Planta (Berl.) 93, 257--268 (1970) 9 by S p r i n g e r - V e r l a g 1970
Growth and Regeneration of Waxes on the Leaves of Eucalyptus 57. D. HALLiM Botany School, University of Melbourne Parkville, Victoria, Australia Received May 16/June 5, 1970
Summary. The relationships of wax morphology to wax chemistry and the effects of light intensity on wax development were investigated using rubbing techniques to produce nearly wax free cuticular surfaces. Wax regeneration took place rapidly on leaves which were in their exponential stage of expansion, but only slowly on those that had fnlly expanded. The pattern of wax development suggested that tube waxes originate as fiat plates that fuse along one edge to form a hollow structure. Introduction A plant having a waxy surface is water repellent. This is important when considering the entry of compounds into leaves and the adhesion of fungicides and insecticides to them when such substances are used for agricultural or horticultural purposes. I t is understandable therefore t h a t m a n y investigations on wax development and wax production have been carried out on agriculturally important plants. Dewey, Gregory and Pfeiffer (1956), for example, reported t h a t wind blown particles of sand damaged the wax deposits on leaves and stems, this leading to an increase in the susceptibility of crop plants to herbicides. W a x production through the cuticle is not fully understood. I t m a y take place by means of pores (Hall and Donaldson, 1962, 1963) or by means of cuticular lamellae (Hallam, 1964). The ultimate pattern of wax arrangement m a y be partly be a result of the exit p a t h w a y through the cuticle. I n an earlier paper (Hallam, 1964) it was reported t h a t there are three main leaf wax types within the genus Eucalyptus, tubes, plates, and a mixed wax of both tubes and plates. A detailed study of over 300 species, to be published elsewhere, has shown t h a t depending on the degree of ornamentation or complexity of the wax types, various forms of each can be related to the t a x o n o m y of the genus. 18b Planta (Berl.), Bd. 93
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In t h e p r e s e n t p a p e r t h e effects of light i n t e n s i t y on w a x developm e n t a n d w a x r e g e n e r a t i o n after m e c h a n i c a l r e m o v a l are r e p o r t e d .
Methods
Carbon Replica Preparation Carbon replicas were prepared by evaporating carbon on to the leaf surface at a vacuum of 3 • 10-4 mm Hg using an Edwards 12 E 6 vacuum coating unit. The carbon replicas, supported with Parlodion and backed with Bedacryl 122 X, were stripped from the leaf surface with a polythene based tape (Polytape 504, Sellotape Pry. Ltd.). The plastics and any remaining waxes were dissolved with a 1:1 vv mixture of acetone:ethyl acetate and followed by immersion in two changes of chloroform. The replicas were picked up on 200 mesh grids and examined in a Siemens Elmiskop 1 electron microscope at 80 kV.
Wax Extractions Waxes were removed from leaves by refluxing for 1 h over petroleum ether (40--60 ~ C BP). After evaporation of the solvent, the residue, composed of wax, oils and pigments was dissolved in a minimum volume of boiling acetone. The wax crystallized out of solution on cooling. Species with plate wax on the leaf surface gave a wax extract that crystallized from solntion as small highly refractive plates. The tube waxes crystallized as a flocculent mass. The wax samples were filtered, washed with cold acetone, and allowed to dry.
