Flora 218 (2016) 24–34

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Flora
journal homepage: www.elsevier.com/locate/flora

Leaf structure and histochemistry of Ficus carica (Moraceae), the fig

tree
Stavroula Mamoucha a , Nikolas Fokialakis b , Nikolaos S. Christodoulakis a,∗
a
Department of Botany, Faculty of Biology, National and Kapodistrian University of Athens, Athens 15701, Hellas (Greece)
b
Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens 15771,
Hellas (Greece)

a r t i c l e i n f o a b s t r a c t

Article history: Heavily “armed” with trichomes, the hypostomatic leaf of Ficus carica L. is investigated with light and
Received 16 February 2015 scanning electron microscopy. Histochemical tests were also applied to trace the secondary matabolites
Received in revised form 26 October 2015 produced in the leaf of this well known, Mediterranean tree with the delicious fruits. Numerous idioblasts
Accepted 14 November 2015
with crystals, laticifers and sized lythocysts appear within the compact mesophyll. Protective trichomes
Edited by Shahin Zarre
Available online 24 November 2015
are located on both surfaces while capitate secretive hairs and stomata appear only abaxially. Stomata,
concerning their magnitude, can be assembled in three size-groups in which the arrangement of their
parastomatal cells differs as well. Histochemical reactions proved positive only for alkaloids within the
Keywords:
Leaf anatomy laticifers and phenolic compounds, especially for the condensed tannins accumulated within the vacuole
Laticifers of the epidermal cells of this simply constructed leaf with unique features among the Mediterranean
Secretory tissues herbs. Comparison of GC–MS and LC–HRMS spectra of different extracts of the nerves and, separately, the
Secreting activity rest of the leaf, revealed that coumarines such as umbeliferone, psoralen, bergapten and xanthotoxol are
Secondary metabolites probably biosynthetically produced within the nerve area where the highly differentiated, main secretive
apparatus of the leaf (laticifers) resides. That is why, these metabolites, are absent from the extracts of
the undifferentiated cells derived from in vitro cultures.
© 2015 Elsevier GmbH. All rights reserved.

1. Introduction
Volumes have been written on the structure and function of the
two main types of vegetation thriving in areas with Mediterranean
climate conditions: the seasonally dimorphic subshrubs and the
evergreen sclerophylls (Mooney et al., 1970; Di Castri and Mooney,
1973; Mooney and Dunn, 1976). Each one of these types of vege-
tation has developed a certain, interesting strategy to evade rather
than escape the unfavorable conditions of the Mediterranean cli-
mate (Eyre, 1968; Margaris, 1981). Interesting anatomical data are
available for many species of either the seasonally dimorphic sub-
shrubs or the evergreen sclerophylls (Christodoulakis and Mitrakos,
1987; Christodoulakis, 1989; Christodoulakis et al., 1990, 2010,
2011, 2014; Nikolakaki and Christodoulakis, 2004, 2006, 2007).
Besides these two main types of vegetation, a large number of
therophytes (Di Castri and Mooney, 1973), survive the stressful
months in the form of seeds and complete their life-cycle during
favorable season (Raunkiær, 1904). In addition, numerous crypto-
∗ Corresponding author. Fax: +30 2107274702.
E-mail address: nchristo@biol.uoa.gr (N.S. Christodoulakis).
phytes and chamaephytes (i.e., Rosmarinus officinalis L., Erica spp.
etc.) along with non-typical Mediterranean plants able indeed to
survive in dry, hot and sunny areas, can be traced in the stressful
Mediterranean environment.
Among the large, drought resistant phanerophytes is a func-
tionally dioecious, deciduous tree of Moraceae, common in
the Mediterranean region, Middle East and Western Asia. It is
extremely tolerant and commonly known as the fig tree (Ficus car-
ica L.) (Tutin et al., 2001). The large, deeply lobed fragrant leaves,
the complex, hidden inflorescence, the multiple fruit and the latex
produced by the plant are known since ancient times. Homer, in his
epic “Odyssey”, brings the main hero back to Ithaca but his father,
Laertis, recognizes him only after Ulysses reminds the old king of
the forty fig trees, a birthday present to him when he was young.
In the ancient city-state of Athens, figs were precious and it was
illegal to export them. A few centuries later, Dioscurides refers to
the latex of the plant (Dioscurides, 2001 77AC) as a treatment for
dental decay.
Lots of information is available for F. carica, concerning the use
of leaves, fruits and roots in traditional medicine for their antibac-
terial, antifungal, anthelmintic, antioxidant and antiviral activities
(Hemant et al., 2013; Debib et al., 2013; Mawa et al., 2013). Leaf

