RESEARCH COMMUNICATIONS
Developmental process of essential oil
glandular trichome collapsing in
menthol mint
Shruti Sharma, N. S. Sangwan and
Rajender S. Sangwan*
Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP,
Lucknow 226 015, India
Essential oil glandular trichomes are the specialized
anatomical and structural characteristic of plants
amassing significant quantities of commercially and
pharmaceutically valuable essential oil terpenoids.
Developmental dynamics of these structures together
with the oil secretory process and mechanisms have a
direct bearing with the secondary metabolite production, sequestration and holding potential of the producer systems. Therefore, in this study, essential-oil
gland trichomes of menthol mint leaf have been
stereologically analysed to discern their anatomical
archetype vis-à-vis volatile oil secretion and sequestration as integrated in the overall ontogeny of leaf.
Cuticular ‘dehiscence’ or decapping, leading to collapsing of the peltate trichomes was found to be a
notable characteristic of the menthol mint oil glands.
Ecophysiological,
evolutionary,
phytopharming and
biotechnological connotations of the novel phenomenon have been hypothesized.
ESSENTIAL oils have been valued historically for their
aesthetic, culinary, flavoural, fragrance and medicinal
properties1–8 . Mints (Mentha species, Lamiaceae) share
the largest volume of volatile oil traded worldwide and
display enormous diversity in commercial and consumer
utility. Menthol mint (Mentha arvensis L.) is one of the
most important among them, owing to preponderance
(> 80%) of menthol in its leaf essential oil6 . Menthol is
probably the most traded single monoterpene at a global
scale6 . Recently, the refreshing, soothing and thermosensation effects of menthol have been traced to a molecular
site (menthol sensitive receptor, a member of long transient receptor potential family of ion channels) of its
action in trigeminal and dorsal root ganglia9 . The commercial, therapeutic, thermosensation and other nutraceutical values of the volatile oils and/or their specific
chemical constituent(s), have given an impetus to understanding of the processes of biosynthesis, as well as secretion of the oil, to envision the metabolic engineering
and molecular modulation for better phytochemical harvests. Varied researches have yielded substantial insights
into the novel DOXP pathway of biosynthesis of monomeric biological isoprene units (isopentenyl pyrophosphate, IPP and dimethyl allyl pyrophosphate, DMAPP)
for plastid limited terpenoid (oil) production (as reviewed
*For correspondence. (e-mail: [email protected])
544
in refs 1, 8, 10–16), metabolic steps and genes involved
in generation of diverse metabolites, and impact of overexpression or suppression of some of the pathway
genes 8,17–19 . Since the pathway of IPP and DMAPP synthesis for monoterpenes has been enzymologically delineated only during last couple of years, a brief but fully
updated metabolic mode of menthol biosynthesis is presented in Figure 1, for ready reference of the readers.
Nevertheless, over and above the biosynthetic machinery, certain oil-producing plants are bestowed with specialized and characteristic anatomical structures called
essential-oil glands, a type of glandular trichomes, capable of secreting and sequestering the secondary metabolic
products in significant quantities1 . Where present, the oil
glandular trichomes are the primary sites of biosynthesis
of essential oil, and the plants that lack such specialized
structures can synthesize and amass only trace quantities
of monoterpenes1,17–19 . Accordingly, developmental dynamics of these structures together with the oil secretory
process(s) and mechanism(s) have a direct bearing with
the oil production/holding potential of the producer system. Therefore, in this study, essential-oil glandular
trichomes of menthol mint leaf were stereologically analysed to discern their anatomical archetype vis-à-vis
volatile oil secretion and sequestration as integrated in
the overall ontogeny of leaf. This communication describes developmental collapsing or ‘dehiscence’ of a mint
oil peltate gland and gives an account of its putative ecophysiological, evolutionary, phytopharming and biotechnological connotations.
