The oleoresin secretory system in seedlings and adult

Flora 206 (2011) 585–594
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Flora
journal homepage: www.elsevier.de/flora
The oleoresin secretory system in seedlings and adult plants of copaíba (Copaifera
langsdorffii Desf., Leguminosae–Caesalpinioideae)
Tatiane Maria Rodrigues a,∗ , Simone de Pádua Teixeira b , Silvia Rodrigues Machado a
a
b
São Paulo State University, UNESP, Institute of Biosciences, Department of Botany, 18618-000 Botucatu, São Paulo State, Brazil
São Paulo University, USP, Faculty of Pharmaceutical Sciences of Ribeirão Preto, 14040-903 Ribeirão Preto, São Paulo State, Brazil
a r t i c l e
i n f o
Article history:
Received 16 July 2010
Accepted 17 October 2010
Keywords:
Anatomy
Canal
Cavity
Copaifera langsdorffii
Ultrastructure
a b s t r a c t
The ecological and economic importance of oleoresin produced by Copaifera langsdorffii is well established. This study aims to investigate the ontogeny, anatomy and ultrastructure of the internal glands
of C. langsdorffii during plant development. Samples were processed for light and electron microscopy
and a specific technique was applied to impregnate endomembranes. Internal secretory glands were
observed in the hypocotyl, epicotyl and eophylls of seedlings, and in the primary stem, pulvinus,
petiole, rachis and leaf blade of adult plants. Canals and cavities show differential distribution. They
arise from ground meristem cells, and the lumen is first formed by schizogenesis followed by later
schizolysigenous development. The dense cytoplasm of epithelial cells shows mitochondria, plastids without thylakoids, polyribosomes and endoplasmic reticulum. A periplastidial reticulum was
also observed. Secretion is released by eccrine, granulocrine and holocrine processes. Lipophilic and
hydrophilic compounds were histochemically detected in both canals and cavities, whereas resin was
detected only in canals. The presence of these substances has been associated with plants’ defences
against dehydration, as well as against attacks from herbivores and pathogens, from seedling stage
onwards.
© 2011 Elsevier GmbH. All rights reserved.
Introduction
Copaifera L. is a tropical genus of Leguminosae, subfamily Caesalpinioideae, characterised by the presence of internal secretory
structures, such as canals and cavities (Metcalfe and Chalk, 1950),
comprising a lumen and a secretory epithelium (Fahn, 1979). These
structures are the sites of synthesis and accumulation of oils and
oleoresins (Plowden, 2003) and have ecological functions and commercial value (Langenheim, 2003). Based on these substances, the
defence mechanisms against herbivores and pathogens (Harbone,
1993) partially ensure the success of this tropical genus in colonising diverse habitats (Langenheim, 2003).
Copaifera langsdorffii Desf., popularly known as copaíba, is an
economically important Brazilian species that is widely distributed
in the Brazilian cerrado and forests (Lorenzi, 1998). The oleoresin
produced by C. langsdorffii is of great commercial and medical interest due to its antimicrobial, anti-inflammatory, anti-ulcerogenic,
anti-tumour, cicatrising and other therapeutic properties (Biavatti
et al., 2006; Cascon and Gilbert, 2000; Pinto et al., 2000; Plowden,
2003; Veiga and Pinto, 2002). The chemical composition of C.
langsdorffii oleoresin is well known, and more than 40 different constituents have been identified (Gramosa and Silveira,
2005).
Despite the great ecological and economic importance of the
substances they produce, knowledge about the secretory structures
of this species is incomplete. There have been a few histoanatomical characterisations of such structures in the stem wood
(Marcati et al., 2001) and in stems and roots of adult plants from
Brazilian cerrado (Rodrigues and Machado, 2009). However, information on the occurrence, distribution and structural features of
the C. langsdorffii secretory system in the initial phases of plant
development is lacking.
To determine whether the structural characteristics of this
plant’s secretory system are different at various developmental
stages, we performed a detailed analysis of the anatomy and
ultrastructure of the internal secretory structures present in both
seedlings and adult plants of C. langsdorffii.
Materials and methods
∗ Corresponding author at: São Paulo State University, UNESP, Institute of Biosciences, Department of Botany, Distrito de Rubião Jr s/n, PO Box 510, 18618-000
Botucatu, São Paulo State, Brazil. Tel.: +55 14 3811 6265; fax: +55 14 38152838.
