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Localisation of furanocoumarins in tissues and on the

surface of shoots in Heracleum sosnowskyi Manden.

Journal: Botany

Manuscript ID cjb-2017-0043.R2

Manuscript Type: Article

Date Submitted by the Author: 06-Jun-2017

Complete List of Authors: Weryszko-Chmielewska, Elżbieta; University of Life Sciences in Lublin, Department of Botany Chwil, Mirosława; University of Life Sciences in Lublin, Department of Botany

Is the invited manuscript for consideration in a Special

Issue? :

N/A

Keyword: trichomes, epidermis, furanocoumarins, histochemistry tests, Heracleum sosnowskyi

https://mc06.manuscriptcentral.com/botany-pubs

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Localisation of furanocoumarins in tissues and on the surface of shoots in Heracleum

sosnowskyi Manden.

Elżbieta Weryszko-Chmielewska, Mirosława Chwil

Department of Botany, University of Life Sciences in Lublin, Akademicka 15,

20-950 Lublin, Poland; e-mail: [email protected]

Abstract: Heracleum sosnowskyi was introduced in Poland as a fodder plant.

Currently, it is regarded as an invasive plant posing a health hazard to humans and animals

and a threat to native flora. The aim of the study was to localise furanocoumarins in the stem

and leaf tissues The investigations were carried out using light, fluorescence, and scanning

electron microscopy as well as histochemical assays. The epidermis of the analysed organs

bears live, non-capitate hairs with variable length, which contain lipids, essential oils,

polysaccharides, tannins, and furanocoumarins. The observations performed with scanning

electron microscopy revealed the presence of a foamy substance and furanocoumarin crystals

on the surface of the trichomes and other epidermal cells, as well as in the parenchyma cells.

Characteristic furanocoumarin autofluorescence was present in the epidermis and on its

surface as well as in the subepidermal parenchyma. Secondary fluorescence was emitted by

furanocoumarins in different leaf petiole tissues: psoralen, bergapten, and xanthotoxin. We

have detected for the first time the presence of furanocoumarins in different tissues of leaves

in H. sosnowskyi. Furanocoumarins were present also abundantly on the epidermal surface of

cells. This explains why the contact with the plant is dangerous to humans and results in

development of photodermatoses.

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Key words: epidermis, trichomes, furanocoumarins, histochemistry tests, Heracleum

sosnowskyi.

Résumé: Heracleum sosnowskyi a été introduit en Pologne comme plante fourragère.

Maintenant, il est considéré comme une plante invasive qui provoque des problèmes de santé

chez les hommes et les animaux et qui constitue un danger pour la flore autochtone. L’objectif

de cette étude a été de localiser des furocoumarines dans les tissus des tiges et des feuilles.

Dans notre investigation, nous avons employé les microscopies photonique, en fluorescence,

électronique à balayage, ainsi que des essais histochimiques. Dans l’épiderme des organes

analysés sont présents des poils vivants de différentes longueurs qui contiennent des lipides,

essences aromatiques, polysaccharides et tanins. Les observations en microscopie

électronique à balayage faites sur la surface des poils et d’autres cellules de l’épiderme ainsi

que sur les cellules du parenchyme ont démontré la présence de substances mousseuses et de

cristaux de furocoumarines. Nous avons observé une autofluorescence caractéristique des

furocoumarines dans l’épiderme et sur sa surface et dans le parenchyme hypodermique. La

fluorescence secondaire a été émise par des furocoumarines, telles que le psoralène, le

bergaptène et la xanthotoxine, dans de différents tissus des pétioles. Pour la première fois

nous avons détecté la présence de furocoumarines dans de différents tissus des feuilles de H.

sosnowskyi. Les furocoumarines ont été présentes sur la surface des cellules de l’épiderme.

Cela explique pourquoi le contact avec la plante est dangereux pour l’homme et génère la

photodermatose.

Mots-clés: épiderme, poils, furocoumarines, essais histochimiques,

Heracleum sosnowskyi.

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Introduction

Depending on the taxonomic interpretation used, the genus Heracleum (Apiaceae)

comprises 60 species growing mainly in the temperate zone of Eurasia (Szweykowscy 2003)

or 120-125 species of Heracleum sensu lato, with 109 growing in Asia (Pimenov and Leonov

2004). In Poland, the hogweed Heracleum sphondylium is a native species with H.

sphondylium subsp. sphondylium and H. sphondylium subsp. sibiricum distinguished as two

subspecies. In the 70s of the 20th century, two species native to the Caucasus and Dagestan

were introduced for cultivation: H. mantegazzianum Sommier and Levier as an ornamental

plant and H. sosnowskyi Manden. as a fodder plant. Currently, these two introduced species

are regarded as invasive plants in many European countries (Pysek and Pysek 1995; Nielsen

et al. 2005; Moravcová et al. 2007; Tokarska-Guzik et al. 2012; Niinikoski and Korpelainen

2015). Heracleum sosnowskyi occurs in many localities in Estonia, the European part of

Russia, Latvia, Lithuania, and Poland (Jahodová et al. 2007; Kabuce and Priede 2010;

Baležentienė and Bartkevičius 2013).