M. arvensis L. cultivar Kalka plants were grown from
suckers at the Experimental Farm of Central Institute of
Medicinal and Aromatic Plants, Lucknow (26.5°N latitude, 80.5°E longitude, 120 m above msl, subtropical,
semi-arid zone with hot summers and cold winters) following standard agronomic practices. A priori, leaves
were tagged at the time of emergence and their developmental pattern was discerned with respect to phasicphysiology of leaf expansion, maturation and senescence,
as described earlier6 . The leaf samples at initiation (early
expansion, 15%), active growth (50% expansion), maturation and senescence were subjected to scanning electron microscopy (SEM) of both the adaxial and abaxial
surfaces. Light microscopy (LM) of fresh samples was
also carried out to display the key events under aqueous
conditions, wherever required.
Ultra-morphological examination of ontogenic features
of essential oil glands per se was carried out through
SEM following the procedure described by Hayat20 for
both adaxial and abaxial leaf surfaces. Briefly, the leaf
samples were cut into 1.0 mm2 squares and thoroughly
washed with double-distilled water to remove any adherent dust particles. The samples were fixed in 5% (v/v)
aqueous solution of glutaraldehyde prepared in 0.05 M
phosphate buffer (pH 7.0) for about 10 h. The fixed
specimens were repeatedly washed in the buffer solution.
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Figure 1.
Isoprenogenic pathway for major volatile secondary metabolites in menthol mint.
Washed specimens were kept in 1% osmium tetraoxide
for 3–4 h, followed by washing with double-distilled
water and then standard dehydration through graded
alcohol series (50, 70, 90 and 100% ethanol, v/v, 30 min
each). Alcohol-dehydrated specimens were subjected to
critical-point dehydration/drying (CPD) in a critical-point
drier at 31°C and 7.3 × 106 Pa to remove any fluid as
vapour. The dried specimens were mounted on a doublesided adhesive tape on metallic stubs with the adaxial and
abaxial surfaces visualizable separately. These were further coated with silver dag and finally coated with gold–
palladium alloy under sputter-coating unit (Polaron,
Model E-5000) at 10–20 kVA. The specimens were electron-microscopically examined by mounting in the SEM
chamber (Phillips, Model 505) and applying accelerating
voltage (20–30 kVA) to obtain optimum image of the oil
glands at 100 × and 800 × magnification.
LM examination of the non-dehydrated, aqueously cut,
glycerine-mounted sections of leaf was done at 40 ×
magnification under a standard phase contrast stereo-binocular compound optical microscope with on-line image
capture facility.
The essential oil glandular trichomes in menthol mint
leaf were noted to exist as epidermal structures in semiCURRENT SCIENCE, VOL. 84, NO. 4, 25 FEBRUARY 2003
depression to the plane of leaf surface and were present
on both adaxial as well as abaxial leaf surfaces, similar
but not identical to several other volatile oil plants of
Lamiaceae21–24 . We had shown earlier, through transmission electron microscopic (TEM) studies, that the oil
glandular trichomes in menthol mint leaf were comprised
of two types of sub-populations: large and multi (8)-cell
secretory head containing peltate glands/trichomes, and
small and single-head celled capitate glands/trichomes6 .
The leaf had abundance (ca. 4 : 1) of peltate glands compared to capitate glands6 . This, together with the relatively much larger oil biogenetic and amassment capacity
of peltate glands 25 , implies their prime role in determining essential oil yield and quality in menthol mint. As a
sequel to the previous quantitative pattern of neogenesis
of oil glands and their ontogenic analysis through transmission electron microscopic investigations in the developing menthol mint leaf; herein, we provide a qualitative
account of scanning electron microscopic analyses-aided
developmental context of stereological features of trichome structures including novel ‘decapping and collapsing’ of the peltate glands in the plant.
