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Flora 248 (2018) 10–21
Contents lists available at ScienceDirect
Flora
journal homepage: www.elsevier.com/locate/flora
Functional traits of floral and leaf surfaces of the early spring flowering
Asphodelus ramosus in the Mediterranean region
T
Chrysanthi Chimonaa, Danae Koukosa, Maria-Sonia Meletiou-Christoua, Emmanuel Spanakisb,
⁎
Apostolos Argiropoulosa, Sophia Rhizopouloua,
a
b
Department of Botany, Faculty of Biology, National and Kapodistrian University of Athens, Athens 15784, Panepistimiopolis, Greece
Department of Materials Science and Technology, University of Crete, P.O. Box 2208, 14 Voutes, Heraklion 71003, Crete, Greece
A R T I C LE I N FO
A B S T R A C T
Edited by Hermann Heilmeier
In this study functional micromorphological traits and water repellent attributes of floral and leaf surfaces of
Asphodelus ramosus are presented. The branched inflorescences of A. ramosus bear numerous short-lived flowers
arranged on tall flowering stalks, while the basal long-tapered leaves are long-lived. The most striking feature of
the short-lived flowers of A. ramosus is the surface structure of the coloured midrib and the white bilateral
sections of tepals, revealing different functionality between the two sections of tepals and the two sides of the
corollas. Structural features of adaxial and abaxial epidermal cells may function predominantly for water repellence of floral tissues expanded during the humid period of the year. The leaves are more water repellent than
the tepals and display declining water retention on their surfaces. It appears that micro- and nano-scale patterns
observed on leaf and tepal surfaces of A. ramosus are linked to hydrophobic properties, which are important
adaptive traits contributing to the seasonal performance of A. ramosus in the field, particularly in regard to water
adhesion.
This article is dedicated to the memory of Prof.
John D. Pantis.
Keywords:
Asphodel
Contact angles
Fatty acids
Surface wettability
Tepal topography
1. Introduction
Recently, major importance has been given to multifunctional traits
of tepal and leaf surfaces, which have greatly increased our knowledge
about functionality of plant tissues exposed to environmental conditions (Feng et al., 2011; Argiropoulos and Rhizopoulou, 2012;
Rhizopoulou et al., 2015; Taneda et al., 2015; Argiropoulos et al., 2017;
Fernández et al., 2017; Bailes and Glover, 2018). The surface relief,
besides serving as a constitutive barrier between plant tissues and environment, is also considered to bear imprint of tissues’ developmental
processes and to contribute to recognition by pollinators by creating
surface stimuli (Abad et al., 2017; Gorb et al., 2017; Moyroud et al.,
2017; Watson et al., 2017). The surface relief is covered by epicuticular
folds and waxes, which form the outermost layer of plant tissues and
influence functional attributes, such as wettability, water adherence
influencing net CO2 assimilation, water drip, mechanical containment
and optical properties of the tissues (Yeats and Rose, 2013; PegueroPina et al., 2015). It has been reported that variability between structures and functions may sometimes impede or facilitate the uptake of
water (Fernández et al., 2014).
The structure and the composition of epicuticular wax vary widely
⁎
among plant species and even among organs of the same species
(Kosma and Rowland, 2016). Long-chain fatty acids and their derivatives (i.e. alkanes, alcohols, esters, ketones) are ubiquitous compounds
of the cuticular layer and exhibit hydrophobic properties (Samuels
et al., 2008; Buschhaus and Jetter, 2011; Domínguez et al., 2017);
qualitative and quantitative profiles of cuticular fatty acids together
with the surface micromorphology provide additional data toward the
classification of plant taxa (Gülz, 1994; Jenks and Ashworth, 1999;
Müller and Riederer, 2005; Choi et al., 2011; De Castro et al., 2015;
Piwowarczyk and Kasińska, 2017; Zareh et al., 2017; Giuliani et al.,
2018; Shayanmehr et al., 2018). Also, epicuticular fatty acids have been
implicated in the role of the cuticle in plant defence (Kachroo and
Kachroo, 2009; Reina-Pinto and Yephremov, 2009; Serrano et al., 2014;
Lim et al., 2017).
There is growing interest in research and industry to obtain a more
comprehensive understanding of surface properties of vegetative and
reproductive plant tissues expanded under ambient conditions. Such
surfaces have great significance for both basic research and a variety of
practical applications, including water-repellent coatings, self-cleaning
and chemical shielding. Apparently, the multifunctional submicron
features of floral and leaf surfaces provided a potential natural template
Corresponding author at: National and Kapodistrian University of Athens, Department of Biology, Institute of Botany, Panepistimiopolis, Athens 15784, Greece.
E-mail address: srhizop@biol.uoa.gr (S. Rhizopoulou).
https://doi.org/10.1016/j.flora.2018.08.003
Received 30 January 2018; Received in revised form 19 July 2018; Accepted 1 August 2018
Available online 16 August 2018
0367-2530/ © 2018 Elsevier GmbH. All rights reserved.
Flora 248 (2018) 10–21
C. Chimona et al.
2. Materials and methods
for man-made applications and have inspired the manufacture of biomimetic materials (Ma and Sun, 2009; Bhushan and Her, 2010; Lee
et al., 2011; Schulte et al., 2011; Telford et al., 2013; Hünig et al., 2016;
Szczepanski et al., 2016; Yuan et al., 2016; Barthlott et al., 2017);
further studies of the properties and inferred functions of surface nanostructures are expected to give rise to more applications.