Results and Discussion
The Relationships el Wax Chemistry to Wax Morphology I t has been suggested t h a t w a x precursors m o v e t h r o u g h t h e leaf cuticle a n d p o l y m e r i z e or oxidize a t t h e surface to t h e i r m o r p h o l o g i c a l form. I t has also been suggested t h a t t h e precursors m o v e t h r o u g h t h e cuticle v i a a long p a t h w a y of a n a s t o m i z i n g euticular lamellae (Hallam, 1964). This w o u l d m e a n t h a t t h e i r f o r m is d e p e n d e n t m o r e on t h e i r chemical c o m p o n e n t s t h a n on t h e w a y in which t h e y are e x t r u d e d . I f this is so, i t should be possible t o crystallize leaf w a x f r o m solution in a f o r m similar to t h a t which a p p e a r s n o r m a l l y on t h e leaf surface. P r e p a r e d s a m p l e s of Eucalyptus globulus a n d E. ovata leaf w a x were dissolved i n h o t a c e t o n e a n d allowed t o recrystallize slowly f r o m solution on t o a microscope slide in a n a t m o s p h e r e s a t u r a t e d w i t h acetone v a p o u r . The acetone was allowed to e v a p o r a t e a f t e r c r y s t a l l i z a t i o n h a d occurred a n d c a r b o n replicas p r e p a r e d of t h e w a x d e p o s i t on t h e slide. T h e t u b e w a x of E. globutus (Fig. 1 a, b) r e c r y s t a l l i z e d in a f o r m t h a t is a t least r e l a t a b l e t o t h a t d e v e l o p e d on t h e leaf n a t u r a l l y . Similarly, t h e p l a t e w a x of E. ovata (Fig. 1 c, d) crystallized as p l a t e s on t h e glass slide (although these were v e r y m u c h larger t h a n t h o s e d e v e l o p e d n a t u r a l l y ) , i n d i c a t i n g a close link b e t w e e n t h e m o r p h o l o g y a n d chemical c o m p o s i t i o n
Fig. 1. a Carbon replica of the leaf w a x on Eucalyptus globulus, b Carbon replica of the leaf w a x of Eucalyptus globulus recrystallized f r o m acetone at - - 5 ~ C. c Carbon replica of the leaf w a x on Eucalyptus ovata, d Carbon replica of the leaf w a x of Eucalyptus ovata recrystallized f r o m acetone a t - - 5 ~ C 18c Planta (Berl.), Bd. 93
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of these waxes. I t is probable t h a t the rapid crystallization taking place under these artificial conditions, as well as the differential solubility of some of the components in cold acetone causes some variation in wax structure when compared with the wax on the leaf. Undoubtedly there are contaminants in the samples also. These findings however provide further evidence that wax m o r p h o l o g y is more probably a process resulting from the chemical oxidation, polymerization or reerystallization of the wax or its precursors, rather than a result of an extrusion process.
Light Intensity and Wax Development Seedlings of Eucalyptus cypellocarpa (tube wax), E. radiata (plate wax) and E. obliqua (sparse plate wax around the stomata) were grown under shade screens in the open so t h a t the plants were subjected to the normal regimes of temperature, rainfall and light intensities similar to those found in their natural environment. Under the shade screens the light intensity was 30, 20, 6.5, 2.5 and 1.2% of full daylight, this measured on an overcast day with a selenium cell. Carbon replicas were made from the lower surface of the first fully expanded leaves of each of the species when three weeks old and examined in the electron microscope. At light intensities above 20% of full daylight, the leaf wax is fully developed and at 30% light, the mierographs showed the wax to be little different to those at 20%. For this reason, only wax from plants grown at 20% of full daylight and below is illustrated. The variation in leaf wax with light intensity on E. cypellocarpa is illustrated in Fig. 2. The wax of the leaves grown at the two lowest light intensities (1.2 and 2.5%) is sparsely arranged on the leaf surface and the tubes lack the complex branching pattern shown at 6.5 and 20 %. At these higher light intensities, the wax structure is similar to that found on E. cypellocarpa grown under full sunlight. Plants of E. radiata grown at the three lowest light intensities (1.2, 2.5 and 6.5% of full daylight) had wax plates which were stellate and with long projections. At the higher light intensities (20 and 30% of full daylight) the plates where rhomboidal and similar to the wax type seen on fully expanded leaves grown in full sunlight (compare Fig. 3 a, b). The leaves of E. obliqua when grown at the lowest light intensity were virtually devoid of wax and showed only a smooth cuticle (Fig. 3 c). At 6.5% light, wax was produced and at 20% the wax was fully developed and essentially similar to t h a t found on fully expanded juvenile leaves grown in full sunlight (see Fig. 3d). W a x was produced in greatest amount in regions surrounding the stomata, this being typical of several species in the section l~enantherae (Normales).