As oil gland neogenesis occurred all through the leaf
growth phase (albeit at slower rates during post-mid545
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expansion)6 , the glands at almost all the stages of their
development could be visualized throughout the leaf ontogeny. In fact, SEM surveillance of the surface of a
developing menthol mint leaf for the oil glandular
trichomes revealed a scenario of developmentally nonsynchronous population of the peltate glands (Figures
2 a, c, 3 a, c). Moreover, temporal span of development
of the oil glandular trichome was much shorter than that
a
c
b
d
Figure 2. SEM of menthol mint (Mentha arvensis) essential-oil glandular trichomes in the presecretory phase. a, Adaxial surface view (100 ×) with abundant nascent peltate glands (og) and nonglandular hair; b, Single peltate gland on the adaxial surface (800 ×) with cuticle (C) tightly appressed to the secretory cells (SC); c, Abaxial surface view (100 ×) with larger number of young
peltate glands; d, Single peltate gland on abaxial surface (800 ×) with foldings at the perimeter.
a
c
b
d
Figure 3. SEM of menthol mint (M. arvensis) essential-oil glandular trichomes in the secretory
phase (left panel) and maturation phase (right panel). a, Adaxial surface view (100 ×) with semi-depressed peltate glands in secretory phase; b, Single peltate gland (800 ×) with sutured (demarcated)
8-celled secretory head assembly; c, Abaxial surface view (100 ×) with both mature and neogenic
peltate glands; d, Single, fully oil-laden, dome-shaped peltate gland (800×) with some micropores
depicted by arrows.
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of leaf6,26. The oil glands underwent their own characteristic ultrastructural modulations as part of the preset
developmental programme integrated within the leaf
ontogenic configuration, as suggested previously6 . The
study specifically stresses upon the qualitative changes
that occur in oil glands during their development in
menthol mint, rather than making quantitative estimates.
Thus, SEM and LM-based stereological structural
scrutiny of the glandular trichomes helped to discern (i)
developmental ultramorphological features of peltate oil
glands, and (ii) archetype of association between their
major population anatomical feature and the leaf ontogenic/physiological phase. Based on the observations, the
glandular ontogeny could be phase-wise categorized as
(i) pre-secretory phase, (ii) secretory phase, and (iii)
post-secretory phase. The post-secretory phase was most
elaborate and further divisible into (a) maturation stages,
(b) dehiscence (decapping) stage and (c) degeneration/collapsing stage, in accordance with the anatomical
dynamics of gland secretory-head development.
In the pre-secretory phase, the neogenic peltate oil
gland in the mint was a rigid structure, almost in a depression on the leaf surface. Owing to nascency and little
oil filling of secretory head cells at the pre-secretory
phase, they appeared as a flat-topped fine contoured
structure observed in the leaf at the youngest stage, as
shown in Figure 2 b and d. Once the constituent [1 + 1 +
8] cellular architecture of the menthol mint glandular
trichomes6 was established, its oil-biogenetic mode of
function sets in progressive oil filling, since the glands
(secretory cell head leucoplasts) are the primary site of
biosynthesis of the monoterpene oils10 . The pre-secretory
phase represents a developmental span up to metabolic
preparedness of the intracellular biosynthetic sites (plastids) leading to the state of ‘switching on’ of expression
of genes and catalytic activities relevant to the biosynthetic steps26,27. A field scan (100 ×) of the leaf surfaces
(both adaxial and abaxial) revealed that the peltate glands
at the early ontogenic stage were flat or depressed on top
(Figure 2 a and c). SEM (800 × magnification) of the
morphology of individual representative glands showed
that the cuticle was close-fit to the cellular head gland
with foldings at the perimeter (Figure 2 d). The secretory
cells appeared to be devoid of oil (top-depressed), but
intact.