Invasive Heracleum species growing in Poland produce high stems (2-5 m), differ in

leaf morphology, produce many seeds, and adapt easily to various environmental conditions,

in particular in areas where soil is disturbed. They colonize abandoned green areas, meadows,

riverbanks, forest edges, and roadsides (Müllerová et al. 2013).

All Heracleum species contain furanocoumarins in their aboveground organs, which

can be components of essential oils and exhibit photoallergic and photocarcinogenic

properties. The most dangerous compounds are psoralen, bergapten, and xanthotoxin (Zobel

and Brown 1990b, Dewick 1997; Jakubowicz et al. 2012). These furanocoumarins have been

detected in many species from the families Rutaceae and Umbelliferae (Zobel and Brown

1990a). They are also present in some representatives of the families Moraceae - Dorstenia

(Abegaz et al. 2004) and Fabaceae - Psoralea (Zobel et al. 1991).

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In the presence of UV radiation, furanocoumarins cause photodermatoses in humans,

which are manifested by redness associated with burns, swelling, blisters, and - consequently

- persistent skin discolouration and scars (Wagner et al. 2002; Jakubowicz et al. 2012; Gulati

and Guttman-Yassky 2014).

Environmental stress increased the production of furanocoumarins in Heracleum

lanatum, Apium graveolens, Ruta graveolens, and Bituminaria bituminosa (Zobel and

Głowniak 1994; Walker et al. 2012). In such circumstances, secretion of furanocoumarins on

the surface of the analysed organs was enhanced as well. Furanocoumarins create a barrier on

the organ surface protecting against the action of bacteria and fungi. Since these secondary

metabolites have the ability to absorb UV rays, they may constitute a shield to protect the

surface of plant organs against this type of radiation (Zobel and Głowniak 1994).

Many researchers report that furanocoumarins, like many other secondary metabolites,

can serve as a defence against insect attack and control oviposition (Zobel and Brown 1995;

Carroll and Berenbaum 2006; Zangerl et al. 2007). Enhanced biosynthesis of these

compounds can be triggered by biotic factors, such as viruses, bacteria, fungi, and insects as

well as abiotic elements, such as trace metals salts, antibiotics, sulphur dioxide, detergents,

herbicides, and synthetic peptides (Głowniak and Kozyra 2001).

When washed away from the plant surface by rainfall, coumarins can also act as

inhibitors of seed germination (Zobel and Brown 1995). The compounds inhibit cell division

and plant growth (Hale et al. 2004).

Furanocoumarins are used in psoriasis therapy (Ceska et al. 1987; Conforti 2009;

Martinez et al. 2010). Diwan and Malpathak (2007) describe that furanocoumarins exhibit a

positive effect in the treatment of leukoderma, vitiligo, psoriasis, and multiple sclerosis and

may be potent anti-HIV agents. Literature data indicate their role as potent antioxidants

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(Diwan et al. 2012). In addition to their diverse activities, furanocoumarins have been shown

to have anticancer properties (Luo et al. 2011; Bronikowska et al. 2012).

Sosnowski’s hogweed, occurring in many regions of Poland, not only poses a threat to

humans and animals but also, as an invasive plant, threatens the native flora and protected

vegetation of National Parks (Zając and Zając 2001; Tokarska-Guzik 2005). The number of

plant species was reported to decline 62-69% in dense Heracleum patches, in comparison

with phytocoenoses without the presence of this plant (Sobisz 2007).

The control of invasive plants in large areas is associated with economic losses. In the

buffer zone of the Góry Stołowe National Park in Poland, H. sosnowskyi occurs over an area

of ca. 30 ha, which poses a serious economic problem (Tokarska-Guzik et al. 2012). Since the

spread of H. sosnowskyi in Poland is a growing problem and cases of photodermatosis are still

widespread (Wojtkowiak et al. 2008; Rzymski et al. 2015), we undertook investigations of the

outer tissues of the stem and leaves in this species, which contain human health-threatening

furanocoumarins. There are only few studies concerning the anatomical aspects of localisation

of furanocoumarins. Zobel and Brown (1989) investigated the histological location of

furanocoumarins in Ruta graveolens shoots: Zobel et al. (1991) in Psoralea bituminosa fruits,

and Diwan and Malpathak (2010) in R. graveolens cell cultures.

This study is a continuation of our previous investigations of the furanocoumarin

secreting structures in H. sosnowskyi (Weryszko-Chmielewska and Chwil 2014). The aim of

the present study was to localise furanocoumarins in the leaf and stem tissues of this species

with the use of light, fluorescence, and scanning electron microscopy as well as histochemical

assays. Special attention was focused on structures present on the epidermal surface of the

leaf, including trichomes and secretions that can be contained therein, as suggested by the

literature on other furanocoumarin-containing species. The goals of this study were to

determine: (i) whether furanocoumarins are released onto the surface of the aboveground

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organs via trichomes or other epidermal cells, and (ii) why even a short contact with the plant

causes skin burns (photodermatoses).

Material and methods

Study material

Leaves of H. sosnowskyi Manden. were sampled from 5 plants in the Botanical Garden

of Maria Curie-Skłodowska University in Lublin (51°15′49.76″N 22°30′49.59″E).