Asphodelus ramosus L. (asphodel), assigned to Xanthorrhoeaceae
(The Plant List, 2016; Safar et al., 2014), is a tuberous-rooted geophyte
and a widespread native species in semi-arid ecosystems of the Mediterranean region (Pantis and Margaris, 1988; Díaz Lifante, 1994; Melián
et al., 2017). Geophytes are an important life form in Mediterranean
ecosystems; it has been argued that the last degradation stage of ecosystems in the eastern Mediterranean is dominated by Asphodelus species (Sakar et al., 2010 and references therein). The basal rosettes of
succulent, dorsiventral, linear leaves of A. ramosus appear in autumn
and carry out photosynthesis during the winter and spring months; the
leaves senesce and dry out during summer (Chimona, 2015). The
leafless flowering stalks of A. ramosus are up to one meter tall, they bear
branched inflorescences composed of tens to hundreds of flowers sequentially expanded during the flowering season that extends from
February to April and wilt during summer (Chimona, 2015;
Rhizopoulou and Pantazi, 2015); anthesis of individual inflorescences
lasts c. one month, while anthesis of individual flowers lasts c. 24 h
(Schuster et al., 1993; Argiropoulos, 2009).
In the literature Asphodelus ramosus L. has been cited as synonym of
Asphodelus albus Mill. and Asphodelus aestivus Brot. that has also been
cited as synonym of Asphodelus microcarpus Viv. (Jordan, 1860;
Narducci, 1957; Schuster et al., 1993; Díaz Lifante and Valdés, 1994;
Rhizopoulou et al., 1997; Lev-Yadun and Ne’eman, 2004; Samuni-Blank
et al., 2014; Polatoğlu et al., 2016; Biscotti et al., 2018; Malmir et al.,
2018). The generic name Asphodelus is derived from the ancient Greek
word ασφόδελoς (asphodelos) (Verpoorten, 1962; Negbi, 1989;
Amigues, 2004; Gledhill, 2008). In English, the meaning of ramosus is
much branched (Gledhill, 2008). Also, Asphodelus ramosus was included
in the 4th volume of the 1st edition of Flora Graeca Sibthorpiana –one of
the most rare and magnificent botanical books ever written– published
in London in 1823 (Digital Flora Graeca, 2016: Flora Graeca published
version, v.4, tabula 335). The original watercolour made by Ferdinand
Bauer (1760–1826) in Oxford in the late 18th century (Digital Flora
Graeca, 2016: Flora Graeca drawings, v.5, folio 99) was based on specimens collected during spring of the year 1787 from Aegean islands of
Greece, where numerous stands of A. ramosus are still growing
(Sibthorp and Smith, 1806; Panitsa et al., 2003; Biel, 2005; Harris,
2008; Sarika et al., 2016; personal observations).
The objective of this work was to study and compare features of
tepal and leaf surface micromorphology, wettability and fatty acid
composition of A. ramosus grown in the field and exposed to rainfall, as
part of a larger study of functional interfaces between plant tissues and
environment, as well as adaptations of wild plants to environmental
conditions (Argiropoulos and Rhizopoulou, 2012; Chimona et al., 2012;
Kolyva et al., 2012; Gkikas et al., 2015; Koukos et al., 2015;
Rhizopoulou et al., 2015; Argiropoulos et al., 2017). Light-, scanning
electron- and atomic force microscopy were used to study morphological traits of tepals and leaves of A. ramosus. Static contact angles of
water droplets on the tissues were measured to evaluate the surface
wettability. Analysis of fatty acid composition in flowers and leaves and
their epicuticular wax was performed using gas chromatography. To the
best of our knowledge, surface topography of aerial parts of flowering
A. ramosus, using high resolution imaging that enables detailed study of
plant surfaces and enhances our understanding of relationships between tissues and environment has not yet been published. Submicron
patterns of plant surfaces despite their ecological and adaptive role in
different habitats have important applications to the rapidly growing
and promising field of research for biomimetics.
2.1. Plant material and study area
The study was conducted on naturally occurring stands of wild
plants of Asphodelus ramosus L., near the campus of the National and
Kapodistrian University of Athens (37° 57´ N, 23° 47´ E, altitude 250 m
a.s.l.). Plant material was identified by Associate Professor T.
Constantinidis (Department of Biology, Section of Systematics and
Ecology, National and Kapodistrian University of Athens, Greece) and
voucher specimens have been deposited in the Herbarium of the
National and Kapodistrian University of Athens (Chimona et al., 2014).
Flowering of A. ramosus was observed in individual plants (n > 35),
from the bud to the open-corolla stage and then to wilting on a daily
basis, during the flowering period of this species that extends from
February to April, for three successive years. Floral longevity was defined as the time between opening and wilting of the corollas and was
monitored on a monthly basis between 2013 and 2016. Average
monthly rainfall ranged from 52 ± 4 mm to 30 ± 2 mm and monthly
temperature from 8 ± 2 °C to 15 ± 3 °C between February and April,
respectively. Climatic data for the research site (Fig. S1) were obtained
from the Hydrological Observatory of the National Technical University
of Athens, which is the nearest meteorological enclosure to the research
site (approximately 1 km distant, http://hoa.ntua.gr/stations/d/356),
and are typical for the research site in comparison with recent years
(Nastos and Polychroni, 2016). Leaves and flowers were randomly
harvested from abundant plants in the morning, during the median day
of flowering of A. ramosus (Rhizopoulou and Pantazi, 2015); the
average daily quantum flux density, obtained from the above mentioned nearby meteorological station, was approximately 12 mol m−2
day−1. Cell dimensions, leaf and tepal areas were measured in 50
samples collected from at least 30 individual plants using scanned digital images of the plant material and the ImageJ processing program
(http://rsb.info.nih.gov/ij/). Floral diameter was measured using a
ruler.