E-mail address: [email protected] (T.M. Rodrigues).
0367-2530/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.flora.2010.10.002
Plant material
Seedlings and young branches of adult plants were used in this
study. Samples of shoots (twigs and leaves) were collected from
586
T.M. Rodrigues et al. / Flora 206 (2011) 585–594
Copaifera langsdorffii plants growing in an area of cerrado vegetation located in the municipality of Botucatu (22◦ 55 S, 48◦ 30 W), in
the state of São Paulo, Brazil, during the growing season (September 2006 through February 2007 and September 2007 through
February 2008). Vouchers were deposited in the Herbarium Irina
Delanova Gemtchújnicov (BOTU) under the number 24911.
Samples of seedlings were collected from 15-day-old individuals. To obtain seedlings, seeds were collected after the spontaneous
dehiscence of the follicle from adult plants in the cerrado. To disinfect, the seeds were washed in alcohol 70% for 1 min and in sodium
hypochlorite 2% for 20 min, rinsed in distilled water (Coelho et al.,
2001) and placed in acrylic boxes lined with wet filter papers. The
boxes with seeds were maintained at 25 ◦ C under white fluorescent
light.
In this work, the term seedling refers to the developmental stage
encompassing the primary root emergence to the expansion of the
first eophyll pair (Oliveira, 2001).
Table 1
Distribution of canals and cavities in the primary vegetative axis of Copaifera langsdorffii seedlings and adult plants.
Organ/region
Epicotyl
Hypocotyl
Eophylls
Cotyledons
Primary root
Shoot
Primary pulvinus
Petiole
Rachis
Light microscopy
For general anatomical characterisation, a portion of the collected samples were fixed in FAA 50 (Johansen, 1940) for 24 h
at room temperature (25 ◦ C), then dehydrated in an ethanol
series and embedded in methacrylate resin (Leica Historesin)
according to Gerrits (1991). The material was sectioned using
a semi-automatic rotary microtome, and the sections (3 ␮m
thick) were stained with Toluidine blue 0.05%, pH 4.3, in 0.1 M
phosphate buffer (O’Brien et al., 1964). Permanent slides were
mounted with Permount (Gerlach, 1969). Other lots of material were freshly sliced using razor blades and stained with
Safrablau (Bukatsch, 1972). Temporary slides were mounted with
glycerin.
For histochemical assays, fresh material was sectioned using
razor blades. Sections (15 ␮m thick) were treated with the following: Sudan IV to detect total lipids (Johansen, 1940), Nadi’s
reagent for essential oils and oleoresin (David and Carde, 1964),
10% aqueous solution of ferric chloride for phenolic compounds
(Johansen, 1940), 0.02% aqueous solution of ruthenium red for
polysaccharides and pectin (Jensen, 1962), Schiff’s reagent (periodic acid-Schiff’s – PAS) for neutral polysaccharides (Amaral et al.,
2001), Wagner’s reagent for alkaloids (Furr and Mahlberg, 1981),
bromophenol blue for proteins (Mazia et al., 1953), and 1% cupric
acetate for resin (Johansen, 1940). For each test, a specific control was performed according to the described technique by each
author.
All the specimens were examined and documented with a light
microscope (BX 40, Olympus) equipped with a digital camera.
Transmission electron microscopy (TEM)
Samples were initially fixed in 2.5% glutaraldehyde solution
in 0.1 M phosphate buffer at a pH of 7.3 for 24 h at 5 ◦ C, postfixed with 1% osmium tetroxide in the same buffer for 1 h
at 25 ◦ C, dehydrated through acetone series and embedded in
Araldite resin (Machado and Rodrigues, 2004). Ultra-thin sections
were obtained with a Diatome diamond knife and poststained
with uranyl acetate and lead citrate (Reynolds, 1963). The material was examined with a Philips EM 301 transmission electron
microscope.
For endomembrane impregnation, samples were initially fixed
as described above and then incubated in solution with zinc, iodide,
TRIS-aminomethanol buffer and osmium tetroxide (ZIO technique)
at 10 ◦ C for 24 h (Machado and Gregório, 2001). Samples were subsequently handled as in the conventional technique.
