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Flora 233 (2017) 1221

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

Salvia uliginosa Benth.: Glandular trichomes as bio-factories of


volatiles and essential oil
Claudia Giuliani a,b, , Roberta Ascrizzi c , Corrado Tani d , Martina Bottoni a,b ,
Laura Maleci Bini d , Guido Flamini c , Gelsomina Fico a,b
a
Department of Pharmaceutical Sciences, University of Milan, Via Mangiagalli 25, I-20133 Milan, Italy
b
Ghirardi Botanic Garden, Department of Pharmaceutical Sciences, University of Milan, Via Religione 25, I-25088 Toscolano Maderno, Brescia, Italy
c
Department of Pharmacy, University of Pisa, Via Bonanno 6, I-56126 Pisa, Italy
d
Department of Biology, University of Florence, Via La Pira 4, I-50121 Florence, Italy

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

Article history: Micromorphological, histochemical and ultrastructural investigations on the indumentum of vegetative
Received 8 March 2017 and reproductive organs were carried out for Salvia uliginosa Benth. (Lamiaceae). Besides non-glandular
Received in revised form 5 May 2017 hairs, two types of glandular trichomes (peltate and short-stalked capitate) are described and compared
Accepted 5 May 2017
with trichomes in other Lamiaceae species. The histochemical ndings evidence a similar, complex chem-
Edited by Alessio Papini
ical nature of the secretory products for both types of trichomes: lipids, terpenes, phenols and avonoids.
Available online 10 May 2017
TEM investigation allows to conrm the preliminary histochemical results.
In addition, we report for the rst time the phytochemical characterization of the leaf and ower
Keywords:
Salvia uliginosa benth
volatile emission proles and the analysis of the essential oil obtained from the aerial parts.
indumentum Leaf volatile prole resulted more complex, presenting a total number of constituents almost double
Micro-morphology compared to that found in the owers (50 vs 27 compounds, respectively). Leaf VOC prole is char-
Hs-spme acterized by 30 exclusive compounds, dominated by 1,8-cineole (3.84%) and trans--cadinene. Flower
Gc/ms VOC prole Exhibits 7 exclusive compounds, among which germacrene D (8.65%) is the most abun-
VOC prole dant one. Sesquiterpene hydrocarbons represent the major chemical class in both proles (90.13% in
Essential oil leaves and 92.18% in owers) and 20 common compounds have been detected: among them -elemene,
-caryophyllene, -humulene and bicyclogermacrene are the dominant constituents, with relative per-
centages in the range of about 5.0017.00% for the leaves and of about 9.00-31.00% for the owers.
49 different compounds were identied in the essential oil obtained from the aerial parts. The extraction
yield is 0,02%. Sesquiterpenes represent the most abundant terpene fraction (89.56%). The main com-
pound is bicyclogermacrene (16.30%) followed by germacrene D (14.81%), -caryophyllene (8.57%) and
-cadinene (8.47%), among the hydrocarbons. Spathulenol (12.66%) dominates the oxygenated sesquiter-
penes.
2017 Elsevier GmbH. All rights reserved.

1. Introduction In the Lamiaceae family, glandular hairs represent the primary


sites for the synthesis of bioactive volatile compounds, which play a
The Salvia genus (family Lamiaceae, subfamily Nepetoideae, crucial role in the seduction towards pollinators or seed dispersers
tribe Menthae) includes about 9501000 species (Dizkirici et al., or in the repulsion strategies against phytophagous insects or pests
2015), some of which are of commercial value. The glandular hairs (Maffei, 2010). In addition, this specialized structures secrete large
and the essential oil proles of various species have been charac- quantities of exudates, forming a continuous layer on the plant
terized and investigated also in relation to their biological activities surface, which may regulate the interactions with the microbiota
(Kintzios, 2000; Fu et al., 2013; Ali et al., 2015). inhabiting the phyllosphere and the anthosphere (Werker, 2000).
As part of a wide-spectrum project on Salvia spp., aimed at
exploring the close relationship between the productivity in sec-
ondary metabolites, their bio-ecological signicance and their
Corresponding author at: Department of Pharmaceutical Sciences, University of potential importance to humans, in this work we carried out a
Milan, Via Mangiagalli 25, I-20133 Milan, Italy. micro-morphological and phytochemical survey of the indumen-
E-mail address: claudia.giuliani@unimi.it (C. Giuliani).

http://dx.doi.org/10.1016/j.ora.2017.05.002
0367-2530/ 2017 Elsevier GmbH. All rights reserved.
C. Giuliani et al. / Flora 233 (2017) 1221 13

