Protoplasma (1997) 198:170-176 PROTOPLASMA Springer-Verlag 1997 Printed in Austria The cellular distribution of antifeedant prenylated anthranoids in the tissues of Vismia guianensis during development B. Monaeelli 1' *, G. Pasqua 1, N. Raseio 2, B. Botta 3, G. Delle Monache 4, A. Vitali 4, and A. Chiappeta 5 1 Dipartimento di Biologia Vegetale and 3 Dipartimcnto di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universit~ "La Sapienza", Rome, 2 Dipartimento di Biologia, Universit~t di Padova, Padua, 4 Centro Chimica dei Recettori, Istituto di Chimica, Universit~t Cattolica S. Cuore, Rome, and 5 Departamento de Antibioticos, Universidade Federal de Pernambuco, Recife, Brasil Received December 16, 1996 Accepted February 7, 1997 Summary. Accumulation and distribution of two types of phenolic antifeedant metabolites, vismiones and ferruginins, in specialized cells were investigated in Vistula guianensis DC. Chemical, light, and electron-microscope analyses revealed that the two types of compounds were produced in different cells and organs and at differ- ent stages of plant development. Vismiones accumulated at various concentrations in the leaf, depending on its developmental stage, as well as in the stem of very young plants. Ferruginins appeared late in the plant's development and accumulated exclusively in secretory ducts in the secondary body of the stem and in secretory cavities of the frnits. Keywords" Vismia guianensis; Antifeedant metabolites; Secretory ducts; Phenolic localization Introduction In recent years, several studies have described the chemical defence of plants against herbivores. These studies have shown that secondary metabolites acting as feeding deterrents accumulate predominantly in those organs and tissues which are most exposed to attack and therefore require the most protection, i.e., leaves, buds, and fruits (Harborne 1988). The com- pounds can be stored at the surface of an organ (epi- dermal cells, trichomes) or internally (secretory cavi- ties and ducts, laticifers) (Fahn !990). * Correspondence and reprints: Dipartimento di Biologia Vegetale, Universit~t "La Sapienza", Piazzale Aldo Moro 5, 1-00185 Rome, Ita- ly Antifeedant metabolites can be produced as a tempo- rary response to injury or pathogen attack, or be con- sistently present, although at different concentrations, depending on the developmental stage of the organ. For instance, the young internodes of paper birch (Betula resinifera) are inedible because of the high concentration of papyriferic acid, whereas this triter- penoid occurs at 25 times lower quantities in the mature internodes (Reichardt et al. 1984). In Coffea arabica, the production of caffeine and theobromine peaks when the young leaf is soft. As the leaf matures the alkaloid content decreases with the increased mechanical durability (Frischknecht et al. 1982). A similar change in defence strategy occurs during fruit development (Keller et al. 1972). In Vismia guianensis DC. (fam. Hypericaceae), a widespread species in Central and South America, the defence strategy involves the production of two types of phenolic compound, i.e., vismiones and ferrug- inins, which provide good antifeedant activity against lepidopterous larvae and orthopterans (Simmonds et al. 1985), but do not show any antibacterial or anti- fungal activity (Cassinelli et al. 1986). Oligophagous insects, such as Locusta migratoria and Spodoptera exempta, are the most susceptible species to vis- miones, with over 60% feeding inhibition. On the other hand, a polyphagous insect, such as Heliothis virescens, is more susceptible to ferruginins (Sim- monds et al. 1985).
Transcript
Protoplasma (1997) 198:170-176
PROTOPLASMA �9 Springer-Verlag 1997 Printed in Austria
The cellular distribution of antifeedant prenylated anthranoids in the tissues of Vismia guianensis during development
B. Monaeel l i 1' *, G. Pasqua 1, N. Raseio 2, B. Botta 3, G. Delle Monache 4, A. Vitali 4, and A. Chiappeta 5
1 Dipartimento di Biologia Vegetale and 3 Dipartimcnto di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universit~ "La Sapienza", Rome, 2 Dipartimento di Biologia, Universit~t di Padova, Padua, 4 Centro Chimica dei Recettori, Istituto di Chimica, Universit~t Cattolica S. Cuore, Rome, and 5 Departamento de Antibioticos, Universidade Federal de Pernambuco, Recife, Brasil
Received December 16, 1996 Accepted February 7, 1997
Summary. Accumulation and distribution of two types of phenolic antifeedant metabolites, vismiones and ferruginins, in specialized cells were investigated in Vistula guianensis DC. Chemical, light, and electron-microscope analyses revealed that the two types of compounds were produced in different cells and organs and at differ- ent stages of plant development. Vismiones accumulated at various concentrations in the leaf, depending on its developmental stage, as well as in the stem of very young plants. Ferruginins appeared late in the plant's development and accumulated exclusively in secretory ducts in the secondary body of the stem and in secretory cavities of the frnits.