Eucalyptus cypellocarpa. Grown under 1.2 % full daylight, b Eucalyptus cypellocarpa. Grown ur~der 2.5% full d~ylight, c Eucalyptus cypellocarpa. Grown under 6.5 % full daylight, d Eucalyptus cypellocarpa. Grown under 20 % ~ul] daylight
Fig. 2. a
Fig. 3. a Eucalyptus radiata, Grown under 1.2% full daylight, b Eucalyptus radiata. Grown under 20% full daylight, c Eucalyptus obliqua. Grown under 1.2% full daylight, d Eucalyptus obliqua. Grown under 20% full daylight
N. D. Hallam: Growth and Regeneration of Waxes
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Wax Regeneration niter Removal To p r o d u c e a wax-free cuticle surface, t h e leaf surface was g e n t l y r u b b e d with c o t t o n wool. A s t u d y of t h e r e g e n e r a t i o n process was m a d e on four species, two w i t h t u b e wax (E. perriniana a n d E. cephaloearpa) a n d two with plates (E. alba a n d E. ovata). The two t u b e w a x species were p a r t i c u l a r l y useful for this s t u d y as t h e juvenile leaves are round, opposite a n d deeussate, t h u s m a k i n g a t w i n leaf m e t h o d of i n v e s t i g a t i o n possible. One p a i r of leaves could be e x a m i n e d j u s t after r u b b i n g a n d t h e opposite leaf could be e x a m i n e d after a suitable t i m e span, so t h a t w a x r e g e n e r a t i o n could be followed n o t only on t h e same p l a n t a n d shoot, b u t also on a leaf of t h e same physiological age. W a x was r e m o v e d from the first four pairs of leaves ranging from 3 m m in d i a m e t e r a t t h e a p e x to 2.5 cm a t leaf pair n u m b e r four, these last leaves being fully e x p a n d e d a n d sclerophyllous. Replicas were m a d e of the u p p e r a n d lower surfaces of one of t h e leaves of each p a i r imm e d i a t e l y a f t e r rubbing. Considerable w a x r e g e n e r a t i o n t o o k place a n d there was no t i m e lag in lower to u p p e r surface r e g e n e r a t i o n as h a d been n o t e d b y J u n i p e r (1960) on Pisum. The leaves were c o n s i d e r a b l y waxier after t w e n t y four hours t h a n those of Pisum, i.e., w a x regeneration t o o k place more q u i c k l y in Eucalyptus. The p l a t e w a x species, E. alba a n d E. ovata do n o t h a v e opposite juvenile leaves a n d small pieces of t h e same leaf were used t h r o u g h o u t , w a x r e g e n e r a t i o n being c o n s i d e r a b l y slower on these species. F o u r shoots were t r e a t e d as a b o v e to p r o v i d e enough m a t e r i a l for successive e x a m i n a t i o n s of t h e leaf surfaces a t 1, 2, 3, 4, 5, 6, 24, a n d Table. Advancing contact angle o/a 2/d droplet o/the abaxial lea/sur/ace Time (hrs)
Leaf from apex
E. perriniana
1
2 3 4 5 6 24 48
E. alba
1
2
3
1
2
3
1
2
3
145 120 105 88 83 76 65 45 38
110 110 115 lO1 90 73 73 52 32
105 98 100 106 94 80 66 61 35
123 111 115 110 99 93 72 36 32
146 134 122 117 108 89 68 31 35
133 138 131 121 110 87 76 40 35
105 110 113 90 88 93 83 75 58
112 123 108 85 73 81 88 71 63
123 116 114 95 82 88 80 78 65
(o) 0
E. cephalocarpa
(o)
(o)
(o)
(o)
(o)
(o)
(o)
(o)
Fig. 4 a--f
N. D. Hallam: Growth and Regeneration of Waxes
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48 hours after w a x removal. This was especially i m p o r t a n t as t h e first leaves were o n l y slightly larger t h a n a 2.3 m m grid. The c o n t a c t angle of a 2 ~1 d r o p p l a c e d on t h e leaf surface a t each of t h e s a m p l i n g stages d u r i n g t h e r e g e n e r a t i o n was o b s e r v e d (see Table). This was done using a h o r i z o n t a l microscope a n d c a m e r a lucida. I n general species h a v i n g t u b e waxes are m o r e w a t e r repellent t h a n those h a v i n g p l a t e waxes. I n Fig. 5, t h e leaf of E. perriniana has h a d t h e w a x r u b b e d off t h e left side of t h e lamina, this resulting in a n easily w e t t e d surface. The u n t r e a t e d r i g h t h a n d side r e m a i n s w a t e r repellent.
