RotanicalJoumalof the Linnean Society, 78: 3 1-40. With 15 figures.

January 1979

Ultrastructure and development of oil cells in

Laurus nobilis L. leaves
R. M A R 0 N AND A. FAHN

The Hebrew University of Jerusalem,Jerusalem, Israel

Accepted for publication August 1978

The oil cell development in Laurus nobilis leaves has been studied. At the early developmental stage,
when the cell wall consists of the outer cellulose wall only, the oil cells differ from the neighbouring
niesophyll cells in their larger size, lower starch content and in their plastid organization. After the
deposition of the lamellated suberin layer and the inner cellulose layer, a wall protuberance (cupule)
is formed on the periclinal wall facing the epidermis. From its reaction with periodic acid-
hexamine-silver nitrate, it is suggested that the cupule is cellulosic. The portion of the inner cellulose
wall layer bearing the cupule seems to contain patches of suberin. Plasmodesmata occur in special
wall protuberances and appear to become occluded with age. The oil produced inside the protoplast
is secreted to the outside ofthe plasmalemma, and accumulates as a drop at the place predetermined
by the cupule. Except at the cupule, the oil drop is surrounded by the plasmalemma.

KEY WORDS: Laurus - oil-cell - wall protuberance - suberin layer - plasmodesma.

CONTENTS

Introduction . . . . . . . . . . . . . . . . . . . . . . . . 31
Material and methods . . . . . . . . . . . . . . . . . . . . . 32
Results . . . . . . . . . . . . . . . . . . . . . . . . . 32
Light microscopy . . . . . . . . . . . . . . . . . . . . . 32
Electron microscopy . . . . . . . . . . . . . . . . . . . . 33
Nature ofthe cupule . . . . . . . . . . . . . . . . . . . . 38
Discussion . . . . . . . . . . . . . . . . . . . . . . . . 38
References . . . . . . . . . . . . . . . . . . . . . . . . 40

INTRODUCTION

Oil cells occur in a number of plant families. In most cases they are isolated
and differ from their neighbours in size, content and in that they usually possess
wall protuberances, often in the shape of a cupule to which an oil drop-usually
termed in the literature ‘oil-sac’-is attached. The wall of a mature oil cell
consists of three layers, the middle one of which is of suberin. Although a
relatively large number of investigations have been made of oil cells (Muller,
1905; Lehmann, 1926; Kisser, 1926; Leemann, 1928; Paech, 1952; SzentpPtery,
Sarkany, Fridvalsky, 8c Nagy, 1966; Ziegler, 1960 and others) a number of
31
0024-4074/79/0 1003 1- 10/$02.00/00 0 1979 The Linnean Society of London
32 R. MARON AND A. FAHN

problems concerning the structure of these cells remain unsolved. Most in-
vestigations were carried out with the aid of the light microscope and only very
few attempts have been made to study oil cells with the aid of the electron
microscope (Scott, 1963; Szentpktery et al., 1966; Amelunxen 8c Gronau, 1969).
For this reason the present investigation into .the oil cells of Laurus nobilis was
undertaken. Special attention was given to the nature of the cupule-like wall
protuberance and to the position of the oil drop in the cell.

MATERIAL AND M E T H O D S

The investigation was carried out on leaf primordia and adult leaves of Laurus
nobilis L. The adult leaves were taken from trees growing on the Hebrew
University campus at Givat Ram, Jerusalem.
The primordia examined were either removed from buds of the trees or from
young seedlings. Seedlings were grown in pots with garden soil in a greenhouse
and in petri dishes, containing water only, in the laboratory.
For observations with the electron microscope, buds three millimetres long
were cut longitudinally into two halves, fixed in 2% glutaraldehyde in 0.1 M
sodium cacodylate buffer (pH 7.2) for two hours at 4OC, rinsed 3-5 times in
buffer solution during one hour, then postfixed in 2% osmium tetroxide in the
cacodylate buffer for two hours at 4OC. After washing for at least one hour with
the buffer solution, the fixed material was dehydrated in ethanol, embedded in
low viscosity embedding medium (Spurr, 1969) and sectioned with glass knives
on an LKB ultramicrotome IIIA. The sections for most observations were stained
on grids with uranyl acetate and lead citrate according to Reynolds ( 19631, unless
stated otherwise. Some sections were mounted on gold grids and treated with
periodic acid-hexamine-silver nitrate reagent (Pickett-Heaps, 1967) for
determination of polysaccharides. For staining of the plasmalemma,
phosphotungstic acid-chromic acid (Roland, Lembi 8c Morrk, 1972)was used.
The sections were examined with a Philips 300 electron microscope.
Sections one micron thick were prepared for light microscope observation and
stained with Paragon C & C Co., multiple stain. Free-hand sections of mature
leaves and primordia were prepared for observation in the light microscope and
for plasmolysis experiments. Microtome sections of material embedded in
paraffin and stained with safranin and fast green were also made.

