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Glandular trichomes of
Rosmarinus officinalis
L.: Anatomical and
phytochemical analyses of leaf volatiles
Yilan Fung Boixab; Cristiane Pimentel Victórioac; Anna Carina Antunes Defaverid; Rosani Do Carmo De
Oliveira Arrudad; Alice Satod; Celso Luiz Salgueiro Lagea
a Laboratório de Fisiologia Vegetal, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do
Rio de Janeiro (UFRJ), Rio, Brazil b Laboratório de Biotecnologia Vegetal, Centro Nacional de
Electromagnetismo Aplicado (CNEA), Universidad de Oriente, Cuba c Colegiado de Ciências
Biológicas, Centro Universitário Estadual da Zona Oeste, Campo Grande, Rio, RJ, Brazil d
Departamento de Botânica, Laboratório de Anatomia Vegetal e Laboratório de Cultura de Tecidos
Vegetais, Rio, Brazil
First published on: 16 June 2011
To cite this Article Boix, Yilan Fung , Victório, Cristiane Pimentel , Defaveri, Anna Carina Antunes , Arruda, Rosani Do
Carmo De Oliveira , Sato, Alice and Lage, Celso Luiz Salgueiro(2011) 'Glandular trichomes of
Rosmarinus officinalis
L.:
Anatomical and phytochemical analyses of leaf volatiles', Plant Biosystems - An International Journal Dealing with all
Aspects of Plant Biology,, First published on: 16 June 2011 (iFirst)
To link to this Article: DOI: 10.1080/11263504.2011.584075
URL: http://dx.doi.org/10.1080/11263504.2011.584075
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Glandular trichomes of Rosmarinus officinalis L.: Anatomical and
phytochemical analyses of leaf volatiles
YILAN FUNG BOIX
1,2
, CRISTIANE PIMENTEL VICTO
´RIO
1,3
,
ANNA CARINA ANTUNES DEFAVERI
4
, ROSANI DO CARMO DE OLIVEIRA ARRUDA
4
,
ALICE SATO
4
, & CELSO LUIZ SALGUEIRO LAGE
1
1
Laborato´rio de Fisiologia Vegetal, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
(UFRJ), Rio de Janeiro – RJ 21941-902, Brazil,
2
Laborato´rio de Biotecnologia Vegetal, Centro Nacional de
Electromagnetismo Aplicado (CNEA), Universidad de Oriente, 53 22 646378, Cuba,
3
Colegiado de Cieˆncias Biolo´gicas,
Centro Universita´rio Estadual da Zona Oeste, Campo Grande, Rio de Janeiro – RJ 23070-200, RJ, Brazil and
4
Laborato´rio
de Anatomia Vegetal e Laborato´rio de Cultura de Tecidos Vegetais, Departamento de Botaˆ nica, Universidade Federal do
Estado do Rio de Janeiro (UNIRIO), Rio de Janeiro – RJ 22290-240, Brazil
Abstract
Rosmarinus officinalis is known for the production of volatile compounds used in medicinal and food preparations. Leaves
of R. officinalis are densely covered with capitate and peltate glandular trichomes where biosynthesis of volatiles mainly
occurs. This study aims to conduct a morphological assessment to identify anatomical characteristics of both leaves and
trichomes, as well as a chemical analysis of leaf volatile compounds, using histochemistry and stem distillation
extraction. Specifically, anatomical and chemical constituents of the secretory structures of R. officinalis leaves were
investigated using light and scanning electron microscopy, in addition to gas chromatography. One peltate and three
types of capitate glandular trichomes were observed on the leaves of R. officinalis. Histochemical tests showed positive
reactions to lipophilic compounds for both capitate and peltate trichomes, with only a slight detection of terpenoids with
carbonyl group in peltate glands. Gas chromatography revealed camphor (23.2%) as the main volatile compound, mostly
accumulating in peltate glandular trichomes. This phytochemical study of volatile compounds, together with anatomical
and histochemical analyses of R. officinalis leaves, demonstrated the importance of leaves as a center of volatile
production in peltate and capitate trichomes, as well as the nature of volatile composition, which is involved in species
survival.
