Glandular Trichomes of Calceolaria adscendens Lidl. (Scrophulariaceae) : Histochemistry, Development and Ultrastructure
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Annals of Botany 83 : 87–92, 1999
Article No. anbo.1998.0778, available on line at http:\\www.idealibrary.com
SHORT COMMUNICATION
Glandular Trichomes of Calceolaria adscendens Lidl. (Scrophulariaceae) :
Histochemistry, Development and Ultrastructure
G. S A C C H E T T I*, C. R O M A G N O LI*, M. N I C O L E T TI†, A. D I F A B I O†, A. B R U N I* and F. P O L I‡
* Department of Biology-Section of Botany, Uniersity of Ferrara, C.so Porta Mare 2, I-44100 Ferrara, Italy,
† Department of Plant Biology, Uniersity ‘ La Sapienza ’, P.le Aldo Moro 5, I-00185 Rome, Italy and
‡ Department of Eolutionary and Experimental Biology, Uniersity of Bologna, Via Irnerio 42,
I-40126 Bologna, Italy
Received : 24 April 1998 Returned for revision : 25 May 1998 Accepted : 3 September 1998
This paper reports the results of a study of the morphology and development of glandular trichomes in leaves of
Calceolaria adscendens Lidl. using light and electron microscopy. Secretory trichomes started as outgrowths of
epidermal cells ; subsequent divisions gave rise to trichomes made up of a basal epidermal cell, a stalk cell and a two-
celled secretory head. Ultrastructural characteristics of trichome cells were typical of terpene-producing structures.
Previous phytochemical studies had revealed that C. adscendens produces diterpenes. Comparison with C.
olckmanni, which produces triterpenes, and has trichomes with eight-celled secretory heads, suggests that there could
be a relationship between the type of glandular trichome and the class of terpene produced. Further work is needed
to test the hypothesis and to develop trichome characters as taxonomic tools.# 1999 Annals of Botany Company
Key words : Calceolaria adscendens Lidl., glandular trichomes, histochemistry, morphology, ultrastructure.
the development and ultrastructure of the glandular
INTRODUCTION
trichomes.
The genus Calceolaria (Scrophulariaceae), native of South
America, includes numerous plant species used in local folk MATERIALS AND METHODS
medicine, particularly in Chile (Navas, 1979). Only the
aerial parts of the plant are used for their digestive, diuretic Plant material
and antibacterial properties (Navas, 1979 ; Mun4 oz, Barrera Seeds of Calceolaria adscendens Lidl. harvested from wild
and Meza, 1981). plants in Chile were germinated in the dark on moistened
Species of Calceolaria are known to contain terpenes, paper. Seedlings were cultivated in soil in pots (25 cm
flavonoids and phenylpropanoid glycosides (Nicoletti et al., diameter, 25 cm high) in a phytotron (HERAEUS, VEPHQ
1988 ; Chamy et al., 1989 ; Wollenweber, Mann and 5\1350) with a photoperiod of 16 h light (15 Wm−#), at
Roitman, 1989 ; Garbarino, Chamy and Piovano, 1992 ; Di 20p1 mC and 80p10 % humidity. As the experimental
Fabio et al., 1995). Detailed phytochemical studies have plants were grown at low irradiance, the results were
revealed variation in the production of terpenes within the confirmed by observing the leaf trichome characteristics of
genus Calceolaria : some species, such as C. adscendens, C. plants grown under field conditions.
glandulosa, C. foliosa and C. hypericina produce diterpenes,
whereas others such as C. olckmanni, C. crassifolia and C.
