Epidermis and epicuticular waxes of Syagrus coronata leaf lets

Raul Dodsworth Machado and Claudia Franca Barros

Abstract: The outer epidermal cell walls of the leaf blade of the licuri palm tree were studied by light microscopy, scanning electron microscopy, and transmission electron microscopy, with special attention to the epicuticular waxes. On the intensely green adaxial surface, the wax adheres in the form of a smooth, flexible, varnish-like layer. On the pruinose, dull, greenish or bluish abaxial side, the wax appears as a thin amorphous layer from which rodlets and columns protrude. Very curved rodlets, in compact rows, border each stoma, sometimes almost completely closing its aperture. Numerous pores, not resolvable with the light microscope, were detected in both cuticular membranes. Comments are presented concerning the possible functions of several configurations of epicuticular waxes.

Key words: epicuticular waxes, wax micromorphology, Syagrus, licuri, epidermal wall.

RCsumC : La paroi Cpidermique externe de la feuille du palmier licuri a CtC etudiCe avec les microscopes photonique et Clectroniques a transmission et a balayage, particulibrement la micromorphologie des cires Cpicuticulaires. La surface adaxiale est couverte par une couche continue, uniforme, flexible, comme un vernis et, par suite, elle est brillante et vert foncC. La surface abaxiale, pruineuse et d'un gris verditre, mate, est couverte par une couche continue et fine de cire amorphe, d'oh sortent longs bitonnets droits ou ICgbrement courbes, et des colonnes de bitonnets fusionnCs. Un rang de bitonnets trks courbCs, en group compact, se place de chaque cBtC des ouvertures stomatales, qu' ils peuvent fermer presque complktement. Des nombreux pores, non rCsolubles par le microscope photonique, se trouvent dans les deux membranes cuticulaires. Un commentaire est prCsentC sur le rB1e possible des cires Cpicuticulaires relation6 avec leur configuration morphologique.

Mots clks : cires $icuticulaires, micromorphologie des cires, Syagrus, licuri, paroi Cpidermique.

Introduction The licuri or ouricuri palm tree, very abundant in the state

In Brazil, two vegetable waxes attained economical impor- tance: carnauba, yielded by the leaves of the palm Coper- nicia cerifera Mart. (now Copernicia prunifera (Miller) H.E. Moore), and licuri, from the leaflets of Syagrus coronata (Mart.) Becc. (Palmae). The carnauba palm tree is abundant in northeastern Brazil, mainly in the states of Piaui, Cearh, and Rio Grande do Norte. Its leaves and waxes were the object of several studies. Machado (1945) pointed out the difference between its stomata and those of carandi (Coper- nicia australis Becc., now Copernicia alba, Morong). The two palm trees are not easily distinguished (Trancoso 1945) and a special key was made by Kuhlmann (1945). Braga Arraes et al. (1966) described details of the leaf anatomy, collected from palms from several places in Cearh, and aspects of the ultrastructure of the outer wall, in which con- centrations of intraparietal wax were found.

Received February 21, 1995.

R.D. Machado. Laboratbrio de Microscopia EletrBnica Hertha Meyer, Institute de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil. C.F. Barr0s.l Area de Botinica Estrutural, Jardim Botinico do Rio de Janeiro, Rua Jardim Botinico 1008, Givea, 22460-000, Rio de Janeiro, Brazil.