tum of mature leaves and owers of Salvia uliginosa Benth. (section full-open owers) by means of light microscopy, as well as both
Uliginosae). scanning electron, and transmission electron microscopy.
Salvia uliginosa, known as bog sage, is a perennial subshrub Five replicates, similar for size, position and developmental
native to South America (Brazil, north-eastern Argentina, Uruguay) stage, were selected from different individuals for each plant part,
and was rst introduced as ornamental at the Royal Botanic Gar- in order to verify the consistency of the trichome morphotypes,
dens of Kew in 1913 (Turrill, 1914), because of its beautiful sky-blue distribution pattern, histochemistry and ultrastructure.
owers, with white beelines on the upper and lower lips. Nowa-
days it is widely cultivated in the gardens of Europe, also in virtue 2.2.1. Light microscopy (LM)
of its high adaptability to different soil types, sun exposures, and Histochemical investigation was performed to evidence the
drainage regimes. Indeed, although both the common name and main chemical classes of metabolites in the trichome secretory
the species epithet refer to the boggy conditions in which the plant products. For each replicate of all the fresh plant parts, hand-made
grows in the wild in its home range, it may well tolerate dry con- sections (4050 m thick) and semi-thin sections (2025 m thick)
ditions. obtained by means of a cryostat, were stained with the follow-
Besides the presence of medium-sized blue owers, this species, ing dyes: Sudan III, IV (Johansen, 1940), Fluoral Yellow (Brundrett
as all the members of section Uliginosae, displays a unique staminal et al., 1991), Nile Red for neutral lipids (Greenspan et al., 1985),
structure. This synapomorphy consist in an appendage that is a Ruthenium Red for polysaccharides other than cellulose (Jensen,
forward-facing or assurgent tooth located on the ventral side of 1962) and Alcian Blue for acidic polysaccharides (Beccari and Mazzi,
the posterior connective branches (Turner, 2009). As most Salvia 1966), Nadi reagent for terpenes (David and Carde, 1964), ferric
species, this plant is bee-pollinated (Wester and Claen-Bockhoff, trichloride for polyphenols (Gahan, 1984), aluminium trichloride
2007). for avonoids (Gurin et al., 1971), Mercuric Bromophenol Blue
In its native range, S. uliginosa (syn. S. lanceolata Larranaga) was for total proteins (Beccari and Mazzi, 1966). Matchings for all of
employed in the folk medicine for its carminative, tonic and stim- the histochemical stains were performed with control procedures.
ulant properties (Pinto, 2012). Autouorescence of the trichome secretory products were also
The glandular indumentum of S. uliginosa has never been evaluated. Observations were performed under a Leitz DM-RB Fluo
previously investigated. With regard to the phytochemical state- Optic microscope equipped with a digital camera Nikon DS-L1.
of-the-art, literature data on the characterization of VOCs lack.
Only one contribution focusing on the essential oil prole of a 2.2.2. Scanning electron microscopy (SEM)
population from Brazil is known (Pinto, 2012): hexadecanoid acid, For each replicate, small segments with a surface of 2530 mm2
n-nonadecane and nonadecanal resulted as the main compounds; were xed in FAA (formaldehyde: glacial acetic acid: 70% ethanol
the biological activity of the essential oil against Staphylococcus 5:5:90 by volume) at room temperature for 7 days. Then the sam-
aureus has been also proved (Pinto, 2012). ples were dehydrated in ascending ethanol series up to absolute
The principal goal of this work was to comprehensively char- with 20-min intervals, transferred to acetone, critical point-dried
acterize the glandular trichomes of both the vegetative and the in a Balzer Union CPD 020 apparatus and sputter coated with gold.
reproductive organs by light, scanning electron and transmission Observations were made under a Philips scanning electron micro-
electron microscopy; histochemical procedures were also used to scope at an accelerating voltage of 20 kV.
localize the secreted substances within and on the surfaces of the
hairs. In addition, we combined the micro-morphology of the secre- 2.2.3. Transmission electron microscopy (TEM)
tory structures with their productivity in secondary metabolites For each replicate of leaves, calices and petals small segments
through the phytochemical characterization of the vegetative and (510 mm2 ) were xed in 2,5% glutaraldehyde in phosphate buffer
oral volatile bouquets and essential oil analysis. 0.1 M pH 7.2 overnight at room temperature. Subsequently they
The overall set of such information, and in particular the chemi- were rinsed twice in the same buffer, post-xed in 2% osmium
cal nature of the volatile substances emitted, may nally contribute tetroxide for 2 h and washed in distilled water two times for 5 min.
to shed light on the biotic interactions established by the examined Then, the material was dehydrated in ascending ethanol series up
species, thus constituting the basis for future insights on the ecolog- to absolute and embedded in Spurr resin. The samples were cut in
ical roles of the secondary metabolites, mainly in relation to insect ultrathin sections (3 m thick) by means of an ultramicrotome and
pollinators. stained with uranyle acetate and lead citrate. Observations were
made under a Philips EM-300 transmission electron microscope.

2. Materials and methods


2.3. Phytochemical investigation

2.1. Plant material


2.3.1. Volatile organic compounds (VOCs)
Three leaves and three full-opened owers were cut and imme-
Salvia uliginosa was cultivated at the Ghirardi Botanic Garden
diately inserted in two separate glass vials of suitable volume for
(Toscolano Maderno, Bs) of the Department of Pharmaceutical Sci-
the sampling.
ences of the University of Milan. Voucher specimens was deposited
HS-SPME Sample analysis Supelco SPME (Solid Phase Micro-
in the Herbarium of the Ghirardi Botanic Garden, University of
Extraction) devices coated with polydimethylsiloxane (PDMS,
Milan, Italy under the accession codes UNIMI 0030/15.
100 m) were used to sampling the headspace. SPME sampling
The sampling for the micro-morphological analysis were carried
was performed using the same new bre, preconditioned accord-
out on plants in full-bloom in June 2016. Collection of plant material
ing to the manufacturer instructions, for all the analyses. Sampling
for the phytochemical investigation (VOCs and essential oil) was
was accomplished in an air-conditioned room (22 1 C) to guar-
performed simultaneously.
antee a stable temperature. The sampling temperature was chosen
to avoid the thermal damage of the supercial glandular hairs
2.2. Micromorphological analysis of the leaves and, thus, any articial-induced volatiles release.
After 30 min of equilibration time, the bre was exposed to the
The glandular indumentum was examined on both vegetative headspace for 30 min. The equilibration time was chosen as the
and reproductive organs (stems, leaves, bracts, oral buds and ideal one after several trials at different intervals. The sampling
14 C. Giuliani et al. / Flora 233 (2017) 1221