Conentional and fluorescence microscopy
scabiosaefolia do not (Garbarino et al., 1992). In recent
years, some possible terpenoid chemotaxonomic genus Small pieces of vegetative apices, and young and mature
indicators have been identified (Nicoletti et al., 1988 ; leaves, were fixed and embedded following the procedures
Chamy et al., 1989 ; Garbarino et al., 1992), and mor- reported in Bruni, Tosi and Modonesi (1987). For his-
phological research has been undertaken on the secretory tological and histochemical investigations, periodic acid-
structures located on the leaves of Calceolaria olckmanni fluorescent Schiff reagent (F-PAS) was used to detect
(Sacchetti et al., 1996). polysaccharides (Bruni and Vannini, 1973). Fresh sections
This paper presents the results of a morphological study of leaves at different stages of development were cut using
of the glandular trichomes of Calceolaria adscendens Lidl., razor blades. These were then stained with : (a) Sudan Black
using conventional light, fluorescence and electron mi- B for lipids (Jensen, 1962) ; (b) NADI reagent for terpenes
croscopy (SEM and TEM) to identify the main chemical (David and Carde, 1964) ; (c) ferrous thiocyanate [Fe(SCN) ]
#
classes of metabolite present in the secretion and to study and concentrated H SO for sesquiterpenes (Cappelletti,
# %
Caniato and Appendino, 1986) ; (d ) antimony trichloride
‡ For correspondence. (SbCl ) for terpenes containing steroids (Hardman and
$
0305-7364\99\010087j06 $30.00\0 # 1999 Annals of Botany Company88 Sacchetti et al.—Glandular Trichomes of Calceolaria adscendens F 1–4. SEM micrographs of Calceolaria adscendens leaf trichomes. Fig. 1. Adaxial surface showing non-glandular and glandular trichomes (arrows). i80. Fig. 2. Abaxial surface showing glandular (arrows) and non-glandular trichomes. i350. Fig. 3. Detail of the leaf tip, abaxial surface. There are no secretory trichomes on the margin nor along the veins. A hydatode (H) can be seen. i290. Fig. 4. Mature secretory trichome showing a short stalk and the two-celled glandular head. The cuticle of the secretory cells is partially ruptured (*). Cuticle blisters, possibly corresponding to sub-cuticular secretory deposits, are present (arrows). i3100. F. 5. Abaxial surface of a leaf of C. adscendens viewed under an optical microscope with UV light (λ l 365 nm). Top view of the two-celled secretory heads of the glandular trichomes. A yellow-orange
Sacchetti et al.—Glandular Trichomes of Calceolaria adscendens 89
Sofowora, 1972) ; and (e) aluminium chloride (AlCl ) and stalk cell wall (Fig. 8), but, as it matured, the stalk cell wall
$
neutral lead acetate for flavonoids (Guerin, Delaveau and became non-reactive to F-PAS (Fig. 9).
Paris, 1971). For all the histochemical methods cited, By TEM, it was observed that, at the initial stages of
control reactions were carried out following the suggestions trichome development, the expanding epidermal cell had an
of the respective authors. Observations were made on a electron-dense cytosol, a prominent nucleus with a clearly
Zeiss Axiophot microscope using both transmitted light and evident nucleolus and a large basal vacuole (Fig. 10). The
epifluorescence (UV-H365 : BP 365\12, FT 395, LP 397 mature trichome had an epidermal basal cell and a stalk cell,
excitation filter). both containing well-developed vacuoles with osmiophilic
material. The secretory head cells, on the other hand, had a
Scanning and transmission electron microscopy (SEM and dense cytosol and poorly-developed vacuoles with small
TEM) osmiophilic droplets (Fig. 11). The mature trichome showed
clear signs of cutinization of the lateral wall of the stalk cell
For SEM, pieces of leaves (approx. 2 mm long), at and numerous plasmodesmata were seen on the transverse
different stages of development, were fixed and processed as stalk walls (Fig. 12). In the secretory head cells, an
reported in Bruni et al. (1987). The samples were viewed endoplasmic reticulum system appeared to wrap around the
with a Stereoscan 360 Cambridge microscope (Electronic plastids, which showed complex tubular structures (Fig. 13).
Microscopy Center, University of Ferrara). In the dense cytoplasm of the secretory cells, normal
For TEM, similar samples were fixed and processed as mitochondria were also present. Although no evidence of
explained in Poli et al. (1989). Observations were made vesicle fusion was detected, a very sinuous plasma membrane
using a Zeiss electron microscope EM 109 N. with invaginations in association with paramural bodies
was observed (Fig. 14). Secretion took place both between
RESULTS the secretory head cells and in the subcuticular space. The
The adaxial and abaxial surfaces of mature leaves of secretion seemed to cause a partial separation of the head
Calceolaria adscendens showed numerous non-glandular cells and slight elevation of the cuticle (Fig. 15).
and glandular trichomes (Figs 1 and 2). No glandular
trichomes were found along the veins or at the leaf margins
DISCUSSION
on the abaxial surface where hydathodes could be seen
(Fig. 3). The mature glandular trichomes were made up of C. adscendens has glandular trichomes with characteristic
a short stalk and a two-celled secretory head. A partial morphology and ontogeny. The secretory trichome has : (1)
rupture of the cuticle and possible sub-cuticular secretion a basal cell which remains epidermal and supplies resources
storage zones were observed in glandular cells (Fig. 4). to the head ; (2) a stalk cell ; and (3) a two-celled secretory
Under UV light, the two secretory head cells showed yellow- head. When the glandular trichome is mature, the stalk cell
orange autofluorescence (Fig. 5). Histochemical reactions shows clear cutinization of the lateral wall, as indicated by
with Sudan Black B and NADI reagent revealed, respec- the negative F-PAS reaction and TEM. This special feature
tively, the lipophilic nature of the secretion and its of glandular trichomes may prevent leakage of the secreted
terpenoid content. Other histochemical tests for terpenoids substance back through the apoplast (Fahn, 1988 ; Serrato-
[Fe(SCN) , H SO and SbCl ] were negative, while the Valenti et al., 1997 ; Ascensa4 o and Pais, 1998). Previous
# # % $
reactions with AlCl and neutral lead acetate, used for morphological studies have shown differences in the
$
checking the presence of flavonoids, were weakly positive presence, distribution and type of glandular trichomes
(Table 1). between members of the Calceolaria. For example, C.