' Author to whom all correspondence should be addressed.

of Bahia (eastern Brazil), was extensively observed by Bondar (1942), the first to point out its potential as a substi- tute for carnauba wax in the manufacture of polishing pastes and candles. It is found in large numbers in Maracas, Milagres, S. Teresinha, and Itiuba, in the "cah-tinga" ecosystem, where a long and irregular dry season is usual. Machado (1959) published a study of the anatomy of the leaf blade and of the micromorphology of adaxial and abaxial epicuticular waxes (different in form and chemical composition). The present paper extends the knowledge based onthese previous observations, by the use of scanning electron microscopy. Glassman (1972) published the taxonomy of genus Syagrus based on leaf anatbmy, and we used his data and correspon- dence to confirm the identity of our material. A large con- tribution to the knowledge of epicuticular waxes was made by Barthlott and co-workers (Barthlott and Frolich 1983; Barthlott and Ehler 1977: Barthlott and Wollenweber 1981; Fehrenbach and ~ar th lo t t , 1988; Frolich and Barthlott 1988; Engel and Barthlott 1988; Benke and Barthlott 1983) cover- ing a diversity of micromorphological aspects and correlat- ing with taxonomy and evolution. In Brazil, epicuticular waxes, mainly in species of the cerrado, were examined by Salatino and co-workers (Amaral et al. 1985; Salatino et al. 1986). The presence of epicuticular waxes is generally inter- preted as influencing transpiration, penetration of water, nutritive solutions, herbicides, pesticides (Price 1982), fungi-

Can. J. Bot. 73: 1947- 1952 (1995). Printed in Canada I ImprimC au Canada

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Fig. 1. Adaxial surface of the leaf blade. An aspect of the extensive wax layer (w), the epidermis underneath (e), and abundant fungal hyphae (h). SEM 315 x . Scale bar = 30 pm. Fig. 2. Adaxial surface. The wax layer was broken on purpose. The thickness of the detached piece of wax (w) is between 1 and 2 pm. The relief of the epidermis (e ) is impressed in the wax. A fungal hypha is seen (h). SEM 7 3 0 x . Scale bar = 10 pm. Fig. 3. Thin section of the epidermal outer wall. The wall is very homogeneous but shows a cuticle (c) and a lighter layer (ce), presumably cellulosic. The median region exhibits small electron-dense granules and the suggestion of fibrilar texture. No clear indication of pectin or cutin concentration is evident. In continuation of the middle lamella (m), electron-dense material extends under the cuticle. TEM 17 000 x . Scale bar = 1 pm. Fig. 4. Abaxial surface. Wax is present in scattered goups of rodlets and columns. At intervals, a continuous amorphous layer (white stars) covers the cuticle showing longitudinal fractures, possibly due to shrinkage of the leaf. Rows of stomata are seen (white arrows), each one with two borders of very curved rodlets. Two stomata at the center have apertures almost completely closed by the wax borders. SEM 2 8 0 x . Scale bar = 30 pm. Fig. 5. Abaxial surface. At left a group of adherent rodlets that are slightly curved (r). At right, a massive column of rodlets fused together (c). At its base, a periclinal lamellation (white arrow) can be seen. On the upper surfaces of the columns and rodlets, small particles are deposited, similar to the ones abundantly spread over the amorphous layer. SEM 1 9 0 0 ~ . Scale bar =

10 pm. Fig. 6. A stoma with its two borders of curved wax rodlets. The stomatal aperture can be seen inside (white arrow). SEM 2000x . Scale bar = 10 pm. Fig. 7. Inner surface of the adaxial cuticular membrane. Pores are very abundant. SEM 11 000 X . Scale bar = 1 pm. Fig. 8. Inner surface of abaxial cuticular membrane. Pores are abundant but more widely spaced than in Fig. 7 . SEM I 1 000 X . Scale bar = 1 pm.

cides, and participating in the defence of plants against insects and parasites.

The occurrence of the two very different forms of epicuticular waxes found over the licuri leaflet, a fact not known to occur in any other plant, is intriguing and should be studied further.

The problem of the production and exteriorization of the wax led to this investigation of the ultrastructure of the outer wall and the existence of passages through the cuticular membrane. An important contribution was recently made concerning the secretion of tubular epicuticular filamentous wax by the cork cells of Sorghum bicolor (Jenks et al. 1994). We present evidence of pores in solvent-extracted cuticular membranes of the licuri leaflet.

