dermal secretory cavities from Eucalyptus . Plant Methods 6 : 20 .
22. Stpiczynska M , Davies KL ( 2008 ) Elaiophore Structure and Oil Secretion in Flowers of Oncidium trulliferum Lindl. and Ornithophora radicans (Rchb .f.) Garay & Pabst (Oncidiinae: Orchidaceae) . Annals of Botany 101 : 375 - 384 .
23. Cutter EG ( 1980 ) Plant anatomy - experiments and interpretation . Part 2. Organs . London: Edward Arnold Publishers.
24. Heinrich G , Schultze W ( 1985 ) Composition and site of biosynthesis of the essential oil in fruits of Phellodendron amurense Rupr . (Rutaceae). Israel Journal of Botany 34 : 205 - 217 .
25. Lawrence BM ( 1992 ) Chemical components of labiate oils and their exploitation . Advances in Labiatae science 399-436.
26. Spring O ( 2000 ) Chemotaxonomy based on metabolites from glandular trichomes . Advances in Botanical research 31 : 153 - 174 .
27. Kelsey RG , Reynolds GW , Rodriguez E ( 1984 ) The chemistry of biologically active constituents secreted and stored in plant glandular trichomes . In: Rodriguez E, Healey PL , Metha J, eds. Biology and chemistry of plant trichomes . New York : Plenum Press. 187 - 241 .
28. Harborne JB ( 1993 ) Introduction to ecological biochemistry . 4th edn. London: Academic Press.
29. Richardson PM ( 1992 ) The chemistry of the Labiatae: an introduction and overview . Advances in labiate science 291-297.
30. Moura MZD , Isaias RMDS , Soares GLG ( 2005 ) Ontogenesis of internal secretory cells in leaves of Lantana camara (Verbenaceae) . Botanical Journal of the Linnean Society 148 : 427 - 431 .
31. Shirley BW ( 2001 ) Flavonoid biosynthesis: a colorful model for genetics, biochemistry, cell biology, and biotechnology . Plant Physiology 126 : 485 - 493 .
32. Buer CS , Imin N , Djordjevic MA ( 2010 ) Flavonoids: new roles for old molecules . J Integr Plant Biol 52 : 98 - 111 .
33. Amelunxen F ( 1965 ) Elektronenmikroskopische Untersuchungen an den Drusenschuppen von Mentha piperita L. Plant Methods 13 : 457 - 473 .
34. Machado SR , Gregorio EA , Yanagizawa Y , Carmello SM ( 1995 ) Ultrastructural aspects of the peltate glandular trichomes of the gynoecium in Zeyheria digitalis (Vell.) Hoehne (Bignoniaceae) . Brazilian Journal of Botany 18 : 197 - 205 .
35. Schnepf E ( 1972 ) Tubulares endoplasmatisches Reticulum in Drusen mit lipophilen Ausscheidungen von Ficus , Ledum, und Salvia. Biochem Physiol Pflanz 163 : 113 - 125 .
36. Bosabalidis A , Tsekos I ( 1982 ) Glandular scale development and essential oil secretion in Origanum dictamnus L. Planta 156 : 496 - 504 .
37. Bourett TM , Howard RJ , O'Keefe DP , Hallahan DL ( 1994 ) Gland development on leaf surfaces of Nepeta racemosa . Int J Plant Sci 155 : 623 - 632 .
38. Franceschi VR , Giaquinta RT ( 1983 ) Glandular trichomes of soybean leaves: cytological differentiation from initiation through senescence . Bot Gaz 144 : 175 - 184 .
39. Turner GW , Croteau R ( 2004 ) Organization of Monoterpene Biosynthesis in Mentha . Immunocytochemical Localizations of Geranyl Diphosphate Synthase, L imonene-6-Hydroxylase , Isopiperitenol Dehydrogenase , and Pulegone R eductase. Plant Physiology 136 : 4215 - 4227 .
40. Zheng B , Yu L , Xing S , Wang F ( 2002 ) Ultrastructure of the secretion of peltate glandular hairs in Ocimum basiliumL . Bull. Bot. Res . 22 : 176 - 180 .
41. Burbulis IE , Shirley BW ( 1999 ) Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway . Proc Natl Acad Sci USA 96 : 12929 - 12934 .
42. Shirley BW ( 1999 ) Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways . Plant Physiology 107 : 142 - 149 .
43. Robards AW , Stark M ( 1988 ) Nectar secretion in Abutilon: a new model . Protoplasma 142 : 79 - 91 .
44. Robins RJ , Juniper BE ( 1980 ) The secretory cycle of Dionea muscipula Ellis . I. Fine structure and the effect of stimulation on the fine structure of the glands . New Phytology 86 : 279 - 296 .
45. Vassilyev AE , Muravnik LE ( 1988 ) The ultrastructure of the digestive glands in Pinguicula vulgaris L. (Lentibulariaceae) relative to their function . I. The changes during maturation . Annals of Botany 62 : 329 - 341 .
46. Tanchak MA , Fowke LC ( 1987 ) The morphology of the multivesicular bodies in soybean protoplasts and their role in endocytosis . Protoplasma 138 : 173 - 182 .
47. Tse YC , Mo B , Hillmer S , Zhao M , Lo SW , Robinson DG , et al ( 2004 ) Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells . The Plant Cell 16 : 672 - 693 .
48. Sacchetti G , Romagnoli C , Nicoletti M , Di Fabio A , Bruni A , et al. ( 1999 ) Glandular trichomes of Calceolaria adscendens Lidl . (Scrophulariaceae) : histochemistry, development and ultrastructure . Annals of Botany 83 : 87 - 92 .
49. Pachno BJ , Kiszkurno MK , S wiatek P ( 2007 ) Functional Utrastructure of Genlisea (Lentibulariaceae) Digestive Hairs . Annals of Botany 100 : 195 - 203 .
50. Smaoui A , Barhoumi Z , Rabhi M , Abdelly C ( 2011 ) Localization of potential ion transport pathways in vesicular trichome cells of Atriplex halimus L. Protoplasma 248 : 363 - 372 .
51. Meyberg M ( 1988 ) Cytochemistry and ultrastructure of mucilage secreting trichomes of Nymphoides peltada (Menyanthaceae) . Annals of Botany 62 : 537 - 547 .