The leaves were collected from the 7th node of each plant. Cross sections for

preliminary analyses were hand made from fresh leaf fragments (margin and central part of

the lamina and petiole). The collected samples were fixed from SEM observations of the

surface of the epidermis.

The epidermal surface and trichome structure were observed using stereoscopic (SM),

bright-field light (LM), fluorescence (FM), and scanning electron (SEM) microscopy.

Microscopy

Stereoscopic microscopy

Preliminary observations of the epidermis and comparative analyses of the trichomes

present on the lamina and petiole were carried out using an SMT 800T stereoscopic

microscope. Photographic documentation was made with a Nikon Coolpix 4500 camera.

Fluorescence microscopy

Hand-made sections (3 – 4) x 5 plants of fresh leaf fragments were prepared. They

were placed in a droplet of auramine O (Heslop-Harrison and Heslop-Harrison 1981). Slides

were observed under a fluorescence microscope equipped with an FITC filter (EXP. 465-495,

DM 505; BA 515-555). A UV-2A (EXP. 378/11; DM 416; BA 416 LP) filter was used for

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localisation of flavonoids in the tissues after addition of aluminium trichloride or magnesium

acetate (Charrière-Ladreix 1976) and furanocoumarins after treatment with 10% KOH in

methanol (Harborne 1973). This filter was also used for analysis of autofluorescence of

furanocoumarins. Microphotographs of the examined cells were taken with the Nikon Eclipse

90i fluorescence microscope coupled with a digital camera (Nikon DS-Mc) and a camera

(Olympus C-7070). The furanocoumarin colour obtained was determined according to Maerz

and Paul (1950).

Bright-field light microscopy

Trichomes, which differed in their structure and length, were observed on the cross

sections of laminas and petioles collected from 5 leaves. Measurements of the trichome length

were performed using the Nikon Eclipse 90i bright-field light microscope. Measurement

details are presented in section 4 - Morphometric measurements.

Scanning electron microscopy

Fragments of leaves were fixed in 4% glutaraldehyde for 6 hours and in 0,1 M

phosphate buffer, pH 7,0 at 4 ºC for 24 hours. Next, the plant samples were dehydrated in a

series of increasing acetone concentrations. After critical point drying in liquid CO2, they

were coater with a layer gold to a thickness of 20 nm using an Emitech K550X sputter coater.

Micromorphological observations of the surface of the epidermal cells of the leaf were carried

out using a Tescan VEGA II LMU scanning electron microscope.

Histochemical assays

The selected groups of biologically active compounds contained in the trichomes and

cells of other tissues were determined based on staining reactions after using relevant

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histochemical assays. Sudan III (Jensen 1962), Sudan IV (Pearse 1985), Sudan Red B

(Brundrett et al. 1991), Neutral Red (Clark, 1981) were used to detect lipids and essential oils.

Nile Blue was used to detect acid lipids and neutral lipids (Jensen 1962), Ruthenium Red for

polysaccharides other than cellulose/pectins (Johansen, 1940), potassium dichromate (Gabe

1968) and vanillin/HCl-test (Gardner 1975) for tannins, aluminium trichloride and magnesium

acetate for flavonoids (Charrière-Ladreix 1976), and 10% KOH in metanol (Harborne 1973)

and Lugol’s solution (Broda 1971) for furanocoumarins. The presence of calcium oxalate

crystals was determined using alizarin (McGee-RusSell 1958).

Morphometric measurements

We compared the length of the three types of trichomes on the surfaces of the petiole,

midrib, and intercostal fields between the leaf veins (abaxial epidermis). The height of the

trichome cell and the height of the epidermal cells surrounding the base of the trichome were

measured separately. Sixteen measurements of each trait were performed in 3-4 sections from

5 leaves different plants. The measurements were carried out with the use of software for

microscope image analysis Nikon NIS-Elements version 3.0 Advance Research. The mean of

the measurements and the standard deviation were calculated using Microsoft Office Excel

version 2013.

Results

Micromorphology and distribution of trichomes

The massive H. sosnowskyi shoots (Fig. 1A, B) had only non-capitate hairs classified

in the literature as non-glandular trichomes. They were situated on the margins of the lamina

(Figs 1C), on both leaf surfaces (Figs 1C, D, F), and they were more abundant on the abaxial

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(Fig. 1F, G) than adaxial surface of the leaf (Fig. 1D). The trichomes were also present on leaf

petioles (Fig. 2A-D) and stems (2E).

We distinguished 3 types of trichomes: Type I, II, and III (Table 1). Type I trichomes

were composed of a long cell surrounded at the base by cells (0,4 – 9 mm) forming a high

rosette (61-485 µm). The cell length in type II trichomes was 121 – 586 µm and epidermal

cells surrounding the base were 20-101 µm high. Only a single cell (51-202 µm) was observed

in type III trichomes and there was no rosette of epidermal cells at the base (Table 1, Fig. 2A,

C). Trichomes were usually conical or cylindrical with a sharp apex (Fig. 2A-F). Trichomes

with a rounded apex were noted infrequently. The largest petiole trichomes usually had a

unicellular body and cushions composed of epidermal cells which were evidently elevated

over the surface of other ca. 485 µm high epidermis cells surrounding the base of the

trichome. The cells of the cushions contained anthocyanin (Fig. 2B, D). The cushions around

the base of the trichomes located on the laminas (up to 60 µm high) were composed of 8-10

cells arranged in rosettes (Fig. 2F). The cuticle of some trichomes was verrucose (Fig. 2G).