2.2. Microscopy
The study was carried out in fully expanded tepals and leaves of A.
ramosus. Tepal samples from the coloured midrib and the white marginal sections of the blades (Fig. S2), and leaf blades were carefully cut
into square pieces (2 mm × 2 mm); the square pieces of the tissues were
washed with a 2% TWIN 20 solution overnight at 4 °C, followed by
immersion in Na-phosphate buffer at pH 7 at room temperature for
30 min, and then fixed in 3% glutaraldehyde in Na-phosphate buffer at
pH 7, at room temperature for 2 h. The samples were then washed three
times with the buffer for 30 min each time, and post-fixed in 1% OsO4
in the same buffer at 4 °C. Dehydration of the material was accomplished with acetone solutions of increasing concentrations; dehydrated
samples were critical-point-dried in a Bal-tec CPD-030 dryer (Balzers,
Liechtenstein), mounted with double adhesive tape on stubs, and
sputter coated with 20 nm gold in a Bal-tec SCP-050. The samples were
then viewed using a JEOL JSM-6390LV Scanning Electron Microscope
(SEM, JEOL Ltd, Tokyo, Japan); software associated with this microscope was used for the production of the digital images. Adaxial and
abaxial segments (5 μm × 5 μm) from the coloured midrib and the
white sections of the tepals were viewed using a tap mapping atomic
force microscope (AFM Multiview-4000, Nanonics Imaging, Jerusalem,
Israel) and representative topographical micrographs at the nanometerscale are presented. Several parameters were analysed and processed,
using the software package WSxM 5.0 (Horcas et al., 2007), to detect
detailed information about tepal surfaces of A. ramosus. The quantitative measurements include the horizontal (Hd) and vertical (Vd) distances (representing widths and heights between folds on the relief),
the surface roughness (Rs), and the surface ratio (Sr) that represents the
density of nanofolds and are presented as mean values of ten specimens
11
Flora 248 (2018) 10–21
C. Chimona et al.
evaporated, as mentioned above. The methyl esters were re-dissolved in
hexane; the aliquots of fatty acid methyl esters in hexane were analyzed
with a Hewlett-Packard 5890 series II gas chromatograph, as mentioned
in paragraph 2.3.
followed by the standard errors (cf. Table 2).
2.3. Lipids and fatty acids
Fully expanded leaves and petals of A. ramosus were selected from
30 individual plants of A. ramosus, dried at 60 °C for 3 days and then
ground up. Extraction of lipids was accomplished using a chloroform–methanol (2:1, v/v) solution; total lipids were quantified according to Meletiou-Christou and Rhizopoulou (2017). Saponification
of lipids and methylation of fatty acids were carried out with 5% (v/v)
H2SO4 in methanol. The fatty acid methyl esters were separated using a
Hewlett Packard 5890 (Series II) gas chromatograph (GC, Wilmington,
DE, USA) with a flame ionisation detector and were identified by
comparing the retention times with those of the standard fatty acid
methyl esters (Analytical standards KIT 611C, PolyScience, IL, USA); a
30 m glass capillary column 0.32 mm in diameter (HP-INNOWax coated
with polyethyleneglycol, film 0.5 μm thick) was used, with nitrogen as
the carrier gas (1 mL min–1). The gas chromatography was performed
under 1 mL min−1 N2 flow rate, at 220 °C injector temperature and
275 °C detector temperature; the oven temperature was programmed at
150 °C for 1 min, followed by temperature gradients of 15 °C min−1 to
200 °C for 5.3 min and 2 °C min−1 to 240 °C for 20 min, then the final
temperature (240 °C) was held for 18 min. Data processing was accomplished with a computer equipped with the HP ChemStations
software. The amounts of fatty acids are presented as percentages of the
total fatty acids.
2.6. Contact angle
Measurements of static contact angle (Θ) were performed on each
side of the same expanded leaves and tepals using the sessile drop
method with an automated tensiometer (Boyce and Berlyn, 1988; Nairn
et al., 2011). Droplets (2 μL) of distilled and deionised water were
gently positioned, using a microsyringe, on plant surfaces mounted
with double adhesive tape on stubs, and images were captured to
measure the angle formed at the liquid–solid interface (Fernández et al.,
2014; Koukos, 2015). The ImageJ processing program was used for the
measurements (Williams et al., 2010). The mean values were calculated
from ten different randomly selected leaves and tepals from five different plants. The criteria for evaluating surface wettability were based
on those of Nosonovsky and Bhushan (2012), where Θ < 90° is called
hydrophilic, while Θ > 90° is called hydrophobic.
2.7. Statistical analysis
The results are presented as means ± Standard Error (SE).
Differences between traits of tepal and leaf surfaces of A. ramosus were
tested by performing multivariate analysis of variance (MANOVA) at
P ≤ 0.05, where the variability within groups and interaction of different factors on traits are examined. More specifically, as independent
variables are considered the tissue (either tepal or leaf), the surface
within tissue (either adaxial or abaxial) and the region within tepal
surface (either white or coloured); in the case of leaves, there is only
one region (central) and differences were not detected. As dependent
variables are considered the dimensions of epidermal cells (length and
width), the structural traits of tepal surfaces (Vd, Hd, Rs, Sr), the
composition of fatty acids found in the considered tissues and cuticular
waxes, and the contact angles of sessile droplets on tissue surfaces.
Wherever significant effects within groups were detected, Duncan’s test
was applied. Statistical tests were performed using the SPSS statistical
programme v. 23.0 (SPSS Inc., Chicago, IL, USA).
2.4. Wax extraction
Epicuticular waxes of fully expanded leaves and tepals of A. ramosus
were removed by dipping the tissues in chloroform at room temperature, while being gently agitated for 3 min and 30 s, respectively
(Bewick et al., 1993; Razeq et al., 2014; Koukos et al., 2015; Huggins
et al., 2018). Microscopic examination of leaf and tepal surfaces confirmed the removal of the wax.