http://dx.doi.org/10.1016/j.flora.2015.11.003
0367-2530/© 2015 Elsevier GmbH. All rights reserved.
 S. Mamoucha et al. / Flora 218 (2016) 24–34
juice is also used for treatment of asthma, cough, sexual disorders,
diarrhea, haematuria, earache, toothache, migraine, eye trouble,
gastric problems and scabies (Vikas and Vijay, 2011). Some data
is also available for the adventitious bud formation and plantlet
regeneration from leaves (Yakushiji et al., 2003), the hypogly-
caemic (Perez et al., 2000) and hepatoprotective activity (Gond and
Khadabadi, 2008) of various extracts, the production of multiple
shoots and plant regeneration from leaf segments (Kim et al., 2007),
the leaf characteristics and their optical properties (Baldini et al.,
1997), the seasonal and diurnal photosynthetic behavior of the
plant under semi-arid climatic conditions (Can and Aksoy, 2007) as
well as the value of the plant as an ancient source of healthy food
and bioproducts (Barolo et al., 2014). Beyond those various pieces
of information stated above, there is no data available – besides the
morphological observations on the leaves of some other species
of the Moraceae family (Wu and Kuo-Huang, 1997) reporting cal-
cium crystals as a common feature of the family and the inadequate
report, with only one low quality picture, by Bercu and Popoviciu
(2014) – on the anatomy of the drought resistant leaf, the secret-
ing structures of it and their activity as well as the composition of
the secondary metabolites produced. The results, after a thorough
investigation on these issues, are reported in this paper.
2. Materials and methods
2.1. Plant material
Mature leaves of F. carica plants growing wild in a natural for-
mation east of Athens metropolitan area (37◦ 57 76N, 23◦ 48 08E,
362 m elevation), were collected in late May 2012 and 2013. Young
(ten days old) leaves were collected from the same plants in early
May 2013, as well.
Some of the leaves collected were cut in to small pieces
(1 mm × 1 mm) and fixed in phosphate buffered 3% glutaraldehyde
(pH 6.8) at 0 ◦ C for 2 h. Some of the pieces were dehydrated in
graded acetone series, critical point dried, coated with gold and
viewed with a JEOL JSM-6360 Scanning Electron Microscope. The
rest of the tissue was post fixed in 1% osmium tetroxide in phos-
phate buffer, dehydrated in graded ethanol series and embedded
in Durcupan ACM (Fluka, Steinheim, Switzerland). Fixation was
repeated, one year later, in early May 2013, and the embedded tis-
sues were sectioned and observed, for cross-checking the results.
Literature for double fixation is cited in detail by (Christodoulakis
et al., 2009, 2010). Semithin sections of plastic embedded tissue
(pet), obtained on an LKB Ultrotome III, were glass mounted and
stained with 0.5 toluidine blue O (in 1% borax solution) as a general
stain for light microscopic observations.
2.2. Histochemistry
Glass mounted semithin sections of plastic embedded tissue
(pet) were stained with: (a) saturated Sudan black B solution in
70% ethanol for the histochemical detection of lipids; (b) saturated
alcian blue solution in 3% acetic acid for the detection of polysac-
charides; and (c) 1% aniline blue black in 70% acetic acid for the
detection of proteins.
Fresh leaves were sectioned by hand or with a cryotome (Leica
CM1850, Germany) at −10 ◦ C and stained with common histo-
chemical reagents: (a) osmium tetroxide for unsaturated lipids;
(b) concentrated H2 SO4 for sesquiterpenes; (c) vanillin/HCl for
flavonoids; (d) antimony trichloride (SbCl3 ) for terpene containing
steroids; (e) Wagner’s reagent for alkaloids; (f) Dittmar’s reagent
for alkaloids; (g) iodine–iodide solution for alkaloids; (h) potassium
bichromate for tannins; (i) nitroso reaction for catechol tannins; (j)
ferric chloride for polyphenols; (k) alcoholic vanillin/HCl (vanillin
25
test) for phenolic compounds; (l) 4-nitrosophenol in concentrated
H2 SO4 for monoterpene phenols; (m) DMB (di methoxy benzalde-
hyde or veratral aldehyde) for phenolic tannin precursors. All stains
were matched by controls. The literature for histochemical reac-
tions is cited in detail by (Christodoulakis et al., 2009, 2010).
2.3. In vitro cell cultures
Pieces of leaf tissue were treated for in vitro cell culture. They
were washed with tap water, counter washed with distilled, ster-
ilised water for 5 min, rinsed with 70% ethanol for 30 s, immersed in
1% aqueous solution of hypochlorous acid (HOCl) and then rinsed
three times with distilled, sterilised water. These pieces of leaf
tissue were then transferred in 10 cm Ø Petri dishes containing
1.1 g/500 mL of Murashige and Skoog medium (Murashige and
Skoog, 1962), 0.8% agar, 2% sucrose, 0.2 mg/ml of culture medium
synthetic auxin (2,4-dichlorophenoxyacetic acid) and 0.2 mg/ml
of the cytokinin benzylaminopurine (BAP) (Coleman and Thorpe,
1977; Jafari et al., 2011). They were incubated at 12 h daylight
conditions, for 2 weeks, at 24 ◦ C. All specimens were viewed with
an OLYMPUS CX41 optical microscope. Original light micrographs
were recorded digitally, using a Nikon D5000, 12.3 megapixel cam-
era. SEM images are digital records from the instrument’s built in
camera.
2.4. Extraction
Mature leaves were dissected in a way that the leaf nerves
were kept intact while the encircled leaf tissue was collected sep-
arately. Dissected nerves, the remaining nerveless leaf pieces and
cell masses (calli) produced after in vitro leaf-cell cultures, were
extracted using solvents of increased polarity, CHCl2 , MeOH and
H2 O, respectively. The extraction was performed using sonication
for 20 min with the above solvents at a ratio 1:10 of dry plant
material to solvent. Then solvent was removed under nitrogen and
the dry residue was stored at −20 ◦ C until used for GC–MS and
LC–HRMS analysis.
2.5. GC–MS, GC–FID and LC–HRMS analysis
GC–MS analysis was carried out using a Hewlett-Packard
5973-6890 operating on EI mode (equipped with a HP 5MS
15 m × 0.25 mm × 0.25 ␮m film thickness capillary column).
Helium was used as carrier gas (2 mL/min). The initial temperature
of the column was 60 ◦ C and then it was heated to 300 ◦ C at a
3 ◦ C/min rate, following an isocratic 300 ◦ C temperature for 20 min.
GC–FID analysis was carried out on a PerkinElmer Clarus 500 gas
chromatograph, fitted with a HP 5MS 15 m × 0.25 mm × 0.25 ␮m
film thickness capillary column. The column temperature was
programmed from 60 ◦ C to 300 ◦ C at a rate of 3 ◦ C/min. The injector
and detector temperatures were programmed at 230 ◦ C and
300 ◦ C, respectively. Helium was used as the carrier gas at a flow
rate 2 mL/min. In addition LC–HRMS analysis was performed on
an Accela High Speed LC System hyphenated to a LTQ-Orbitrap
XL hybrid mass spectrometer, using an ESI ionization probe, in
negative mode (Thermo Scientific, Bremen, Germany). Separations
were carried out on an Ascentis C18 column (150 × 2.1 mm i.d.,
3 ␮m). After optimization, a 25 min LC gradient elution program
was developed enabling the efficient separation of the majority
of the components. The flow rate was set to 400 ␮L/min and the
solvent system was (A) water, 0.1% Formic Acid (FA, 0.1% v/v)
and (B) Acetonitrile, 0.1% Formic Acid (FA, 0.1% v/v). The elution
program was: 5% B at 0 min; 100% B at 20 min; 100% B for 1 min;
5% B in 1 min and hold for 3 min. The injection volume was 10 ␮L.
The HRMS data were acquired with mass range of 120–1200 m/z.
ESI conditions: capillary temperature 350 ◦ C; capillary voltage
26 S. Mamoucha et al. / Flora 218 (2016) 24–34