Secretions in tissues and on the leaf surface observed in SEM

A foamy substance (FIG. 3A-C) or crystalline mass (Fig. 3D, E), most likely an

accumulation of secretions, was visible on the surface of the trichomes or at their base. A

foamy secretion (Fig. 4A, B) and crystal complexes (Fig. 4E, F) were also present on the

surface of the epidermis of the leaf as well as stems (Fig. 3C). A foamy mass was also present

among accompanied the crystal complexes (Fig. 4D, E). Similarly, the surface of the cross

sections of the H. sosnowskyi petioles exhibited numerous crystals with a similar morphology

(Fig. 4F, G). Identification of the crystals with Lugol’s solution confirmed the coumarin

origin of the crystallised substance (purple-grey staining). Treatment with alizarin applied to

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detect the presence of calcium oxalate crystals yielded a negative result, confirming the

absence of this compound.

Histochemical analyses

Several classes of chemical compounds were identified inside the trichome cells. We

detected the presence of lipids, essential oils, polysaccharides (pectins), tannins, and

furanocoumarins. The presence of secondary metabolites, such as essential oils, flavonoids,

tannins, and furanocoumarins, was also detected in the petiolar and laminar tissues.

Microscopic observations of the trichomes in the fresh plant material revealed the

presence of viable transparent protoplasts with numerous granularities (Fig. 5A). Staining of

the petiole cross sections with Sudan III, Sudan IV (Fig. 5B), and Sudan Red B revealed the

presence of lipids (orange or reddish staining) in the epidermal cells and glandular epithelial

cells (Fig. 5G). The use of Nile Blue confirmed the presence of acid lipids (blue staining) in

some trichomes (Fig. 5C) and neutral lipids (pink staining) in others (Fig. 5D). The presence

of lipids or essential oils in the trichomes was confirmed by the Neutral Red reaction (red

staining) (Fig. 5E).

After staining with Ruthenium Red, which detects pectins, we observed a positive

reaction in the trichomes (crimson staining). The presence of tannins both in the trichomes

(Fig. 5F) and in the epidermal cells, subepidermal parenchyma, and around vascular bundles

and secretory canals in the petioles (Fig. 5H) was detected with the use of potassium

dichromate (brown staining). The presence of tannins in these tissues was also confirmed by

their fluorescence emitted after application of the vanillin-HCl test (red staining) (Fig. 6D).

The yellow-greenish staining visible in these tissues after their treatment with

aluminium trichloride (Fig. 5I) and magnesium acetate indicated the presence of flavonoids in

the basal cells of the trichomes and in the subepidermal parenchyma cells of the petioles. The

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presence of flavonoids in the parenchyma of the petiole was confirmed by yellow secondary

fluorescence induced by magnesium acetate (Fig. 5J) and aluminium trichloride (Fig. 5K),

which were used as fluorochromes for flavonoid detection.

Characteristic autofluorescence of furanocoumarins in the Heracleum tissues was

observed under the fluorescence microscope. The typical blue psoralen fluorescence was

visible in the epidermal cells and on their surface as well as on the surface of the trichomes

(Fig. 6A, B). In turn, the yellow fluorescence of the subepidermal cells indicated the presence

of bergapten (Fig. 6A).

The content of the basal zone of the trichomes and the adherent secretion exhibited

green fluorescence after the auramine treatment (Fig. 6C). After the treatment of the fresh

preparations with a 10% KOH solution in methanol, intense and diverse fluorescence of

furanocoumarins was observed in the petiolar tissues of the leaf (Fig. 6 E). The epidermis and

its surface as well as the epithelial cells around the secretory canals exhibited blue

fluorescence (psoralen). The collenchyma and subepidermal parenchyma were characterised

by orange fluorescence typical of xanthotoxin, whereas the parenchyma surrounding the

secretory canals and vascular bundles exhibited yellow fluorescence characteristic for

bergapten (Fig. 6E). See Table 2 for a summary of the histochemistry of the H. sosnowskyi

tissues.

