American Journal of Plant Sciences, 2011, 2, 165-174
doi:10.4236/ajps.2011.22018 Published Online June 2011 (http://www.SciRP.org/journal/ajps)
Leaf Traits and Histochemistry of Trichomes of
Conocarpus lancifolius a Combretaceae in
Semi-Arid Conditions
Amina Redha1, Naemah Al-Mansour2, Patrice Suleman3, Mohamad Afzal4, Redha Al-Hasan5
Faculty of Science, Department of Biological Sciences, Kuwait University, Kuwait City, Kuwait.
Received February 22nd, 2011; revised March 24th, 2011; accepted March 28th, 2011.
ABSTRACT
Leaf traits, structure and water status of Conocarpus lancifolius, a Combretaceae were investigated under semi-arid
conditions. The leaf traits examined included leaf area and thickness, stomatal distribution, sclerophylly, succulence
and relative water content. Additionally, the types of secretory structures, histochemistry of trichomes, and chemical
nature of the cuticlular waxes were evaluated. Leaves showed xerophytic characteristics including a high degree of
sclerophylly, thick cuticle and outer epidermal cell wall, low relative water content and high trichome density on
younger leaves. The species has two types of trichomes; a secretory, short-stalked capitate trichome and a non-secre-
tory trichome w i t h a bulb ous base and a poi nt ed ti p . The leaves also have a pair of extrafloral nectaries on both sides of
the distal end of the petiole, 3 - 4 pairs near the leaf apex and two secretory ducts or cavities on mature leaves that se-
creted polysaccharides, epicuticlar waxes and polyphenols. Compared to young leaves mature leaves had almost 3
times total cuticular wax deposit or load. The most abundant fatty acids were palmitic, stearic, nondecanoi c, behenic
and arachidic acids. The leaf traits and structures are discussed in relation to semi-arid habitat.
Keywords: Leaf Morphology, Trichomes, Succulence, Sclerophylly, Cuticular Wax
1. Introduction
Most plants in arid and semi-arid conditions survive ex-
treme environmental conditions by developing structures
to tolerate their habitats. These plants commonly referred
to as xerophytes have common features which include:
small ratio of leaf area to volume [1,2], a reduction in
leaf cell size and number [3], thick cuticle and epidermal
cell walls [2] and increased density in vascular tissues
and sclerenchyma, which contribute to sclerophylly [4,5].
Xeromorphic leaves may be succulent, covered with
trichomes and may develop water-storing cells. Xero-
morphism however, is not limited to xerophytes and not
all xerophytes develop xeromorphic features. Some
plants that grow well in wet conditions have xeromorphic
leaf features while others in dry habitats have mesomor-
phic characters [6]. Thus, leaf features partially explain
how xerophytes thrive in arid habitats.
The plant species, Conocarpus lancifolius, was intro-
duced into Kuwait as part of the “Greenery Program” and
has turned out to be a “miracle ornamental plant” be-
cause it thrives extremely well under the semi-arid con-
ditions.
C. lancifolius is Combretaceae (a family of trees,
shrubs and lianas), a native plant on the coastal areas of
Yemen, Somalia and Djibouti. In semi-arid conditions
and sandy soils, it is a fast growing tree and produces
large amounts of biomass particularly in the hot summer
(≥45˚C) with drip irrigation. It can withstand a range of
ambient temperatures (15˚C - 50˚C) but appears to be
slightly frost sensitive. As an evergreen, it grows up to
several meters in height with a single or multiple stems.
Currently, no serious herbivores attack the species and it
appears to be totally devoid of plant pathogens. Its adap-
tation or tolerance to abiotic and biotic factors is cur-
rently being investigated.
The species is vegetatively propagated from twigs, but
two phenotypes or morphotypes of the species exists in
the habitat: 1) plants with slightly gray, small green
leaves and 2) those with larger glossy, dark green leaves.
Phenotypes of this nature may be due to genotype dif-
ferences or response to environmental conditions [7].
The existence of “silver and green” leaves within Cono-

Leaf Traits and Histochemistry of Trichomes of Conocarpus lancifolius a Combretaceae in Semi-Arid Conditions 166
carpus erectus in islands of central Bahamas has been
reported [8]. In semi-arid conditions, it appears C. lan-
ciflolius may have morphological and structural adapta-
tions to withstand low water potential.