Fig. 5. Perfoliate leaf of Eucalyptus perriniana. The wax on the left hand side of the leaf has been removed by rubbing and is therefore easily wetted. The untouched right hand side of the leaf is covered with a "bloom" of tube wax and is water repellent
The t h i c k l a y e r of t u b e w a x on species such as E. perriniana a n d E. cephalocarpa is a l m o s t completet:g:removed b y r u b b i n g (Fig. 4a) a n d only b r o k e n t u b e s remain. I n E. alba however, t h e e p i d e r m a l cells are d o m e d a n d t h e w a x r e m a i n s u n t o u c h e d in t h e areas of t h e a n t i c l i n a l walls of t h e e p i d e r m a l cells (Fig. 6a). I n t h e t h r e e species e x a m i n e d , w a x r e g e n e r a t i o n follows t h e decrease in c o n t a c t angle t h a t occurs b e t w e e n 4 - - 6 hours a f t e r r u b b i n g t h e leaf. A f t e r t h r e e hours, Eucalyptus alba (Fig. 6a) shows w a x r c g r o w t h as small u n d i f f e r e n t i a t e d platelets. The r e g e n e r a t i o n proceeds r e l a t i v e l y slowly c o m p a r e d w i t h t h e t u b e w a x species a n d even a f t e r 48 hours (Fig. 6b) t h e r e g e n e r a t e d w a x p l a t e l e t s
Fig. 4. a Eucalyptus cephalocarpa. Leaf surface immediately after wax removal. b Eucalyptus cephalocarpa. Leaf surface 1 h after wax removal, c Eucalyptus cephalocar2a. Leaf surface 2 h after wax removal, d Eucalyptus cephalocarpa. Leaf surface 4 h after wax removal, e Eucalyptus cephalocarpa. Leaf surface 5 h after wax removal, f Eucalyptus cephalocarpa. Leaf surface 24 h after wax removal
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Fig. 6 a ~nd b
Growth and Regeneration of Waxes
267
are still smaller than the original wax between epidermal cells. Eucalyptus cephalocarpa showed a faster regeneration than E. alba, an almost complete cover of compoundly branching wax tubes being produced in a 24 hour period. Even 2 hours after rubbing (Fig. 4 c) it is apparent that the tubes originate from the cuticle as narrow plates, these appearing to roll over and fuse their edges to form a hollow tube. This is more apparent on the four (Fig. 4d) and five hour (Fig. de) electron micrographs. After twenty four hours (Fig. 4f) the wax had regenerated almost completely the leaf surface being covered with compoundly branching tube wax virtually identical to that on leaves of comparable age that had not been rubbed. This regeneration is of the order of 15.0 • 10-s g/cm 2 of wax in a twenty four hour period. I n view of the pattern of development of the waxes on species such as E. perriniana and E. cephalocarpa; that is to say, plate waxes that fuse along their free edges to form hollow structures, it is felt that the term " t u b e " , is more appropriate than the term "rod" or "fibril". These investigations have demonstrated that leaf waxes of the genus Eucalyptus can be recrystallized from solution in a pattern relatable to that which is normally developed on the leaf surface. This suggests a close link between wax chemistry and wax morphology. While the distribution of wax over the leaf surface is markedly influenced by light intensities under which the leaf is growing, the wax morphology is not substantially altered. This is probably one of the reasons for the greater susceptibility of eucalypt seedlings to fungal pathogens when grown in low light regimes. Experiments involving the removal of surface wax by rubbing demonstrated a rapid wax regrowth and indicated that tube waxes are hollow structures that reach their mature form quickly. Plate waxes originate as small unornamented platelets and take longer to develop their final morphology. I n contrast to previous accounts, no evidence of pores in the cuticle could be found immediately after wax removal or after treatment with organic solvents. The author is grateful to Professor T. C. Chambers and Professor J. S. Turner for their interest in this project and providing electron microscope facilities, to Miss Margaret Waite for skilled technical assistance and to Mrs. Caroline Myers and Mrs. Margot ttindle for help with photography. This project was financed by a G. J. Coles Post Graduate Award.