Discussion

Types of trichomes

In the present study, we have shown the presence of simple unicellular (non-capitate)

trichomes composed of elongated, viable cells in the H. sosnowskyi epidermis of the leaves

and stems. The longest trichomes were surrounded at the base by a multicellular rosette

formed by epidermal cells. In accordance with the morphological typology proposed by Payne

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(1978), the trichomes observed in H. sosnowskyi can be classified as “subulate”, (awl-

shaped), and “attenuate” with a long, gradual taper. Cronquist (1981) and Judd et al. (2008)

describe various sorts of hairs in Apiaceae (Umbelliferae). As suggested by Metcalfe and

Chalk (1972), trichomes in representatives of the family Umbelliferae are nearly always non-

glandular and can be divided into 5 types: (i) simple unicellular, (ii) unicellular, bladder-like,

(iii) dendroid, (iv) stellate, (v) small glandular hairs with 2- and 4-celled heads. The trichomes

in H. sosnowskyi examined in the present study belong to the first type specified by the

authors; additionally, we distinguished three subtypes differing in the length and the presence

or absence of a rosette at the trichome base. They are presented in Table 1. Epidermal cells in

this type of trichome can have a rosette-like arrangement around the base of the hair; in such a

case, they are referred to as “lateral” cells (Braune et al. 1979). In our previous study

(Weryszko-Chmielewska and Chwil 2014), we observed some unicellular hairs on the

epidermis of H. sosnowskyi leaves; they were surrounded by epidermal cells arranged in a

rosette-like pattern and elevated above the surface of this tissue.

Hegi (1975) describes that the lower surface of Heracleum leaves bears either rough

hairs or felt trichomes. As shown by our observations, the H. sosnowskyi trichomes can be

regarded as the rough type. Similar findings concerning trichomes were reported by Arora et

al. (1982), who examined leaves of other Heracleum species. They distinguished only

unicellular thread-like trichomes of different lengths on the epidermis of H. sibiricum and H.

mantegazzianum. The trichomes in these Heracleum species were also surrounded by 6-8

crown epidermal cells.

Secretory activity of Heracleum trichomes

The histochemical tests demonstrated that the trichomes present on the H. sosnowskyi

shoots contained neutral and acid lipids, polysaccharides, flavonoids, tannins, and

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furanocoumarins. We found differences in the content of these compounds for the individual

types of trichomes, which is shown in Table 2.

Under the light microscope, we observed irregularly shaped crystal complexes on the

surface of trichomes located on the leaves of the species. SEM revealed remnants of a

flocculent secretion on the outer trichome walls. Blue autofluorescence of the surface of the

trichomes, which is characteristic for some furanocoumarins (psoralen), was visible under the

fluorescence microscope. Based on the histochemical assays as well as the LM, FM, and SEM

observations, it can be assumed that the trichomes are viable and exhibit secretory properties.

Similarly, in their morphological, histochemical, and ultrastructural investigations of

non-glandular trichomes in 3 species from the family Lamiaceae and 4 species from the

family Verbenaceae, Tozin et al. (2016) demonstrated that the trichomes not only protected

plants against biotic and abiotic stresses but also, were capable as viable structures of

producing and secreting biologically active substances. As suggested by the authors, the

trichomes are involved in chemical interactions between plants and their environment. Other

studies have demonstrated that trichomes can accumulate toxic compounds (trace metal),

which are removed in this way from the plant (Choi et al. 2001; Lavid et al. 2001).

Crystals on the surface of epidermis cells

Phytochemical studies conducted by other authors showed that furanocoumarins not

only were present in plant organs but were also transported onto their surface. In species

examined previously, different percentages of the total furanocoumarin content on the surface,

of the epidermis of leaves were reported e.g. in Ruta graveolens 37 -56%, Heracleum lanatum

82%, and H. mantegazzianum 1,9% (Zobel and Brown 1990b). Furanocoumarins were shown

to be able to crystallise (Zobel and Brown 1989; Gupta et al. 1993).

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Scanning electron microscopy showed numerous crystal complexes and a foamy

secretion on the surface of the epidermal cells of H. sosnowskyi laminas. The crystals were

also visible under the light microscope. Cronquist (1981) reports that “crystals are rarely

formed” in Apiaceae. Based on the negative result of the alizarin assay, we excluded the

presence of calcium oxalate crystals. Numerous crystal complexes, similar to those observed

on the surface of the epidermis, were also present in cross sections of H. sosnowskyi petioles

within the parenchyma tissue, as shown by the SEM analyses.

Small furanocoumarin crystals on the surface of Ruta graveolens leaves were observed

under a light microscope and SEM (Zobel and Brown 1988, 1989; Zobel and Głowniak

1994). Phytochemical analysis conducted by these authors revealed the presence of

xanthotoxin, psoralen, and bergapten on the surface of rue leaves. Using SEM, Ceska et al.

(1986) found long, needle-like furanocoumarin crystals, with angelicin as the main

component, on the surface of Pastinaca (parsnip) roots (Apiaceae). They also detected other

furanocoumarins such as 5-methoxypsoralen, 8-methoxypsoralen, psoralen, bergapten, and

xanthotoxin in the mass of the examined crystals. Similarly, Zobel et al. (1991) observed

complexes of needle-like furanocoumarin crystals on the surface of cotyledons in Psoralea

seeds.

We assume that the numerous crystals on the surface of the H. Sosnowskyi leaves

consist of furanocoumarins as well. We attempted to find the reason for such an abundant

occurrence of the compound on the leaves of H. Sosnowskyi. Other authors found the greatest

amount of furanocoumarin during the flowering period in two Heracleum species (Pira et al.

1989; Zobel and Brown 1990b).