Secretory trichome development started with an out- olckmanni has trichomes with eight-celled secretory heads,
growth of an epidermal cell. Initially, the nucleus of this cell mainly located on the adaxial surface, on the leaf margins
was centrally located, but it migrated towards the apex of and along the main veins (Sacchetti et al., 1996), whereas in
the outgrowth, leaving the basal (epidermal) part of the cell C. adscendens they were distributed on both surfaces without
occupied by a large vacuole (Fig. 6). Thereafter, the first any appreciable variation in density.
periclinal division generated a plasma-rich upper cell and a Phytochemical studies of species of Calceolaria have
vacuolated lower cell (Fig. 7). The latter cell remained an shown the presence of flavonoids, phenylpropanoid glyco-
epidermal cell, but a periclinal division of the upper cell gave sides and terpenes. In C. adscendens, the presence of
rise to a short stalk cell and to a mother glandular head cell flavonoid compounds was revealed by AlCl and neutral
$
(Fig. 8). By an anticlinal division, the mother head cell then lead acetate, whereas the lipophilic nature of the secretion
generated a two-celled secretory head (Fig. 9). In the early and the presence of terpenes were demonstrated by positive
stages of trichome development (i.e. the stage where the reactions with Sudan Black B and NADI. The production
trichome was a three-celled hair), the F-PAS reaction of terpenes by C. adscendens, reported by Nicoletti et al.
showed the presence of polysaccharides on the trichome (1988) is thus confirmed at the ultrastructural level.
autofluorescence was emitted under UV excitation. i300. F 6–9. Longitudinal sections of glandular trichomes at different stages of
development. F-PAS staining. Fig. 6. Trichome initial. Outgrowth of a single epidermal cell (E). i380. Fig. 7. Two-celled trichome. The first
periclinal division generates a plasma-rich upper cell and a vacuolated lower cell. i400. Fig. 8. Three-celled trichome after the second periclinal
division, showing a basal cell, a stalk cell and the mother cell of the head (M). i400. Fig. 9. Mature trichome showing the two-celled secretory
head. Note that the lateral walls of the stalk cell (arrows) are clearly non-reactive to F-PAS, used for the detection of polysaccharides. i360.90 Sacchetti et al.—Glandular Trichomes of Calceolaria adscendens F 10–12. TEM micrographs of glandular trichomes of C. adscendens. Fig. 10. Glandular trichome at an early stage of development. Note the electron-dense cytosol, the large basal vacuole (V) and the nucleus (N) with a prominent nucleolus (Nu). i4100. Fig. 11. A mature secretory trichome showing a basal epidermal cell (B) and a stalk cell (S), both with large vacuoles (V) containing osmiophilic droplets (arrowheads). The
Sacchetti et al.—Glandular Trichomes of Calceolaria adscendens 91
T 1. Reactiity of the glandular trichomes with different histochemical indicators
Staining Target compounds Cell of the stalk Cells of the head Secretion
Sudan Black B Lipids jk jj jj
NADI reagent Terpenes k jj jj
Fe(SCN) Sesquiterpenes k k k
#
Concentrated H SO Sesquiterpenes k k k
SbCl
# % Steroidal terpenes k k k
AlCl
$ Flavonoids k jk jk
$
Neutral lead acetate Flavonoids k jk jk
k, Negative ; jk, weakly positive ; jj, positive.
The presence of a tight network of endoplasmic reticulum,
A C K N O W L E D G E M E N TS
numerous plastids (leucoplasts) and mitochondria in the
secretory head cells showed that these cells had the typically- This work was supported by grants from the Consiglio
active metabolism of similar glandular systems (presumably Nazionale delle Ricerche (CNR), Ministero dell’Universita'
involved in biosynthesis, transport and secretion). In e della Ricerca Scientifica e Tecnologica (MURST) of Italy
particular, the proximity between plastids and endoplasmic and INTERREG. The authors wish to thank Eileen N.
reticulum is typical of terpene-secreting systems (Figueiredo Cartoon for the English translation of this manuscript.
and Pais, 1994) as are poorly-developed plastids with
tubular structures, and a few poorly-developed dictyosomes
in the secretory head cells (Bosabalidis and Tsekos, 1982 ; LITERATURE CITED
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