In recent years, several studies have described the chemical defence of plants against herbivores. These studies have shown that secondary metabolites acting as feeding deterrents accumulate predominantly in those organs and tissues which are most exposed to attack and therefore require the most protection, i.e., leaves, buds, and fruits (Harborne 1988). The com- pounds can be stored at the surface of an organ (epi- dermal cells, trichomes) or internally (secretory cavi- ties and ducts, laticifers) (Fahn !990).
* Correspondence and reprints: Dipartimento di Biologia Vegetale, Universit~t "La Sapienza", Piazzale Aldo Moro 5, 1-00185 Rome, Ita- ly
Antifeedant metabolites can be produced as a tempo- rary response to injury or pathogen attack, or be con- sistently present, although at different concentrations, depending on the developmental stage of the organ. For instance, the young internodes of paper birch (Betula resinifera) are inedible because of the high concentration of papyriferic acid, whereas this triter- penoid occurs at 25 times lower quantities in the mature internodes (Reichardt et al. 1984). In Coffea arabica, the production of caffeine and theobromine peaks when the young leaf is soft. As the leaf matures the alkaloid content decreases with the increased mechanical durability (Frischknecht et al. 1982). A similar change in defence strategy occurs during fruit development (Keller et al. 1972). In Vismia guianensis DC. (fam. Hypericaceae), a widespread species in Central and South America, the defence strategy involves the production of two types of phenolic compound, i.e., vismiones and ferrug- inins, which provide good antifeedant activity against lepidopterous larvae and orthopterans (Simmonds et al. 1985), but do not show any antibacterial or anti- fungal activity (Cassinelli et al. 1986). Oligophagous insects, such as Locusta migratoria and Spodoptera exempta, are the most susceptible species to vis- miones, with over 60% feeding inhibition. On the other hand, a polyphagous insect, such as Heliothis virescens, is more susceptible to ferruginins (Sim- monds et al. 1985).
B. Monacelli et al.: Distribution of antifeedant compounds in Vismia guianensis 171
L O OH OH r
' V ~ ~ OMe AcO 1
/• . / "-.5/" OMe
H ~- ~rH
OH 0 OH /
3
OH 0 OH
6 R=H
0 OH
OH
8
OH OH 0
o OH ? 2~-"~1
H ~ M e 2
oH o o %
H ~ H
oH o 62;~
4
o . o ,0%
~ " ~ ~ OMe
0
7
o .
H
CH2 I~
CH2R R R'
9 H H 10 OH H l l OH OH
Fig. 1. Constituents of Vistula guianensis: vismione A (1), vismione B (2); bianthrone from vismione A (3); bianthrone from vismione B (4); 2-prenylphyscion (5); physcion (6); anthraquinone from vismio- ne B (7); ferruanthrone (8); ferruginin (9); y-hydroxyferruginin (10); y-dihydroxyferruginin (11); madagascin (]2)
Table 1. Prenylated anthranoid content in Vistula guianensis
Values are percent of fresh weight Vis Vismiones; Fer ferruginins
by various C5 or C10 substituents. The compounds are generally defined as prenylated anthranoids and are closely related biogenetically (Delle Monache 1985). The occurrence of prenylated anthranoids is limited to three genera: Vismia, Harungana, Psorospermum, members of the family Hypericaceae (Delle Monache 1985). The cellular distribution of the above metabo- lites have not previously been studied. We chose to focus our study on Vismia guianensis because of the presence of vismione A (Fig. 1, structure 1), which has strong activity against certain tumours, such as ovarian carcinoma and melanocarcinoma B 16 (Cassi- nelli etal. 1986). In a previous publication, we showed that vismione A is not produced in calli or in cell-suspension cultures (Pasqua et al. 1991), but that its production requires the differentiation and devel- opment of leaves, since it is produced in regenerated plants (Pasqua et al. 1995). In the present study, we carried out chemical, histochemical, and ultrastruc- rural analyses to establish how production depends on specific organs and their developmental stage, and to determine the sites of accumulation.