Only a few studies have been conducted on C. lanci-
folius in particular and the genus, in general [8,9]. A high
correlation was observed between drought, salinity and
polyamine accumulation in Conocarpus lancifolius [9].
There has been no detailed characterization of the leaf
morphology and structure of this species in any habitat.
The objective of this study was to examine the leaf
traits C. lancifolius in semi-arid conditions. Additionally,
we studied the histochemistry of trichomes, and compo-
sition of cuticular layer in order to understand their con-
tribution to the species adaptation to a semi arid habitat.
2. Materials and Methods
2.1. Leaf Morphology and Characteristics
Young and mature leaf samples from 7-year-old trees
were used for comparative study. Samples were taken
from the same trees during the period of rapid growth in
the summer. Leaf angle or orientation to the main axis
was measured at mid-day during summer. Leaf mor-
phology and structure were studied visually, with dis-
secting, light microscopes and scanning electron micros-
copy (SEM).
Additionally, the following leaf parameters were also
measured: leaf area (LA; cm2), measured with a portable
leaf area meter (CI—203 CID Inc. Camas, WA USA),
fresh weight (FW; g), fresh weight of fully saturated leaf
(SFW; g) of leaves immersed for 48 h at 4˚C in
de-ionized water, dry mass (DW; g), after drying at 70˚C
to a constant weight. The sclerophyll index, leaf mass per
unit area (Sc = DW/LA; g·m2) and specific leaf area
(SLA) was calculated as m2 leaf area per kg dry weight
(SLA = m2·kg–1). Leaf thickness (LT) was measured on
the cross-sectional leaf cuts with scanning electron mi-
croscopy (SEM).
2.2. Stomatal Characteristics
Mature leaves were collected at midday and late in the
evening to determine opened and closed guard cells.
Stomatal distribution was determined on transpiring
leaves by staining them with ethanol:crystal violet, 0.5%
(w/v) immediately after removal from plant. Leaf sam-
ples were preserved and processed for SEM. Both light
and scanning electron microscopy (SEM) were used to
determine stomatal density of both adaxial and abaxial
surfaces.
Impressions of epidermal cells and stomatal com-
plexes were made by applying clear nail polish on both
the adaxial and abaxial surfaces of various leaves. The
nail polish was removed and the number of stomata and
type of stomatal complex were determined using a light
microscope.
2.3. Leaf Water Relations
Leaves were harvested every 14 days in summer and the
following leaf parameters were determined. The relative
water content (RWC = (FW – DW)/SW – DW) × 100,
leaf succulence Su = (FW – DW)/LA; mg·H2O·cm−2) and
leaf water content (LWC) was measured as a rough esti-
mate of leaf density; (LWC = 1 − DW/FW), [10].
2.4. Leaf Structure and Histochemistry
2.4.1. Light Microscopy
Leaves at different stages of development were used for
bright field microscopy. Both cleared and fresh leaves
were hand-sectioned. Clearing was done in glacial acetic
acetic acid: 95% ethanol (1:3) and heated in an oven at
65˚C until most of the pigments were removed. The
leaves were then placed in 85% lactic acid and heated at
65˚C. Microtome sections were made from leaf tissues
fixed in formaldehyde: glacial acetic acid: 70% ethanol
mix (1:1:18 v/v) and embedded in paraffin.
2.4.2. Sc anning El e ct ron Micr os copy (SEM)
For SEM, leaves at various stages of development were
fixed in glutaraldehyde, dehydrated in ethanol series,
dried to the critical point with carbon dioxide and coated
with gold. The samples were examined in a JEOL
JSM-6300 SEM at 20 kV. Composition of secretory prod-
uct of trichomes on fresh and gold coated leaf samples
was carried out using LEO ZEISS, SUPRA 50 VP FE-
SEM, (Carl Zeiss Nts, Oberkochen, Germany), voltage
15 kV, with Roentec’s x-flash SDD detector (Roentec,
Bruker’s Berlin, Germany), and a energy-dispersive
x-ray spectroscope. Point analysis was done qualitatively
and quantitatively with QuanTax 1.5 software.
2.4.3. Histoch em ical Inves tigation
The following histochemical tests were carried: Ruthe-
nium Red for polysaccharides i.e. pectins and other mu-
cilage other than cellulose [11]; Sudan 7B for suberin
and cutin [12]; Sudan III for lipids; ferric chloride and
vanillin HCl for phenolics [13,14]; Auramine O for cutin
and suberin localization [15].