Keywords: Glandular trichomes, histochemistry, leaf anatomy, terpenoids, volatile organic compounds
Introduction
Rosmarinus L. (Lamiaceae) is native to the Mediter-
ranean regions and can be found in countries with a
temperate climate (Rosua 1986). Most species of
this genus are sources of volatile compounds used in
both medicinal and food preparations. Specifically,
Rosmarinus officinalis L., a herbaceous and perennial
plant, also known as rosemary and ‘‘alecrim’’, is used
in food flavoring, and in folk medicine to treat hepatic,
intestinal, renal, and respiratory infections (Lorenzi &
Matos 2008). Additionally, its antimicrobial and
antifungal activities have been widely demonstrated
(Soliman et al. 1994). Among the volatile compounds
of R. officinalis L., terpenoids mainly contribute to
its medicinal activity, as well as to aroma and taste
(Atti-Santos et al. 2005). The aroma is a complex
chemical mixture containing mono- and sesquiterpe-
nic families of hydrocarbons, ketones, alcohols,
aldehydes, esters, and phenols. Different chemotypes
are suggested for R. officinalis because of intraspecific
constituent variation that depends on the geographic
region of collection. Camphor, a–pinene, 1,8 cineole
and myrcene have been described as the main volatile
chemotypes (Diab et al. 2002; Pintore et al. 2002;
Atti-Santos et al. 2005; Boix et al. 2010).
Correspondence: Cristiane Pimentel Victo´rio, Laborato´rio de Fisiologia Vegetal, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro, Bloco G, sala G2-050, Rio de Janeiro – RJ 21941-902, Brazil. Tel. þ55-21-2562-6330. Email: cristianevictorio@uezo.rj.gov.br
Plant Biosystems, 2011; 1–9, iFirst article
ISSN 1126-3504 print/ISSN 1724-5575 online ª2011 Societa` Botanica Italiana
DOI: 10.1080/11263504.2011.584075
Downloaded By: [Victório, Cristiane Pimentel] At: 13:00 16 June 2011
Several natural compounds in plants are mainly
synthesized in specialized cells of leaves, flowers, and
roots. Among the Mediterranean species, many
interesting compounds have been identified in the
leaves, e.g. in the halophyte Crithmum maritimum L.
(Cornara et al. 2009) and the common Nerium
oleander (Ibrahim et al. 2009). Glandular structures
are known to be primary sites of secondary metabolite
biosynthesis, secretion, and storage, and these
structures generally consist of either simple subcuta-
neous glands or trichomes (Fahn 1979; Weiss 1997).
Furthermore, recent studies have demonstrated the
production of secondary metabolites in plastids from
secretory cells, which is followed by their transloca-
tion to the peripheral portion of the cytoplasm, and
subsequent movement through the walls to the
subcuticular space (Machado et al. 2006; Biswas
et al. 2009). Glandular trichomes may occur on
leaves, stems, and even parts of flowers, depending on
the species (Wagner 1991; Dayan and Duke 2003);
their distribution and structure on the leaf surface are
mainly associated with volatile metabolite production
(Bosabalidis 2002; Sharma et al. 2003). Peltate and
capitate glandular trichomes are the most common,
with peltate trichomes being the main site of volatile
production and storage (Sharma et al. 2003).
According to Fahn (1979), the Lamiaceae present
two types of glandular trichomes with the same basic
morphology, consisting of a basal region, a stalk, and
a head. The capitate glandular trichome is formed by
a head with one secretory cell and a stalk containing
two cells, while the peltate type is formed by a head
with eight secretory cells, one basal epidermal cell,
and a wide unicellular stalk cell. Trichomes are also
considered important for discriminating among taxa,
and they play a key role in the systematics of the
Lamiaceae (Salmaki et al. 2009).