Leaflet blade
Type of secretory reservoir
Cortex
Pith
Cortex
Pith
Mesophyll
Midrib cortex
Midrib pith
–
–
Cortex
Pith
Cortex
Pith
Cortex
Pith
Cortex
Pith
Mesophyll
Midrib cortex
Midrib pith
CV; CN
CN
CV; CN
CN
CV
CV
CN
–
–
CV; CN
CN
CV; CN
CN
CV; CN
CN
CV; CN
CN
CV
CV
CN
CV: cavity; CN: canal; –: cavities or canals absents.
Results
Distribution, morphology and histochemistry of the secretory
system
Internal secretory structures are present in the aerial vegetative
organs of Copaifera langsdorffii from the seedling to adult stage.
In the seedlings, glands in different developmental stages were
observed in the cortex and pith of epicotyl and hypocotyl and in
the mesophyll of eophylls (Fig. 1A–C). In adult plants, glands were
present in the cortex and pith of the primary stem, pulvinus, petiole and rachis and usually in the mesophyll of the expanded leaves
(Fig. 1D–H) and leaf primordia. Glands in the leaflet blade were visible as translucent dots in the internerval areas immersed in the
spongy parenchyma, with some reaching the palisade parenchyma
(Fig. 1H). Glands were present in the cortex and pith of the midribs
of leaflets (Fig. 1I). No glands were observed in cotyledons or during
the primary growth of the roots of either seedlings or adult plants.
In cross sections, mature secretory glands are comprised of a
secretory epithelium delimiting a wide, round lumen where secretions accumulate (Fig. 1J–K). However, these glands have different
shapes in longitudinal sections and can be classified as either
spherical secretory cavities (Fig. 1L) or elongated secretory canals
(Fig. 1M). The cortex of epicotyl, hypocotyl, primary stem, pulvinus,
petiole, rachis and midrib predominantly contain cavities, while
canals are observed in the pith of these regions. Cavities are also
observed in the mesophyll of eophylls, leaf primordia and expanded
leaves. The differential distribution of cavities and canals in the primary body of adult plants and seedlings of C. langsdorffii is detailed
in Table 1.
The secretory glands of the petiole and rachis cortex have a
droplet shape in cross section (Fig. 1J). In the region where the
lumen is narrower, the epithelial cells are smaller, turgid and round
(Fig. 1J).
The epithelial cells of the secretory glands in all the organs
analysed show a variety of shapes, from rounded to tabular and
tangentially elongated (Fig. 1J–K). The epithelium of the secretory glands is delimited by a sheath of tangentially elongated cells
with dense cytoplasms and voluminous nuclei, intercalated with
rounded parenchymal cells (Fig. 1J–K). The sheath cells can divide
periclinally (Fig. 1K), resulting in cells which are added to the secretory epithelium.
T.M. Rodrigues et al. / Flora 206 (2011) 585–594
587
Fig. 1. (A–M) Photomicrographs of Copafiera langsdorffii vegetative organs. (A, C, E, F and H–M) Toluidine blue. (B, D and G) Safrablau. (A) Transverse section of epicotyl
(150 ␮m). (B) Transverse section of hypocotyl (150 ␮m). (C) Transverse section of eophyll (150 ␮m). (D) Transverse section of primary stem (150 ␮m). (E) Transverse section
of pulvinus (500 ␮m). (F) Transverse section of petiole (150 ␮m). (G) Transverse section of rachis (150 ␮m). (H) Transverse section of leaf blade (100 ␮m). (I) Transverse
section of midrib (100 ␮m). (J) Transverse section of petiole showing cavity with droplet shape in the cortex of (50 ␮m). (K) Transverse section of rachis showing canals in
the pith (50 ␮m). The head arrow indicates cell division in the sheath. (L) Longitudinal section showing rounded cavities in the cortex of pulvinus (150 ␮m). (M) Longitudinal
section showing longated canals in the pith of stem (150 ␮m). The arrows indicate the location of the glands.
588
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Table 2
Results of histochemical tests to secretory cavities and canals present in aerial vegetative organs of Copaifera langsdorffii seedlings and adult plants.