A mycelium of a pathogenic fungus was visible on the leaves of the examined plants,

as shown in the photographs. One of the functions of furanocoumarins in plants is protection

of tissues against parasite invasion (Głowniak and Kozyra 2001). Krasawa et al. (1990) found

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that the level of furanocoumarins in Apium graveolens plants infected by fungi was

considerably higher than that in healthy plants. In the case of Pastinaca plants infected by

fungi, Głowniak et al. (1999) demonstrated that the concentration of xanthotoxin, which has

high antifungal activity, was higher on the surface of fruits than inside their tissues.

It seems that the flowering phase in the examined H. sosnowskyi plants may have

caused the release of substantial amounts of crystalized furanocoumarins on the surface of the

epidermis.

Localisation of furanocoumarins in the plant

Furanocoumarins produced in plant tissues have cytotoxic properties and, since they

are dangerous for the plant, they are excreted (Podbielkowska et al. 1994; Zobel and Brown

1995). Their extrusion to the surface of the plant can serve as a barrier against herbivores,

insect attack, bacteria, and fungi. When furanocoumarin excretion on the surface of the

epidermis from deeper tissues is impossible, the compounds are transported to intercellular

spaces (Zobel and Brown 1995). For example, we detected autofluorescent furanocoumarins

in the intercellular spaces of the H. sosnowskyi leaf.

Zobel and Brown (1990a) demonstrated various concentrations of 3 furanocoumarins:

psoralen, xanthotoxin, and bergapten on the surface of Heracleum montegazzianum and H.

lanatum leaves. Xanthotoxin was present at the highest concentration in both species. It was

followed by psoralen and bergapten in H. montegazzianum and by bergapten and psoralen in

H. lanatum. Our histochemical assays have shown the presence, of these three

furanocoumarins in H. sosnowskyi. We observed blue autofluorescence in the trichomes and

on the surface of the leaf, which is typical of psoralen, as indicated by Zobel and Brown

(1989). The subepidermal parenchyma in the leaf petioles exhibited orange fluorescence

characteristic for xanthotoxin, whereas yellow fluorescence, which is typical of bergapten,

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was detected in the parenchyma surrounding the secretory canals. We also observed green

fluorescence on the surface of the epidermis and inside its cells. In their investigations of Ruta

graveolens leaves, which exhibited green fluorescence, Zobel and Brown (1989) suggested

that it was induced by the co-occurrence of the aforementioned furanocoumarins.

In plant material obtained from in vitro Ruta graveolens cultures, Diwan and

Malpathak (2010) demonstrated that individual furanocoumarins were located in different

tissues. Based on the characteristic autofluorescence, they found that the psoralen, bergapten,

and xanthotoxin accumulation sites were spatially separated from each other. The authors

found that psoralen emitting blue fluorescence was located in root initials and bergapten was

identified in the epidermis of shoot initials based on its yellow autofluorescence.

In epidermal cells, furanocoumarins are deposited in the layer of epicuticular wax and

on its surface (Zobel and Brown 1989, 1990a). The authors of those studies describe that

vaporised furanocoumarins are detectable in the atmosphere surrounding rutaceous and

umbelliferous plants. There are many known cases of photodermatoses triggered by direct

skin contact with H. sosnowskyi leaves; however, photodermatoses were also found to

develop without contact with plants but in their close proximity, where the concentration of

coumarin volatile compounds in the air was high. These substances can easily penetrate the

skin through the epidermis (Jakubowicz et al. 2012) and cause photodermatosis.

Conclusions

As shown in our study, there are only simple hairs different lengths on the leaves of

H. sosnowskyi. The base of these trichomes is often surrounded by epidermal cells arranged in

a rosette-like pattern. The trichomes are found in high density on the surface of veins on the

abaxial surface of the leaves.

In the present study, we have shown for the first time secretion of furanocoumarins by

H. sosnowskyi trichomes, whose surfaces were covered by a foamy substance or crystals.

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Since one of the functions of trichomes is accumulation and disposal of redundant metabolites

into the environment, furanocoumarins can also be removed via this route.

Using scanning electron microscopy, we have demonstrated the presence of

furanocoumarin crystals not only on the epidermis of the leaf but also in the parenchyma of

the petiole in H. sosnowskyi. The investigations conducted with the use of the fluorescence

microscopy have shown that furanocoumarins (psoralen, xanthotoxin, bergapten) were

contained in different H. sosnowskyi tissues. In this study, we have documented the presence

of extremely numerous furanocoumarin crystals on the outer walls of the trichomes and

epidermal cells of the leaf, which confirms the release of substantial amounts of these

compounds on the surface of the plant and sheds light on the cause of the serious risk posed to

humans who come in contact with the plant. The release of furanocoumarins from plant

organs may suggest a great potential of H. sosnowskyi in protection against biotic and abiotic

environmental threats, e.g. phytophages, bacteria, fungi, or ultraviolet radiation.

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Figure captions:

Fig. 1. General habit of the plant and distribution of trichomes on the adaxial and abaxial leaf

surfaces in Heracleum sosnowskyi. (A) Flowering plants produce large umbels composed of

white flowers. (B) Smaller inflorescences grow in the axils of pinnate leaves. (C) Margins of

laminas and veins are covered by trichomes. (D, E) Unicellular trichomes (arrows) are

abundantly located on the margins of the lamina. (F, G) numerous trichomes on the margins of

the lamina and veins, hyphae of a pathogenic fungus visible among the trichomes (double

arrows). [Colour online.]