−35 V; tube lens −110 V. Nitrogen was used as sheath gas (45 arb) Table 1
Chemical compounds in leaves of fig tree investigated here, histochemical reagents
and auxiliary gas (10 arb). Based on spectrometric features such
used and the result of their application (pet = plastic embedded tissue, ft = fresh
as suggested Elemental Composition (EC), Ring Double Bond tissue).
equivalents (RDBeq) and data from literature, the identification of
Compounds investigated Reagent used Reaction
constituents was performed.
General Stain (pet) Toluidene Blue “O” +
Polysaccharides (pet) Alcian blue −
Proteins (pet) Aniline blue black −
3. Results
Lipids + phenolics (pet) Sudan black B +
Lipids (ft) OsO4 Minor +
3.1. Leaf anatomy Terpenes (ft) Concentrated H2 SO4 ++
Flavonoids (ft) Vanillin/HCl Epidermal tissues ++
Steroids (ft) Antimony trichloride Epidermal tissues +
The leaves (Fig. 1) of F. carica posses a compact double lay-
Alkaloids Wagner’s reagent Epidermal tissues ++
ered palisade parenchyma, just below the single layered adaxial (ft) Dittmar’s reagent Minor +
epidermis (Fig. 2). The continuity of the palisade tissue is often dis- Tannins (ft) Potassium bichromate Epidermal tissues +
rupted by the extensions of the bundle sheaths the terminal cells of Phenols Ferric chloride Trichomes only +
which come in contact with the epidermal cells (Fig. 2). Along the (ft) Vanillin test Mesophyll +++
4-Nitrosophenol ++
first layer of palisade cells, numerous crystal bearing idioblasts are
Phenolic tannin precursors Dimethoxy-benzaldehyde −−−
observed (yellow arrows in Figs. 2 and 3). Large bundles traverse (ft) (DMB)
the mesophyll, most of the times hosting a laticifer just above the
xylem elements, surrounded by the sheath extension cells (black
arrow in Figs. 2, 6 and 7). The spongy parenchyma is rather loose and medium ones. The largest among the stomata are usually open and
numerous, globular lithocysts can be observed, adjacent to the cells can easily be distinguished by fluorescent microscopy due to the
of the lower epidermis. Within these lithocysts, a sized cystolith is epicuticular phenolics accumulated over the ledges of their guard
accommodated in a peculiar, inverted position, “hanging upwards” cells (Fig. 18).
from a small stalk (Fig. 15). The tip of this stalk protrudes outside the The leaves of F. carica start their life as compact leaflets with
sheath of the lithocyst forming a spiked appendage (red arrows in a very dense indumentum out of which only the trails of the leaf
Figs. 11 and 15). The large cystoliths are different in structure com- nerves seem to protrude (Fig. 13). Big unicellular hair, the size of
pared to the crystals within the idioblasts of the palisade tissue as those present on the mature leaf, produce a very “aggressive” struc-
it can easily be demonstrated with polarized light (compare Figs. ture as they appear embedded in a thin, compact leaflet at a stage
3 and 4). Stomata appear only on the lower (abaxial) surface. The of development when the mesophyll has not been discerned yet
lower epidermal cells are apparently smaller compared to those of into palisade and spongy parenchyma (Fig. 13). Gradually the leaf
the upper epidermis (Figs. 2, 3 and 6). expands, trichomes appear to scatter in larger surface and small,
Within the mesophyll, laticifers form a network which follows capitate, secretive hairs develop on the nerve surface of the lower
the “route” of the conductive bundles. They appear on the adaxial epidermis (Figs. 9, 10 and 14). Secretive hairs possess a four-celled
side of each major bundle, at the section of the mesophyll where the head (Fig. 14). The produced metabolites are accumulated within
palisade parenchyma is located. They do not appear in the spongy their head cells. Young leaves do not seem to possess any litho-
parenchyma (black arrows in Figs. 5 and 6). The network of the non- cysts, on the mature leaves, however, we can easily observe the
articulated, multinucleate, branched laticifers (Fahn, 1979) extends tiny spikes of the abaxial surface, projecting from the middle of a
through the petiole to the stem and the whole plant axis (black characteristic, small, rounded dome. They indicate the presence of
arrows in Fig. 8). an underlying lithocyst (Figs. 11 and 15).
Both surfaces are well equipped with trichomes. They are of two
types: a) The spiny, unicellular hairs, with their tumescent lower 3.2. Histochemical investigations
end embedded within the mesophyll (Figs. 2 and 16) and supported
by a rosette like arrangement of epidermal cells (Fig. 10) which, Finally, histochemical investigations in sections from plastic
when sectioned axially, appear to possess, at the dilated lower part embedded tissue revealed the accumulation of numerous granules,
of the cell, a hanging structure, resembling a cystolith (black arrows within the cells, retaining a dark brown/black color after staining
in Fig. 16); and b) the small, capitate secretive hairs (green arrows with Aniline blue–black. The large lithocysts (indicated with white
in Figs. 9 and 10), with their short stalk and a multicellular head. arrows in Figs. 2–4 and 6) retain a brilliant light blue color after
The formers appear on both surfaces being numerous on the adaxial staining with Alcian blue (for sugars). No sign of reaction is observed
epidermis, while the latter secretive hairs are located on the pro- after staining with Sudan black B except the starch granules, within
jecting trails of the leaf nerves formed on the lower surface of the the chloroplasts, appearing brightly white against a dark blue back-
leaf only. ground. Application of histochemical reagents in sections obtained
Stomata present an interesting variation in size and structure. from fresh tissue, revealed the accumulation of condensed tannins
Some of them are really large, resembling hydathode-like struc- within the dilated lower part of the trichomes as well as within the
tures. They lie on the top of a projection formed by the epidermal epidermal cells. Brilliant reactions are given for terpenes (Fig. 19),
cells (yellow arrows in Figs. 9 and 17). The rest of the stomata are after the application of sulphuric acid, and flavonoids (Fig. 20) after
obviously smaller and the epidermal cells round them are arranged applying the vanillin–HCl reagent. Wagner’s reagent (Fig. 21) seems
in a flat formation. They appear to be of the anomocytic type. The to have effect mainly on the epidermal tissue while vanillin test
length of the pore formed between the guard cells was measured gives a vivid color (Fig. 22). A weak reaction is observed in the
(Fig. 12) in 40 to 50 stomata in each of the 10 randomly selected mesophyll after the application of OsO4 for lipids. Our data, on sec-
fields, at 300× magnification. Most stomata (about 60%) appear to ondary metabolite tracing for all histochemical reagents applied, is
have the length of their stomatal pore extending from about 12 to presented in Table 1.
14 ␮m. A considerable number (about 30%) has a length of about Since our observations indicate that the capitate trichomes are
21 to 25 ␮m, and only 10% of the stomata have gigantic dimensions located on the nerves, at the abaxial side of the leaf (green arrows
with the length of their pore measuring from 32 to 42 ␮m. That in Figs. 9, 10 and 25), and the laticifers are accommodated within
means threefold the size of the small ones and twofold that of the the nerves, at the abaxial side of the bundles, just above the xylem,
 S. Mamoucha et al. / Flora 218 (2016) 24–34 27