~ig. 6. a E.~calyptus alba. Wax platelets developing 3 h after wax removal by rubbing. The epidermal cells are domed and some wax remains untouched between the cells as in the upper right hand side of the picture, b Eucalyptus alba. Regenerating wax plates 48 h after removal. The new wax plates are oriented in rows surrounded by the original untouched plates
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References Dewey, O.R., Gregory, P., Pfciffer, 1~. K.: Factors affecting the susceptibility of peas to selective dinitroherbicides. Brit. Weed Control Conf. Proc. 1, 313---327 (1956). Hall, D. M., Donaldson, L. A. : Secretion from pores of surface wax on plant leaves. Nature (Lond.) 194, 1196 (1962). - - - - The ultrastrueture of wax deposits on plant leaf surfaces. 1. Growth of wax on leaves of Tri/olium repens. J. Ultrastruct. Res. 9, 259--267 (1963). Hallam, N. D. : Sectioning and electron microscopy of eucalypt leaf waxes. Aust. J. biol. Sci, 17, 587--590 (1964). Juniper, B.E.: Growth, development and the effect of the environment on the ultrastructure of plant surfaces. J. Lima. Soc. (Bot.) 56, 413--419 (1960). N. D. Hallam Unit of Experimental Agronomy Department of Agricultural Science University of Oxford Oxford OXI 3 PF, England
Growth and Regeneration of Waxes on the Leaves of Eucalyptus 57. D. HALLiM Botany School, University of Melbourne Parkville, Victoria, Australia Received May 16/June 5, 1970
Summary. The relationships of wax morphology to wax chemistry and the effects of light intensity on wax development were investigated using rubbing techniques to produce nearly wax free cuticular surfaces. Wax regeneration took place rapidly on leaves which were in their exponential stage of expansion, but only slowly on those that had fnlly expanded. The pattern of wax development suggested that tube waxes originate as fiat plates that fuse along one edge to form a hollow structure. Introduction A plant having a waxy surface is water repellent. This is important when considering the entry of compounds into leaves and the adhesion of fungicides and insecticides to them when such substances are used for agricultural or horticultural purposes. I t is understandable therefore t h a t m a n y investigations on wax development and wax production have been carried out on agriculturally important plants. Dewey, Gregory and Pfeiffer (1956), for example, reported t h a t wind blown particles of sand damaged the wax deposits on leaves and stems, this leading to an increase in the susceptibility of crop plants to herbicides. W a x production through the cuticle is not fully understood. I t m a y take place by means of pores (Hall and Donaldson, 1962, 1963) or by means of cuticular lamellae (Hallam, 1964). The ultimate pattern of wax arrangement m a y be partly be a result of the exit p a t h w a y through the cuticle. I n an earlier paper (Hallam, 1964) it was reported t h a t there are three main leaf wax types within the genus Eucalyptus, tubes, plates, and a mixed wax of both tubes and plates. A detailed study of over 300 species, to be published elsewhere, has shown t h a t depending on the degree of ornamentation or complexity of the wax types, various forms of each can be related to the t a x o n o m y of the genus. 18b Planta (Berl.), Bd. 93
258
N.D. Hallam: Growth and Regeneration of Waxes
In t h e p r e s e n t p a p e r t h e effects of light i n t e n s i t y on w a x developm e n t a n d w a x r e g e n e r a t i o n after m e c h a n i c a l r e m o v a l are r e p o r t e d .
Methods
Carbon Replica Preparation Carbon replicas were prepared by evaporating carbon on to the leaf surface at a vacuum of 3 • 10-4 mm Hg using an Edwards 12 E 6 vacuum coating unit. The carbon replicas, supported with Parlodion and backed with Bedacryl 122 X, were stripped from the leaf surface with a polythene based tape (Polytape 504, Sellotape Pry. Ltd.). The plastics and any remaining waxes were dissolved with a 1:1 vv mixture of acetone:ethyl acetate and followed by immersion in two changes of chloroform. The replicas were picked up on 200 mesh grids and examined in a Siemens Elmiskop 1 electron microscope at 80 kV.
Wax Extractions Waxes were removed from leaves by refluxing for 1 h over petroleum ether (40--60 ~ C BP). After evaporation of the solvent, the residue, composed of wax, oils and pigments was dissolved in a minimum volume of boiling acetone. The wax crystallized out of solution on cooling. Species with plate wax on the leaf surface gave a wax extract that crystallized from solntion as small highly refractive plates. The tube waxes crystallized as a flocculent mass. The wax samples were filtered, washed with cold acetone, and allowed to dry.
