Asteraceae

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Oct 17, 2002 - chemical tests: toluidine blue (TBO) (O'Brien and. McCully, 1981) as a generic dye for DNA, cytoplasm, and some cell wall components; ...
Israel Journal of Plant Sciences

Vol. 51

2003

pp. 185-192

Secretory structures of Santolina ligustica Arrigoni (Asteraceae), an Italian endemic species ANNAMARIA PAGNI,* ROMINA ORLANDO, ANTONIO MASINI, AND DANIELA CICCARELLI Department of Botanical Sciences, University of Pisa, Via Luca Ghini 5, 56100 Pisa, Italy (Received 17 October 2002 and in revised form 22 January 2003)

ABSTRACT Santolina ligustica Arrigoni (Asteraceae) shows two different types of secretory structures: secretory ducts and glandular hairs. Ducts consist of a lumen varying in width surrounded by one or two layers of secretory cells. The size of the ducts varies in length and width, depending on the organ in which they are present. Hairs are multicellular and biseriate. They consist of two basal, two stalk, and six head cells. Ducts are located in the receptacle, interfloral and involucral bracts, in the stigma, in the leaves, stem, and root. They are absent in the corolla. Glandular hairs are present on the corolla, on the involucral and interfloral bracts, and in the stem and leaves. The histochemical analyses of the secretion of ducts and hairs show the presence of lipids, sesquiterpene lactones, alkaloids, and flavonoids. Although similar, the two secretions differ in the lipid composition: ducts produce both essential oils and resins. while hairs produce essential oils. Pectin-like substances and tannins seem to be produced by glandular hairs only.

INTRODUCTION Santolina L. grows spontaneously in Italy and consists of two groups of endemic species: four diploid schizoendemic species in the Italian peninsula (S. ligustica Arrigoni, S. leucantha Bertol., S. etrusca (Lacaita) Marchi and D' Amato, S. neapolitana Jordan and Fourr.) and two polyploid apoendemic species (S. insularis (Genn. ex Fiori) Arrigoni, S. corsica Jordan and Fourr.) in Sardinia (Arrigoni, 1979). Studies on karyology and on pollen morphology revealed elements that were helpful for the understanding of the phylogenetic relationship among the taxonomic units (Marchi and D'Amato, 1973; Arrigoni and Mori, 1980; Mariotti Lippi et al., 1999). All species of the Santolina genus are aromatic plants; most of them are used in folk medicine (Corsi et al. 1980; Ballero and Fresu, 1991; Senatore and De Feo, 1994; Ballero et d., 1995). In the past, S. ligustica was used in popular medicaments to sterilize laundry and, above all, as a sub-

stitute for chamomile; during the Second World War, it was also used as a substitute for tobacco (Tomcelli et al., 1999). Chemical analyses of some of these species showed pharmacologically-active secondary metabolites (Flamini et al., 1994; Senatore and De Feo, 1994; Poli et al., 1997). In spite of the use and chemical characterization, few morpho-anatomical data on the structures secreting secondary metabolites exist. Only two species of Santolinu have been investigated: S. leucantha (Pagni, 1995; Pagni and Masini, 1999), an endemic species of the Apuan Alps (northwestern Tuscany), and S. insularis, an endemic species of Sardinia (Sacchetti et al., 1997), in which only the internal secretory system was studied. S. ligustica is a diploid threatened species (Torricelli et al., 1999) living on aphiolithic substrata in open and sunny surroundings of eastern Liguria (Mariotti, 1982). *Author to whomcorrespondence should be addressed. E-mail: [email protected]

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It has small capitula (diameter 4-6 mm) with white tubular flowers, surrounded by several rows of involucral bracts (Arrigoni, 1977). Studies have been conducted on S. ligustica concerning its chromosomal number (Arrigoni and Mori, 1980), area of distribution (Mariotti, 1982; Torricelli et al., 1999), and pollen morphology (Mariotti Lippi et al., 1999). There are no morpho-anatomical data on its secretory structures. This paper reports the distribution, morphology, and histochemistry of the secretory structures of S. ligustica using light, fluorescence, and scanning electron microscopy. The aim was to investigate the secretory system of the plant, to indicate the main classes to which the compounds, present in the secretion. belong, and to increase the morpho-anatomical knowledge about the Italian units of Santolina.