Materials and methods

Fully mature leaves were collected from plants in the Botani- cal Garden of Rio de Janeiro. For scanning electron micros- copy, fragments of the leaf blade with sides not exceeding 5 mm were processed, one part after boiling in chloroform to remove the wax, another part dried in air at room tempera- ture so as not to alter the wax. In both cases the samples were glued to aluminum stubs and covered with a gold layer close to 25 nm in thickness, using a Balzer's sputtering apparatus. For observation and microscopy a JEOL 25-S-I1 or Zeiss 940 scanning electron microscope was used at 15 kV accelera- tion. When it was necessary to detach the cuticular mem- branes, hot Jeffrey's mixture (Johansen 1940) was used. For transmission electron microscopy, leaf fragments were fixed in a mixture of 4 % glutaraldehyde, 2% paraformaldehyde, and 0.1 M buffer phosphate, followed by postfixation in 1 % osmium tetroxide and embedding in Epon. Thin sections were obtained with a diamond knife and a MT-2B Porter- Blum ultramicrotome.

Observations were carried out with a Zeiss 900 or 902 transmission electron microscope in the Laboratory Hertha Meyer of the Institute of Biophysics at the Federal University of Rio de Janeiro (UFRJ). Part of the work was carried out at the Laboratory of Microscopy of the Rio de Janeiro Botan- ical Garden, part in the Laboratory Hertha Mayer, and part in the Laboratory of Cell Biology of the Oswaldo Cruz Foun-

dation, Manguinhos, Rio de Janeiro (Zeiss 940 scanning electron microscope).

For v i s ~ a l i z i n ~ ~ ~ o s s i b l e pores, cuticular membranes were boiled several times in chloroform and prepared by the usual methods for scanning electron microscopy. Counts were made at a suitable magnification, in squares 3.5 X 3.5 cm, corresponding to 4 pm2 in the specimen. Twenty-five counts of stomata and trichomes were made in 1-mm squares using a camera lucida.

Results

The adaxial epidermis of Syagrus coronata in surface view presents polygonal cells, isodiametric in certain areas, slightly elongated in others, sometimes obliquely to the long axis of the leaf. Stomata are only found over the median vein. Multicellular trichomes occur on the young leaflet at an average density of 2/mm2. In the fresh leaf, the adaxial sur- face is intensely green, an effect intensified by the uniform, continuous flexible and adherent varnish-like wax layer. At the top left of Fig. 2, part of an adaxial wax plate is seen, detached on its thickness appearing tb be between 1 and 2 pm. The cuticle relief is imprinted on the wax plates (Figs. 1 and 2). Fungal hyphae are abundant, sometimes in tangled masses (Fig. 1).

In the fresh leaf, the abaxial surface appears dull, pruinose, greenish gray or bluish. The epidermal cells are polygonal, some of them isodiametric, others elongated, par- ticularly over costal areas. Over intercostal regions, numer- ous tetracytic stomata occur, the stomatal apertures parallel to the longitudinal direction of the leaflet at a density of 225/mm2. In the young leaf, pluricellular trichomes appear, 5/mm2. The epicuticular wax is scattered (Fig. 4); some groups are formed by a few straight or slightly curved rodlets ( ~ i ~ : 5) with longitudinal striae: while others form massive columns (Fig. 5) of merged straight rodlets. At the base of some columns, periclinal parallel lamellae can be seen (Fig. 5). Bordering the stoma, a dense row of heavily curved rodlets is found (Fig. 6); these rodlets are variably distant from each other and sometimes almost close the stomatal aperture (Fig. 4, center). Between stomata, wax rodlets, and columns, the sur- face is covered by a rough thin layer (amorphous layer) over

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which numerous small particles are scattered (Figs. 5 and 6). On the top of the columns (Fig. 5) is found what could be a remnant of the amorphous layer and its small particles.