Although the phytochemical aspects of R. officinalis
have been well studied, the presence, and the
secretory structures, of volatile compounds remain
to be elucidated. Based on the importance of
R. officinalis in the fragrance and food industries,
many investigators have analyzed its volatile com-
pounds using different extraction methods (Table I).
The aim of this study was to analyze the glandular
trichomes of R. officinalis for their chemical composi-
tion and production of volatile compounds using
imaging techniques in order to determine leaf
anatomy and histochemical content. In addition, leaf
volatile extraction was carried out by simultaneous
distillation–extraction, elution with dichloromethane,
and gas chromatography.
Material and methods
Plant material
Varieties of R. officinalis, which are grown in
commercial fields near Rio de Janeiro, were sown in
a greenhouse in 2009 (Figure 1). Voucher specimens
were deposited in the Herbarium of Rio de Janeiro
Botanical Garden (Brazil) under catalogue number
RB 471608. Anatomical analyses were performed
from leaf samples fixed in F.A.A. (formalin, acetic
acid, ethanol 70%, Johansen 1940), while volatile
composition and its localization in leaf tissues were
investigated using fresh leaves.
Scanning electron microscopy
For scanning electron microscopy (SEM), fixed
leaves were dehydrated in graded ethanol series,
submitted to critical-point drying with CO
2
(Leica
EM CPD-030), mounted on stubs, and coated with
a thin layer of gold (Denton Vacuum Desk IV, LLC).
The samples were analyzed with a JEOL-JSM 6390
LV scanning electron microscope (JEOL, Tokyo,
Japan).
Table I. Summary of studies on volatile compounds of Rosmarinus officinalis.
Sample origin
Extraction
method
Analytical
method
Total
identified Main volatiles Reference
Spain Supercritical
fluid extraction
GC/FID,
GC/MS
33 a-pinene, 1,8-cineole,
camphor, verbenone, and
borneol
Santoyo et al. (2005)
Parana´
a
Supercritical
carbon dioxide
23 camphor, verbenone, Genena et al. (2008)
Rio Grande
do Sul
a
Hydrodistillation 20/28 a-pinene and 1,8-cineole Atti-Santos et al.
(2005); Cassel et al.
(2009)
Algeria Hydrodistillation
and microwave
hydrodiffusion
and Gravity
33 a-pinene, camphene,
limonene, camphor, and
verbenone
Bousbia et al. (2009)
a
Brazil
2Y. F. Boix et al.
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Light microscopy
Fully developed leaves were fixed in F.A.A. for 48 h
and preserved in 70% ethanol. For light microscopy,
sections of the leaf blade were taken from the
intercostal region, dehydrated in a graded ethanol–
butanol series, embedded in paraffin, sectioned
transversely and longitudinally using a rotary micro-
tome at a thickness of 12 mm. The sections were
stained with 1% fucsin in 50% ethanol and 1%
Alcian blue, and mounted on synthetic resin. The
epidermis was studied using sections of the leaf
blade dissociated in acetic acid/hydrogen peroxide
(Franklin 1945) and stained in 1% aqueous
safranin.
Histochemical staining was employed to observe
the leaf tissues responsible for the production and
accumulation of lipophilic compounds and terpe-
noids. Fresh leaf blades sectioned transversally were
subjected to the following histochemical tests: Sudan
black B (Pearse 1980), Sudan III and Sudan IV
(Johansen 1940) for lipophilic compounds; and 2,4-
dinitrophenylhydrazine for terpenoids with a carbonyl
group (C¼O) (Ganter & Jolle´s 1969). Observations
were carried out and captured by light microscopy
using an Olympus BX-41 microscope. Sudan tests
were carried out by placing leaf sections in 70%
ethanol for 1 min, staining in 0.03% filtered solutions
of each Sudan type in 70% ethanol for 30 min at
408C in water bath, and washing rapidly with 70%
ethanol. For terpenoids with a carbonyl group,
sections were treated with saturated and filtered
solutions of 2,4-dinitrophenylhydrazine in HCl 2 N
for 30 min.