time was experimentally determined to obtain an optimal adsorp- they are distributed on the whole lamina and appeared particularly
tion of the volatiles, to avoid both under- and over-saturation of the abundant at the edges (Fig. 2c).
bre and of the mass spectrometer ion trap. Once sampling was n- In both types of glandular trichomes, the apical region of the
ished, the bre was withdrawn into the needle and transferred to immature hairs appear wrinkled or slightly smooth, indicative of
the injection port of the GCMS system. All the SPME sampling and the close adhesion of the cuticle to the outer periclinal wall of the
desorption conditions were identical for all the samples. Further- secretory cells. At maturity, the cuticle surface becomes smoother
more, blanks were performed before each rst SPME extraction and due to the accumulation of the secretion products within the devel-
randomly repeated during each series. Quantitative comparisons of oping storing chamber formed by detachment of the cuticle.
relative peaks areas were performed between the same chemicals Non-branched, uniseriate non-glandular trichomes were
in the different samples. observed on the whole plant surface (Fig. 2aj, l). Two main types
can be distinguished. Type I hairs are multicellular, sharply pointed,
with a basal wider cell and a gradually minor cell diameter toward
2.3.2. Essential oils (EOs)
the apical region (see Fig. 2b,c); they present distinct articulations
Plant aerial parts at blooming were dried at room temperature
between cells and display different length and a variable number
and in the dark and stored under the same conditions until the
of cells, ranging from one to ve-six. Their cuticle is smooth or
hydrodistillation process.
slightly papillate; an evident crown of swollen epidermal cells
The essential oil hydrodistillation was performed in a standard
encircles the basal cell of type I hairs (Fig. 2c). These trichomes
Clevenger apparatus for 2 h.
can be perpendicular to the plant surface, but generally they tend
GC/MS Analysis The GC/EI-MS analyses were performed with
to lean toward the apex of the organ bearing them. Longer type
a Varian CP-3800 apparatus equipped with a DB-5 capillary col-
I hairs are situated on stems, leaves (both along the margins and
umn (30 m X 0.25 mm i.d., lm thickness 0.25 m) and a Varian
over midribs and major veins), oral peduncles and on the veinal
Saturn 2000 ion-trap mass detector. The oven temperature was
system of calyces. Shorter type I hairs occur on the interveinal
programmed rising from 60 C to 240 C at 3 C/min; injector tem-
regions of leaves and calyces and over the corolla adaxial surface.
perature, 220 C; transfer-line temperature, 240 C; carrier gas, He
Type II hairs, exclusively observed on the corolla abaxial side,
(1 ml/min).
consist of two-four cells with an equal diameter and present
Volatiles analysis The essential oils were injected after dilution
a rounded apex; they are erect and possess a wrinkled cuticle
in n-hexane HPLC grade at 5%. The identication of the constituents
(Fig. 2k).
was based on the comparison of their retention times (tR ) with
those of pure reference samples and their linear retention indices
3.1.2. Histochemistry of the glandular trichomes
(LRIs) determined relatively to the tR of a series of n-alkanes. The
Various histochemical dyes were used to detect the produc-
mass spectra were compared with those listed in the commercial
tion of major chemical compound classes in glandular trichomes
libraries NIST 2014 and ADAMS and in a home-made mass-spectral
(Table 2, Fig. 3ah). Staining with Ruthenium Red showed that the
library, built up from pure substances and components of known
cell walls of the secretory heads of peltate and capitate trichomes
oils, and MS literature data (Stenhagen et al., 1974; Masada, 1976;
contained unesteried pectins (Fig. 3a). Acidic polysaccharides,
Rdel et al., 1982; Davies, 1990).
detected by Alcian Blue, were present in the cytoplasm of the secre-
tory cells of both type of glandular trichomes, as well as in the
3. Results neck cells; the secretory material resulted not stained by the above
mentioned hydrophilic procedures (Table 2).
3.1. Micromorphological investigation Total proteins were present in all cells of both types of glandular
trichome (Table 2), whereas the response of the secretory material
3.1.1. Trichomes morphotypes and distribution pattern stored in the subcuticular spaces to Mercuric Bromophenol Blue
SEM analysis revealed that the glandular indumentum of S. uligi- was invariably negative.
nosa consist of peltate and capitate hairs (Fig. 1ab). Both hair types The secretory head of peltate and capitate trichomes stained
are distributed over both vegetative and reproductive organs and yellow-greenish with the Fluoral Yellow-088 test, indicating the
co-occur with non-glandular trichomes (Fig. 2an). presence of total lipids (Fig. 3b, f). NADI reaction resulted in an
Peltate trichomes are formed by one basal epidermal cell, a stalk intense dark-violet staining of the secretory product within sub-
cell and a broad head of four secretory cells arranged in a single disc cuticular space, typically in the form of droplets, as in the peltate
(Fig. 1a). They are endowed with a large apical subcuticular space trichomes (Fig. 3c), thus conrming the presence of terpenes in all
in which the secretion is stored. trichome types (Table 2).
Peltate trichomes display a distribution pattern that includes The head and the secretory products of both types of glandu-
stems, leaves, oral peduncles, calyces and corollas (Table 1). On lar trichome contained also phenolic compounds, as indicated by
stems and peduncles their occurrence and actual density can not the greenish-brown staining with Ferric trichloride (Fig. 3g). In par-
be adequately assessed because of the general pubescence due to ticular, the intense response to Aluminium trichloride, proved by
the abundant non-glandular trichomes completely covering the the brilliant blue staining, indicate the production and release of
epidermal surface. They occur on the interveinal regions of leaf avonoidic compounds (Fig. 3d, h).
and sepal abaxial surfaces, whereas they lack on the adaxial ones
(Fig. 2af, i). On petals their distribution is limited to the abaxial 3.1.3. Ultrastructure of the glandular trichomes
side, both over the corolla tube and at the upper and lower lips TEM analysis on both glandular trichome types at the active
(Fig. 2jl). secretory phase allows to evidence the major cellular compart-
The capitate hairs are short-stalked trichomes and consist of ments involved in the secretion process (Fig. 4).
one basal epidermal cell, one neck-stalk cell and a round head of In mature peltate trichomes (Fig. 4a), the most striking ultra-
two broad cells surrounded by a medium-wide subcuticular space structural features of the secreting cells is the presence of several
(Fig. 1b). They are widespread on the whole plant surface, with electron-dense plastids occasionally containing starch granules.
the exception of the stem and the calyx adaxial side (Fig. 2al). Generally they display large globular intraplastidial bodies and
They are mainly located along the veinal system, especially on the scarce tubular membrane elements (Fig. 4b, c). Abundant dilated
abaxial side of leaves and calyces (Fig. 2e, i). On the leaf adaxial side cisternae originated from the smooth endoplasmic reticulum (SER)
C. Giuliani et al. / Flora 233 (2017) 1221 15