The secretory phase was considered as one of the active
monoterpene oil biosynthesis, most represented in the
rapid expansion phase of leaf development6 . As shown in
Figure 3 b, the glandular head was progressively filled
with the monoterpene essential oil. The glands attained
larger size with top surface extended or distended to assume a ballooning appearance, with intercellular cell wall
regions well-marked as constrictions (Figure 3 b). This
surface feature signifies that the secretory cells are fully
laden with the essential oil. The cuticle was still tightly
apposed to the secretory cells at the perimeter, but
CURRENT SCIENCE, VOL. 84, NO. 4, 25 FEBRUARY 2003
stretched over the cell volume region due to oil-fill pressure from underneath leading to the demarcation of the
individual cell boundaries (Figure 3 b). This phase appeared to represent the developmental progression towards
cessation of oil biogenesis and its cellular secretion. The
termination of secretory phase was manifested by glands
possessing stereologically semi-depressed appendaged
structure on the epidermal face (Figure 3 a and b). Some
glands in this representative scan visualizable in the presecretory phase (more or less top-depressed) as depicted
in Figure 3 a, represented the relatively small late-neogenic population of peltates. Their quantitative proportion in the scan area under reference was about 25%.
The post-secretory phase was considered as the developmentally marked ontogenic sequence of events
subsequent to oil-filling. A priori examination of the
characteristic features of the peltate gland development
allowed sequential categorization of the features into
ontogenic stages. These included (a) maturation, (b) cuticular decapping or dehiscence (c) secretory cell-complex collapsing.
The maturation stage entailed creation of subcuticular
space over the 8-celled secretory cell complex and progressive efflux/sequestration of monoterpene oil into the
subcuticular space7 . At this stage, secretory head complex lost the top-view characteristic morphological demarcation (sutured 8-cell assembly as shown in Figure
3 b) of the previous stage and appeared as a fully inflated
smooth ball (Figure 3 d) due to sequestration of oil into
the subcuticular space above the secretory head. The internal pressure (up-thrust) from within the secretory cells
was manifested on the cuticular surface as loss of demarcations (inter-secretory cell-wall junctions) underneath
the 8-cell assembly in the top-view of the sphericalshaped secretory head. The novel and interesting sequence of events in the subcuticular excretion of the oil
from 8-celled secretory head complex was more explicitly captured in the LM pictures of the freshly-cut section
of the leaf (Figure 4 a–d). In the field view of the SEM
scan of the leaf around mid-expansion stage, mature
glands appeared quite abundant (as gross quantitative
estimate constituted 80% of the glands in the area under
reference in the Figure 3 c).
The trans-plasma membrane exocytosis of monoterpene oil (lipid) to subcuticular space appeared polarized
or directional lipid transfer, whether it occurred in a
membrane vesiculated form or vaculolated exocytosis
form or molecularly mediated through lipid-transfer proteins known to occur as a large family in plants. Our previous observation of transmembrane migrating (probably
coalescing) oil droplet in TEM micrographs6 may be
evocative of the phenomenon.
During the cuticular dehiscence stage, the end of secretion of oil from secretory cell complex into subcuticular space was followed by the beginning of rupture of
the cuticular covering over the secretory head (Figure
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4 f ). Among mints, this rupture was a most notable characteristic of peltate-type glands of the menthol mint. The
rupture of cuticle began along the equatorial plane of the
oil gland to finally progress to complete cuticular ‘decapping’ (Figure 4 d and f ). Consequently, subcuticular
sequestered monoterpene oil became bare to environment
and, thereby, far more freely facile to volatilization. It
was ascertained that ‘cuticular dehiscence’ over the peltate glandular head was a physiologically-defined preset
gland developmental phenomenon, rather than a mechanical smash-up occurring during the non-aqueous
processing for SEM. For this, untreated (non-fixed),
freshly-cut sections of leaf were made and observed
under LM. Thus, the studies have demonstrated the fascinating sequence of events of natural developmental
collapsing of peltate trichomes in menthol mint as a
sequence of events comprising: (i) detachment of operculum from the peltate gland top to generate subcuticular
space; (ii) progressive movement of the monoterpene oil
from secretory cell complex into the subcuticular space
until fullness; (iii) appearance of a fissure in the cuticle
along the equatorial line of weakness, and (iv) finally,
a
b
c
d
e
f
Figure 4. (a–d) LM (40 × magnification) and (E, F) SEM studies of
menthol mint (M. arvensis) leaf at different developmental stages of
peltate oil glands. a, Large subcuticular space (CS) visible on the top
of secretory cell (SC) cluster; b, Small drop of oil (O) accumulated in
the subcuticular space; c, Almost the entire subcuticular space engorged with volatile oil; d, Rupture of oil-gland cuticle along equatorial plane (CL, cuticular lid); e, Single peltate gland (800 ×) at the postsecretory phase; f, Peltate gland (800 ×) depicting rupture of cuticular
lid.
