Planta

PIanta (Berl.) 129, 123 126 (1976)

9 by Springer-Verlag 1976

In situ Assembly of Cuticular Wax Caroline Sargent* Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond Surrey, TW9 3DS, U.K.

Summary. Cytochemical reactions within the primary cuticle (cutinised layer) indicate that the lamellae are formed from polar lipids. The electron microscope shows that the lamellae are involved in wax formation and it is suggested that the polar lipids provide in situ precursors for the synthesis of cuticular wax.

Introduction

Recent studies of the ultra structure of the cuticle indicate that the cuticular membrane (Sitte and Rennier, 1963) is composed of an outer lamellate region of alternating electron dense and lucent material and an inner region where osmiophilic fibrillae traverse an electron lucent matrix (Chafe and Wardrop, 1972; Juniper and Cox, 1973). These regions are said to correspond directly with the cutinised and cuticularised layer defined by Esau (1953) (Juniper and Cox, 1973), although the terminology was questioned by Sitte and Rennier (1963), who introduced the usage cuticle proper and cuticular layer. The outer lamellate layer develops early in ontogeny whilst the inner layer does not differentiate until extension growth of the underlying epidermal cell is complete (Sargent, 1976) and for this reason the present author prefers the terms primary and secondary cuticle. The lamellate structure occurring in the primary cuticle was described by Hallam (1964) (Martin and Juniper, 1970) who suggested that the lamellae might provide an anastomosing pathway through which wax or wax precursor could reach the surface. Fisher and Bayer (1972) described the lamellae as wax plates. More recently Juniper and Cox (1973) have claimed that the chemical nature of the lamellation is not understood. They showed that the electron dense/ lucent contrast is produced by reaction with osmium * Present address: Botany Department, Imperial College, Prince Consort RD., London, S.W. 7.

tetroxide, however Wardrop (personal communication) has suggested that the lamellae are artefacts of glutaraldehyde fxation. Surface replica techniques have shown additional structure in the cuticle. Depressions may occur in the primary cuticle beneath the epicuticular wax (Mueiler et al., 1954; Hall, 1967) but there is little evidence to indicate that these penetrate the cuticle. Hall (1967) has also described the occurrence of 40 nm diameter channels in freeze fracture replicas of the cuticle. He suggested that wax may be extruded in liquid form through these channels although he did not show any direct connection with the surface, the published micrographs indicating that the channels were restricted to the secondary cuticle. Osmiophilic fibrils which may or may not be related to the channels have been described in this region by O'Brien (1967) who termed them pectic fibrils and by Martin and Juniper (1970) who suggest they may be cellulosic. Other terms used to describe the structures include fibrillar matrix (Hallam, 1970), pectic fingers (Fisher and Bayer, 1972) and micro-channels (Chafe and Wardrop, 1972). Juniper and Cox have concluded that the fibrils may be cellulosic, pectic or of a wax precursor nature. Sargent (unpublished) has shown that the material is unlikely to be cellulosic but believes that the fibrils require more precise definition. This paper is concerned with the cytochemistry and fine structure of cuticular components in relation to wax extrusion. Methods and Materials Leaf material at different stages of development was taken from the following plants: Libertia elegans Poepp. (Iridaceae) HK 000-69 19245 Bobartia gracilis Klatt. (Iridaceae) HK 75-1475. Gordonia axillaris (Ker) D. Dietr. (Theaceae) HK 38257 The material is presently under cultivation at the Royal Botanical Gardens, Kew and voucher specimens have been preserve&

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C. Sargent: In situ Assembly of Cuticular Wax

Fig. 1. Libertia elegans Poepp. x 120,000, Glut. Os.Pb.Ur. The micrograph shows the relationship between wax platelets (wx) and the lamellae of the primary cuticle (C). The lamellae, polar fatty acids in myeloid form, with a period of 6 7 nm, are actively involved in the synthesis of wax

Fig. 4. Bobartia gracilis Klatt. x 200,000 Glut. Os.Pb.Ur. The micrograph shows the primary cuticle resting directly on the pectic lamella of the primary cell wall prior to differentiation of the secondary cuticle, The differential staining of the lamellae may be due to slow penetration by osmium tetroxide

Fig. 2. Libertia elegans Poepp. • 89,000 Glut. Os.Pb.Ur. The micrograph shows the lamellate primary cuticle (c) prior to wax formation. The number of Iamellae at this stage is invariabiy greater than the number found beneath differentiated wax.