Fig. 1. ab. Glandular trichomes morphotypes in Salvia uliginosa, LM: a. peltate trichome. b. short capitate trichome.

Table 1
Distribution pattern of the indumentum in Salvia uliginosa.

Trichome type Stem Leaves Calyx Corolla

adaxial abaxial adaxial abaxial adaxial abaxial

non-glandular type I +++ +++ +++ +++ ++


non-glandular type II ++
peltate + + + +
short capitate ++ + + + +

Table 2
Results of the histochemical investigation of the glandular trichomes of Salvia uliginosa.

Staining procedure Target compounds Observed colour Trichome types

peltate short capitate

Fluoral Yellow-088 Total lipids Yellow to orange + ++


Nile Red Neutral lipids Golden-yellow + ++
Nadi reagent Terpenes Violet-blue + +
FeCl3 Polyphenols Emerald-green + +
AlCl3 Flavonoids Blue-green + +
Ruthenium red Polysaccharides/unesteried pectins Pinkish to red +a +a
Alcian blue Acid polysaccharides Pale-blue +a +a
Hg Bromophenol Blue Total proteins Dark Blue +a +a

Results: () absent; () scarce, (+) intense, and (++) very intense.


Symbols indicate the staining response of the secretory products stored in the subcuticular space
a
Refers exclusively to the staining response of the cytoplasm of the glandular cells.

are also present, generally in close association with plastids (Fig. 4c, occurring only at the apical region (Fig. 4a, g). In the subcuticular
d). Golgi stacks and rough endoplasmic reticulum (RER) occur occa- space the secretion products present a heterogeneous appearance,
sionally. Numerous plasmodesmata are present at the tangential however generally globose electron-dense inclusions immersed in
walls of all the trichome cells, especially between the neck cell and an electron-opaque granular matrix are observed (Fig. 4d).
the secreting cells (Fig. 4e), indicating that also the basal cells and No pre-arranged openings for the released of the secretion were
the neck cells take part in the secretion process. The cytoplasm of observed; the secretion products are presumably extruded through
neck cell displays large lipidic droplets (Fig. 4e). the intact outer wall or after cuticle breakage, copiously owing out
With the aging of trichomes plastids become ameboid or on the head, along the stalk and on the epidermis. Materials with
cup-shaped (Fig. 4e) and several autophagic vacuoles containing various electron-density degrees are occasionally observed across
myelin-like gures are observed. the intact outer periclinal cell walls (Fig. 4i).
In full-active short capitate trichomes, the cytoplasm of the
glandular cells is dense and is characterized by several l vacuoles,
mithocondria, multishaped plastids surrounded by SER cisternae, 3.2. Phytochemical investigation
and lipidic droplets (Fig. 4). Golgi stacks and RER cisternae generally
lack. 3.2.1. VOCs emission
In both peltate and short-capitate trichomes the storing cham- The spontaneous emission prole of S. uliginosa revealed a total
ber is enclosed by a thick cuticle layer and by the external portion of of 57 different compounds: 50 were detected in the leaf samples
the pectic-cellulosic layer of the outer periclinal walls of the glan- and 27 in the ower ones (Table 3).
dular cells (Fig. 4f, h). The cuticle remains rmly adherent to the cell The leaf samples are dominated by sesquiterpene hydrocarbons,
wall at the basal part of the secretory cell, with cuticle detachment reaching 90.13%; oxygenated monoterpenes and monoterpene
hydrocarbons followed in comparable relative percentages, 3.84%
16 C. Giuliani et al. / Flora 233 (2017) 1221