Reagent
Cupric acetate
Nadi’s reagent
Sudan IV
Wagner’s reagent
Bromophenol blue
Ruthenium red
Ferric chloride
PAS
Substance
Resin
Terpenes
Total lipids
Alkaloids
Proteins
Pectic substances
Phenolic compounds
Non-cellulosic polysaccharides
Color
Green to turquoise
Blue to purple
Red
Brown to reddish
Blue
Pink
Dark green to brown
Pink
Cavities
Canals
Lumen
Epithelial cells
Sheath cells
Lumen
Epithelial cells
Sheath cells
−
+
+
+
−
+
+
+
−
+
+
+
+
+
+
+
−
−
+
+
−
+
+
+
+
+
+
+
−
+
+
+
+
+
+
+
+
+
+
+
−
−
+
+
−
+
+
+
+: positive; −: negative.
Histochemical tests of all the analysed organs similarly detected
the presence of alkaloids, proteins, pectic substances, noncellulosic polysaccharides, phenolic compounds, total lipids and
terpenes in the cavities and canals. The cupric acetate test, however, showed that resin is only found in medullar canals. The results
obtained by histochemical tests are summarised in Table 2.
Ontogenesis and development of the secretory system
In C. langsdorffii seedlings and adult plants, internal glands arise
and differentiate early in the shoot apex, in the region immediately
below the apical meristem (Fig. 2A) and in the newly produced
leaf primordia (Fig. 2A–D). The first indication of the origin of the
internal gland is the appearance of a cluster of cells with dense
cytoplasm in the ground meristem (Fig. 2A). Each cluster is initiated
from one cell that divides first in the periclinal plane (Fig. 2A and
C). These cells then divide in the anticlinal plane, giving rise to four
cells. Successive cell divisions occur in different planes, increasing the number of cells that constitute the cluster (Fig. 2C). When
the cluster reaches four to six cells, a discrete intercellular space
is formed in the central region (Fig. 2C). As development proceeds,
this space widens and gives rise to the central lumen of the secretory reservoir (Fig. 2C–D); the surrounding cells organise around
this lumen and constitute the secretory epithelium (Fig. 2D).
Secretory canals and cavities can arise in different regions of
young shoots and leaves. The occurrence of cell clusters that give
rise to secretory canals or cavities is common in the cortex and pith
of the stem and in the mesophyll of the leaf.
Ultrastructure of the secretory system
Ultrastructurally, the middle lamella between the central
cluster of cells is swollen in some regions and exhibits loose
fibrillar material (Fig. 3A). Later in development, the swollen middle lamella dissolves to form the central lumen. The precursor
cells of the internal glands are characterised by very thin walls
(Fig. 3A) with plasmodesmata (Fig. 3B), sinuous plasmalemma,
spherical nucleus, abundant and electron-dense cytoplasm and
small vacuoles (Fig. 3A–B). The cytoplasm of these cells contains
polyribosomes, mitochondria with developed cristae, hyperactive dictyosomes, smooth endoplasmic reticulum and plastids
(Fig. 3A–E). The plastids are polymorphic, without an inner membrane system and with granular homogenous matrix (Fig. 3A and
D–E). The presence of periplastidial reticulum is a remarkable feature of these cells (Fig. 3D).
Notably, a sheath of distinctive cells is present around each cluster of precursor cells (Fig. 4A). The sheath cells are tangentially
elongated and characterised by thicker walls, conspicuous nucleus
and abundant and slightly electron-dense cytoplasm. Oil droplets,
rough endoplasmic reticulum, mitochondria, typical chloroplasts
with an organised endomembrane system and vacuoles are also
observed in these cells (Fig. 3E). Plasmodesmata present in the cell
walls guarantee connections between these and neighbouring cells
(Fig. 3E).
Over the course of the differentiation process, the plasmalemma
of the secretory cells becomes strongly sinuous and gives rise to
periplasmic spaces of variable sizes, where paramural bodies are
observed (Fig. 3F). An increase in the mitochondria, dictyosomes,
smooth endoplasmic reticulum and plastid populations is notable
(Fig. 3F).