Prenylated anthranoids have recently been tested on Leptinotarsa decemlineata (Coleoptera) and Blattella germanica (Blattoidea). Vismiones were active, whereas ferruginins are practically inactive against these insects (E. Caprioli and F. Delle Monache unpubl, data). Both vismiones (tetrahydroanthracenes) and ferrug- inins (dihydroanthracenes) are characterised by a non-aromatic A and C ring, respectively (Fig. i), and
Material and methods Chemical analysis
The following samples were examined: leaves and branches at dif- ferent developmental stages (Figs. 2 a, 3 a, and 4 a), fruits (Fig. 4 e), and exudates obtained by scarification of the stem and fruits. The samples were ground and extracted exhaustively with cold methanol (MeOH). The cooled methanolic extracts were concentrated to a small volume and diluted with CHC13 and H20. The mixture was extracted three times with CHCI3 and then three times with ethyl
172 B. Monacelli et al.: Distribution of antifeedant compounds in Vismia guianensis
Fig. 2. a Seedlings 10 days after germination, b Light micrograph, cross section of a leaflet (less than 1 cm in length): small phenolic inclu- sions are visible in all tissues of the lamina, e Ultrastructure of epidermal and palisade tissues: osmiophilic globules are visible in the vacuoles. Bars: in b, 50 ~m; in c, 5 ~,tm
acetate (EtOAc). The CHC13 extracts were monitored by thin-layer chromatography and then purified on a silica gel column. The eluants were: CH2C12-hexane (3:2) and CH2C12 for the vismione pool and CH2Cl2-hexane-EtOAc (7:2:1) for the ferruginin pool. The pure compounds were identified by comparison with authentic samples (1H and 13C nuclear magnetic resonance, thin layer chro- matography). The percentage of vismiones relative to the fresh weight of the organs was calculated as the cumulative amounts of vismione A or B (Fig. 1, structures 1 and 2) and the corresponding artefacts (Pasqua et al. 1995) such as bianthrones (Fig. 1, structures 3 and 4) and anthraquinones (Fig. 1, structures 5 and 7, derived from vismlone A). The percentage of ferruginins was estimated as single ferruginins (Fig, 1, structures 9-12). Thin layer chromatography was used to compare the EtOAc extracts in various solvent systems with samples of compounds 1-12 (Fig. 1). Since prenylated anthxanoids were not present, the extracts were not processed further.
Light and electron microscopy
Seedlings (Fig. 2 a) and young plants (12 leaves stage) (Fig. 3 a) grown in pots, and adult plants, grown under natural conditions in the Recife (Brasil) area, in vegetative (Fig. 4 a) and reproductive states were used for microscopical studies. Samples from leaves, brancbes~
and fruits (Fig. 4 e) were fixed overnight in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 6.9), post-fixed in 1% osmium tetroxide in the same buffer for 2 h, and dehydrated in a gradual series of ethyl alcohol and propylene oxide. Staining with uranyl acetate was carried out while dehydrating with 75% alcohol. The samples were embedded in an Epon-araldite mixture. For light microscopy, thin sections (1 gin) were cut with an Ultracut Reichert- Jung ultram~crotome and stained with 1% toluidine blue and 1% Na tetraborate (1:1 by volume) to detect the presence of phenol com- pounds. For trm~smission electron-microscopy (TEM), ultrathin sec- tions (approximately 60 nm thick), cut with the same were post- stained with lead citrate and examined with a Hitachi 300 electron- microscope operating at 75 kV.
Results
Secondary metabolite accumulation
The results of the chemical analyses of the organs at various stages of plant development and of the exu- dates of the stem and fruit are shown in Table 1. In the CHC13 extract, vismione A (Fig. 1, structure 1)
Fig. 3. a Young plant, 12 leaves stage, b Light micrograph, cross section of young leaf. Phenolic inclusions are visible in the palisade tissue; isodiametric secretory cavity is visible in the mesophyll, e Ultrastmcture of epidermal and palisade tissues. Small phenolic globules are visible in the vacuoles of epidermic cells. The vacuoles of some palisade cells are particularly electron-dense for phenolic accumulation_ d TEM micro- graph of a secretory cavity in the leaf mesophyll showing secretory cells (sc) and lumen (L) filled with slightly electron-dense material, e TEM micrograph showing phenolic deposits in the vacuoles of abaxial epidermal and parenchyma cells. Cross section of a type 1 secretory duct near the midrib is also visible (encircled), f Light micrograph, cross section of the primary stem showing small phenolic globules in medullar ray vacuoles. Type 1 secretory ducts in the phloem are also visible (circles). g TEM micrograph showing phenolic globules in the vacuoles of paren- chymatous cells of the stem cortex, h Ultrathin section of a type 1 secretory duct. The lumen (L) is surrounded by four secretory cells (sc). Bars:
in b and f, 50 [xm; in c, d, e, g, and h, 5 ~tm
B. Monacelli et al.: Distribution of antifeedant compounds in Vismia guianensis 173
174 B. M onacelli et al.: Distribution of antifeedant compounds in Vismia guianensis
Fig. 4. a Vegetative Vismia guianensis plant, b Light micrograph, type 1 ducts (arrow) and type 2 duct (T2) are visible in the secondary phloem. e Light micrograph, longitudinal section of a type 2 secretory duct (T2) showing phenolic products in the epithelium, d Light micrograph, cross section of the stem showing secondary structure. A type 2 secretory duct (arrow) is located in the phloem region. Secretory idioblasts in the phloem and medullar parenchyma are also visible, e Vismia guianensis fruits, f Light micrograph, cross section of the fruit mesocarp showing a secretory cavity with phenolic compounds in the epithelium. Bars: in b, c, and f, 20 ~tm; in d, 100 ~tm
and vismione B (Fig. 1, structure 2) were isolated together with the corresponding bianthrones (Fig. 1, structures 3 and 4) and anthraquinones (Fig. 1, struc- tures 5 and 7). Other components, such as physcion (Fig. 1, structure 5), terpenoids, fats, and chlorophylls, were present. Leaves of plants at the seedling stage contained 0.44% vismiones while the percentage in mature leaves fell to 0.20%, in both the vegetative and repro- ductive stages (Table 1). In the stem, the vismiones were only present during the young stage (Table 1).