Fig. 2. al. Trichomes distribution pattern in Salvia uliginosa, SEM: a. General view of the leaf adaxial surface. bc. Particulars of leaf adaxial surface showing short capitate
trichomes (arrow) and non-glandular trichomes of type I: middle region (b) and leaf margin (c). d. General view of the leaf abaxial surface. e-f. Particulars of the leaf abaxial
surface showing peltate (asterisk), short capitate trichomes (arrow) and non-glandular trichomes of type I: veinal system (e), interveinal region (f). g. General view of a oral
bud. h. Particular of the oral pedicel with abundant non-glandular trichomes of type I. i. Calyx abaxial surface with non-glandular type I, peltate (asterisk) and short capitate
trichomes (arrow). j. General view of the mature corolla. k. Particular of the abaxial surface of the upper lip with non-glandular type II and peltate trichomes (asterisk). l.
Particular of the adaxial surface of the lower lip with non-glandular type I and short capitate trichomes (arrow).

and 3.68%, respectively. The principal compounds in the whole sample is bicyclogermacrene (43), accounting for 31.26%. -
headspace is -muurolene (see Table 3, 40, 31.43%), followed caryophyllene (30, 25.66%), -elemene (20, 13.98%), -humulene
by other sesquiterpene hydrocarbons: bicyclogermacrene (43, (35, 9.26%) and germacrene D (41, 8.65%) are other relevant
16.91%), -caryophyllene (30, 12.91%), -humulene (35, 5.93%) and sesquiterpene hydrocarbons. The two most abundant monoterpene
-elemene (20, 5.09%). Monoterpene hydrocarbons are only repre- hydrocarbons in the ower samples are (E)--ocimene (11) and
sented by 1,8-cineole (9, 3.84%). (Z)--ocimene (10), which represent 2.21% and 1.72%, respectively.
The ower headspace is mainly rich in terpene hydrocar- 20 compounds are common to both leaf and ower headspaces.
bons, of which sesquiterpene ones account for 92.18%, followed As a whole, the minor common compounds occurred in similar
by monoterpenes (6.42%). The most abundant compound in this relative percentages; the major ones, corresponding to the domi-
C. Giuliani et al. / Flora 233 (2017) 1221 17

Fig. 3. ah. Histochemistry of the glandular trichomes of Salvia uliginosa, LM. ad. Peltate trichome: Alcian Blue (a); Fluoral Yello-088 (b); Nadi reagent (c); AlCl3 (d). eh.
Short capitate trichome: Alcian Blue (e); Fluoral Yello-088 (f); FeCl3 (g); AlCl3 (h).

nant constituents of leaf and ower bouquets, occurred in different The non-terpene derivatives (apocarotenoids included) account
relative abundances that vary in the range of 515% points. - for about 0.87% of the essential oil.
muurolene (40) is the principal compound of the leaf headspace
(31.43%), where it shows a noteworthy increment in terms of rela-
tive abundance in comparison to the ower sample (0.31%). 4. Discussion
30 and 7 exclusive compounds were found in leaf and ower
volatile prole, respectively. The compounds exclusively found in The glandular trichomes observed on the leaves and owers
leaves are in general present at traces levels or in amounts lower of Salvia uliginosa correspond to the two main trichome types
than 1%; the most represented ones are 1,8-cineole (9, 3.84%) and observed in the family Lamiaceae: peltate and capitate (Werker,
trans--cadinene (48, 1.86%). In the ower volatile prole the char- 2000).
acteristic compounds are represented with relative abundances In S. uliginosa, the peltate hairs, occurring on the vegetative and
lower that 0.6%, with the exception of germacrene D (41, 8.65%). reproductive organs, are formed by one epidermal cell, and by one
stalk-neck cell supporting a wide four-celled head. The presence
of a four-celled head is a character observed in other members
3.2.2. Essential oil prole of Salvia (Eiji and Salmaki, 2016); however, within the genus, the
Salvia uliginosa essential oil composition is reported in Table 4: most common head cell disposition consists in six up to eight cells
a total of 49 different compounds were identied. The extraction arranged in a single circle (Eiji and Salmaki, 2016). In most Lami-
yield is 0,02%. aceae a different pattern prevails: four central cells with a variable
Sesquiterpenes represent the most abundant terpene fraction number of peripheral cells ranging from six up to 16 (Werker, 1993,
(89.56% of the total terpene relative abundance): hydrocarbons 2000). This last pattern has been also documented in Salvia aurea
account for 69.39% and oxygenated ones reach 20.17%. The most (Serrato-Valenti et al., 1997).
abundant compound is bicyclogermacrene (see Table 4, 31, 16.3%), The micromorphological investigation proved evidence that S.
followed by germacrene D (29, 14.81%), -caryophyllene (22, 8.57%) uliginosa bears only one type of capitate trichome, whereas differ-
and -cadinene (35, 8.47%) among the hydrocarbons. Spathulenol ent morphotypes of capitate hairs generally co-occur in the same
(39), accounting for 12.66%, dominates among the oxygenated plant, also over the same organ (Giuliani and Maleci Bini, 2008).
sesquiterpenes. Monoterpene hydrocarbons represent 6.62% of the This evidence is not consistent with the greater number of capi-
total oil and the main compounds are limonene (9), (Z)--ocimene tate morphotypes reported by Gupta and Bhambie (1980) for eight
(10) and (E)--ocimene (12), with similar relative abundances, Salvia species, by Werker et al. (1985a) for S. sclarea and S. dominica,
slightly lower than 2%. and by Werker et al. (1985b) for S. ofcinalis and S. fruticosa. The co-
18 C. Giuliani et al. / Flora 233 (2017) 1221