2.5. Analysis of Cuticular Wax
Leaf cuticle was analyzed for fatty acids that could con-
tribute to resistance to stress factors. Cuticular wax was
extracted from fresh young and mature leaves [16] using
5 g of leaf tissue. Extractions were carried out in tripli-
cates. The extracts were evaporated under reduced pres-
sure to 2 ml, dried under a stream of nitrogen and deri-
vatized.

Leaf Traits and Histochemistry of Trichomes of Conocarpus lancifolius a Combretaceae in Semi-Arid Conditions 167
3.2. Leaf Water Relations, Sclerophylly and
Succulence
The chemical constituents of each extract were ana-
lyzed using gas chromatography coupled with mass
spectrometry (GC-MS DFS Thermo Finnigan, Breman,
Germany). The TR-5 column (30 m × 0.25 mm i.d fused
silica capillary column) from Thermo Electron Corp.,
Osterode, Germany, with helium as carrier gas was at
50˚C for 3 min, increased 6˚C· mi n–1 up to 250˚C and
then to a final temperature of 300˚C at10˚C·min–1. The
individual component peaks and retention times were
identified by comparing their MS with standards from
Nist and Willey 275 database and quantified with X-
Caliber software.
The leaf water status, sclerophylly and succulence data
are shown in (Table 1). Sclerophylly was greater in ma-
ture leaves and leaf veins were relatively obscure on the
adaxial surface of mautre leaves. The leaf RWC was be-
tween 82.4% - 90.3% in the summer.
3.3. Microscopy
A cross section of the leaf showed a thick cuticular layer
(6 - 8 µm) and a thick adaxial epidermal cell wall. Beneath
both epidermal tissues was a layer of hypodermal-like
cells that stained positively for tannins and phenolic
compounds. Mesophyll cells were interspersed with wa-
ter storage cells and cells containing druses. Two types
of trichomes were found on the leaf surfaces: a secretory
and a non-secretory trichomes (Figures 1(a) and (b)). The
non-secretory trichome was a long stalked cell with a
bulbous base with a tapered end. The secretory trichome
had capitate end with 4 pairs of cells and 3 - 4 stalked cells
(Figure 2(a)). At the base of glandular trichome are sec-
retory cells with dense cytoplasm and a few chloroplasts
(Figure 2(b)). The developmental stages of this trichome
are shown in (Figure 2(c)). Leaves turned glossier at
maturity and the total number of trichomes declined.
Trichome density on the abaxial surfaces was higher at
all stages of leaf growth and development. The Fe-SEM
point analysis of the secretory product from the short
3. Results
3.1. Leaf Morphological Characteristics
C. lancifolius leaves were simple, leathery, petiolate and
arranged in pairs alternating with each other at an angle
less than 180°. The mature leaf area was 7 - 28 cm2,
glossy in appearance with relatively fewer trichomes on
both surfaces. Leaves were amphistomatous with rela-
tively same number of stomata on both adaxial and
abaxial surfaces (Table 1). The stomata complex was
anomocytic, with guard cells embedded in crypts formed
by deposits of cuticular wax. Compared to mature leaves,
younger and expanding leaves were glaucous, had
slightly raised stomata, more trichomes and deposits of
various crystalloids on their surfaces. Other leaf trait dif-
ferences are shown in (Table 1).
Table 1. Leaf traits, morphology and water conte nt of C. lancifolius.

Leaf Traits and Histochemistry of Trichomes of Conocarpus lancifolius a Combretaceae in Semi-Arid Conditions 168
(a)
(b)
Figure 1. (a) SEM micrographs of secretory and non-secretory trichomes on a young leaf of C. lancifolius. (b) Stomata sur-
ounded by cuticular wax and a basal port ion of a long non-se cr e t ory tric home. r

Leaf Traits and Histochemistry of Trichomes of Conocarpus lancifolius a Combretaceae in Semi-Arid Conditions 169
trichome showed a high proportion of it was organic in
nature. About 12% of it was phosphates and sulphates of
magnesium, potassium and calcium (data not shown).
The distal end of leaf petioles had a pair of extrafloral
nectaries and on the blade margins, 3-4 pairs near the leaf
apex (Figures 3(a) and (b)).