Results and Discussion
The Relationships el Wax Chemistry to Wax Morphology I t has been suggested t h a t w a x precursors m o v e t h r o u g h t h e leaf cuticle a n d p o l y m e r i z e or oxidize a t t h e surface to t h e i r m o r p h o l o g i c a l form. I t has also been suggested t h a t t h e precursors m o v e t h r o u g h t h e cuticle v i a a long p a t h w a y of a n a s t o m i z i n g euticular lamellae (Hallam, 1964). This w o u l d m e a n t h a t t h e i r f o r m is d e p e n d e n t m o r e on t h e i r chemical c o m p o n e n t s t h a n on t h e w a y in which t h e y are e x t r u d e d . I f this is so, i t should be possible t o crystallize leaf w a x f r o m solution in a f o r m similar to t h a t which a p p e a r s n o r m a l l y on t h e leaf surface. P r e p a r e d s a m p l e s of Eucalyptus globulus a n d E. ovata leaf w a x were dissolved i n h o t a c e t o n e a n d allowed t o recrystallize slowly f r o m solution on t o a microscope slide in a n a t m o s p h e r e s a t u r a t e d w i t h acetone v a p o u r . The acetone was allowed to e v a p o r a t e a f t e r c r y s t a l l i z a t i o n h a d occurred a n d c a r b o n replicas p r e p a r e d of t h e w a x d e p o s i t on t h e slide. T h e t u b e w a x of E. globutus (Fig. 1 a, b) r e c r y s t a l l i z e d in a f o r m t h a t is a t least r e l a t a b l e t o t h a t d e v e l o p e d on t h e leaf n a t u r a l l y . Similarly, t h e p l a t e w a x of E. ovata (Fig. 1 c, d) crystallized as p l a t e s on t h e glass slide (although these were v e r y m u c h larger t h a n t h o s e d e v e l o p e d n a t u r a l l y ) , i n d i c a t i n g a close link b e t w e e n t h e m o r p h o l o g y a n d chemical c o m p o s i t i o n
Fig. 1. a Carbon replica of the leaf w a x on Eucalyptus globulus, b Carbon replica of the leaf w a x of Eucalyptus globulus recrystallized f r o m acetone at - - 5 ~ C. c Carbon replica of the leaf w a x on Eucalyptus ovata, d Carbon replica of the leaf w a x of Eucalyptus ovata recrystallized f r o m acetone a t - - 5 ~ C 18c Planta (Berl.), Bd. 93
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N. D. Hallam: Growth and Regeneration of Waxes
of these waxes. I t is probable t h a t the rapid crystallization taking place under these artificial conditions, as well as the differential solubility of some of the components in cold acetone causes some variation in wax structure when compared with the wax on the leaf. Undoubtedly there are contaminants in the samples also. These findings however provide further evidence that wax m o r p h o l o g y is more probably a process resulting from the chemical oxidation, polymerization or reerystallization of the wax or its precursors, rather than a result of an extrusion process.
Light Intensity and Wax Development Seedlings of Eucalyptus cypellocarpa (tube wax), E. radiata (plate wax) and E. obliqua (sparse plate wax around the stomata) were grown under shade screens in the open so t h a t the plants were subjected to the normal regimes of temperature, rainfall and light intensities similar to those found in their natural environment. Under the shade screens the light intensity was 30, 20, 6.5, 2.5 and 1.2% of full daylight, this measured on an overcast day with a selenium cell. Carbon replicas were made from the lower surface of the first fully expanded leaves of each of the species when three weeks old and examined in the electron microscope. At light intensities above 20% of full daylight, the leaf wax is fully developed and at 30% light, the mierographs showed the wax to be little different to those at 20%. For this reason, only wax from plants grown at 20% of full daylight and below is illustrated. The variation in leaf wax with light intensity on E. cypellocarpa is illustrated in Fig. 2. The wax of the leaves grown at the two lowest light intensities (1.2 and 2.5%) is sparsely arranged on the leaf surface and the tubes lack the complex branching pattern shown at 6.5 and 20 %. At these higher light intensities, the wax structure is similar to that found on E. cypellocarpa grown under full sunlight. Plants of E. radiata grown at the three lowest light intensities (1.2, 2.5 and 6.5% of full daylight) had wax plates which were stellate and with long projections. At the higher light intensities (20 and 30% of full daylight) the plates where rhomboidal and similar to the wax type seen on fully expanded leaves grown in full sunlight (compare Fig. 3 a, b). The leaves of E. obliqua when grown at the lowest light intensity were virtually devoid of wax and showed only a smooth cuticle (Fig. 3 c). At 6.5% light, wax was produced and at 20% the wax was fully developed and essentially similar to t h a t found on fully expanded juvenile leaves grown in full sunlight (see Fig. 3d). W a x was produced in greatest amount in regions surrounding the stomata, this being typical of several species in the section l~enantherae (Normales).