Fig. 4. ai. Ultrastructure of the glandular trichomes of Salvia uliginosa, TEM. a-f. Peltate trichome. a. General view of a mature peltate trichomes at the active secreting stage.
b. Glandular cell cytoplasm of a full active peltate trichome. c. Particular of a glandular cell cytoplasm with electron-dense plastids and well-developed SER. d. Particular of
SER and of the subcuticular space containing lipidic droplets immersed in a granular matrix. e. Cytoplasm of the neck cells of a mature peltate trichome, note the numerous
plasmodesmata (arrows). f. Particular of the outer periclinal wall of the glandular cells, note the detachment of the cuticular layer delimiting the subcuticular space. g-i.
Short capitate trichome. g. Secreting cell cytoplasm of short capitate trichomes at the active secreting stage. h, i. Particulars of the cytoplasm of the secreting cell, note the
osmiophilic secreted materials (asterisks) close to the outer periclinal cell wall.
Symbols: cl, cuticular layer; ld, lipidic droplets; p, plastids; pl, pectic-cellulosic layer; m, mithocondria; Ser, SER; ss, subcuticular space; v, vacuoles.

occurrence of different capitate morphotypes is also conrmed in a similar, complex composition positive to Fluoral Yellow-088,
the detailed analysis of the glandular indumentum of 46 different Nadi reagent, FeCl3 and AlCl3 . Therefore, secretion is constituted of
species of Salvia of Iranian origin (Eiji and Salmaki, 2016). lipophilic substances, in particular terpenes, phenolic compounds
Salvia uliginosa short capitate trichomes, widespread on the and avonoids.
whole plant, consist of two secretory head cells supported by one- Although peltate trichomes were generally considered typical
celled stalk, and of a basal epidermal cell. This kind of capitate hair essential oil producers in Lamiaceae, containing the bulk of the
has been already found in Salvia species (Eiji and Salmaki, 2016) and terpenes produced (Hallahan, 2000; Werker, 2000), capitate tri-
correspond to type II described by Werker et al. (1985a). In addition, chomes of S. uliginosa seemed to produce great amounts of terpenic
these trichomes match the type described for several representa- substances as well. Moreover, a copious production of phenolic
tives of the genus Stachys (Giuliani and Maleci Bini, 2008). and avonoidic substances is demonstrated in both peltate and
The histochemical analysis showed that the secretion prod- short capitate hairs. Chemical complexity of this nature has previ-
ucts of both types of glandular trichomes are characterized by ously been reported for Salvia sclarea and S. dominica (Werker et al.,
C. Giuliani et al. / Flora 233 (2017) 1221 19

Table 3 Table 4
HS-SPME proles of the leaves and owers of Salvia uliginosa. Compositions of the essential oils obtained from the aerial parts of Salvia uliginosa.