As leaves matured, two secretory cavities develop on
the adaxial surface. The first is a large, cavity located
between the mid-vein and branch veins (Figures 4(a)
and (b)). The cavity is surrounded by two layers of
epithelial cells that secreted polysaccharides and other
substances (Figure 4(c)). The second was smaller,
dome-like structures along the mid-vein and randomly on
the leaf blade. On the surface of these dome-like glands
are sparsely distributed stomata. These glands ruptured
or developed a single pore at the top to release secretory
material, mostly epicuticlar waxes and phenolic com-
pounds (Figure 5).
(a)
(b)
(a)
(c)
(b)
Figure 2. (a) SEM micrographs of secretory trichome of C.
lancifolius. (b) Light microscopy of a section through a leaf
and short-stalked secretory trichome (arrow) and secretory
cells; “c”.
Figure 3. (a) Extrafloral nectaries on the petiole of C. lanci-
folius leaf. (b) Extrafloral nectaries on the leaf margin near
the leaf apex of C. lancifolius (arrows).

Leaf Traits and Histochemistry of Trichomes of Conocarpus lancifolius a Combretaceae in Semi-Arid Conditions 170
(a)
(b)
(c)
Figure 4. (a) Leaf surface showing secretory cavities. (a)
Light micrograph of abaxial surface showing a hollow sec-
retory cavities located between the mid-vein and branch
veins (arrow) (×4). (b) SEM of a hollow secretory cavity. (c)
Light micrograph of a section through a hollow secretory
cavity; EC = epithelial cells and S = secretory substance
(×10).
Figure 5. SEM of a pore of a collapsed dome-like secretory
gland with secretions and scale-like platelets of cutin and
waxes around it.
3.4. Histochemistry
Histochemical analysis of leaf tissues and trichomes with
a number of stains showed the following positive results.
Toluidine blue and Vanillin stained the epidermal and
hypodermal cells shades of blue and reddish colors, re-
spectively. Ferric chloride was positive for polyphenols
and Ruthenium red showed the presence of polysaccha-
rides other than cellulose. The capitate end and the bul-
bous base of trichomes stained red with Sudan III (Fig-
ure 6), an indication of the presence of lipids, waxes and
terpenes. Sudan 7B and Auramine O turned the contents
of the nectaries orange to red. Secreted material from
extrafoliar nectaries on the petiole and leaf tips also
turned reddish brown with Fehling’s solution and brick
red with Barfoed’s test.
3.5. Composition of Leaf Cuticular Waxes
The major components of the cuticle were alkanes, fatty
Figure 6. Bright field micrograph of short-stalked trichome
with capitate head stained with Sudan III (×10).

Leaf Traits and Histochemistry of Trichomes of Conocarpus lancifolius a Combretaceae in Semi-Arid Conditions 171
acids and phenols. The composition of the cuticle was
very similar in young, expanding and mature leaves
(Figure 7). The alkanes comprised of C17-C32 chain
lengths, the most abundant were pentamethylicosane
(C20), tetracosane (C24), hexacosane (C26), dotriacontane
(C32). Fatty acid methyl esters (FAME) detected in the
wax are shown in Table 2. The predominant saturated
fatty acids were palmitic acid and stearic acid although;
nondecanoic acid, arachidic acid and behenic acid were
found in significant amounts. The most prevalent pheno-
lic compound was dimethylbenzylphenol.
4. Discussion
Plants that thrive in semi-arid habitats have adaptive fea-
tures or mechanisms to mitigate extreme environmental
factors such as water deficit [17] low nutrition [18] high
Figure 7. Percent composition of cuticular wax of young and old leaves of C. lancifolius.
Table 2. GC-MS analysis of fatty acid methyl esters extracted from cuticular layer of young and mature leaves of C. lanci-
folius.

Leaf Traits and Histochemistry of Trichomes of Conocarpus lancifolius a Combretaceae in Semi-Arid Conditions 172
temperatures and solar radiation [19]. In this study leaf
traits of C. lancifolius growing in semi-arid conditions
showed xerophytic characteristics. These included a thick
cuticle, thick adaxial epidermal cell wall and sclero-
phylly.
A few plants showed morphological differences simi-
lar to the polymorphism of Conocarpus erectus [8,20].