Eucalyptus cypellocarpa. Grown under 1.2 % full daylight, b Eucalyptus cypellocarpa. Grown ur~der 2.5% full d~ylight, c Eucalyptus cypellocarpa. Grown under 6.5 % full daylight, d Eucalyptus cypellocarpa. Grown under 20 % ~ul] daylight
Fig. 2. a
Fig. 3. a Eucalyptus radiata, Grown under 1.2% full daylight, b Eucalyptus radiata. Grown under 20% full daylight, c Eucalyptus obliqua. Grown under 1.2% full daylight, d Eucalyptus obliqua. Grown under 20% full daylight
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Wax Regeneration niter Removal To p r o d u c e a wax-free cuticle surface, t h e leaf surface was g e n t l y r u b b e d with c o t t o n wool. A s t u d y of t h e r e g e n e r a t i o n process was m a d e on four species, two w i t h t u b e wax (E. perriniana a n d E. cephaloearpa) a n d two with plates (E. alba a n d E. ovata). The two t u b e w a x species were p a r t i c u l a r l y useful for this s t u d y as t h e juvenile leaves are round, opposite a n d deeussate, t h u s m a k i n g a t w i n leaf m e t h o d of i n v e s t i g a t i o n possible. One p a i r of leaves could be e x a m i n e d j u s t after r u b b i n g a n d t h e opposite leaf could be e x a m i n e d after a suitable t i m e span, so t h a t w a x r e g e n e r a t i o n could be followed n o t only on t h e same p l a n t a n d shoot, b u t also on a leaf of t h e same physiological age. W a x was r e m o v e d from the first four pairs of leaves ranging from 3 m m in d i a m e t e r a t t h e a p e x to 2.5 cm a t leaf pair n u m b e r four, these last leaves being fully e x p a n d e d a n d sclerophyllous. Replicas were m a d e of the u p p e r a n d lower surfaces of one of t h e leaves of each p a i r imm e d i a t e l y a f t e r rubbing. Considerable w a x r e g e n e r a t i o n t o o k place a n d there was no t i m e lag in lower to u p p e r surface r e g e n e r a t i o n as h a d been n o t e d b y J u n i p e r (1960) on Pisum. The leaves were c o n s i d e r a b l y waxier after t w e n t y four hours t h a n those of Pisum, i.e., w a x regeneration t o o k place more q u i c k l y in Eucalyptus. The p l a t e w a x species, E. alba a n d E. ovata do n o t h a v e opposite juvenile leaves a n d small pieces of t h e same leaf were used t h r o u g h o u t , w a x r e g e n e r a t i o n being c o n s i d e r a b l y slower on these species. F o u r shoots were t r e a t e d as a b o v e to p r o v i d e enough m a t e r i a l for successive e x a m i n a t i o n s of t h e leaf surfaces a t 1, 2, 3, 4, 5, 6, 24, a n d Table. Advancing contact angle o/a 2/d droplet o/the abaxial lea/sur/ace Time (hrs)
Leaf from apex
E. perriniana
1
2 3 4 5 6 24 48
E. alba
1
2
3
1
2
3
1
2
3
145 120 105 88 83 76 65 45 38
110 110 115 lO1 90 73 73 52 32
105 98 100 106 94 80 66 61 35
123 111 115 110 99 93 72 36 32
146 134 122 117 108 89 68 31 35
133 138 131 121 110 87 76 40 35
105 110 113 90 88 93 83 75 58
112 123 108 85 73 81 88 71 63
123 116 114 95 82 88 80 78 65
(o) 0
E. cephalocarpa
(o)
(o)
(o)
(o)
(o)
(o)
(o)
(o)
Fig. 4 a--f
N. D. Hallam: Growth and Regeneration of Waxes
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48 hours after w a x removal. This was especially i m p o r t a n t as t h e first leaves were o n l y slightly larger t h a n a 2.3 m m grid. The c o n t a c t angle of a 2 ~1 d r o p p l a c e d on t h e leaf surface a t each of t h e s a m p l i n g stages d u r i n g t h e r e g e n e r a t i o n was o b s e r v e d (see Table). This was done using a h o r i z o n t a l microscope a n d c a m e r a lucida. I n general species h a v i n g t u b e waxes are m o r e w a t e r repellent t h a n those h a v i n g p l a t e waxes. I n Fig. 5, t h e leaf of E. perriniana has h a d t h e w a x r u b b e d off t h e left side of t h e lamina, this resulting in a n easily w e t t e d surface. The u n t r e a t e d r i g h t h a n d side r e m a i n s w a t e r repellent.