The fully differentiated glands show wide lumina containing flocculated material and osmiophilic accumulations, as well
as a locally biseriated secretory epithelium (Fig. 4A). The secretory epithelial cells are thin-walled with dense and abundant
cytoplasm (Fig. 4A). The vacuoles become more numerous and
can coalesce, giving rise to larger ones (Fig. 4B–C). Electrondense accumulations are seen adhering to the inner surface
of the tonoplast (Fig. 4C). Aspects suggesting plastid divisions
are frequent (Fig. 4D). A smooth endoplasmic reticulum with
enlarged edges filled with electron-dense material is common
(Fig. 4D), and these dilated edges give rise to vacuoles with electron dense contents (Fig. 4D–E). Small electron dense globules
are also observed in some mitochondria (Fig. 4E). Deposits of
osmiophilic material intermixed with fibrillar wall material can be
observed in the periclinal wall of epithelial cells facing the lumen
(Fig. 4F). This material is released by the disintegration of the cell
wall.
In a later developmental stage of the internal glands of C.
langsdorffii, cells become crowded with plastids with periplastidial reticulum, smooth endoplasmic reticulum, hyperactive
dictyosomes, mitochondria and vesicles with very electron dense
content (Fig. 4G). In the periplasmic space, secretion material is
visible (Fig. 4H). Functional epithelial cells are adjacent to senescent epithelial cells (Fig. 4I). The latter show loose sinuous walls,
retraction of the protoplast with accumulated secretion in the
periplasmic space, and a very electron-dense cytoplasm with
poorly defined organelles (Fig. 4H–I). Disintegration of the middle
lamellae starts from the anticlinal walls and progresses toward the
whole cell over the course of its extension (Fig. 4I). These processes
culminate with protoplast fragmentation, and the cell contents are
released toward the lumen.
The ZIO technique was used to impregnate endomembranes in
epithelial cells and sheath cells (Fig. 5A–E) to facilitate observation
of the organelles. In the epithelial cells, deposition of ZIO reaction products was seen in dictyosomes and endoplasmic reticulum.
In dictyosomes, the lumen and edges of cisterns and the released
vesicles showed impregnation with the metal (Fig. 5A). Reaction
products accumulated in the lumen of cisterns and in the bulbous
edges of the smooth endoplasmic reticulum (Fig. 5B–C); vesicles
and membranes of some vacuoles located near the endoplasmic
reticulum were also impregnated (Fig. 5C). In addition, vesicular structures with or without reaction products were observed
T.M. Rodrigues et al. / Flora 206 (2011) 585–594
589
Fig. 2. (A–D) Photomicrographs of vegetative apex of Copaifera langsdorffii stained with toluidine blue. (A) Longitudinal section showing glands in the subapical region
(arrows) and in the leaf primordial (150 ␮m). (B) Transverse section showing glands (arrows) in different developmental stages in the leaf primordia (150 ␮m). (C) Detail
of the previous figure showing clusters constituted by two, four and numerous initial secretory cells in leaf primordium (50 ␮m). (D) Detail showing differentiated cavities
with secretory epithelium and lumen in leaf primordium (50 ␮m).
between the plasmalemma and the cell wall (Fig. 5D). ZIO impregnation revealed a greater abundance of dictyosomes and small
vesicles in sheath cells’ cytoplasm compared to those of epithelial
cells (Fig. 5E).
Discussion
The morphological differences between canals and cavities were
determined for the first time by Col (1903), who postulated that
cavities are shorter and wider and canals are longer and narrower.
In this work, structural analysis showed that secretory canals and
cavities are present in the vegetative aerial axis of Copaifera langsdorffii independent of the developmental stage and are widely
scattered in adult plants and seedlings. The occurrence of both
canals and cavities in C. langsdorffii corroborates revision studies
by Metcalfe and Chalk (1950), who reported the widespread occurrence of canals and cavities in Caesalpinioideae members.
Interestingly, C. langsdorffii oil cavities occur in the cortex, while
oleoresin canals are more common in the pith. Although the presence of resin is a common characteristic of the members of the
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Fig. 3. (A–F) Electronmicrographs (TEM) of glands in initial stage of development in the stem of Copaifera langsdorffii. (A) Initial secretory cells grouped in a cluster. Observe
the thin walls with swollen middle lamellae in certain regions (arrowheads), the very electron dense cytoplasm and the voluminous nucleus (2.3 ␮m). (B) Plasmodesmata
(arrows) connecting secretory cells of the cluster (0.4 ␮m). (C) Detail of cluster cells with abundance of smooth endoplasmic reticulum with dilated cisterns, dyctiosomes
with adjacent vesicles and poliribosomes (0.5 ␮m). (D) Detail of cluster cell showing developed plastids without inner membrane system. Note the presence of periplastidial
reticulum (0.4 ␮m). (E) Detail showing oil droplet inside the chloroplasts in sheath cells. The arrows indicate plasmodesmata connecting sheath cell and epithelial cell (0.5 ␮m).