Figs. 1–8. The leaf of the fig tree. Fig. 1: The lobed leaves and the young, complex inflorescence consisting of a hollow fleshy structure lined with numerous unisexual
flowers. Fig. 2: Cross section from a plastic embedded mature leaf. The thick upper epidermis, the numerous crystal containing idioblasts (yellow arrows), the loose spongy
parenchyma, the laticifer (black arrow) within the nerve of the leaf, the lithocyst with the sized cystolith (white arrow) and the long protective hair can be observed. Fig.
3: The two layered palisade hosts the idioblasts with the spiny crystals. They may appear in pairs. The texture of the cystolith contained within the lithocyst, (below right)
seems to be completely different. Fig. 4: The same section as in Fig. 3 viewed with polarized light. The difference between the crystals and the cystolith becomes obvious.
Fig. 5: Paradermal section from a plastic embedded leaf. At the border (upper left) the epidermal cells can be observed followed by the palisade parenchyma (pp). Numerous
crystal containing idioblasts can be observed within the palisade (yellow arrows). A laticifer (la–black arrow) is obvious at the margin between the palisade and the spongy
parenchyma (sp–bellow, right). Fig. 6: A detail of a laticifer, just above the xylem of the conductive bundle (black arrow) and one of the big lithocysts at the abaxial site of
the leaf, as observed in a cross section. Fig. 7: An oblique section of a laticifer (black arrow) just above the xylem elements of the conductive bundle. Idioblasts with spiny
crystals are always present (yellow arrows). Fig. 8: A paradermal section of a laticifer (black arrow) from a fresh leaf, stained with a very weak lugol solution. Yellow arrows
point at the spiny crystals. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