To observe the vascular system, fixed leaves were
placed in 5% NaOH and heated in an oven at 408C
until most of the pigment was removed. Afterwards,
the pieces were cleared with 5% chloral hydrate
for 2 h until they became transparent, and stained
with 0.5% acid fucsin in 50% ethanol. To make a
permanent preparation, the material was de-
hydrated in a graded ethanol/t-butanol series, and
then mounted on synthetic resin.
Collection of volatiles by gas chromatography flame
ionization detector (GC-FID) and gas chromatography
mass spectrometry (GC-MS)
Fresh leaves (5 g) of plants were homogenized with
70 mL of distilled water and subjected to simulta-
neous distillation–extraction (SDE) for 2 h using 2
mL of dichloromethane as an organic collecting
solvent (Godefroot et al. 1981).
Analytical GC/FID was carried out on a Varian
Star 3400 gas chromatograph fitted with a DB-5/MS
column (30 m 60.25 mm, film thickness 0.25 mm).
Temperature increase was programmed from 60 to
2708Cat38Cmin
71
.A1-mL aliquot of the sample
was injected at 2708C, splitless mode. Hydrogen was
used as the carrier gas at a flow rate of 1 mL min
71
.
GC/MS analyses were performed using a Shimadzu
Model GC MS-QP 5000 apparatus under the
following conditions: column, HP-5 fused silica
capillary column (30 m 60.25 mm, film thickness
0.25 mm); carrier gas, helium at 1 mL min
71
;
injection of 1 mL; split ratio 1:40; injector tempera-
ture, 2708C; interface 2008C; column temperature,
60–2708Cat38Cmin
71
; mass spectra, 70 eV. The
identification of the constituents was achieved by
comparison of retention indices (RI) calculated for all
volatile contents using a homologous series of n-
alkanes (C
8
–C
28
) recorded under identical operating
conditions, comparing MS data and GC data with
those of standard samples obtained by a computer
library search of the National Institute of Standards
and Technology (NIST) and from the literature
(Adams 1995).
Figure 1. Rosmarinus officinalis. A. Plants in greenhouse; B. Herbaceous habit. Scale bars ¼4cm.
Trichomes of Rosmarinus officinalis leaves 3
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Figure 2. Leaf surface of Rosmarinus officinalis imaged by scanning electron microscopy. (A) Distribution of glandular trichomes on the
adaxial epidermis; (B) Distribution of non-glandular and glandular trichomes and revolute leaf margin (asterisks) on the abaxial epidermis;
(C) Detail of the abaxial leaf epidermis showing the high density of trichomes, predominantly located in epidermis depressions (arrow); (D)
Non-glandular, capitate and peltate glandular trichomes on the abaxial epidermis; (E) Capitate glandular trichomes (long and short stalk) on
the adaxial epidermis; (F) Detail of peltate (p) and capitate (c) glandular trichomes and cuticle striations; (G) Secretory head of peltate
trichome showing eight radially disposed cells and broken cuticle (arrow); (H) Detail of the rupture in the cuticle of a peltate glandular
trichome. Scale bars: A–D ¼100 mm; E–H ¼10 mm.
4Y. F. Boix et al.
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Results
In front view, both leaf surfaces of R. officinalis are
composed of common epidermal cells, stomata, and
glandular trichomes, the latter being numerous in the
abaxial epidermis where non-glandular trichomes
also occur (Figure 2A–E). Glandular trichomes occur
in greater numbers in grooves (Figure 2C–D). Both
glandular and non-glandular trichomes are pluricel-
lular and composed of a thick indumentum in the
abaxial epidermis (Figure 2B, C). Several types of
glandular trichomes can be recognized. One capitate
type exhibits a unicellular secretory head (Figure 2F
and Figure 3A), a basal cell, and a short bicellular
stalk (Figure 3B and Figure 4G). A second capitate
type also has a unicellular secretory head and a basal
cell, but a bicellular stalk, both long and short
(Figures 3C and 4H). The third capitate is long-
stalked and can be distinguished from the others by
having three stalk cells (Figures 3D and 4A). The
capitate trichomes were located randomly on both
leaf surfaces; they were more numerous than the
peltate trichomes (Figure 2D). At the same time,
however, the peltate trichome (Figures 2F, 3A, and
4F) is more voluminous and has a secretory head with
eight radially disposed cells (Figure 2G). Its stalk is
formed by one basal epidermal cell and a large stalk
cell. Particularly in this last type, the cuticle becomes
detached from secretory cell walls to create a space for
the accumulated secretion. The rupture of the cuticle
of peltate glandular trichomes was observed through
SEM analysis of R. officinalis leaves (Figure 2H).