(F) Two cluster cells showing middle lamellae with fibrillar feature. Note the occurrence of paramural bodies (0.4 ␮m). CL: chloroplast; DI: dyctiosome; EP: epithelial cell;
ER: endoplasmic reticulum; MI: mitochondria; ML: middle lamellae; NU: nucleus; OL: oil droplet; PB: paramural bodies; PL: plastid; PR: polyribosome; SH: sheath cell; VA:
vacuole.
subfamily Caesalpinioideae (Langenheim et al., 1982), such compartmentalisation of resin exclusively in the canals, as observed
in the aerial vegetative primary organs of C. langsdorffii, is a novel
finding that indicates a high degree of specialisation in the glands
in this species from seedling stage onwards.
The formation of secretory canals and cavities is similar in
the different organs in C. langsdorffii. Our analyses showed that
the internal glands of C. langsdorffii result from successive divisions of the ground meristem cells or parenchyma cells, located
in the meristematic or differentiated shoot regions (shoot axis
and leaves). Similar data about the origin of the oil cavities were
reported by Teixeira and Rocha (2009) in Dahlstedtia. Therefore,
our data differ from most studies that have reported the protodermal origin of internal glands in legumes (Paiva and Machado, 2007;
Turner, 1986).
The initiation and differentiation of the internal glands of C.
langsdorffii take place early in shoot development. The presence
of initial cells immersed in the ground meristem, in the subapical
T.M. Rodrigues et al. / Flora 206 (2011) 585–594
591
Fig. 4. (A–I) Electronmicrographs (TEM) of differentiated glands in the stem of Copaifera langsdorffii. (A) Differentiated gland showing locally biseriated epithelium. The *
indicates secretion with fibrillar aspect in the lumen (8.3 ␮m). (B) Coalescence of vacuoles in epithelial cells (1.1 ␮m). (C) Vacuole with osmiophilic content adhered to the
inner surface of the tonoplast (1.0 ␮m). (D) Epithelial cells showing endoplasmic reticulum dilated cisterns and vesicles on the edges with osmiophilic content. The arrows
indicate plasmodesmata between epithelial cell and sheath cell (1.1 ␮m). (E) Epithelial cell showing vacuoles with electron dense material. Note the occurrence of dark
globules in the mitochondria (0.7 ␮m). (F) Epithelial cell abundant polyribosome and smooth endoplasmic reticulum with dilated cisterns. Note the accumulation of electron
dense material mixed to fibrillar material in the cell surface (0.25 ␮m). (G) Epithelial cells crowded with organelles (0.7 ␮m). (H) Detail showing accumulation of secretion in
the periplasmic space of epithelial cell (0.4 ␮m). (I) Active epithelial cells alongside darker senescent epithelial cell (1.6 ␮m). EP: epithelial cell; ER: endoplasmic reticulum;
LU: lumen; MI: mitochondria; OL: oil droplet; PL: plastid; SH: sheath cell; VA: vacuole; VE: vesicle.
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T.M. Rodrigues et al. / Flora 206 (2011) 585–594
Fig. 5. (A–E) Electronmicrographs (TEM) of differentiated glands in the stem of Copaifera langsdorffii treated with ZIO. (A) Detail of epithelial cell showing dictyosomes with
cisterns and adjacent vesicles fully impregnated with the metal (0.2 ␮m). (B) Smooth endoplasmic reticulum in epithelial cell with cisterns impregnated with the metal
(0.15 ␮m). (C) Smooth endoplasmic reticulum with dilated edges (arrows) impregnated with the metal in epithelial cell (0.2 ␮m). (D) Epithelial cell showing paramural
bodies (arrows) with and without impregnation with metal in the periplasmic space (0.7 ␮m). (E) Sheath cell showing dictyosomes with cisterns and vesicles impregnated
with metal. Note the occurrence deposits of the reaction products in the vacuole and the partially impregnation of the tonoplast (0.4 ␮m). DI: dictyosome; ER: endoplasmic
reticulum; VA: vacuole.
region, can explain the presence of glands in the cortex and pith of
C. langsdorffii. Maturation of glands before that of neighbouring tissues has been observed in many different plant species (Curtis and
Lersten, 1986; Fueyo, 1986; Joel and Fahn, 1980; Monteiro et al.,
1995).