we may assume that the secreting apparatus of the fig leaf is con-
fined, almost totally, on or within the nerves. Therefore, we tried
to investigate the leaf, separating the major nerves from the rest of
the leaf tissue (Fig. 23). The excised leaf tissue contained actually
the abaxial and adaxial epidermis, the mesophyll in the middle and
the sized lithocysts (white arrows in Figs. 24–25). Now we have
three types of extracts to analyze and compare: the extract from
the leaves, an extract form the nerves and the extract from the calli
(in vitro cultures) (Figs. 26–29).
3.3. GC–MS, GC–FID and LC–HRMS analysis
GC–MS analysis of the volatile compounds of CH2 Cl2 extract
of the nerves (Fig. 30A) in comparison with the CH2 Cl2 extract
of the rest of the leaf (Fig. 30B) revealed that there were no
major qualitative differences between them but rather quantita-
tive. The differences in volatile secondary metabolites produced
were mainly in two peaks with retention time (Rt) 41.5 min and
49.5 min that correspond to the furanocoumarins psoralen (2) and
bergapten (3), respectively (Fig. 33). Compound 2 seems to be in
28 S. Mamoucha et al. / Flora 218 (2016) 24–34

Figs. 9–12. Scanning electron microscopy of the leaves of fig tree. Fig. 9: The abaxial surface of the leaf. The pointed protective trichomes, a small stoma (light blue arrow),
a large stoma (yellow arrow) and a capitate trichome (green arrow) located on one of the nerves can be observed. Fig. 10: The rosette-like arrangement of the epidermal
cells round the base of the large protective trichome (black arrow) and the capitate trichomes (green arrows) accommodated on the nerves are clearly demonstrated in this
view of the lower surface of the leaf. Fig. 11: A medium sized stoma (yellow arrow) and the spiked appendage (orange arrow) in the middle of the domed outer wall of
the lithocyst are demonstrated. Fig. 12: In this micrograph the various sizes of the stomata are measured. In the middle, one of the large stomata appears on the top of an
epidermal swelling. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