2.5. Fatty acid composition of extracted epicuticular wax
The chloroform solution containing the extracted waxes was evaporated by heating gently under a constant stream of nitrogen gas. The
waxes were then re-dissolved in 5 mL methanol and transmethylated
with 5 mL elemental sodium–methanol solution (2 M), followed by
2 mL 37% HCl drop-wise, while gently agitating for 20 min; after
transfer into a separating funnel 30 mL distilled water and 10 mL petroleum ether were added. The upper phase of petroleum ether containing the methyl esters of the fatty acids was collected and the petroleum ether was evaporated under nitrogen gas. The flask was washed
with petroleum ether, and the content was transferred into a test tube
that was heated gently under a constant stream of nitrogen gas, then the
petroleum ether evaporated. The test tube was then washed with
chloroform and the contents were transferred into a vial and
3. Results
3.1. Floral and leaf traits
The long-tapered and channelled basal leaves of A. ramosus
(20–40 cm in length, 2–4 cm in width) emerge during October and grow
close to the ground (up to 15–20 cm in height). The rapidly extended
inflorescences stalks (approximately 20 cm per day) reach a height of
80–100 cm, in February. Flowering of A. ramosus averages a seven-week
period (from February to April) and produces hundreds of successively
open flowers on branched inflorescences; approximately five
Fig. 1. Fully expanded, open flower of Asphodelus ramosus; (A) adaxial view of tepals and filaments, (B) abaxial view of tepals, (C) view of the coloured midrib along
the expanded tepal highlighted using red dotted line (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article).
12
Flora 248 (2018) 10–21
C. Chimona et al.
epidermal cells (Table 1) were significantly different between tepals
and leaves (F = 4.21, P < 0.05), as well as between white and coloured regions within tepal tissues (F = 18.78, P < 0.05). Concerning
the adaxial and the abaxial surfaces of tepals and leaves, significant
differences were not detected (F = 4.26, P = 0.78).
High resolution imaging of tepal epidermises of A. ramosus reveals
detailed patterns of a relief at the nanometer scale (Fig. 6). The tepal
surfaces of A. ramosus possess different nanosculptures between sections and sides, as indicated by projections in the shape of peaks and
cavities that vary in height, density and arrangement (Table 2). In the
white section of tepals, the adaxial (Fig. 6E, F) epidermal cells possess
smaller Vd and Hd than those of the abaxial (Fig. 6G, H) epidermal
cells. The epidermal cells of the adaxial coloured midrib section
(Fig. 6A, B) possess smaller vertical (Vd) and horizontal (Hd) distances
than those of the abaxial (Fig. 6C, D) section (Table 2). Also, Hd is
approximately 3-fold higher than Vd in both the bilateral white and the
coloured midrib sections (Table 2) of the adaxial tepal surface, while,
on the abaxial tepal surface, Hd is 6-fold and 3-fold higher than Vd in
the white and the coloured section, respectively (Table 2). Actually,
structural features of the epidermal relief of tepals possess more extensive nanometric area on the abaxial coloured midrib and white
sections in comparison with the narrower area on the adaxial white and
coloured midrib sections. The abaxial epidermal cells of both the white
bilateral and the coloured midrib sections of tepals exhibit 2-fold higher
roughness (Rs) than the adaxial epidermal cells (Table 2). The density
of cuticular forms on the epidermal relief is represented by Sr and is
different between the adaxial and the abaxial coloured midrib and the
white bilateral sections of tepals (Table 2). Taken together, significantly
different nanometric structural features were detected in tepals of A.
ramosus (Vd: F = 10.01 at P < 0.05, Hd: F = 67.67 at P < 0.05, Rs:
F = 57.16 at P < 0.05 and Sr: F = 5.32 at P < 0.05). In the case of
tepals, there were significant interactive effects between region within
tissue and surface within region for the dependent variables Vd
(F = 7.71, P < 0.05) and Rs (F = 5.31, P < 0.05). Linear regressions
among nanometric estimates (Table S1) of the examined tepal tissues
reveal that Rs was correlated with Hd (R2 = 0.95), Vd (R2 = 0.84) and
Sr (R2 = 0.55); also, Sr was positively correlated with Vd (R2 = 0.80).
Table 1
Dimensions of epidermal cells of tepals and leaves of Asphodelus ramosus.
Means ± standard errors (n = 10) followed by the same uppercase (region
within tissue) and lowercase (surface within region) superscript for each dependent variable are not significantly different (P > 0.05). Superscript asterisks
indicate comparisons between tepals and leaves (tissue); values followed by a
different number of asterisks are significantly different (P < 0.05), whereas
values followed by the same number of asterisks are not significantly different
(P > 0.05).
Tissue
Surface
Region within tissue
Length
(μm)
tepal
tepal
tepal
tepal
leaf
leaf
adaxial
abaxial
adaxial
abaxial
adaxial
abaxial
white
white
coloured
coloured
central
central
40.14
41.07
55.11
56.03
98.71
84.23
±
±
±
±
±
±
Width
(μm)
3.25Cc*
4.18Cc*
4.72Bc*
5.93Bc*
2.26Aa**
7.59Ab**
34.40
34.90
21.84
22.14
25.36
23.25
±
±
±
±
±
±
4.36Dd*
4.87Dd*
2.98Ee*
3.74Ee*
6.04Ee*
6.08Ee*
Table 2
Nanometric structural features of the adaxial and the abaxial tepal surfaces of
Asphodelus ramosus. Means ± standard errors (n = 10) followed by the same
uppercase (region within tepal) and lowercase (surface within region) superscript letter for each dependent variable are not significantly different
(P > 0.05). Vd: vertical distance, Hd: horizontal distance, Rs: roughness, Sr:
surface ratio.