Dendroid non-glandular trichomes are sustained by
one cell, followed by a bifurcated series of cells
(Figure 2D; Figure 4B and E).
The normal epidermal cells have straight anticlinal
cell walls and are covered by a striated cuticle. On
the opposite surface, these cells have sinuous cell
walls, and the cuticle covering them shows conspic-
uous striations, especially around stomata where they
are radially arranged (Figure 4A–C). Diacytic
stomata are abundant on the abaxial surface
(Figure 4C) where they occur in depressions of the
leaf blade delimitated by rib projections. In trans-
versal section, guard cells are projected outwards,
but protected by numerous non-glandular tri-
chomes. The leaf midrib region is slightly depressed
on the upper surface, but stands out on the abaxial
surface (Figure 4D). In this region and at the leaf
margin, epidermal cells are elongated and trichomes
are scarce. The leaf margin is also very revolute
(Figures 2B and 4D).
In transversal section, the epidermis is uniseriated
and covered by a thick cuticle on the adaxial surface,
and a thin cuticle on the abaxial one. Epidermal cells
are flattened and exhibit thick cell walls on the upper
surface and thin ones on the lower surface. Oil
droplets could be found in the cytoplasm of these
cells and also trapped between the cell walls and
cuticle. The leaf mesophyll structure is dorsiventral
(Figure 4D). A hypoderm with no chloroplasts and
thick cell walls occurs under the adaxial epidermis.
The palisade parenchyma has two or three layers of
cells with thin walls. Spongy parenchyma has up to
layers separated by wide intercellular spaces. Hy-
dathodes were observed in the epidermal margin of
leaves (Figure 4Nand O). The vascular system
consists of collateral bundles surrounded by a
parenchyma sheath which delimits the chlorenchyma
(Figure 4D).
Histochemical testing showed the presence of
deposits that accumulated in the subcuticular space
of peltate and capitate trichomes. These contain
lipophilic compounds as shown by strong positive
reaction to Sudan tests (Figure 4I–M). Epidermal
cells, as well as parenchyma and chlorenchyma
cells, also exhibited lipophilic droplets, which were
detected by these tests. Sudan staining seemed
to be more intense in the secretory cells of
peltate trichomes (Figure 4K and M). Staining with
Figure 3. Schematic morphology of glandular trichomes of Rosmarinus officinalis. (A) Peltate; (B) Capitate trichome with bicellularshort stalk;
(C) Capitate trichome with bicellular long stalk. Scale bars ¼50 mm.
Trichomes of Rosmarinus officinalis leaves 5
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6Y. F. Boix et al.
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2,4- dinitrophenylhydrazine gave a slightly positive
reaction (orange–brown color) in peltate glandular
trichomes, which suggests that terpenoids with a
carbonyl group are produced mainly by these
structures. Based on GC analysis, camphor is the
main terpenoid with a carbonyl group produced by
the R. officinalis leaves collected in Rio de Janeiro
(Figure 5).
Numerous compounds were detected by SDE in
the region where monoterpenoids elute. The relative
area of each constituent, and its retention index, is
indicated in Table II. The main volatile compounds
were a-pinene (11.2%), b-pinene (8.5%), myrcene
(9.5%), 1,8 cineole (13.4%), camphor (23.2%), and
verbenone (8.2%), giving a total of 87.7% mono-
terpenes. Among the identified compounds, only
three were sesquiterpenes, namely cedrene (4.2%),
santalene (1.7%) and bisabolol (0.3%). The percen-
tage of terpenoids with a carbonyl group, such as
camphor and verbenone, was above 30% (Figure 5).