significant higher concentration in the extract of the nerves than
in the extract of the leaves (Fig. 30A and B) while compound 3 had
smaller differences but was always more abundant in the nerves.
Indeed quantitative analysis using GC–FID showed that the nerves
extract constituted of 27.8% of compound 2 and 8.7% of compound
3 while the rest of the leaves extract constituted from just 9.6%
and 5.0% of compounds 2 and 3, respectively. On the contrary an
unidentified compound bearing a non-coumarin moiety, in Rt 14.7
(abundance 2.1%), was observed only in the leaves but not in the
nerves.
Further investigation by HPLC–HRMS was performed by com-
paring the metabolic profile of the methanolic extracts of the
leaves (Fig. 31B, ) with the nerves (Fig. 31A). The methanolic
extracts of the nerves, generally had more rich profile in terms
of non-volatile secondary metabolites. Comparing the two chro-
matograms, this difference could be located at the areas with Rt
5.50–6.67 min and Rt 10.25–10.98 min. The high resolution mass
measurements, in combination to the proposed molecular formu-
las, MS/MS fragmentations and RDB equivalents, revealed that the
major compounds with Rts 5.50, 5.63, 6.35 and 6.67 correspond to
coumarins. Compound eluted at Rt 6.67 min with pseudomolecu-
lar ion on a negative mode at m/z 161.0244 and molecular formula
C9 H5 O3 was identified as umbelliferone (1). This simple coumarin
is generally considered as precursor of all other furanocoumarins
(Del Rio et al., 2014) and it was located clearly only in the nerves
of the plant material. Compound eluted at Rt 5.50 min has a pseu-
domolecular ion at m/z 365.0868 and according to the suggested
molecular formula and RDB equivalents, it corresponds to a glycosi-
lated coumarin. Its main MS/MS fragment at m/z 203.0346 confirms
this assumption and the fragment corresponds to furanocoumarin
xanthotoxol (4), after the detachment of the glycosyl moiety.
Compound eluted at Rt 6.35 is an isomer, assuming that the
differentiation could be either different glycosilation position, or
another aglycon structure. Finally, coumarin eluted at Rt 5.63, is
not a major one and has been identified as a glycosilated umbel-
liferone derivative. Compounds at Rts 10.25, 10.77 and 10.98 min
are also unique for the nerves and completely absent from the leaf
extract, however, data from HRMS measurements and data from
literature were not enough for their identification. In addition, the
comparison of the H2 O extracts (Fig. 32) showed that a major peak,
corresponding to a glucosylated coumarin (Rt 6.63 min and molec-
ular formula C17 H17 O9 ) exists at significant higher concentrations
in the nerves (Fig. 32A) than leaves (Fig. 32B). On the contrary the
major peak of the leaves extract (Rt 5.47 min and molecular formula
C26 H28 O14 ) corresponds to a bis-glycosylated flavonoid which is
present in the nerves as well but in smaller quantities.
In comparison to the above metabolic profiling of different
extracts also the cell masses (calli) produced after in vitro leaf-
cell cultures were extracted and compared to the extracts from
the plant material. The CH2 Cl2 extract (Fig. 30C) contained all
metabolites produced in the leaf or the nerve except the peaks
that correspond to the furanocoumarins psoralen and bergapten. In
addition comparison of the HR–LCMS metabolite profiling of MeOH
(Fig. 31C) and H2 O extract (Fig. 32C) with the respective of the nerve
or the leaf revealed that the MeOH extract had remarkable metabo-
lites that are clearly different from what is produced in the nerve
or the leaf while the H2 O extract was very poor in metabolites and
no coumarins were observed.
Overall it seems that the simple coumarin umbeliferone
that is considered biosynthetically the precursor of linear fura-
nocoumarins, is localized basically in the nerves and several other
linear coumarins such as psoralen, bergapten, xanthotoxol (Fig. 33)
and their glucosylated derivatives were found in higher quantities
in the nerves. Thus we believe that furanocoumarins are biosyn-
thetically produced in the nerves of the leaves and most probably
they are transferred and accumulated in the capitate trichomes
residing on the nerves only, at the abaxial side of the leaf.
 S. Mamoucha et al. / Flora 218 (2016) 24–34 29