The inner surface of the isolated adaxial cuticular mem- brane shows a relief formed by anticlinal walls and their flanges. Globular projections are also visible. After thorough extraction with choloroform, abundant densely grouped pores are observed at a concentration of 25/pm2, with a diameter of approximately 75 nm (Fig. 7).

The inner surface of abaxial cuticular membranes clearly depicts the stomata and the penetration of anticlinal walls. Some globular projections are also seen, as are small densely grouped pores at a concentration of 12/pm2, with a diameter close to 85 nm (Fig. 8), more widely spaced than the pores on the adaxial cuticular membranes.

In thin sections (Fig. 3) the outer wall is very homogene- ous and does not show evidence of concentration of pectin or cutin. With a total thickness of ca. 5 pm, it shows a 0.12-pm cuticle and a lighter 0.2-pm inner layer (probably cellulosic). The intermediate region exhibits very small electron-dense granules and a suggestion of fibrilar texture. In continuation of the middle lamella (Fig. 3), a darker layer almost reaches the surface, and adjacent electron-dense material extends under the cuticle. No lamellae were visible in the cuticle.

Discussion

It is generally agreed that the epicuticular wax minimizes transpiration. However, the question is not so simple. Leaf transpiration is mainly dependent on stomata1 apertures. Rizzini (1976) found that cuticular transpiration in several brazilian species varied from 0.3 to 11 % of the total transpi- ration. In the amphistomatic waxy leaf of the carnauba palm, the extensive, continuous, adherent wax layer completely covers both surfaces, including stomata. Over the stomata, the wax layer is continuous, showing only a difference in tex- ture (Machado 1945; Braga Arraes et al. 1966). In this case, it is reasonable to suppose that the wax layer (thickness ca. 20 pm) restricts total transpiration. In the "caaf-ua~d" plant Calathea lutea (Aubl.) Schult (Marantaceae), the extensive waxy layer of the abaxial surface has perforations over the stomata. In the licuri, a continuous, uniform, adax- ial wax layer covers the astomatous surface and must be effective in controling cuticular transpiration. At the abaxial face, the compact groups of curved rodlets bordering the stomata must influence the passage of moisture and gases (Fig. 6). Cuticular transpiration, on the other hand, must be modified by the amorphous layer. MCrida et al. (1981) found that soluble cuticular lipids are responsible for the impermea- bilization of the epidermal outer wall.

Inside the epidermal outer wall of several plants (Meyer 1938; Sitte and Rennier 1963) and also in licuri (Machado 1959), a lipid component is constantly present, as shown by the disappearance of birefringence when it was heated to temperatures above the melting point of wax. The same result can be obtained by the use of wax solvents, as shown by Sitte and Rennier (1963).

In several works, the existence of discontinuities in the cuticular membrane were related to the excretion or absorp- tion of substances. However, such channels present very different diameters, extensions, and even structures, often

not clearly described. Large apertures in the outer wall of leaf epidermal cells, in which a portion of cytoplasm pene- trates but does not reach the exterior, were found in several plants in the Myrtaceae (Fontenelle et al. 1994). These struc- tures are similar to the ones referred to by Haberlandt (1928) as "minute papillose processes" in Drosera (Droseraceae). In Eugenia sulcata and in Eugenia excelsa (Myrtaceae), those penetrations could be seen in the light microscope, measuring more than 1 pm in diameter and 4 pm in depth (Fontenelle et al. 1994). Over them, the wall is thinner but not perforated. Complete perforations were found in the gland cuticle of Drosera by Chafe and Wardrop (1973) in which they showed the protrusion of material. Sometimes a rather large incomplete opening is named an ectodesm (Franke 1961). Some of his illustrations represent mushroom-like structures, visible in cross sections of the wall at 640x . Perhaps the term ectodesm should be reserved for the struc- tures described by Schnepf (1959) and by Lyshede (1978). They are not precisely channels but thin bands with a texture slightly different from that of the wall. In their illustrations the diameter can be estimated as ca. 270 nm. This structure Esau (1977) calls a teichode, explaining it as a "linear space in the outer epidermal wall in which the fibrillar structure is more loose and open than elsewhere in the wall." Cuticular channels, on the other hand, appear to be tubules with a defined wall. Miller (19866) clearly showed that transcuticu- lar canalicules open on the outer surface of isolated dewaxed leaf cuticular membranes of Hoya carnosa (Asclepiadaceae).