Region
within
tepal
Surface
within
region
Vd
(nm)
white
white
coloured
coloured
adaxial
abaxial
adaxial
abaxial
362
430
338
667
±
±
±
±
Hd
(nm)
7De
9Dd
5Ce
8Cc
1,100
2,690
1,031
2,195
Rs
(nm)
±
±
±
±
12Ab
15Aa
11Bb
13Ba
124
264
135
257
±
±
±
±
Sr
4Eg
6Ef
3Eg
4Ef
1.23
1.31
1.30
1.41
±
±
±
±
0.02Gj
0.02Gi
0.02Fi
0.04Fh
actinomorphic corollas (20–25 mm in diameter) with six tepals
(10–20 mm in length) expand per day, per each inflorescence branch.
Each individual flower exhibited a 24 h life span (Fig. 1A). The tepals of
A. ramosus are characterized by a midrib (central), conspicuous coloured line-like mark along the white blades (Fig. 1A–C); therefore,
each tepal of A. ramosus consists of two distinct portions, i.e. the coloured midrib and the bilateral white sections (Fig. S2). Concerning the
dimensions of epidermal cells of both tepals and leaves, it is likely that
the widths are not statistically different, whereas the opposite holds
true for the lengths (Table 1).
3.3. Fatty acids
Detailed analyses revealed differences between fatty acids of tepals
and leaves. The fatty acid determination of total lipids (Table 3) indicates the abundance of three fatty acids, i.e. linolenic acid
(18:3Δ9,12,15), palmitic acid (16:0) and linoleic acid (18:2Δ9,12), accounting for approximately 73% in tepals and 80% in expanded leaves
of A. ramosus. The contribution of linolenic acid and palmitic acid was
higher in leaves by approximately 20%, in comparison with that of
tepals; while, the contribution of linoleic acid was higher in tepals by
approximately 25% in comparison with that of leaves. Also, the contribution of long chain fatty acids, i.e. arachidic acid (20:0) and behenic
acid (22:0) was detected in tepals, while lignoceric acid (24:0) equally
contributed to tepals and leaves (Table 3).
The fatty acid composition of the isolated epicuticular wax fraction
from leaves and tepals of A. ramosus ranged from palmitic acid (16:0) to
lignoceric acid (24:0) (Table 3); the tepal wax was found to contain
unusually high content of C20, C22 and C24 fatty acids, while in the leaf
wax substantially higher content of C24 (lignoceric acid) was detected.
Also, fatty acids of longer chain length, i.e. cerotic (26:0) and melissic
(30:0) were detected in leaf wax, albeit at small percentages ranging
from 2.3% to 3.7%, respectively. In tepal wax, arachidic (20:0), behenic
(22:0) and lignoceric (24:0) acids contributed from approximately 20%
to 29%, followed by linoleic (18:2), linolenic (18:3), palmitic (16:0)
and palmitoleic (16:1) acids that contributed substantially smaller
percentages, ranging from approximately 4% to 12% (Table 3). The
contribution of myristic (14:0), oleic (18:1) and linolenic acid was
different (P < 0.05) in the studied tissues (F = 7.71, F = 10.22 and
3.2. Surface micromorphology
Representative scanning electron micrographs of the coloured
midrib section of tepals of A. ramosus reveal that it consists of elongated, rectangular epidermal cells (Table 1), oriented longitudinally
and in parallel to the tepal’s long axis (Fig. 2), covered by corrugated
cuticular striations, densely arrayed on their adaxial (Fig. 2A, B) and
abaxial (Fig. 2C, D) surfaces. Numerous stomata can be observed in the
dense epicuticular striae of the abaxial coloured midrib section (Fig. 3)
of tepals; the density of stomata was found to be substantially higher on
the abaxial surface than on the adaxial surface (82 ± 5 and 27 ± 3
stomata mm–2, respectively). In contrast, the adaxial (Fig. 4A, B) and
the abaxial (Fig. 4C, D) surfaces of the white bilateral sections of tepals
‒consisting of convex lenticular epidermal cells (Table 1) and covered
by undulated striated relief‒ lack stomata (Fig. 4).
The adaxial (Fig. 5A, B) and the abaxial (Fig. 5C, D) leaf surfaces of
A. ramosus consist of elongated epidermal cells (Table 1), with a protruding papillae structure at their center (Fig. 5); cuticular ridges
commence from the center and extend to the periphery of adaxial
epidermal cells (Fig. 5B). Numerous stomata observed on the adaxial
and abaxial epidermises are covered by waxy platelets (Fig. 5). Traits of
13
Flora 248 (2018) 10–21
C. Chimona et al.
Fig. 2. Scanning electron micrographs of adaxial (A, B) and abaxial (C, D) rectangular, elongated epidermal cells of the colored midrib of tepals of Asphodelus
ramosus. Scale bars: 10 μm (A, C) and 5 μm (B, D).
121.8 ± 4.8°) and the abaxial (Fig. 7F, Θ: 119.3 ± 2.4°) leaf surfaces.
The contact angles (Θ) of tepals and leaves are significantly different
(F = 56.8, P < 0.05). In the case of leaves, significant difference was
not estimated between the adaxial and the abaxial surfaces (F = 4.49,
P = 0.74). In the case of tepals, there were significant differences between surfaces within region (F = 4.83, P < 0.05), and significant
interaction effects between region within tepal and surface within region (F = 4.26, P < 0.05).
4. Discussion
Topography of the adaxial and abaxial tepal surfaces of Asphodelus
ramosus revealed different traits between the two sides of the corolla.