Fig. 5. Perfoliate leaf of Eucalyptus perriniana. The wax on the left hand side of the leaf has been removed by rubbing and is therefore easily wetted. The untouched right hand side of the leaf is covered with a "bloom" of tube wax and is water repellent
The t h i c k l a y e r of t u b e w a x on species such as E. perriniana a n d E. cephalocarpa is a l m o s t completet:g:removed b y r u b b i n g (Fig. 4a) a n d only b r o k e n t u b e s remain. I n E. alba however, t h e e p i d e r m a l cells are d o m e d a n d t h e w a x r e m a i n s u n t o u c h e d in t h e areas of t h e a n t i c l i n a l walls of t h e e p i d e r m a l cells (Fig. 6a). I n t h e t h r e e species e x a m i n e d , w a x r e g e n e r a t i o n follows t h e decrease in c o n t a c t angle t h a t occurs b e t w e e n 4 - - 6 hours a f t e r r u b b i n g t h e leaf. A f t e r t h r e e hours, Eucalyptus alba (Fig. 6a) shows w a x r c g r o w t h as small u n d i f f e r e n t i a t e d platelets. The r e g e n e r a t i o n proceeds r e l a t i v e l y slowly c o m p a r e d w i t h t h e t u b e w a x species a n d even a f t e r 48 hours (Fig. 6b) t h e r e g e n e r a t e d w a x p l a t e l e t s
Fig. 4. a Eucalyptus cephalocarpa. Leaf surface immediately after wax removal. b Eucalyptus cephalocarpa. Leaf surface 1 h after wax removal, c Eucalyptus cephalocar2a. Leaf surface 2 h after wax removal, d Eucalyptus cephalocarpa. Leaf surface 4 h after wax removal, e Eucalyptus cephalocarpa. Leaf surface 5 h after wax removal, f Eucalyptus cephalocarpa. Leaf surface 24 h after wax removal
266
iN. D. H~llam:
Fig. 6 a ~nd b
Growth and Regeneration of Waxes
267
are still smaller than the original wax between epidermal cells. Eucalyptus cephalocarpa showed a faster regeneration than E. alba, an almost complete cover of compoundly branching wax tubes being produced in a 24 hour period. Even 2 hours after rubbing (Fig. 4 c) it is apparent that the tubes originate from the cuticle as narrow plates, these appearing to roll over and fuse their edges to form a hollow tube. This is more apparent on the four (Fig. 4d) and five hour (Fig. de) electron micrographs. After twenty four hours (Fig. 4f) the wax had regenerated almost completely the leaf surface being covered with compoundly branching tube wax virtually identical to that on leaves of comparable age that had not been rubbed. This regeneration is of the order of 15.0 • 10-s g/cm 2 of wax in a twenty four hour period. I n view of the pattern of development of the waxes on species such as E. perriniana and E. cephalocarpa; that is to say, plate waxes that fuse along their free edges to form hollow structures, it is felt that the term " t u b e " , is more appropriate than the term "rod" or "fibril". These investigations have demonstrated that leaf waxes of the genus Eucalyptus can be recrystallized from solution in a pattern relatable to that which is normally developed on the leaf surface. This suggests a close link between wax chemistry and wax morphology. While the distribution of wax over the leaf surface is markedly influenced by light intensities under which the leaf is growing, the wax morphology is not substantially altered. This is probably one of the reasons for the greater susceptibility of eucalypt seedlings to fungal pathogens when grown in low light regimes. Experiments involving the removal of surface wax by rubbing demonstrated a rapid wax regrowth and indicated that tube waxes are hollow structures that reach their mature form quickly. Plate waxes originate as small unornamented platelets and take longer to develop their final morphology. I n contrast to previous accounts, no evidence of pores in the cuticle could be found immediately after wax removal or after treatment with organic solvents. The author is grateful to Professor T. C. Chambers and Professor J. S. Turner for their interest in this project and providing electron microscope facilities, to Miss Margaret Waite for skilled technical assistance and to Mrs. Caroline Myers and Mrs. Margot ttindle for help with photography. This project was financed by a G. J. Coles Post Graduate Award.
~ig. 6. a E.~calyptus alba. Wax platelets developing 3 h after wax removal by rubbing. The epidermal cells are domed and some wax remains untouched between the cells as in the upper right hand side of the picture, b Eucalyptus alba. Regenerating wax plates 48 h after removal. The new wax plates are oriented in rows surrounded by the original untouched plates
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References Dewey, O.R., Gregory, P., Pfciffer, 1~. K.: Factors affecting the susceptibility of peas to selective dinitroherbicides. Brit. Weed Control Conf. Proc. 1, 313---327 (1956). Hall, D. M., Donaldson, L. A. : Secretion from pores of surface wax on plant leaves. Nature (Lond.) 194, 1196 (1962). - - - - The ultrastrueture of wax deposits on plant leaf surfaces. 1. Growth of wax on leaves of Tri/olium repens. J. Ultrastruct. Res. 9, 259--267 (1963). Hallam, N. D. : Sectioning and electron microscopy of eucalypt leaf waxes. Aust. J. biol. Sci, 17, 587--590 (1964). Juniper, B.E.: Growth, development and the effect of the environment on the ultrastructure of plant surfaces. J. Lima. Soc. (Bot.) 56, 413--419 (1960). N. D. Hallam Unit of Experimental Agronomy Department of Agricultural Science University of Oxford Oxford OXI 3 PF, England