Hall (1967) demonstrated pores in the cuticle of many plants. The pores, as revealed by replicas, were cup-shaped with somewhat diffuse borders. Their size, as estimated from his illustrations of Eucalyptus pores, was below the resolving power of the light microscope. Miller (1982, 1983, 1985, 1986a, 19866) clearly showed pores and canalicules in the cuticular membrane of 52 species in several families. The canalicules may be tapered, with a diameter of 2.5 to 12.5 pm on the outside, and 0.8 to 1.6 pm on the inside. They are present in both the inner and in the outer sides of the cuticular membrane (Miller 1982). The length of the canalicules depends on the thickness of the cuticular mem- brane, varying from 5 to 25 pm. In Hoya carnosa the pores are 6540/mm2 in the adaxial face and 4680/mm2 in the abaxial (Miller 19866). Jenks et al. (1994) demonstrated the secre- tion of epicuticular wax filaments by cork cells in the sheath of Sorghum bicolor, but even at a relatively high magnifica- tion in the SEM, no pores were visible. In the licuri cuticular membrane, the important fact is the presence of numerous pores on both sides (Figs. 7 and 8). Their diameter is near 85 nm at the abaxial side, and 75 nm at the adaxial. This measurement is not completely exact because the extremity of the (possible) canalicules can be tapered, corresponding to the fact that the profiles of their apertures are not sharp. Their density is 25/pm2 on the adaxial side, and 12/pm2 on the abaxial side. It is reasonable to think that they may facili- tate wax extrusion, as suggested by Lyshede (1982). It was not possible to follow the trajectory of the possible canali- cules.

In the licuri leaf, the observed characteristics suggest that the wax may reach the exterior in part by diffusion, forming the thick layer of the adaxial surface, and the amorphous layer of the abaxial. The orientation of the wax molecules

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agrees with that described by Sargent (19766) in the pericli- nal lamellae inside the cuticle of other plants and to which she ascribes a role in wax extrusion. Another part of the wax could pass through the pores assembling in filaments o r rib- bons. which in turn could unite to form rodlets and columns. The iemnants seen over the columns suggest that the diffu- sion process precedes the extrusion. The ultrastructure of the outer wall o f licuri leaf euidermal cells in thin section under the transmission electron microscope appears to belong to type 6 of Holloway (1982) in which the internal structure is mainly amorphous, as seen in material fixed in glutaral- dehyde - osmium.

Sargent (1976a, 1976b) found lamellae in some primary cuticles, which she thinks may be related to the origin of epicuticular waxes. No similar structure was seen in the cuti- cle of licuri leaflets.

Acknowledgements

The authors are indebted to the National Council for Scien- tific and Technological Development (CNPq), the Institution for Financing Studies and Projects (FINEP), Dr. Wanderley d e Souza for facilities in the laboratory Hertha Meyer of Institute of Biophysics Carlos Chagas Filho, Dr. Maria Nazareth Leal d e Meirelles for the use of the S E M of the Oswaldo Cruz Foundation, Dr. Cecilia G o n ~ a l v e s Costa for reading the manuscript and giving helpful suggestions, Mr . Anthony French Colson for reading the English text, Mr . Luiz Raul Dodsworth Machado for the final revision, Mr . Paulo Rogtrio Ferreira Dias for photographic work, and Mr. Sebastiio da Cruz for help in preparing samples for SEM.

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