The ornamentation of the cuticular relief of epidermal cells of A. ramosus is characterized by large horizontal distances that increase the
surface area of epidermal cells of tepals of the short lived flowers of A.
ramosus; this may promote both optical and water repellent properties
of the exposed floral tissues to wet conditions (Bird and Gray, 2003;
Gkikas et al., 2015; Aparecido et al., 2017). In particular, the white
sections of tepals consisting of rectangular epidermal cells with irregular surfaces (Fig. S3D) may increase reflectance at different angles
and therefore floral advertisement (Lee, 2007; Gkikas et al., 2015). The
vertically and longitudinally oriented dense cuticular folds on the epidermal cells of tepals of A. ramosus may be linked to the underlying cell
expansion of floral tissues (Martens, 1936; Antoniou Kourounioti et al.,
2013; Huang et al., 2017). Also, the tightly appressed nanoridges on
tepals (Figs. 2 and 4), which indicate rapid expansion of tissues, were
observed on the petals of short-lived flowers of other early spring
flowering wild Mediterranean species (Li-Beisson et al., 2009;
Argiropoulos and Rhizopoulou, 2012).
The flowering season of A. ramosus partly coincides with elevated
precipitation and relatively low temperatures in the Mediterranean
region (Fig. S1). In tepals, the abaxial white section appears to have
higher surface roughness and Sr –representing the density of nanofolds–
and deeper cavities (Fig. 6G, H) than the adaxial white section (Fig. 6E,
F); this contributes to the firmness of contact with water droplets
(Fig. 7B, E). It appears that the adaxial white surfaces are more susceptible to wetting under humid conditions than the abaxial white
surfaces of tepals (Fig. 7). Also, the roughness of the abaxial midrib
surface is approximately 2-fold higher than that of the adaxial midrib
Fig. 3. Scanning electron micrograph of the abaxial coloured midrib section of
tepals of Asphodelus ramosus displaying stomata and remnants of secretion in
the form of small globular structures on striations. Scale bar: 20 μm.
F = 106.69, respectively). Also, the contribution of linoleic
(F = 198.96), arachidic (F = 236.93), behenic (F = 116.33) and lignoceric (F = 212.71) acid varied considerably (P < 0.05) between
epicuticular waxes of the studied tissues. Concerning the contribution
of palmitic, linoleic, linolenic, arachidic, behenic and lignoceric fatty
acids, there were significant interactive effects (P < 0.05) between the
studied tissues and epicuticular wax (F = 27.39, F = 13.72, F = 68.91,
F = 58.85, F = 48.76 and F = 65.60, respectively).
3.4. Contact angles
The contact angles (Θ) of water droplets on tepal- and leaf-surfaces
of A. ramosus shown in Fig. 7 reveal hydrophobic properties of the
examined tissues. In tepals, the adaxial coloured midrib (Fig. 7A, Θ:
98.9 ± 3.2°) and white bilateral (Fig. 7B, Θ: 101.3 ± 1.9°) sections
are less hydrophobic than the abaxial coloured (Fig. 7D, Θ:
110.7 ± 0.9°) and white (Fig. 7E, Θ: 105.2 ± 1.7°) sections (Fig. 8).
The highest contact angles were detected on the adaxial (Fig. 7C, Θ:
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C. Chimona et al.
Fig. 4. Scanning electron micrographs of adaxial (A, B) and abaxial (C, D) lenticular, convex epidermal cells of the white section of tepals of Asphodelus ramosus. Scale
bars: 20 μm (A), 10 μm (B, C) and 5 μm (D).
surfaces, as well as the contact area between floral surfaces and insects
(Scholz et al., 2010; Rands et al., 2011; Prüm et al., 2012; Gorb et al.,
2017; Sharma et al., 2018). Apparently, traits of tepal surfaces of A.
ramosus are linked to hydrophobicity, which seems to be a prerequisite
for the stability of these short-lived tissues that are exposed to spring
rains.
A. ramosus is one of the earliest spring flowering plants in the
Mediterranean region; therefore, its flowering is an early-season source
of floral rewards and floral micromorphology may affect pollinators’
perception, given that insect activity is largely limited by low temperature, while slippery floral surfaces discourage grip of flowers (Lee,
2007; Rejšková et al., 2010; Costa et al., 2017; Gorb et al., 2017).
surface. Consequently, the radius of the water droplets retained in between the projections of the abaxial epidermal relief of the white sections of tepals would be bigger than that of the adaxial white sections
(Wagner et al., 2003; Polymeni et al., 2010). The surface water repellence of the ephemeral tepals of A. ramosus with the thin epidermises, which could be easily infected and impaired, provides protection against injury and contributes to the removal of residues from
the tissues’ surface (Kerstiens, 1996; Shepherd and Griffiths, 2006).
Scanning electron microscopy analyses showed that epicuticular
wax crystals were not present on the tepal surfaces of A. ramosus
(Figs. 2, 4 and S5); this may be advantageous for the short-lived tepals,
because epicuticular wax crystals can alter the water status of tepal
Fig. 5. Scanning electron micrographs of the adaxial (A, B) and abaxial (C, D) epidermal cells of leaves of Asphodelus ramosus. Scale bars: 50 μm (A, B) and 10 μm (C,
D).
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C. Chimona et al.
Fig. 6. Atomic force micrographs of tepal surfaces of Asphodelus ramosus; three-dimensional and plane profiles of adaxial (A and B, respectively) and abaxial (C and
D, respectively) coloured midrib section, as well as of adaxial (E and F, respectively) and abaxial (G and H, respectively) white section.
16
Flora 248 (2018) 10–21
C. Chimona et al.
Table 3
Fatty acid composition (%) of total lipids and epicuticular wax of fully expanded leaves and tepals of Asphodelus ramosus; the values represent
means ± standard errors (n = 3). Values followed by the same uppercase
(tissue and wax) and lowercase (tepals and leaves) superscript letter are not
significantly different (P > 0.05).