Fig. 2. Different types of trichomes on the epidermis of H. sosnowskyi leaves and stem. (A)

Trichomes (arrow) of various lengths on the surface of the petiole. (B - D) Different types of

trichomes on the surface of the leaf; multicellular base with anthocyanin (double arrows). (E)

trichomes on the epidermis of the stem. (F) un-stained trichome from a fresh leaf with visible

epidermal cells at the base (arrows) arranged in a rosette. (G) verrucose cuticle of the trichome

surface (asterisk). [Colour online.]

Fig. 3. Trichomes with secretions on their surface and at their base. (A) Foamy secretion on the

surface of the trichomes on the stem. (B, C) Foamy secretions on the surface of the trichomes on

the leaves. (D) Crystals at the base of a trichome (asterisk). (E) trichome with green fluorescence

and furanocoumarin crystals after treatment with auramine (asterisk). [Colour online.]

Fig. 4. Secretion (furanonocoumarins) with a foamy and crystalline structure on the surface of

the epidermis. (A, B) Foamy secretions on the cells (asterisks). (C) crystals (arrow) and foamy

secretion (asterisk) on the surface of the epidermis of the stem. (D - F) Crystal aggregates

(arrows) and foamy secretions (asterisks) on the surface of the epidermis. (G, H) Crystal

complexes in petiole sections (arrows).

Fig. 5. Control leaf trichome, petiole trichomes, and cross sections of the petiole subjected to

histochemical assays and fluorescence microscopy. (A) viable trichome with granularities visible

in the cytoplasm. (B) Trichome stained red with Sudan IV (lipids). (C, D) Trichomes stained

with Nile Blue: (C) blue indicating the presence of acid lipids (two asterisks), (D) pink indicating

the presence of neutral lipids (asterisk). (E) Content of trichomes stained red with Neutral Red

(lipids/essential oils). (F) Trichome stained brown with potassium dichromate (tannins). (G)

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Petiolar tissues treated with Sudan Red; the content of lipids in epithelial cells around the

secretory canals; epidermal cells around the secretory canals; epidermal cells were stained red

revealing the presence of lipids/essential oils (arrows). (H) Petiolar tissues treated with

potassium dichromate: epidermis and parenchyma cells stained brown (tannins). (I) Petiolar

tissues stained greenish yellow after application of aluminium trichloride (flavonoids). (J)

Yellow secondary fluorescence induced by magnesium acetate indicating the presence of

flavonoids (double arrows) in the parenchyma cells of the petiole. (K) Subepidermal parenchyma

cells exhibiting yellow secondary fluorescence after treatment with aluminium trichloride

indicating the presence of flavonoids. [Colour online.]

Fig. 6. Fragments of petioles with primary and secondary fluorescence of tissues. (A, B)

Epidermal cells and trichomes exhibiting blue autofluorescence typical of psoralen (arrow): (A)

and yellow autofluorescence of the subepidermal parenchyma typical of bergapten (two arrows)

(A); blue autofluorescence on the surface of the trichome indicating the presence of psoralen (B).

(C) Parenchyma of the petiole cells exhibiting red secondary fluorescence of tannins

(arrowheads); after the application of vanillin-HCl. (D) Petiolar tissues with intensified

fluorescence after treatment with KOH: light blue indicating the presence of psoralen (white

arrows), orange - xanthotoxin (two asterisks), yellow - bergapten (double arrows). [Colour

online.]

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Fig. 1. General habit of the plant and distribution of trichomes on the adaxial and abaxial leaf surfaces in Heracleum sosnowskyi. (A) Flowering plants produce large umbels composed of white flowers. (B) Smaller

inflorescences grow in the axils of pinnate leaves. (C) Margins of laminas and veins are covered by

trichomes. (D, E) Unicellular trichomes (arrows) are abundantly located on the margins of the lamina. (F, G) numerous trichomes on the margins of the lamina and veins, hyphae of a pathogenic fungus visible among

the trichomes (double arrows). [Colour online.]

209x296mm (300 x 300 DPI)

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Fig. 2. Different types of trichomes on the epidermis of H. sosnowskyi leaves and stem. (A) Trichomes (arrow) of various lengths on the surface of the petiole. (B - D) Different types of trichomes on the surface of the leaf; multicellular base with anthocyanin (double arrows). (E) trichomes on the epidermis of the stem.

(F) un-stained trichome from a fresh leaf with visible epidermal cells at the base (arrows) arranged in a rosette. (G) verrucose cuticle of the trichome surface (asterisk). [Colour online.]

209x296mm (300 x 300 DPI)

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Fig. 3. Trichomes with secretions on their surface and at their base. (A) Foamy secretion on the surface of the trichomes on the stem. (B, C) Foamy secretions on the surface of the trichomes on the leaves. (D) Crystals at the base of a trichome (asterisk). (E) trichome with green fluorescence and furanocoumarin

crystals after treatment with auramine (asterisk). [Colour online.]