548
removal of the cuticular cap or disc from the top of the
gland.
Let us now consider secretory cell-complex degeneration. Loss of protective cuticular covering probably triggered the loss of structural homeostasis. The degenerative
phase of leaf senescence was marked by collapsing of the
glandular structure with only a glandular ‘ruin’ remaining
finally, besides only a few very late neogenic glands still
intact. The dehisced oil glands exhibited advanced cellular disorganization with shrunken and shriveled
vestige of the withered gland (Figure 5 c and d). The
disorganized gland assumed semi-projected form to
planar stereology with respect to leaf surface, and lost
transparency under simple microscopic view due to loss
of oil. This could be raison d’être for the general lack of
sight of any kind of surface scars of remnant dehisced
and collapsed oil gland. SEM scan also revealed that the
capitate oil glands which were far less in occurrence
compared to peltate-type in menthol mint6 , did not follow
the developmental feature of decapping (Figure 5 a and
b) unlike peltate-type. Similarly, Rosmarinus officinalis
capitate glands neither undergo cuticular rupture nor
excrete their metabolites from the secretory cell21 .
The observed phenomenon of gland collapsing or dehiscence in menthol mint may (at least partially) explain
our previous observation6 of lower essential-oil content
in a mature menthol mint leaf compared to that in the
actively growing leaf. Probably, the pattern of oil content6
may be better explained as physical loss of exocytotically
sequestered oil accompanying cuticular dehiscence,
rather than in terms of biogenetic turnover or developmental dynamics of the terminal oxido-reductive (pulegone reductase, menthol dehydrogenase, etc.) enzymes
(unpublished data). It could also be one of the modes of
environmental modulations in the yield and composition
of essential oil2–4,11,12. If menthol mint monoterpenes are
ranked in order of free volatility based on vapour pressure at ambient temperature, many volatile substances
like menthone, menthofuran, isomenthone and neomenthol may share vapour pressure more than their actual
proportion in the oil, leading to enrichment of relatively
less volatile monoterpenes (like menthol) in the remnant
essential oil.
It is speculated here that evolutionarily, the peltate
glands might represent an advanced structure of ecological significance in accumulation and functionality of
volatile secondary metabolites. Lack of programmed
gland/cuticular dehiscence in capitate glands together
with their limited oil-sequestration capacity might put
them as a sort of ‘primitive’ oil glands or secretory trichomes which originated from non-glandular trichomes
in a phylogenetic sequence. The more number of nonglandular hairs/trichomes on adaxial than the abaxial
surface, i.e. in just reverse density to that of glandular
trichomes6 , might numerically support this notion. In
fact, it is known that even a single gene change can transCURRENT SCIENCE, VOL. 84, NO. 4, 25 FEBRUARY 2003
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a
c
b
d
Figure 5. LM and SEM of capitate and peltate glandular trichomes of menthol mint. a, Single
capitate gland (cg, 40 ×) under light microscope; b, Magnified view (460 ×) of a single capitate
gland with single secretory cell (sc) and prominent stalk cell (s) under SEM; c, Complete disorganization and disarray of cellular architecture of peltate gland (800 ×) under SEM; d, Peltate gland
(800 ×) showing secretory cell complex degeneration (collapsing).