l.r.i. Compounds Relative Abundance (%) l.r.i.a Compounds Relative


Abundance (%)
Leaves Flowers
1 931 -thujene tr
1 931 -thujene tr 2 941 -pinene 0.13
2 941 -pinene tr 3 976 sabinene 0.12
3 976 sabinene tr 4 980 1-octen-3-ol tr
4 982 -pinene tr 1.23 5 982 -pinene 0.34
5 987 3-octanone tr 6 993 myrcene 0.33
6 993 myrcene 0.2 0.54 7 1005 -phellandrene 0.13
7 1005 -phellandrene 0.13 0.12 8 1027 p-cymene tr
8 1032 limonene tr 9 1032 limonene 1.69
9 1034 1,8-cineole (=eucalyptol) 3.84 10 1042 (Z)--ocimene 1.95
10 1042 (Z)--ocimene 1.73 1.72 11 1045 phenyl acetaldehyde tr
11 1052 (E)--ocimene 1.32 2.21 12 1052 (E)--ocimene 1.79
12 1090 cis-linalool oxide (furanoid) tr 13 1062 -terpinene tr
13 1101 linalool 0.32 14 1101 linalool 0.13
14 1129 allo-ocimene 0.3 0.6 15 1133 (E)-2,6-dimethyl-1,3,5,7- 0.14
15 1140 nopinone tr octatetraene
16 1143 camphor tr 16 1351 -cubebene 0.81
17 1189 -terpineol tr 17 1372 -ylangene 0.24
18 1204 decanal tr 18 1376 -copaene 0.82
19 1205 verbenone tr 19 1384 -bourbonene 0.51
20 1244 carvacrol methyl ether 0.36 20 1390 -cubebene 0.49
21 1340 -elemene 5.09 13.98 21 1410 -gurjunene tr
22 1351 -cubebene 0.43 22 1420 -caryophyllene 8.57
23 1372 -ylangene 0.32 23 1429 -copaene 1.15
24 1376 -copaene 1.04 0.15 24 1441 aromadendrene 1.68
25 1384 -bourbonene 0.37 0.15 25 1456 -humulene 4.58
26 1390 -cubebene 0.61 0.11 26 1462 cis-muurola-4(14),5-diene 0.27
27 1392 -elemene 0.6 0.27 27 1470 trans-cadina-1(6),4-diene 0.22
28 1395 sativene 0.18 28 1477 -muurolene 4.63
29 1410 -gurjunene 0.16 0.11 29 1481 germacrene D 14.81
30 1414 -ylangene 0.14 30 1485 -selinene 0.19
31 1420 -caryophyllene 12.91 25.64 31 1495 bicyclogermacrene 16.3
32 1429 -copaene 2.24 0.42 32 1498 -muurolene 0.38
33 1441 aromadendrene 1.37 0.71 33 1507 (E,E)--farnesene 0.63
34 1447 cis-muurola-3,5-diene 0.16 34 1511 cis--cadinene 3.88
35 1456 -humulene 5.93 9.26 35 1524 -cadinene 8.47
36 1461 alloaromadendrene 0.29 36 1534 cadina-1,4-diene 0.41
37 1461 alloaromadendrene tr 37 1538 -cadinene 0.35
38 1462 cis-muurola-4(14),5-diene 1.08 38 1575 germacrene D-4-ol 0.19
39 1470 trans-cadina-1(6),4-diene 0.18 39 1576 spathulenol 12.66
40 1477 -muurolene 31.43 0.31 40 1583 globulol 0.13
41 1481 germacrene D 8.65 41 1590 viridiorol 0.27
42 1485 -selinene tr 42 1606 humulene epoxide II 0.26
43 1492 valencene tr 43 1615 humulane-1,6-dien-3-ol 0.36
44 1495 bicyclogermacrene 16.91 31.26 44 1628 1-epi-cubenol 0.23
45 1498 -muurolene 0.4 45 1640 epi--cadinol 1.74
46 1505 -amorphene 0.53 46 1642 epi--muurolol 2.47
47 1507 (E,E)--farnesene 1.05 0.63 47 1661 cis-calamenen-10-ol 0.11
48 1513 trans--cadinene 1.86 48 1682 (Z)--santalol 1.75
49 1515 cycloisolongifol-5-ol 0.59 49 1845 hexahydrofarnesyl acetone 0.87
50 1524 -cadinene 4.46 0.13
51 1534 cadina-1,4-diene 0.3 Monoterpene 6.62
52 1538 -cadinene 0.38 hydrocarbons
53 1556 germacrene B 0.11 Oxygenated 0.13
54 1575 germacrene D-4-ol 0.77 monoterpenes
55 1576 spathulenol tr Sesquiterpene 69.39
56 1581 caryophyllene oxide 0.14 hydrocarbons
57 1815 2-ethylhexyl salicylate 0.12 Oxygenated 20.17
sesquiterpenes
Monoterpene hydrocarbons 3.68 6.42 Apcarotenoids 0.87
Oxygenated monoterpenes 3.84 0.68 Non-terpene derivatives tr
Sesquiterpene hydrocarbons 90.13 92.18
Oxygenated sesquiterpenes 0.91 0.59 Total 97.18
Non-terpene derivatives tr 0.12 a
Linear retention indices on a DB5 column.
Total 98.56 99.99

(Werker, 1993; Giuliani and Maleci Bini, 2008), whereas only traces
1985a), S. aurea (Serrato-Valenti et al., 1997) and S. blepharophylla of pectic polysaccharidic substances are generally found in the
(Bisio et al., 1999). secreted material of peltate hairs (Werker, 1993; Giuliani and
In both trichomes types, the secreted material gave invariably Maleci Bini, 2008). In S. uliginosa only the wall and the cytoplasm of
negative responses to the hydrophilic stainings, indicating the lack the head cells stained positively to Alcian Blue and Ruthenium Red:
or the scarce production of polysaccharides. On the contrary, abun- in the protoplast, polysaccharides can act on water retention, con-
dant non-cellulosic and acidic polysaccharides have been reported tributing to the maintenance of the water potential in the tissues
in the secreted material of capitate hairs in various Lamiaceae (Werker, 2000).
20 C. Giuliani et al. / Flora 233 (2017) 1221