Apart from leaf area and color, these morphotypes had
relatively the same leaf traits for young and matures
leaves. It is possible that these phenotypic differences in
C. lancifolius plants could be a response of individual
plants to stress factors.
Mature leaves were also less glaucous and apparently
did not lose their capacity for wax deposition. Wax de-
posit and/or composition restrict water loss and influence
water permeability coefficient which in turn affects epi-
dermal permeability and drought tolerance [21]. In this
study, there was apparently no significant difference in
the cuticular wax composition between young, expanding
and mature leaves. Thus, it appeared the wax load cou-
pled with adaxial epidermal wall thickness might be con-
tributing to drought tolerance. In addition to restricting
water loss from leaves, the epicuticlar waxes may serve
to reflect excessive light thus, reducing the possibility of
photoinhibiton [22-24]. The following leaf traits: leaf
angle, stomata in depressions created by the cuticular
wax deposits, thick outer epidermal cell wall with a thick
cuticular layer could help reduce water loss.
The relative water content (RWC) was 82.4 - 90.3, this
range of RWC values correlated with changes in tissue
composition and some alterations in the relative rate of
photosynthesis and respiration [25]. The range of sclero-
phylly was 57.1 - 83.2 g·m–2 and succulence of 289.4 -
362.5 g·m–2 for mature leaves. Both parameters exceeded
the 39 g·m–2 and 56 g·m–2 for sclerophylly and succu-
lence respectively, recorded for mesomorphic leaves of
deciduous trees and shrubs [5]. Sclerophylly in this habi-
tat could be a consequence of drought stress [17] and/or
the low nutrient-sandy soils [18].
Leaf surfaces had two types of trichomes: a secretory
and a non-secretory trichome. The developing leaves and
young leaf primodia had more trichomes per unit area
than mature and fully expanded leaves [26,27]. The num-
bers of trichomes appeared to be established early during
leaf differentiation and continued to increase with leaf
growth [26,28]. During cell enlargement of leaves, no
new trichomes were produced and those present became
spatially distant from each other or were dropped. The
short-stalked secretory trichome developed from epider-
mal initials. The high density of these trichomes may
have a role in insulation and protection of young and
expanding leaves.
Morphology and secretory characteristics of extrafloral
nectaries are diverse in plants [29-31]. The only report of
extrafloral nectaries on the genus Conocarpus is on
Conocarpus erectus, on which a pair of nectatries on the
petiole was described [29,31]. C. lanciflolius however,
has three additional extrafloral nectaries, 3 - 4 pairs on
margins near the leaf tip, a pair between the mid-vein and
branch veins and randomly distributed dome-like secre-
tory structures on the abaxial leaf surface.
Histochemically, the non-secretory trichome showed a
positive reaction to Sudan 7B and Aoramine O, an indi-
cation of the presence of suberin-cutin in and on the cell
wall. The reaction of sudan III showed the capitate head
of the short trichomes contained lipids, waxes and ter-
penes, which may have protective functions [16]. Pheno-
lic substances and some polysaccharides were detected
by ferric chloride and Toliudine Blue in the epidermal
cells and the cells contiguous to them. These phenolic
compounds could deter herbivory and/or ameliorate the
effect of the intense summer solar radiation. Fehling’s
and Bardfoed’s tests on the secretory material from the
nectaries on the petiole and leaf margins indicated the
presence of reducing sugars and monosaccharides, re-
spectively. These sugars probably attracted a number of
beneficial insects that were observed on the plants par-
ticularly in summer.
Palmitic, stearic, nondecanoic, arachidic and behenic
acids were detected in the wax of this species for first
time and are among fatty acids methyl esters reported in
the wax of some mangrove leaves [32,33]. The descrip-
tion of trichomes, extrafloral nectaries and phytochemi-
cal compounds which are adaptive features of species in
arid environments, detected in the cuticle could serve as
additional taxonomic characters.
The ecophysiological and biochemical studies that we
have initiated may provide more insight in the species
capacity to adapt to stress factors in a semi-arid habitat.
5. Acknowledgements
We thank Kuwait Foundation for the Advancement of
Science (KFAS) for funding this project (# 2007/1207/07),
Research Administration, Kuwait University for partially
funding this project (SL04/08) and the EM unit, KU for
their assistance in microscopy. In addition, we thank Mrs.
Jacquilion Jose and Mrs. Divya Saju who worked as Re-
search Assistants on this project.
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