Discussion
SEM analysis allowed us to visualize the general
appearance of the leaf surface, and the distribution,
size, and shapes of trichomes of R. officinalis. Light
microscopy enabled the elucidation of trichome
types on the leaf epidermis. In addition, the
localization of lipophilic compounds and terpenoids
was made possible by histochemical staining.
Trichomes showed the same anatomical and
morphological patterns as those of other members
of the Lamiaceae as described by Fahn (1979), who
used Mentha piperita as the model species. However,
an additional capitate trichome, not previously
reported in the studies of Marin et al. (2006) on
R. officinalis, containing both bicellular and tricel-
lular stalk cells, was presently observed. The
presence of tricellular stalk cells in glandular
trichomes was observed only in the adaxial epider-
mis; they occur in a sparse and rare distribution.
Through staining techniques, the presence of cutin
in stalk cells of glandular trichomes, as described by
Fahn (1979), was not observed. Non-glandular
trichomes were more abundant on abaxial leaf
surface grooves, which coalesce to thicken the
mechanical layer that preserves the integrity of
glandular trichomes and protect the plant from
herbivores and desiccation (Machado et al. 2006).
The present study confirms that the distribution of
glandular trichomes varies substantially in quantity
in relation to their location on a leaf surface.
Cuticle rupture of peltate glandular trichomes in the
leaves of R. officinalis conforms to the same sequential
patterns as those described by Sharma et al. (2003) for
the glandular trichome development of Mentha
arvensis. Specifically, volatile biosynthesis of the apical
oil gland first causes cell lysis. This event is followed by
rupture of the cuticle, which releases volatiles, and,
finally, new cuticle is generated.
Histochemical tests, combined with phytochem-
ical analysis, enabled us to locate the secondary
metabolites in glandular trichomes and plant tissues.
Our results indicated that glandular trichomes of
R. officinalis leaves are involved in producing
Table II. Percentages of terpenoids in GC-FID analyses of
volatiles produced by Rosmarinus officinalis after fresh leaves were
extracted by SDE.
Constituents
a
Formula RT
RI
calculated
Relative
area
b
(%)
a-pinene C
10
H
16
3.893 935 11.2 +1.25
a-camphene C
10
H
16
4.232 951 4.2 +0.27
Verbenene C
10
H
16
4.299 953 0.1 +0.02
b-pinene C
10
H
16
4.905 978 8.5 +1.5
Myrcene C
10
H
16
5.241 988 9.5 +1.5
a-phellandrene C
10
H
16
5.625 1003 0.2 +0.01
a-terpinene C
10
H
16
5.935 1015 0.6 +0.03
1,8 cineol C
10
H
18
O 6.523 1034 13.4 +0.41
g-terpinene C
10
H
16
7.270 1057 2.0 +0.28
p-mentha-
2.4(8)-diene
C
10
H
16
8.178 1082 0.8 +0.03
Linalool C
10
H
18
O 8.883 1099 0.5 +0.15
Chrysanthenone C
10
H
14
O 9.586 1122 0.3 +0.02
Camphor C
10
H
16
O 10.791 1153 23.2 +1.61
Pinocarvone C
10
H
14
O 11.204 1162 0.5 +0.11
Menthol C
10
H
20
O 11.630 1171 1.1 +0.09
Unknown – 11.742 1174 0.4 +0.06
Terpinen-4-ol C
10
H
18
O 11.963 1179 1.0 +0.11
Unknown – 12.739 1194 2.0 +0.10
Verbenone C
10
H
14
O 13.292 1208 8.2 +0.57
Unknown – 15.746 1266 0.2 +0.05
Bornyl acetate C
12
H
20
O
2
16.392 1280 1.1 +0.52
Cedrene C
15
H
24
22.053 1411 4.2 +1.11
Santalene C
15
H
24
23.515 1447 1.7 +0.27
Epi-a-bisabolol C
15
H
26
O 32.348 1677 0.3 +0.01
Total identified 95.4
Note: RI, retention index.
a
Arranged in order of retention time
(RT) on the DB-5 column;
b
Values are means +S.D. obtained in
triplicate.