form non-secretory hair into a secretory one28 . As an
evolutionary link in the transition from capitate to peltate
glands, some plant species possess capitate glands with
more than one secretory head cell, and capitate glands in
some plant species possess exocytotic mechanisms of
emission of volatiles through rudimentary rupture
mode 21,29. Under certain external damage such as wounding
(environmental or pathogenic), even mint glands display
intense browning (due to oxidation of phenylpropanoids/flavonoids) of phytochemical inclusions (unpublished data). Peltate glands of a labiate (Ocimum species)
leaf exhibit high expression of phenylpropanoid-pathway
genes 30 . From an eco-physiological standpoint, the
mechanism should have an advantage over the damageinduced synthesis of volatiles in plants lacking specialized oil glands. The volatile-oil plants synthesize them
constitutively in trichomes and release them to the environment as a proactive defence strategy or other useful
eco-physiological features (thermo-tolerance, allelo- or
allure impact, herbivory inhibition).
A strong organoleptic perception of volatiles sensed on
a walk besides a menthol mint plantation or on encountering the wind coming through the plantation appears to
be manifestation of the physiological peltate-gland collapsing. Such a sniffing scent is poor in peppermint
plantation (shown to have low volatilization26,27 and even
marketed as a dry herb without excerbative economic
losses) or in an aromatic grass (Cymbopogon species)
farm, as their leaves have been shown to keep the
CURRENT SCIENCE, VOL. 84, NO. 4, 25 FEBRUARY 2003
monoterpene oil in ‘deeply buried’ parenchymatous oil
cells1 . However, analogous, glandular decapping has been
noted in a few non-mint aromatic species22,31. Cuticular
elevation was observed in menthol mint, but cuticular
exudation like that in type-II capitate glands of Salvia22
could not be seen, although some micropore in peltates
was apparent but not prominent (Figures 3 d and 4 e).
Two possible mechanisms for the release of glandular
secretions are possible: (i) failure of the cuticle along an
equatorial line of weakness and subsequent detachment
of the cuticular cap, and (ii) the passage of volatile components through the minute pores in the cuticular structure. The apical region of the gland appeared smooth,
indicating that initially the cuticle was closely attached to
the secretory cell emphasizing the cell outlines, but subsequently it becomes detached from the secretory cell
walls. In the small chamber so formed, the secreted material accumulates at first. Although the exact biophysical
mechanism of the cuticle/gland dehiscence cannot be
narrated until it is further examined, it is hypothesized to
proceed as shown in Figure 6. It has been reported that
cuticle is less than half thick on the lateral sides compared to the apex of the secretory head26,27. This implies
that in case of upthrust force of oil and its vapour pressure, decapping would begin and occur along the equatorial line because of a thin and weak cuticle.
Storage secretory structures amassing secondary metabolites may have diverged from primarily originated cell
to either form central ducts for wider internal circulation
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Figure 6. Proposed bio-mechanistic sequence of developmental collapsing of epidermal peltate gland trichomes in menthol mint. Upper
two drawings are in structural essence based on our previous observations in menthol mint 6 .
of the defence secondary metabolites as in certain resinous and latex plants, or emerged to epithelial locations as
glandular trichomes. It is further speculated that within
the trichome structural and developmental hierarchy, it
appears that they evolved from non-rupturing single secretory cell capitate-types to two-celled rupturing-type
capitate glands to the complex peltate with well-marked
dehiscence mechanism evolved in some species and remained up to micropores in others. Regarding menthol
mint, the phenomenon could be a pristine paradigm of
biotechnological opportunities as suppression of the
process for better taming/trapping of terpenes within the
trichomal milieu. Isolation of mutants in this regard
could be a promising approach for understanding the developmental programming of the phenomenon and isolation of the relevant gene(s).
550
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ACKNOWLEDGEMENTS. We thank the Director, CIMAP for facilities and encouragement. S.S. thanks CSIR for a fellowship. Financial
support in the form of a research grant provided by Department of Science
and Technology, Govt. of India to R.S.S. and N.S.S. is acknowledged.
Received 4 November 2002; revised accepted 16 January 2003
CURRENT SCIENCE, VOL. 84, NO. 4, 25 FEBRUARY 2003