Figs. 13–18. The leaf epidermis of the fig tree. Fig. 13: Cross section of a leaf primordium presenting the central nerve (cn) and the numerous, protective trichomes already
well developed on this premature leaf. Fig. 14: A high magnification of the capitate secretive trichomes of the abaxial epidermis (ep). The single cell stalk and the four-celled
head (arrows) can be observed. Fig. 15: A cross section of a lithocyst from a fresh leaf. This cell develops within the spongy parenchyma (sp) with the cystolith (cy) “hanging
upwards” and the characteristic spiked end protruding from the outer part of the cell wall. Fig. 16: Detail of the abaxial side from a cross section of an epoxy embedded leaf.
Two protective trichomes can be seen with the developing, amorphous mass within their lumen. Fig. 17: The adaxial epidermis from a cross section of a fresh leaf. One of
the large (yellow arrow) and one of the medium sized (white arrow) stomata can be seen. The large stoma appears on the top of an epidermal swelling. Fig. 18: Same as in
Fig. 17, observed with fluorescence microscopy. The cutin over the guard cells is clearly visible. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)

Figs. 19–22. Histochemical investigations. Fig. 19: concentrated H2 SO4 for terpenes, Fig. 20: vanillin/HCl for flavonoids, Fig. 21: Wagner’s reagent for alkaloids, Fig. 22:
vanillin test for phenolics.

4. Discussion L. or even Olea europaea L. (Christodoulakis and Mitrakos, 1987).

Besides, the well established feature of the Mediterranean plants
The lobed leaves of F. carica are harsh in texture but appear less to reduce their surface to volume ratio (small leaves), seems to
equipped in mechanical tissue compared to the typical Mediter- have been turned over by F. carica. Both Mediterranean evergreen
ranean leaves like those of Quercus coccifera L., Ceratonia siliqua sclerophyllous shrubs and, in particular, the seasonally dimorphic
30 S. Mamoucha et al. / Flora 218 (2016) 24–34

Figs. 23–25. The leaf dissected. Fig. 23: The whole leaf of a fig tree as dissected to obtain nerves and nerveless leaf tissue. Fig. 24: Close-up of the lower leaf surface. Minor
nerves and the lithocysts (white arrows) can be observed. Fig. 25: A detail of the lower surface. The lithocysts (white arrows) and the capitate trichomes (green arrows),
confined on the nerves, can be observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figs. 26–29. In vitro cultures and histochemistry. Fig. 26: Untreated cell mass (callus) produced after in vitro culture of leaf tissue. Fig. 27: callus cells treated with vanillin/HCl
for flavonoids. Fig. 28: callus cells treated with potassium bichromate for tannins. Fig. 29: callus cells treated with DMB for phenolic tannin precursors.

subshrubs take advantage of the small leaf areas which prevent
leaves from overheating. F. carica, although an “authentic” Mediter-
ranean plant by habitat, has a leaf of great magnitude, with a
blade area more than ten to twenty times larger than the blades of
the larger among the evergreen sclerophylls (Arbutus andrachne L.,
Arbutus unedo L., Nerium oleander L.) (Christodoulakis and Mitrakos,
1987). This is probably the reason for which the leaves of F. carica
are not positioned leveled to the ground but more or less perpen-
dicularly (Fig. 1) so that the sun rays fall on the leaf sideways.
The leaves are compact with the palisade tissue to occupy less
than 50% of the leaf. Numerous idioblasts with spiny crystals appear
preferably between the palisade cells while the big lithocysts,
with their content hanging “upwards” (Fig. 15), find their space
within the spongy parenchyma. The content of these sized cells
seems rough, unstratified and rather water soluble. It reacts with
Alcian blue (for sugars) something that is in agreement with Fu-
gen et al. (1991) reporting that the majority of cystoliths are mainly
composed of polysaccharides while the ultrastructure of their com-
ponents exhibit no orderly arrangement and no stratification.
On the lower surface of the fig leaves, two types of stomata
were observed. They occur in different sizes. The rare, protrud-
ing, big stomata are actinocytic while the small ones that also vary
in magnitude, of the anomocytic type. The magnitude of the large
stomata, their distribution and arrangement, their large substom-
atal chamber (Fahn, 1979), the fact that they appear in the “open”
condition as well as the cutin localized over the ledges of their guard
cells (Karabourniotis et al., 2001), indicate that these apertures,
although looking as typical epithem, may function as the pores
of the hydathodes (open water stomata). Hydathodes scattered
all over the surface of the leaf were reported for Ficus diversi-
 S. Mamoucha et al. / Flora 218 (2016) 24–34 31

Fig. 30. Metabolic profiling of the CH2 Cl2 extracts by GC–MS. A: for the nerves B: for the leaf and C: for the in vitro produced calli.
32 S. Mamoucha et al. / Flora 218 (2016) 24–34

Figs. 31–32. Fig. 31: Metabolic profiling of the MeOH extracts by LC–HRMS of A: nerves B: leaf and C: calli. Fig. 32: Metabolic profiling of the H2 O extracts by LC–HRMS of
A: nerves B: leaf and C: calli.