According to Evert (2006), secretory canals and cavities can
originate from schizogenesis (cell separation), lysigenesis (cell
death) or schizolysigenesis (a combination of both processes). In
all the organs of C. langsdorffii studied, the internal glands originated schizogenously, as described for different legume genera
(Langenheim et al., 1978; Marcati et al., 2001; Metcalfe and Chalk,
1950; Solereder, 1908; Turner, 1986), although schizolysigenesis
occurs throughout their development, as discussed below.
At the ultrastructural level, precursor cell features are very
similar to those of mature glands, suggesting that the process of
producing secretions begins before lumen formation. Some of the
ultrastructural features of the epithelial cells in C. langsdorffii, such
as the abundance of smooth endoplasmic reticulum and polymorphic plastids without inner membranes, are associated with lipid
synthesis (Fahn, 1979, 2000). In fact, lipid droplets are abundant
in the cytoplasm of such cells and in the lumen of the glands in
early developmental stages. The profusion of plastids without inner
membranes is the main feature that distinguishes epithelial cells
from the adjacent parenchyma cells, which have typical chloroplasts. The presence of such plastids and the proliferation of smooth
endoplasmic reticulum have been described in many structures
that secrete lipophilic substances, mainly monoterpenes (Cheniclet
and Carde, 1985; Machado et al., 2006; Monteiro et al., 1999; Paiva
et al., 2008; Turner et al., 1999). The association of plastids with the
periplastidial reticulum as observed in the precursor and differentiated epithelial cells of C. langsdorffii internal glands indicates the
production of resin (Bhatt and Ram, 1992; Carmello et al., 1995;
Dell and Mccomb, 1978; Fahn, 1988; Paiva et al., 2008). It has also
been related to the transport of this substance and its precursors
(Benayoun and Fahn, 1979; Langenheim, 2003).
Several ultrastructural characteristics can be associated with the
production and liberation of terpenes by the epithelial cells of C.
langsdorffii. First, the presence of fewer oil droplets in the epithelial
cells of mature glands may be related to the high metabolic activity
of these cells (Fahn and Evert, 1974), and these substances can be
utilised in the production of the terpenes. Second, the presence of
osmiophilic granules in the mitochondria of epithelial cells and the
electron-dense content in vacuoles and cisterns of smooth endoplasmic reticulum suggest that these organelles are involved in the
T.M. Rodrigues et al. / Flora 206 (2011) 585–594
secretion process and in terpene transport (Fahn and Evert, 1974;
Joel and Fahn, 1980).
The simultaneous presence of both polyribosomes and dictyosomes in precursor and differentiated secretory cells of C.
langsdorffii can be associated with the synthesis and elimination
of lytic enzymes involved in the middle lamellae dissolution and
cell wall degradation that occur during the formation and expansion of the lumen (Carmello et al., 1995; Hall et al., 1984; Machado
and Carmello-Guerreiro, 2001; Paiva and Machado, 2007). Furthermore, dictyosomes are involved in the synthesis of hydrophilic
compounds (Evert, 2006; Fahn, 2000; Paiva and Machado, 2007;
Schnepf, 1969), which were detected by the histochemical tests in
C. langsdorffii.
The greater abundance of dictyosomes and vesicles near the
plasmalemma of the epithelial cells of C. langsdorffii glands suggests the intensive production and liberation of polysaccharides
by a granulocrine process. In fact, this process is thought to occur
by the fusion of vesicles to the plasmalemma and by the presence
of numerous paramural bodies in these cells, in a way consistent
with what is known about other species (Carmello et al., 1995;
Nair et al., 1983; Venkaiah, 1992). Conversely, the presence of
lipid droplets dispersed in the peripheral cytoplasm, near the plasmalemma of epithelial cells and in the lumen of the secretory
glands, suggests an eccrine process of secretion, where material
crosses the porous wall (via loosely arranged cellulose microfibrils)
and accumulates at the cell surface. The absence of plasmodesmata in the periclinal cell walls facing the lumen indicates that this
is the only way that the secretion reaches the lumen (Nair et al.,
1981).