Fatty acid
Leaf tissue
Myristic (14:0)
Palmitic (16:0)
Palmitoleic
(16:1)
Stearic (18:0)
Oleic (18:1)
Linoleic (18:2)
Linolenic (18:3)
Arachidic (20:0)
Behenic (22:0)
Lignoceric (24:0)
Cerotic (26:0)
Melissic (30:0)
7.6 ± 0.3f
20.6 ± 1.2d
3.7 ± 0.9g
15.8 ± 1.2Dd
2.4 ± 0.1g
3.5 ± 0.3g
15.5 ± 0.4Dd
44.5 ± 1.3b
3.6 ± 0.4g
5.7 ± 0.6f
20.9 ± 0.6Cd
35.8 ± 1.2Bc
4.6 ± 0.5F
4.1 ± 0.6F
5.8 ± 0.8Ff
5.9 ± 1.0Ff
Leaf wax
1.0 ± 0.2Gh
1.6 ± 0.3h
5.7 ± 0.6f
85.8 ± 2.2Aa
2.2 ± 0.4
3.7 ± 0.7
Tepal tissue
Tepal wax
5.1 ± 0.2F
4.1 ± 0.4
12.4 ± 2.2De
8.0 ± 1.2E
20.5 ± 2.1Cd
21.2 ± 1.6Cd
28.7 ± 1.5Cc
Fig. 8. Rain droplets suspended on adaxial and abaxial tepal surfaces
Asphodelus ramosus; in the concaveness of droplets, inverted images of flowers
of A. ramosus can be seen indicating lens-like droplets.
Although tepal micromorphology may be a tactile cue for insects to
approach sites containing rewards, at the same time properties of the
tepal surfaces protect the flowers from becoming altered or infected
(Buschhaus et al., 2015; Piwowarczyk and Kasińska, 2017; Takkis et al.,
2018). Features of the adaxial epidermal surface of tepals may also
contribute to firmness of contact and distribution of the spherical pollen
grains of A. ramosus (average length of polar axis: 38 μm, of equatorial
longitudinal axis: 64 μm and equatorial transverse axis: 67 μm), assisting pollination and reproduction of this self-incompatible species
(Díaz Lifante, 1996; Judd, 1997; Kosenko, 1999; Vardar et al., 2013). In
addition, the sequential expansion of the short-lived flowers of A. ramosus in the same inflorescence, which lasts about two months
(Chimona, 2015), may increase the amount of outcross pollen, when
several flowers are mostly visited (and most probably pollinated) by
bees (Obeso, 1993; Weryszko-Chmielewska and Chwil, 2006; Lazaro
et al., 2016).
Qualitative and quantitative profiles of tissue- and epicuticular-fatty
acids varied between leaves and tepals. There was a marked dominance
of the lignoceric acid (24:0) in the composition of the epicuticular wax
of leaves (approximately 86%) and tepals (approximately 29%); this
may be highly characteristic of the above ground plant parts of A. ramosus (Müller and Riederer, 2005). The relatively large amounts of
long-chain fatty acids (i.e. arachidic, behenic and lignoceric), which are
components of the epicuticular wax of tepals of A. ramosus (Table 3),
may act as short-range attractant and ovipositional stimulant to pollinators (Eigenbrode and Espelie, 1995; Mitra et al., 2017). Although
short-chain fatty acids are not common epicuticular lipid components,
tepal epicuticular wax of A. ramosus contains palmitic, palmitoleic, linoleic and linolenic acid. In concentrating our attention here on fatty
acids, it is important to note that research has previously been performed on wax components of flowers and leaves of Asphodelus, such as
alkanes, alkene and related aldehydes and ketones (Rudall and Chase,
1996; Reynaud et al., 1997; Çalış et al., 2006; Malmir et al., 2018).
Also, Chimona et al. (2014) have reported on secondary metabolites
isolated from tepals of A. ramosus and assigned to epicuticular wax
constituents, which may influence the wettability and adhesion properties of the floral tissues that mediate interactions between flowers and
Fig. 7. Sessile drops on tepal and leaf surfaces of Asphodelus ramosus: adaxial coloured midrib (A) and white (B) tepal sections, adaxial leaf side (C), abaxial colored
midrib (D) and white (E) tepal sections, and (F) abaxial leaf side.
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C. Chimona et al.
Aparecido et al., 2017). The water-repellent platelets covering the exposed stomatal pores of A. ramosus (Fig. 3) may constitute a feature that
prevents the formation of water films above the stomata (Neinhuis and
Barthlott, 1997; Pandey and Nagar, 2003; Fernández et al., 2014);
given the fact that CO2 diffuses slower in water than in air, the presence
of water-repellent plant surfaces, exposed to conditions of seasonal
precipitation and air humidity, ensures CO2 supply for photosynthesis
(Smith and McClean, 1989; Peguero-Pina et al., 2015).
The leaf water repellent properties may also impede the deposition
of dust and soil particles on the leaves of the geophyte A. ramosus that
grow close to the ground (Farmer, 1993). In addition, the bent towards
the ground fully expanded leaves of A. ramosus, which do not retain
water droplets, assist removal of water droplets aiding in guiding and
gathering water to the ground above and around the underground parts
of the plant, thus enhancing soil water deposition (Holder, 2012;
Rosado and Holder, 2013; Wang et al., 2014; Koukos, 2015); the leaf
functional traits may incorporate in ecohydrological process of soil
wetness at the onset of the dry period in the Mediterranean region.
pollinators (Jetter et al., 2006; Yeats and Rose, 2013).
The most striking element of the short-lived flowers of A. ramosus is
the coloured midrib section and the bilateral white sections on the tepal
blades (Fig. 1). Tepal anatomy combined with the nanostructures suggests that the coloured midrib and the bilateral white sections have
different optical properties. In fact, the tepals of A. ramosus consist of a
mesophyll that varies in thickness from 140 ± 6 μm in the bilateral
white sections to 550 ± 10 μm in the coloured midrib section (Fig.