209x296mm (300 x 300 DPI)

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Fig. 4. Secretion (furanonocoumarins) with a foamy and crystalline structure on the surface of the epidermis. (A, B) Foamy secretions on the cells (asterisks). (C) crystals (arrow) and foamy secretion (asterisk) on the surface of the epidermis of the stem. (D - F) Crystal aggregates (arrows) and foamy

secretions (asterisks) on the surface of the epidermis. (G, H) Crystal complexes in petiole sections (arrows).

209x296mm (300 x 300 DPI)

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Fig. 5. Control leaf trichome, petiole trichomes, and cross sections of the petiole subjected to histochemical assays and fluorescence microscopy. (A) viable trichome with granularities visible in the cytoplasm. (B)

Trichome stained red with Sudan IV (lipids). (C, D) Trichomes stained with Nile Blue: (C) blue indicating the

presence of acid lipids (two asterisks), (D) pink indicating the presence of neutral lipids (asterisk). (E) Content of trichomes stained red with Neutral Red (lipids/essential oils). (F) Trichome stained brown with potassium dichromate (tannins). (G) Petiolar tissues treated with Sudan Red; the content of lipids in

epithelial cells around the secretory canals; epidermal cells around the secretory canals; epidermal cells were stained red revealing the presence of lipids/essential oils (arrows). (H) Petiolar tissues treated with

potassium dichromate: epidermis and parenchyma cells stained brown (tannins). (I) Petiolar tissues stained greenish yellow after application of aluminium trichloride (flavonoids). (J) Yellow secondary fluorescence induced by magnesium acetate indicating the presence of flavonoids (double arrows) in the parenchyma cells of the petiole. (K) Subepidermal parenchyma cells exhibiting yellow secondary fluorescence after

treatment with aluminium trichloride indicating the presence of flavonoids. [Colour online.]

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138x178mm (300 x 300 DPI)

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Fig. 6. Fragments of petioles with primary and secondary fluorescence of tissues. (A, B) Epidermal cells and trichomes exhibiting blue autofluorescence typical of psoralen (arrow): (A) and yellow autofluorescence of the subepidermal parenchyma typical of bergapten (two arrows) (A); blue autofluorescence on the surface

of the trichome indicating the presence of psoralen (B). (C) Parenchyma of the petiole cells exhibiting red secondary fluorescence of tannins (arrowheads); after the application of vanillin-HCl. (D) Petiolar tissues

with intensified fluorescence after treatment with KOH: light blue indicating the presence of psoralen (white arrows), orange - xanthotoxin (two asterisks), yellow - bergapten (double arrows). [Colour online.]

209x296mm (300 x 300 DPI)

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Table 1. Length of protective trichomes on the epidermis of Heracleum sosnowskyi Manden leaves.

Analysed trait

Type of protective trichomes

I II III

Length of the trichome

Height of the epidermal cells

forming a rosette at the base of

the trichome


Height of the epidermal cells

forming a rosette at the base

of the trichome


min.-max. mean±SD min.-max. mean±SD min.-max. mean±SD min.-max. mean±SD min.-max. mean±SD

µm

Epidermis of the petiole 2302.80-9009.20 3675.14±1526.47 202.00-484.80 306.16±99.22 121.20-585.8 313.10±137.60 20.20-101.00 51.76±24.43 70.70-202.00 111.73±31.83

Abaxial lamina

epidermis

midrib 393.90-1030.2 651.45±187.04 60.60-161.60 109.21±29.32 142.06-363.60 246.82±57.49 30.30-50.50 35.98±7.35 71.12-131.30 90.27±23.46

intercostal fields - - - - 141.40-515.10 321.94±105.60 31.02-60.60 36.08±9.01 50.50-111.10 79.54±20.16

Types of protective trichomes

I - long. bristly. sharply pointed trichomes located on high multicellular bases

II - medium-length sharply pointed trichomes surrounded by a several-celled base

III - short. blunt trichomes

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Botany

Draft

Table 2. Results of histochemical tests of trichomes and epidermis and parenchyma cells in

H. sosnoswskyi leaves.

Staining procedure Target compound Colour observed

Intensity of colour staining in

trichomes

(type II and III)

epidermal

cells

parenchyma

cells

Sudan Red B*

lipids

red + + -

Sudan III* orange + + -

Sudan IV* red ++ + -

Nile Blue*

acid lipids blue ++ + -

neutrallipids pink + + -

Neutral Red*

Lipids, essential

oils

red + + -

Ruthenium Red* polysaccharides crimson + + +

Potassium*

dichromate* tannins

brown + + +

Vanillin + HCl** red + + +++

Magnesium

acetate

**

flavonoids

yellow - greenish + + ++

** yellow - + +++

Aluminium

trichloride

* yellow - greenish + + +

** yellow + + +

Autofluorescence**

furanocoumarins

blue ++ +++ +

orange - - ++

Auramine O** green + ++ -

KOH**

blue + ++ +

orange - - +++

yellow - - ++

* - light microscopy, ** - fluorescence microscopy

Page 36 of 35


Botany

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