Some features of the sheath cells that surround the secretory
canals and cavities, such as conspicuous nucleus, dense cytoplasm,
abundant oil droplets in the cytoplasm, vacuoles and plastids, and
the interconnection with the epithelial cells by many plasmodesmata, indicate the participation of these cells in the production of
substances that can serve as secretion precursors. In addition, the
presence of chloroplasts with electron-dense inclusions indicates
that these cells are able to perform photosynthesis to provide the
precursors and energy necessary for the synthesis of the secreted
substances (Fahn, 1988; Monteiro et al., 1999; Raatikainen et al.,
1992).
The presence of a sheath around the epithelium of oil cavities was described in other species belonging to different taxa
(Bosabalidis and Tsekos, 1982; Machado and Carmello-Guerreiro,
2001; Monteiro et al., 1995, 1999). The production of new cells by
a meristematic sheath enables regeneration of the epithelium and
maintenance of the secretory activity. In this kind of developmental
pattern, cells of the ground parenchyma maintain their meristematic potential through their ability to divide and differentiate into
epithelial cells (Fueyo, 1986; Machado and Carmello-Guerreiro,
2001; Monteiro et al., 1995, 1999; Wittler and Mauseth, 1984).
Thus, in C. langsdorffii, the lumen of the secretory canals and
cavities originates by schizogenesis, and the development of the
internal secretory glands occurs by lysigenesis and schizogenesis,
i.e., a schizolysigenous process. This lysigenesis, and the later liberation of epithelial cell contents, provides materials for secretion
and is characteristic of a holocrine secretion mechanism.
The impregnation of cell membranes by the ZIO technique
allowed the clear identification, localisation, and the determination
of the relative abundance of organelles in the epithelial and sheath
cells. As demonstrated by Machado and Gregório (2001), the ZIO
method also provides information about the relationship among
different organelles in the same cell or secretory structure. In the
present work, the images provided by the ZIO method indicate that
vesicles and multivesicular bodies containing dense material in C.
langsdorffii glands originate from the smooth endoplasmic reticulum with bulbous edges.
593
Although the chemistry of the ZIO impregnation technique is
not yet fully understood, it has been suggested that Zn2+ can link
or substitute the Ca2+ -binding sites in the membranes and reduce
osmium to produce an electron-opaque deposit of zinc osmate
(Gilloteaux and Naud, 1979). Other authors have suggested that
the impregnated material must be lipidic (Maillet, 1962; Niebauer
et al., 1969). However, the chemical nature of the structures that
are preferentially impregnated by the ZIO technique has not yet
been precisely determined.
Our results showed that the internal glands of C. langsdorffii produce polysaccharides, alkaloids, proteins and phenolic substances
in addition to essential oils and total lipids. The presence of resin
only in secretory canals, a characteristic common to the members
of Caesalpinioideae subfamily (Langenheim et al., 1982), was confirmed by histochemistry. In general, the substances detected in
the internal glands (mainly terpenes, phenolic substances and alkaloids) have been associated with resistance to microbial attacks
and protection against herbivores (Harbone, 1993; Taiz and Zeiger,
1998). Due to their hydrophilic properties, the polysaccharides can
act to maintain the high water potential of the adjacent cells and
protect the organs against desiccation damage, as suggested by
Sawidis (1998), in Malvaceae mucilage idioblasts.
The substances produced by the internal glands probably also
play an important role in the protection and development of C.
langsdorffii seedlings. This species is epigeous-phanerocotyledonar
(Guerra et al., 2006) and is thus able to explore the multiple possibilities of the cerrado environment, mainly those related to light
conditions (Franco, 2002). However, the epigeic exposition of the
cotyledons makes the seedlings more vulnerable to biotic and
abiotic adverse conditions. The presence of structures producing
terpenes from the very early stages of shoot development onwards
provides a defensive system in this critical stage of the plant’s life
cycle.
Acknowledgements
We thank FAPESP (Fundação de Amparo à Pesquisa do Estado
de São Paulo – Proc. DR 05/60086-0) and CNPq (Conselho Nacional
de Desenvolvimento Científico e Tecnológico – Proc. 470643/20064) for financial support, and the technical team of the Electron
Microscopy Center of UNESP, Botucatu, for helping in the sample
preparation. Machado, S.R. and Teixeira, S.P. received grants from
the CNPq council.
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