Fig. 33. The structure of major furanocoumarins identified in F. carica. 1: umbelliferone, 2: psoralen, 3: bergapten, 4: xanthotoxol.

folia Reinw. ex Miq. (Lersten and Peterson, 1976). The presence
of hydathodes, although reported for some typical Mediterranean
therophytes (e.g., Cichorium intybus L., Papaver rhoeas L.) seems a
rather unexpected characteristic for the Mediterranean environ-
ment.
The secreting structures of the leaf, besides the numerous
idioblasts, are the laticifers and the capitate trichomes on the lower
surface of the leaf. The latter appears uniform in shape all over the
lower leaf surface. The metabolites produced by the head cells (Fig.
14) are accumulated, in maturity, within these cells in the form of
dark granules. The metabolic profiling of the methanol and aqueous
extracts derived from nerves dissected and separated from the rest
of the leaf area, indicates that the majority of the secondary metabo-
lites produced in the leaves, most probably furanocoumarins, are
produced and retained within the nerves (laticifers) or, at least, they
are confined close to the nerve area (i.e., retained within the capi-
tate trichomes). As no other deviations were observed, and having
in mind that compound 2 (psoralen) (Fig. 31) is one of the precur-
sors of the biosynthesis of furanocoumarins, the present analysis
implies that either an accumulation of furanocoumarins occurs
in the nerves or their biosynthesis through the shikimate path-
way takes place there (Del Rio et al., 2014). They seem to exist
in far smaller quantities within the rest of the leaf. Those fura-
nocoumarins were, strangely, not observed in extracts from the calli
produced in vitro, in spite the existing aspect for the biosynthetic
totipotency of the callus cells (Bourgaud et al., 2001; Ramachandra
Rao and Ravishankar, 2002), but under the specific lab conditions
these calli were cultured. It has been well documented that cal-
lus masses, even of a second generation, are able to synthesize
and accumulate most of the secondary metabolites detected in
parent plants. These metabolites can be isolated for their high com-
mercial (as food additives, pigments) and pharmaceutical value
(terpenes, steroids, alkaloids) (Dronne et al., 1999; Nikolakaki and
Christodoulakis, 2004).
The absence of fouranocoumarins from the secondary metabo-
lites produced from the in vitro cultured cells is probably due to the
fact that callus cells generally originate from the parenchymatic
leaf tissues (palisade and spongy parenchyma). The cells of these
tissues, being rather undifferentiated, can regain their meristem-
atic activity and start dividing to produce calli (cell masses). On
the other hand, the highly differentiated cells of the main secretive
apparatus of the leaf (laticifers and capitate trichomes) can hardly
turn to meristematic cells thus remaining unable to produce cell
masses. Eventually, the special metabolites synthesized within the
 S. Mamoucha et al. / Flora 218 (2016) 24–34
laticifers or the head cells of the trichomes are absent from the
products of the in vitro produced cell masses.
Coumarins are, along with flavonoids, fragrant organic com-
pounds of the benzopyrone chemical class, highly valued for their
pharmaceutical properties. They are proposed to be secondary
metabolites useful as appetite-suppressors, discouraging animals
from grazing plants (Link, 1959). Moreover, furanocoumarins,
which are toxic compounds of various flavors, found primarily in
species of the Apiaceae and Rutaceae, have adverse affects on wide
variety of organisms, ranging from bacteria to mammals. Some of
the furanocoumarins are photoactive, i.e., their toxicity is enhanced
in the presence of ultraviolet radiation. One of the better-studied
models of toxicity involves the binding of furanocoumarins to DNA.
However, furanocoumarins have also been shown to interact with
proteins and lipids (Berenbaum et al., 1986, 1992; Berenbaum and
Zangerl, 1992; Zangerl and Berenbaum 1992, 1997). Taking into
account all the above properties of coumarins and their sequels,
we may suggest that the fig tree takes special care to avoid insects
and other herbivores rather than adopting the common features the
mediterranean plants employ against the stressing climatic condi-
tions. This particular “behavior” probably has to do with the need
of protection, from a wide spectrum of herbivores, of the precious
false or multiple fruit (infructescence or scion) of the tree.
5. Conclusion
Concluding we may say that a number of non common and
rather peculiar – for the Mediterranean plant life – structural char-
acteristics, compose the leaf of one of the common trees of the
Mediterranean region. Some new secondary metabolites produced
within the leaf were identified and the site of their production and
accumulation was localized. Finally, F. carica seems to be a unique
case since the cell masses produced after in vitro cultures of leaf
tissue actually abstain from producing many metabolites detected
in the parent leaves.
Acknowledgements
This work was supported by IKY – State Scholarship Foundation
– 41 Ethnikis Antistaseos Ave, PO Box 142 34, Nea Ionia, Athens,
Greece. The authors express their deep appreciation to Dr. Aika-
terini Termentzi for recording the LC–HRMS spectra.
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