MATERIALS AND METHODS

Scanning electron microscopy (SEM) All the material was fixed in glutaraldehyde (2% in phosphate buffer solution at pH 7.4), dehydrated in a gradient of an alcohol and acetone mixture, critical point dried, and sputter-coated with gold. The samples were examined at 15 KV using a Cambridge Stereoscan 90 SEM.

Fluorescence microscopy Fresh and fluorochrome-treated sections were used. Aluminum chloride was used as a fluorochrome for flavonoids (Guerin et a]., 1971). A Leica DM LB fluorescence microscope with group A filter (BP 340-380; dichroic mirror 450, LP 430 stop filter) was used.

RESULTS S. ligcistica showed two different types of secretory structures: secretory ducts and glandular hairs.

Materials

Ducts

The plants were collected at Lavaggiorosso (La Spezia, Italy; 44'1 1'36"N, 9'36'45"E), 300 m above sea level. Exsiccata in the herbarium of University of Pisa.

Ducts consisted of a lumen surrounded by one or two layers of secretory cells without thickened walls (Fig. 1A). Their cavity originated schizogenously; the lumen, initially small, enlarged as the duct developed and filled up with a secretion (Fig. 1B). The cells in the epithelial layers were flattened at the end of development. The sizes of the ducts varied in length and width. depending on the organ in which they were located (Fig. lC, D).

Light microscopy Observations were carried out on capitula, leaves, stems, and roots at different stages of development. We examined in each organ: (a) longitudinal and cross sections (20 pm thick) of fresh material obtained with Leitz 1720 Digital Cryostat at -14 'C; (b) longitudinal and cross section (3 pm thick) of material fixed in FAA (Sass, 1958). embedded in LR White acrylic resin (Sigma) and cut with Leica 2055 microtome; (c) isolated flowers, capitulum bracts, and leaves laciniae clarified in Javel's water prior to staining or clarified after treatment with 8% KOH. All the material was submitted to the following histochemical tests: toluidine blue (TBO) (O'Brien and McCully, 1981) as a generic dye for DNA, cytoplasm, and some cell wall components; Alkanna tincture (Faure, 1914) for total lipids; Nile blue (Cain, 1947) for neutral and acid lipids; Nadi reagent (David and Carde, 1964) for essential oils and resins; Wagner and Dittmar reagents (Furr and Mahlberg, 198 1) for alkaloids; potassium bichromate (Faure, 1914) for tannins; Delafield hematoxy lin (Faure, 19 14) and Ruthenium Red (Jensen. 1962) for pectin-like substances; brilliant blue Coomassie R250 (Fisher, 1968) for proteins; and concentrated sulphuric acid (Geissmann and Griffin, 197 1) for sesquiterpene lactones. Standard control procedures were carried out at the same time. I.rrciel Jourrial uf Plant Sciences 51

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Distribution of the ducts In the capitulum, ducts were located in the receptacle, bracts, and stigma. They were absent in the corolla. In the receptacle, ducts were numerous, ellipsoidal, with a large lumen, and spread in the parenchyma in the shape of a ring, outside the vascular bundles (Fig. 1E). No anastomosis could be observed. In the involucral and interfloral bracts, there was one, sometimes two, ducts running along the longitudinal axis, parallel to the vein (Fig. IC, F). In the stigma, there was one duct per lobe as long as the stigmatic lobe (Fig. 2A). In the vegetative parts of the plant, ducts were widespread. In the leaves, there were one, two, or more elongated ducts arranged around the vein (Figs. ID, 2B), parallel to the longitudinal axis of the lacinia; the size of their lumen was sometimes very large. In the stem, a large number of ducts were located in the cortical parenchyma, outside the vascular bundles (Fig. 2C). They generally had a small lumen, were very elongated, and showed no anastomosis.

Fig. 1. Secretory ducts of Santolinu ligustica: morphology and distribution. (A) Cross section of receptacle showing a duct with oneltwo layers of secretory cells (TBO). Scale bar = 20 pm. (B) Cross section of receptacle showing a duct filled up with a lipid secretion (Alkanna tincture). Scale bar = 30 pm. (C) Capitulum bract showing a duct after treatment with KOH. Scale bar = 30 pm. ( D ) Leaf lacinia showing a duct after treatment with KOH. Scale bar = 30 pm. ( E ) Cross section of receptacle showing a duct (arrow) outside the vascular bundles (double arrows) (TBO). Scale bar = 7.5 ym. (F) Cross section of interfloral bract with two ducts decorring parallel to the vein (TBO). Scale bar = 50 pni.