Figure 4. Anatomical aspects and details of Rosmarinus officinalis in light microscopy. (A) Capitate glandular trichome on the adaxial
epidermis (arrow); (B) Non-glandular trichome on the adaxial epidermis (arrow); (C) Diacytic stomata; (D) Transversal leaf section showing
revolute leaf margin (arrow) and grooves in the mesophyll (asterisks); (E) Abaxial epidermis showing non-glandular (ng), capitate (c), and
peltate (p) glandular trichomes; (F) Peltate glandular trichome; (G) Short-stalked capitate glandular trichomes; (H) Long-stalked capitate
glandular trichome; (I–M) Histochemical characterization of the secretions of leaf glandular trichomes; (I) Sudan III; (J and K) Sudan IV; (L
and M) Sudan black B. (N and O) Hydathodes along the margin of leaves. Scale bars ¼20 mm, J ¼10 mm, O ¼100 mm.
3
Trichomes of Rosmarinus officinalis leaves 7
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lipophilic compounds and secreting volatile terpe-
noids, thus strengthening their role as a defense
mechanism. Our observation of terpenoids with a
carbonyl group only in peltate trichomes is consistent
with previous histochemical analyses for R. officinalis
leaves (Marin et al. 2006), which gave evidence of
phenolic compounds only in peltate trichomes. In
studies with Zeyheria montana (Bignoniaceae), pel-
tate glandular trichomes constitute the main site of
lipophilic compound biosynthesis/accumulation;
these then can play a protective role in fruit
development, ensuring the reproductive success of
the species (Machado et al. 2006). Although
glandular trichomes are the main site of terpenoid
accumulation, the histochemical tests showed posi-
tive reactions to lipophilic compounds in cells of
other plant tissues, increasing the chances of plant
defense. The high concentration of camphor, and its
presence on peltate glandular trichomes, indicated
that this trichome type is the main site of volatile
production and accumulation in R. officinalis, and
that R. officinalis from Rio de Janeiro is similar to
rosemary, which is a camphor chemotype (Price &
Price 1999). Its constituent is a terpenoid with a
carbonyl group, which agrees with specific histo-
chemical tests performed with peltate glandular
trichomes. Moreover, peltate trichomes exhibit more
head cells, a factor which may contribute to
increased production of volatiles.
Xeromorphic features were also observed in
R. officinalis leaves. These features included reduc-
tion in leaf area, as an adaptation to increase water
resistance and reduce nutritional deficit, and the
presence of specialized pores called hydathodes
responsible for guttation, in agreement with Fahn
and Cutler (1992). Such xeromorphic characteristics
are probably conservative, and do not change in
R. officinalis from different regions. The results
presented here agree with the anatomical and
morphological observations reported by Rotondi
et al. (2003) using native samples collected in the
Mediterranean macchia ecosystem. The high density
of non-glandular trichomes on the foliar epidermis
influences light reflection and may also decrease leaf
temperature (Gutschick 1999; Press 1999), as well as
photoinhibition (Lin & Ehleringer 1983).
Overall, the phytochemical study of volatile com-
pounds, associated with anatomical and histochem-
ical analyses, of R. officinalis leaves demonstrated
the importance of leaves in volatile secondary
metabolite production in glandular trichomes that
permit the reproduction and survival of the species in
nature.
Acknowledgments
We thank CAPES/Brazil-Cuba for providing a
fellowship to the first author, Mr. Elivaldo Lima
(Museu Nacional/UFRJ) for assistance with scan-
ning electron microscopy, Jefferson Ferra˜o for help
with the figures, and Siannah M. Ma´s Diego, MSc.,
(CNEA/UO) for revision of the English text.
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