Fig. 5. Gordonia axillaris (Ker.) D. Dietr. x 55,000 Glut. Ruthenium red. Pb.Ur. The primary cuticle (C) is composed of a large number of lamellae having a period of about 9 n m Islands of lamellar material (/) are found in the underlying cell wall (w). The secondary cuticle (c) is beginning to differentiate at this stage of development. Abbreviations to Figures: w x = W a x ; C = P r i m a r y cuticle ; C' = Secondary cuticle ; W = Primary cell wall ; L = Lamellar islands; PI = Pectic lamella

Fig. 3. Libertia elegans Poepp. x 55,000 Glut. Lanthanum nitrate, Pb.Ur. In the absence of osmium tetroxide or ruthenium red the lamellae of the primary cuticle are ill defined. Lanthanum nitrate is used to increase the definition of wax platelets (wx). The pectic lamella (p/) is unstained

For electron microscopy the material was fixed in 3% glutaraldehyde. Some was also postfixed or stained in osmium tetroxide or ruthenium red (Luft, 1971). Some samples were floated cuticle side down in 0.5% lanthanum nitrate after fixation. Other techniques were standard. The material was examined in an AEI EM 6 electron microscope.

Results

In the absence of osmium tetroxide or ruthenium red neither the lamellae of the primary cuticle nor

the fibrillar component of the secondary cuticle show sufficient contrast with the electron microscope to determine detail (Fig. 3), Epicuticular wax is ill defined, but lanthanum nitrate applied externally outlines the wax plates without penetrating the cuticle (Fig. 3). The cuticular lamellae react with osmium tetroxide (Figs. 1, 2 and 4) and with ruthenium red (Fig. 5) and the number and period of the tamellae varies between species. In Bobartia gracilis, (monocotyledon) (Fig. 4) there are up to twenty lamellae with a period of 6.5-7nm in the primary cuticle prior to wax formation. The period decreases toward the

C. Sargent: In situ Assembly of Cuticular Wax

centre of the primary cuticle where penetration of osmium tetroxide is poor. At this stage, the section being taken from within the sheathing base of the previous leaf, differentiation of a fibrillar secondary cuticle has not occurred and the primary cuticle (C) rests directly on the primary cell wall (W). In Gordonia axillaris (dicotyledon) (Fig. 5) a large number of cuticular lamellae are developed and islands of lamellar material (L) are found in the underlying cell wall. The period of the lamellae is variable but where formation is most regular is close to 9 nm. Ruthenium red penetrates the cuticle evenly and the poor electron density may be improved by concurrent use of osmium tetroxide (Luft, 1971). The fibrillar secondary cuticle (C I) stains densely in Gordonia, but does not enlarge to the same extent as in the monocotyledons observed. In Libertia ixioides (Figs. 1, 2 and 3) the number of 6-7 nm lamellae in the primary cuticle decreases from a maximum of about 12 (Fig. 2) at the onset of wax differentiation (Fig. 1; Sargent, 1976). The differentiated wax plates are in direct continuity with the cuticular lamellae although no internal lamellar structure is observed; the limiting osmiophilic layer is continuous with the outer electron dense lamella of the primary cuticle.

Discussion

Ruthenium red is a basic inorganic compound which reacts with acidic radicals. Luft (1971) has shown that the standard staining of pectic acid by ruthenium red is not specific but characteristic of a range of acidic reactions. Osmium tetroxide is know to reduce lipidic material (Palade, 1952). The acidic components on the cuticle with which both these reagents react are fatty, pectic and amino acids and phenolic, tannic and other aromatic compounds (Huelin, 1957; Baker etal., 1964; Kolattukudy, 1970). Of these the amino acids and aromatic compounds occur in quantities too small (Huelin, 1957) to account for the extensive reaction observed, although they may contribute in some measure. Pectic acid is restricted to the cuticular layer (Sitte and Rennier, 1963) or secondary cuticle and is unlikely to account for staining in the primary cuticle but may react to provide the electron density of the fibrillae (Juniper and Cox, 1973). Fatty acids remain the only likely components having sufficient reactivity and occurring in sufficient quantity to account for the electron density of the lamellae. Polar lipids or polar fatty acids have been shown to form discrete lamellar structures or myeloid forms. The myeloid forms are polarised bimolecular lipid leaflets which are reduced by and accumulate osmium tetroxide in a layer at the hydrophilic poles (FreyWyssling and Mfihlethaler, 1965). Ruthenium red