In the root, ducts were located in the neighborhood of the endodermis, in groups of three or four outside the phloem bundles. These ducts differed from those located in other organs since they were not surrounded by specialized epithelium: they originated by the splitting off of two cortical cells from two endodermal cells (Fig. 2D).

Glandular hairs Hairs were multicellular and biseriate. When mature, they consisted of five pairs of cells: two basal ones, two stalk cells, and six head cells (Fig. 2E). The basal cells of the hairs of the capitulum organs generally projected above the surface of the surrounding epidermal cells Pugrzi e f al. /Secretory structure3 c!f Santolina ligustica Arrigoni

Fig. 2. Secretory ducts and glandular hairs of Santolina l i g ~ ~ ~ t imorphology ca: and distribution. (A) Ducts (arrows) in the stigma as long as the stigmatic lobe (Alkanna tincture). Scale bar = 75 prn. (B) Cross section of leaf showing a duct between two vascular bundles (TBO). Scale bar = 50 pm. (C) Cross section of stem showing a duct (arrow) located outside the vascular bundles (b) (TBO). Scale bar = 40 ym. (D) Cross section of root showing three ducts (arrows), each one originated from the splitt~ngoff of two cortical from two endodermal cells (double arrows) (Alkanna tincture). Scale bar = 30 pm. (E) Biseriate rnulticellular hair consisted of two basal cells, two stalk cells, and six head cells. The two pairs of subapical cells contained numerous chloroplasts (arrows) (Nadi reagent). Scale bar = 25 yrn. (F) Distribution of glandular hairs (arrows) in the corolla (SEM). Scale bar = 450 ym.

(Fig. 2E). This was not the case with the hairs of the vegetative organs, which were most often sunken in a depression in the epidermis (Fig. 3D). The stalk, too, could vary in length: it was very well developed in bracts and corolla hairs and very short in stem and leaf Israel Journal o f Plant Sciences 51

2003

hairs. The secreted material gathered in a subcuticular space originated by the detachment of the cuticle from the top cells. In mature hairs, the upper cells were colorless, while the two pairs of subapical cells were green, containing

Fig. 3. Histochemistry of secretory structures of Suntolina ligustica. (A) Secretory duct showing lipid production (stigma, Alkanna tincture). Scale bar = 50 Fm. (B) Duct showing secretion stained positive for essential oils (stem, Nadi reagent). Scale bar = 20 Fm. (C) Duct showing drops (arrows) stained positive for resins (receptacle, Nadi reagent). Scale bar = 30 ym. (D) Glandular hair showing secretion positive for essential oils (leaf, Nadi reagent). Scale bar = 20 ym. (E) Secretory duct showing drops stained positive for lipids (receptacle, Nile blue). Scale bar = 30 Fm. (F) Glandular hair showing lipid production (corolla, Nile blue). Scale bar = 25 ym. (G) Duct showing strong yellow self-fluorescence (stigma). Scale bar = 35 ym. (H) Fluorescence of glandular hairs in the corolla showing flavonoid production (aluminum chloride). Scale bar = 25 ym.

Pagni et al. /Secretoq~structures oj'santolina ligustica Arrigoni

numerous chloroplasts (Fig. 2E). The hairs underwent a very early ontogenesis. in fact, fully differentiated and secreting hairs can be observed in organ primordia.

Distribution of the hairs In the capitulum, hairs were located on the involucral and interfloral bracts and on the corolla. In the bracts, they were few, located on the abaxial surface and partly hidden among protective hairs. In the corolla, they were numerous, arranged in longitudinal rows on the outer surface (Fig. 2F). On the stem, they were very few and scattered. On the leaves, they were mainly located on the laciniae, where they were spread all over the surface.

Histochemistry The results of the histochemical test are reported in Table I. Ducts were positive for the presence of alkaloids, lipids, essential oils, resins, sesquiterpene lactones, and flavonoids (Fig. 3A-C, G). In the root and stem, ducts stained positive for essential oils, while in the leaves and capitulum, they were positive for both essential oils and resins. Tests for pectin-like substances and proteins were negative. Testing for tannins was uncertain because the natural color of the secretion was the same as that of the reaction with potassium bichromate. Glandular hairs gave a positive reaction for the presence of alkaloids, lipids, essential oils, pectin-like substances, sesquiterpene lactones, tannins, and flavonoids (Fig. 3D-F, H). Testing for resins and proteins gave a negative response. The ducts showed a strong yellow self-fluorescence (Fig. 3G) which did not change after soaking in aluminum chloride. Hairs were poorly self-fluorescent; fluorescence increased after soaking in aluminum chloride (Fig. 3H).