In the examined species, the trichomes responsible for both As most of species included in Salvia genus, S. uliginosa is bee-
the terpene and avonoid production and storage, as evidenced pollinated (Wester and Claen-Bockhoff, 2007). At the Ghirardi
with the histochemical stainings and conrmed by TEM analysis, Botanical Garden S. uliginosa plants were exclusively visited by
are both peltate and capitate trichomes, widespread on the whole honeybees (Giuliani and Fico, personal observations).
plant epidermis. The ultrastructural observations on the cytoplasm Besides visual signalling, bees are also able to perceive chemi-
of the secreting cells showed large electron-dense plastids and a cal cues in a multimodal pattern related to their ability to recover
well-developed periplastidial SER, which are considered cellular food resources (Wright and Schiestl, 2009). Floral scents are blends
compartment responsible for the synthesis and transfer of terpenic of many different chemical compounds (Dobson 2006), present at
substances (Fahn, 2000; Hallahan, 2000). Moreover, SER elements diverse concentrations (Maffei et al., 2011). Honeybees, in particu-
are responsible for the intracellular transport of polyphenols (Fahn, lar, are able to detect subtle differences in the overall intensity or
2000). concentration of single compounds in a scent (Wright et al., 2005).
The limited production of polysaccharidic compounds is sup- Previous studies have demonstrated that some of the volatiles
ported by the occasional occurrence of Golgi stacks and RER, produced by S. uliginosa owers are involved in the attraction of
responsible for the synthesis and transport of such substances honeybees, e.g. -caryophyllene (Blight et al., 1997) and germar-
(Schnepf, 1974; Ascensao et al., 1995; Fahn, 2000). crene D (Lusebrink et al., 2015), the latter exclusively emitted from
The secreted substances copiously accumulate in the subcuticu- S. uliginosa owers. Therefore, a possible role of this volatile in
lar space. In both hair types, ultrastructural observations conrmed the seduction strategies towards honeybees may be hypothesized.
that the secretory material is characterized by a heterogeneous Moreover, since -caryophyllene was simultaneously detected in
composition: indeed, materials with different electro-density the leaf headspace, we can suggest a synergistic action leaf-ower
degrees and appearance are observed in the subcuticular space, in attracting potential pollinators (Vitalini et al., 2011).
thus indicating the presence of different chemical phases. An This report is the rst contribution on the composition of the
emulsion-like appearance has been described for the secretion essential oil of S. uliginosa out of its native range.
products of peltate trichomes in other Lamiaceae species (Giuliani The genus Salvia, as most of the aromatic plants of the Lamiaceae
and Maleci Bini, 2008). family, are included within the subfamily Nepetoideae (El-Gazzar
In these types of trichomes, two potential mechanisms for the and Watson, 1970). However, several studies highlighted that in
extrusion of the secretory products have been hypothesized: (i) via Salvia there are also oil-poor species (Malencic et al., 2004).
pores in the intact cuticular structure, generally described in liter- Indeed, S. uliginosa presents a very low yield of essential oil,
ature only in capitate trichomes (Werker, 1993); (ii) by breakage of 0.02%, as documented in other members of the genus (Malencic
the cuticle, common to members of the Lamiaceae (Werker, 1993, et al., 2004). This result may be related to the fact the glandular tri-
2000). chomes synthesized compounds with low volatility which cannot
The volatile components may pass through minute pores of the be recovered by hydrodistillation (Bicchi et al., 1985).
intact cuticle. Also avonoids may cross the cell walls and the cuti- Noteworthy differences resulted from the comparison of liter-
cle, crystallizing on the surface of trichomes and on the epidermis ature data (Pinto, 2012). In fact, this author reported an essential
of the organs bearing them (Wollenweber, 1994). However it can oil particularly rich in non-terpene compounds, mainly aliphatic
be argued that the most part of the secretion is extruded after the and carbonyl derivatives, such as hexadecanoic acid (30.1%),
rupture of the cuticle, as it generally occur (Giuliani and Maleci Bini, n-nonadecane (17.5%), nonadecanal (4.5%). The only two sesquiter-
2008). penes detected at percentages higher than 1% were spathulenol
With regard to non-glandular trichomes, two distinct morpho- (1.9%) and -muurolol (1.1%). Notably, sesquiterpene hydrocar-
types were described: type I, widespread on the whole plant, and bons, which represented the main chemicals of the essential oil
type II, occurring exclusively on petals. The presence and dis- reported in our study, are completely absent in Pinto (2012)
tribution of non-glandular hairs among Salvia species is widely investigation. This behaviour may be related to the very different
documented and both simple and branched trichomes have been environmental and geographical conditions of growth of the plants.
described (Eiji and Salmaki, 2016). Our results showed that only In addition, the analysis reported by Pinto (2012) is incomplete,
simple trichomes are present in S. uliginosa, as documented since only 64% of the total oil has been identied, therefore this
in almost all the New World species investigated so far, since evidence could furtherly explain the high levels of diversity in the
branched trichomes have been mainly observed in taxa of European oil composition with our samples.
or Asian origin (Eiji and Salmaki, 2016). The results obtained in the present study agree with previ-
About the phytochemical investigation on the vegetative and ous reports that oil-poor species of Lamiaceae possess oil rich in
ower bouquets, this work represents the rst contribution on the sesquiterpene compounds (Lawrence, 1992; Senatore et al., 1997).
characterization of the VOC proles in S. uliginosa.
The obtained volatile proles show high level of variability.
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