Ferruginins, like vismiones, occurred in the CHC13 extract. Ferruginins were never found in the leaves but were accumulated in the branches of the sec- ondary body of the plant at low percentages (0.16%). In branches of reproductive plants, the percentage increased to 1.0%. A larger amount of ferruginins was produced in the fruits (5.2%). The analyses of fruit and stem exudates revealed that fenuginins were the only phenolic compounds present (21% and 51%, respectively). In the ferruginin pool, ferruanthrone (Fig. 1, structure 8) and ferruginins a, b, c, and d (structures 9-12) were
B. Monacelli et al.: Distribution of antifeedant compounds in Vismia guianensis 175
isolated. Ferruginins a, b, c, and d were present at the following percentages: 10%, 74%, 15%, and l%, respectively.
Histological and ultrastructural features
In young leaves, phenolic compounds, stored in the vacuoles and forming black globules of different sizes (Fig. 2 c) were detected in all tissues of the leaf lami- na (Fig. 2 b). In mature leaves, few cells of the pal- isade tissue showed vacuoles enriched with phenolics (Fig. 3 b, c). Notably, phenolic compounds were neither present in the secretory cavities (Fig. 3 b, d) nor in the ducts (Fig. 3 e, h). The secretory cavities were located in the mesophyll (Fig. 3 b) with a uniseriate epithelium (Fig. 3 d). They were filled with lipidic substances that were only slightly electron-dense (Fig. 3 d). The secretory ducts were localized in the phloem region of the midrib and in the outer parenchyma under the midrib (Fig. 3 e). These ducts were sur- rounded by uniseriate epithelium and referred to as "type 1". They were found to be numerous and unconnected, never a component of the leaf lamina. In the young stem, phenolic compounds were accu- mulated in the epidermis, in parenchymatous cells of the cortex (Fig. 3 g), and in the medullar rays (Fig. 3f). Type 1 ducts were also present in the stem, in the both primary and secondary stages of development, located in the outer cortical parenchyma and in the phloem (Fig. 3 f and 4 b). As in the leaves, the ducts were surrounded by four cells, which formed a rosette in transverse section (Fig. 3 h), and did not contain phenolic compounds. A different type of duct, referred to as "type 2", was present only in the secondary body of the stem and exclusively localized in the secondary phloem (Fig. 4 b, d). These ducts were less numerous but generally larger in diameter than the type 1 ducts (Fig. 4 b). Type 2 ducts were bounded by a double layer of cells: sheath and epithelium (Fig. 4 b). The inner layer, the epithelium, contained phenolic products (Fig. 4 b, c). Duct lumina always appear empty because the pheno- lics leak out during sample preparation for histologi- cal analyses. The type 2 ducts, like type 1, were always unbranched and unconnected. In the secondary body of the stem phenolic com- pounds were also accumulated in secretory idioblasts in the phloem and medullar parenchyma (Fig. 4 d).
In fruits (small berries of ca. 1.5 cm diameter; Fig. 4 e), phenolic compounds were accumulated in the secretory cavities of the mesocarp. These cavities (Fig. 4 f) showed the same anatomical structure as the type 2 ducts of the stem. Type 1 ducts were not pre- sent in the fruit.