Table 1 Histochemistry of secretory structures of Santolina ligusticu -

Ducts

Glandular hairs

Lipids Essential oils Resins Pectin-like substances Sesquiterpenes lactones Proteins Tannins Alkaloids Flavonoids

+, positive; uncertain: -, negative +_,

Israel Jounzal of Plant Scieizces 51

2003

DISCUSSION Santolirla ligustica has an internal and an external secretory system, which consist of secretory ducts and glandular hairs, respectively. The ducts show much variability in width and length of the lumen, number of epithelial layers, and frequency. The wide variability in the internal secretory structures of the Asteraceae has been highlighted and discussed by Lersten and Curtis ( 1 987). Later studies (Lersten and Curtis, 1988; Poli et al., 1995) confirmed such variability. In S. ligustica, ducts are found all over the plant, but are most frequent in the capitulum, with the exception of the corolla. Their structure and distribution are similar to those of S. leucantha (Pagni and Masini, 1999) and of S. insularis (Sacchetti et al., 1997). However, in the latter species, unlike in S. ligwtica and S. leucantha, the ducts do not seem to be located in the stigma. In addition, the internal secretory system of the species is similar to that of many other composites belonging to the tribe Anthemideae (Bremer, 1994), such as Artemisia campestris subsp. rnaritima (Ascensao and Pais, 1988) and Artemisia nitidrr (Corsi and Nencioni, 1995). The morphology of the hair of the species examined is widespread in the Asteraceae and has already been shown in other species, such as Chrysanthemum nzorijoliunz (Vermer and Peterson, 1979), lnula viscosa (Werker and Fahn. 1981), and Artemisia canzpestris (Ascensao and Pais, 1987).The hairs of S. ligwstica, like those of S. leucantha (Pagni, 1995; Pagni and Masini, 1999), have the same structure everywhere in the plant and differ only during the development of the basal and stalk cells. These cells often look very elongated in flower hairs: a similar pattern has been observed in the capitulum hairs ofArtemisia niticlcl (Corsi and Nencioni, 1995). Our research revealed the presence in mature hairs of two non-chlorophyllic apical cells and two pairs of chlorophyllic cells, as also observed by Werker and Fahn (198 1) in Itzula 11iscosa and by Ascensao and Pais (1 987) in Artemisia campesrris. Histochemical tests demonstrated that the secretions of glandular hairs and secretory ducts, while similar, are not identical. Hairs contain an oleoresin, whose main constituents are sesquiterpenic lactones, terpenoids, essential oils, alkaloids, tannins, and pectin-like substances. Fluorescence microscope observations would also suggest the presence of flavonoids. Ducts have no pectin-like substances and, in addition, have different terpenoid compositions depending on their location. The strong natural self-fluorescence in ultraviolet light, masking the action of aluminum chlo-

ride, prevented us from checking whether there were any flavonoids, even if. as was assumed for S. leucantha (Pagni and Masini, 1999),they are likely to be present in these structures. Many of the secondary metabolites tested using histochemical reactions were consistent with those found by the chemical analyses in S. neupoliruna (Senatore and De Feo, 1994). Cheniclet and Carde (1985) reported the composition of essential oils of Saiztoli~zapini~atu showing a major percentage of sesquiterpenes in ducts and a prevalence of monoterpenes in trichomes. These results are in accordance with our histochemical tests showing the presence of essential oils and resins in ducts and only essential oils in glandular hairs. A conlparison between the secreting structures of S. ligc~sricuand those of S. leucaizthu showed a strong resemblance between the two species. not only for their morphology and histochemistry but also for their frequency and distribution. This observation confirms a close phylogenetic relationship between the two species belonging to the diploid group. as already suggested by the karyological analyses (Arrigoni, 1977; Arrigoni and Mori, 1980). The differences of the distribution of the ducts between S. ligusticcl and S. insularis could be explained because these two species belong to the diploid and the polyploid groups, respectively. Anatomical analysis of the glandular hairs of S. insularis could complete the comparison among these different taxa.

ACKNOWLEDGMENT This investigation was supported by grants from the University of Pisa.

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Pagni et 01. /Secretory structures ofsantolina ligustica Arrigoni

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