S3C) and two epidermises approximately 30 ± 8 μm in thickness
(Argiropoulos, 2009), which yield correspondingly different spectral
signature (Fig. S4). The highest absorption of both the white (Fig. S4C)
and the coloured sections of tepals (Fig. S4D) was detected at 320 nm,
while in the latter the absorption spectrum had a minor, yet consistent
peak at 670 nm, presumably due to the presence of chlorophyll (Fig.
S4D); moreover, the coloured midrib section possesses stomata (Figs. 3
and S3B), it contains chloroplasts (Fig. S3A) and its chlorophyll content
was investigated approximately 2.30 μg g–1 d.w. (Argiropoulos, 2009).
Tepals and petals are potentially assimilatory organs, able to perform
photosynthesis, which is commonly regarded as a mechanism to partially compensate for the costs of plant reproduction (Obeso, 2002;
Aschan and Pfanz, 2006; De la Barrera et al., 2009; Ohmiya et al.,
2014). The presence of chloroplasts in the coloured midrib tepal segments may increase the capture of light. Flowers with such visible
marks on the corolla (Fig. 1), apart from the function in attracting and
guiding pollinators with visual displays to the nectar rewards, may
provide the floral tissues with photosynthetic assimilates (Dafni and
Giurfa, 1999; Aschan and Pfanz, 2003; Ščepánková and Hudák, 2004;
Sawidis et al., 2008; Caro and Allen, 2017). Also, the dominance of
palmitic, linoleic and linolenic fatty acids in tepals of A. ramosus
(Table 3) may be indicative of photosynthetic efficiency, because the
above mentioned fatty acids are frequently abundant in photosynthetic
tissues (Koukos et al., 2015; Meletiou-Christou and Rhizopoulou, 2017;
Ozturk et al., 2018). Although the contribution of tepals to the carbon
budget of A. ramosus seems to be negligible in comparison to that of the
leaves, due to the relatively small size of the coloured midrib segments,
it may be substantial in relation to the entire floral biomass of A. ramosus. With regard to floral advertisement, the dark midrib of tepals
may enhance colour contrast between the flowers occurring on the tall
inflorescence stalk and the background against which they are exposed;
this is a cue that enhances the perceived signals and the detection of
flowers by pollinators (Menzel and Shmida, 1993; Lunau et al., 1996;
Jacobs et al., 2016).
The white section of the tepal surfaces of A. ramosus yielded higher
transmission than reflectance of light in the visible wavelength range
(Fig. S4 A), while the coloured section of tepal surfaces exhibited higher
reflectance (Fig. S4B). It has been argued that different optical properties of floral tissues may be responsible for patterned distribution of
temperature between central and marginal portions of inflorescences’
flowers in temperate zone species (Rejšková et al., 2010). The white
areas of tepals exhibit reflectance in the UV wavelength range (Fig.
S4A, B), which is in agreement with earlier results (Chittka et al., 1994;
van der Kooi et al., 2016). Also, low reflectance in the UV wavelength
range indicates the presence of (unidentified) UV-absorbing pigments,
which may protect the short-lived tissues against harmful short-wave
radiation; actually, UV-B transmission through the tepals of A. ramosus
was not detected (Fig. S4A, B).
The results indicate that the amphistomatic leaves of A. ramosus are
more water repellent than the tepals (Fig. 7); it appears that there is a
higher investment in energy-consuming traits promoting hydrophobic
properties on the longer-living photosynthetic leaves of A. ramosus that
expand during autumn, endure throughout spring and wilt in the beginning of the drought period. It has been argued that wetness of leaf
surfaces can lead to photosynthetic suppression, because water droplets
may temporarily occlude stomata until they evaporate or drain from the
leaf surfaces (Brewer et al., 1991; Ishibashi and Terashima, 1995;
Pandey and Nagar, 2003; Hanba et al., 2004; Peguero-Pina et al., 2015;
5. Conclusions
The topography of the adaxial and abaxial white bilateral and coloured midrib sections of tepal surfaces of A. ramosus reveal features
linked to the ephemeral performance of flowers arranged around tall
flowering stalks. Adaxial tepal surfaces differ from the abaxial tepal
surfaces. In particular, the surface microsculpture (from a few hundred
nanometers to a few micrometers wide) may contribute to hydrophobic
properties and water status of the short-lived floral tissues potentially
differing between adaxial and abaxial surfaces, as well as between
white and coloured sections of tepals. Also, nanometric structural features of the epidermal relief render larger surface tepal areas than the
projected tepal areas. The leaf hydrophobic properties contribute to
both the maintenance of photosynthetic activity and removal of soil
particles, whenever the leaves touch the ground. It is likely that the
tepal- and leaf-surfaces of A. ramosus perform pheno-morphological
features related to eco-physiological functions and needed to adapt
their lifespan to the seasonality of their habitat.
Author contributions
Conceived and designed the experiments: CC, DK, MSMC, SR.
Sampling of plant tissues: AA, CC, DK. Performed the experiments: AA,
CC, DK, ES. Analysed the data: AA, CC, DK, ES, MSMC, SR. Contributed
reagents/materials/analysis tools: CC, DK. Contributed to writing and
editing of the manuscript: CC, DK, MSMC, SR. Contributed to the revision of the manuscript: CC, SR.
Acknowledgments
We thank the anonymous reviewers for suggestions and comments
on the manuscript. This research has been co-financed by European
social funds and Greek National funds through the Operational Program
Education and Lifelong Learning of the National Strategic Reference
Framework – Research Funding Program: Heracletus II.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.flora.2018.08.003.
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