Discussion
The chemical, histochemical, and ultrastructural analyses indicate the following hypothesis about the cellular distribution of vismiones and ferruginins. In spite of the possibility of common biogenesis (Delle Monache 1985), vismiones and ferruginins were pro- duced in different types of cells, never found simulta- neously in the same organ, and differed in their peri- od of production. Vismions and ferruginins have very similar lipophilic features and according to literature they can be stored in the vacuoles (Luckner et al. 1980). The chemical analyses showed that the highest con- centration of vismiones occurred in the leaf at early stages of development. As soon as the leaf reached maximum development, and its nutritional value fell, vismione content decreased. Thus, the increase in phenolic compounds in the vacuoles of some cells of the mature leaf palisade is not due to vismiones. A small percentage of vismiones was only found in the young stem. We presume that vismiones are local- ized in the epidermis and outer parenchyma since they were chemically undetectable when these tissues disappeared in the secondary body. According to the chemical studies ferruginins appeared late in the plant's development and only in the secondary body of the stem and in fruits. In con- trast to vismiones, ferruginins seemed to depend on the differentation of particular secretory ducts (type 2) and are produced by epithelial cells that surround the lumen ducts. This hypothesis was confirmed by the finding that fen'uginins were the only phenolic compounds isolated from the exudates of both stem and fruits. Other secretory ducts (type 1) located in the leaves and stem did not contain phenolics and therefore nei- ther vismiones nor ferruginins. The simultaneous presence of two types of ducts, which differ in their anatomy, secretory content, and distribution, is quite rare. To date, only one similar example in Ambrosia trifida L. (Asteraceae) has been reported (Lerston and Curtis 1988). Secretory ducts also occur in other genera of the
176 B. Monacelli et al.: Distribution of antifeedant compounds in Vismia guianensis
Hypericaceae, always in the phloem, but sometimes also in the pith, pericycle and outer part of the prima- ry cortex (Metcalfe and Chalk 1983). Their anatomy, precise distribution, and secretory content needs to be studied further. In conclusion, a peculiar defence strategy seems to be activated in Vismia guianensis by the accumulation of two different kinds of compounds, vismiones and fer- ruginins, to protect the most edible organs during the biological cycle.
Acknowledgements This research was supported by funds from the National Research Council (C.N.R.) of Italy.
References Cassinelli G, Geroni G, Botta B, Delle Monache G, Delle Monaehe
F (1986) Cytotoxic and antitumor activity of vismiones isolated from Vismieae. J Nat Prod 49:929-931
Delle Monache F (1985) Chemistry and biological activity of the secondary metabolites of Vismieae. Rev Latinoam Quim 16: 5-15
Fahn A (1990) Plant anatomy, 4th edn. Pergamon, Oxford Frischknecht PM, Eller BM, Baumann TW (1982) Purine alkaloid
formation and CO2 gas exchange in dependence of development
and of environmental factors in leaves of Coffea arabica. Planta 156:295-301
Harbome JB (1988) Introduction to ecological biochemistry, 3rd edn. Academic Press, London
Keller H, Wanner H, Baumann TW (1972) Kaffeinsynthese in Frtichten und Gewebekulturen yon Coffea arabica. Planta 108: 339-350
Lerston NR, Curtis JD (1988) Secretory reservoirs ducts of two kinds in giant ragweed (Ambrosia trifida, Asteraceae). Am J Bot 75: 1313-1323
Luckner M, Diettrich B, Lerbs W (1980) Cellular compartmentation and channelling of secondary metabolism in microorganisms and higher plants. In: Reinhold L, Harborne JB, Swain T (eds) Progress in phytochemistry, vol 6. Pergamon, Oxford, pp 103-142
Metcalfe CR, Chalk L (1983) Anatomy of the dicotyledons, 2nd edn. Claredon, Oxford
Pasqua G, Monacelli B, Finocchiaro O, Botta B, Delle Monache G (1991) Vistula guianensis: in vitro culture, plant regeneration and production of vismione A. G Bot It 125:1004-1005
- Monacelli B, Cuteri A, Spuntarelli F, Rascio N, Botta B, Delle Monache G, Scurria R (1995) Accumulation of vismione A in regenerated plants of Vismia guianensis DC. Protoplasma 189: 9-16
Reichardt PB, Bryant JP, Clausen TP, Wicland GD (1984) Defense of winter-dormant Alaska paper birch against snowshoe hares. Oecologia 65: 58-69
Simmonds M, Blaney W, Delle Monache F, Mac-quhae M, Marini Bettolo GB (1985) Insect antifeedant properties of anthranoids from the genus Vistula. J Chem Ecol 1 I: 1593-1599