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reduces lipid in a similar way (Luft, 1971). These forms have a period which alters with the method of fixation (Gay et aL 1971) with the nature of the lipid and with absorption of water. When sufficient water is absorbed the sign of optical birefringence alters (Frey-Wyssling and M/ihlethaler, 1965). The period of all primary cuticular lamellae measured, 6-9 nm, lie within the range expected for a polarised double molecule of long chain fatty acid, allowing for variability in carbon number, distortion by fixation or hydration and the possible occurrence of protein (enzyme) within the structure. The variable optical signs reported for the primary cuticle (Sitte and Rennier, 1963) may be due to the degree of hydration of the myeloid form. The results strongly indicate that the primary cuticle is composed of polar fatty acids. Kolattukudy (1970) has shown that all major wax components may be derived from cuticular fatty acids, the chain lengths of the cuticular waxes and fatty acids being closely related. The pathway of synthesis is not entirely understood, but the wax hydrocarbons may be derived from fatty acids either by decarboxylation or by condensation. Since Kolattukudy (1965) has shown that ~4C-labeled waxes may be retrieved from the cuticle after short (10 sec) immersion in solvent it suggests that synthesis occurs at, or very close to, the epicuticular position or that the waxes are exceedingly mobile in the solvent. Previous investigations have failed to show conclusively any mechanism whereby liquid, paracrystalline or crystalline wax may be extruded from the surface of the cuticle (Martin and Juniper, 1970, Hallain, 1970). Despite the numerous hypotheses concerning pores or channels in continuum with the epicuticle, electron micrographs indicate that these structures terminate in the secondary cuticle without penetrating the primary cuticle. The micrographs of Libertia indicate that the primary cuticular lamellae are involved in wax formation, and since the lamellae are probably composed of fatty acids which are known to be wax precursors (Channon and Chibnall, 1929; Martin and Juniper, 1970; Kolattukudy, 1970) it is proposed that wax formation occurs through biochemical transformation in the outer lamellae of the primary cuticle. Synthesis is likely to be mediated by proteins (enzymes) within the lamellae which could occur without altering the gross structure of the myeloid form (Frey-Wyssling and Mfihlethaler, 1965). Since first reported by de CondoIle (1827) it has been shown repeatedly that cuticular wax will reform only a limited number of times after mechanical removal. It is suggested that this limit is imposed by the thickness of the generating primary cuticle, and by the quantities of polar lipids (lamellae) within that cuticle. The electron density of the fibrillae suggests

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the movement of additional precursors through the secondary cuticle (Juniper and Cox, 1975, Sargent, 1976) although the application of radiographic techniques will be required to establish this. Since the primary cuticle differentiates before development of the secondary cuticle begins, some fatty acids or wax precursors are in situ prior to secondary growth and movement across the secondary cuticle may be unnecessary. The author is grateful to Dr. John Levy, Dr. John Gay and Dr. David Cutler for reading the manuscript.

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C. Sargent: In situ Assembly of Cuticular Wax faces. II. Cuticular pores and wax formation. J. Ultrastruct. Res. 17, 34-44 (1967). Gay, J.L., Greenwood, A.D., Heath, hB. : The formation and behavionr of vacuoles (vesicles) during oosphere development in Saprolegnia. J. gen. Microbiol. 65, 233-241 (1971) Hallam, N.D. : Leaf wax fine structure and ontogeny in Eucalyptus demonstrated by means of a specialised fixation technique. J. Microsc. 92, 137 144 (1970) Huelin, F.E.: Studies in the natural coating of apples. IV. The nature of cutin. Aust. J. biol. Sci. 12, 175-181 (1957) Jarvis, L.H., Wardrop, A.B.: The development of the cuticle in Phormium tenax. Planta (Berl.) 119, 101 112 (1974) Juniper, B.E., Cox, G.C.: The anatomy of the leaf surface: The first line of defence. Pestic. Sci. 4, 543-561 (1973) Kolattukudy, P.E.: Biosynthesis of cuticular lipids. Ann. Rev. Plant Physiol. 21, 163-192 (1970) Luft, J.H. : Ruthenium red and violet. II. Fine structural localisation in animal tissues. Anat. Rec. 171, 309-416 (1971) Martin, J.T., Juniper, B.E.: The cuticles of plants. London: Edward Arnold 1970 Mueller, L.E., Carr, P.H., Loomis, W.E.: The submicroscopic structure of plant surfaces. An. J. Bot. 41, 593 600 (1954) O'Brien, T.P.: The fine structure of the oat coleoptile. 1. The epidermal cells of the extreme apex. Protoplasma (Wien) 63, 385-416 (1967) Palade, G.E.: A study of fixation for electron microscopy. J. exp. Med. 95, 285~97 (1952) Sargent, C.M.: The occurence of a secondary cuticle in Libertia elegans (Iridaceae). Ann. Bot. (1976) (in press) Sitte, P., Rennier, R. : Untersuchungen an cuticulfiren Zellwachsschichten. Planta (Berl.) 60, 19-40 (1963)

Received 14 September; accepted 10 November 1975