RESULTS
Light microscopy
In the leaves of Luurus nobilis the oil cells occur below the adaxial and abaxial
epidermis among the pallisade cells and in spongy parenchyma. Oil cells can first
be distinguished in the third leaf primordium. In such a primordium oil cells at
several developmental stages are already present. With the light microscope the
oil cells can be distinguished from the neighbouring cells b their larger size, by
F
their intense absorption of stains and by their low content o starch grains. In the
differentiated oil cell (Fig. 11, a cupule-shaped protuberance, which in the
following account will be referred to as the cupule, occurs on the periclmal wall
adjacent to the epidermis. In the oil cells of sections of fresh leaves an oil drop may
be observed attached to the cupule. The oil drop stains with Sudan IV.
 O I L CELLS I N LAURUS LEAVES

Figure 1. Schematic drawing of an oil cell of a leaf. Cu, cupule; E, epidermal cell; 0 , oil d r o p ; SL,
suheriti wall layer.

Electron microscopy
Four main stages in the development of oil cells can be distinguished :
Stage 1. Cells in which only the outer cellulose wall layer is resent.
E
Stage 2. Cells in which the outer cellulose wall layer and a su erin layer is present.
Stage 3. Cells in which the inner cellulose wall layer and cupule is formed.
Stage4. Cells in which the oil drop attains its maximum volume, the cytoplasm
becomes very dark and organelles can no longer be distinguished.
Stage I . The cell wall consists only of'the outer cellulose layer (Figs 2-4). The
vacuole is large. The cytoplasm contains many ribosomes grouped in polysomes.
A few short profiles of the endoplasmic reticulum (ER) are present. Mitochondria
and Golgi bodies are common. The variously-shaped plastids contain few
thylakoids. Very few plastids contain starch grains. Characteristic of the plastids
is the occurrence of rows of small electron translucent vesicles, mainly in the
periphery of the organelle (Figs 2 , 3). In some electron micrographs connections
between such vesicles and the inner membrane of the plastidal envelope can be
seen. Small multivesicular bodies are present inside some plastids (Figs 4, 12).
Stage 2. The suberin layer is formed (Figs 5 , 6, 12). Plasmodesmata occur in
wall protuberances. In the plasmodesmata plasmalemma and desmotubuli can
clearly be seen (Fig. 8). The suberin layer is lamellated. The volume of the
vacuole is first relatively small but towards the final stage of suberin deposition it
increases and a very large central vacuole is formed. The polysomes are very
numerous (Fig. 12). The number of rough ER cisternae increases and they tend to
occur in stacks in a parallel array (Fig. 12). The number of Golgi bodies
decreases. In some plastids the small multivesicular bodies are present (Fig. 12).
Plasmalemma invaginations containing vesicles and disorganized portions of
membrane structures have been observed (Fig. 6).
Stage 3 . The inner cellulose wall layer, the texture of which appears looser
than that of the outer layer, is produced. The deposition of this layer starts at the
place where the cupule is to be formed. Here the suberin layer is thinner and the
3
34 R. MARON AND A. FAHN

Figures 2-5. Fig. 2. Portions of a very young oil cell and of two neighbouring rnesophyll cells (MC).
l'he oil cell wall still consists only ofthe outer cellulose layer. The oil cell contains a large vacuole (V)
and plasticls (PI in wliich rows of small translucent vesicles can be seen. The plastids of the Inesophyll
cells (MC) are filled with starch grains 6). ER, Endoplasmic reticulum; G, Golgi body; M,
rrlirochorltirion. x 13,000. Bar=0.77 p m . Fig. 3. Young oil cell, showing plastids (P) with rows of
\mall electron translucent vesicles. MC, Ordinary inesophyll cell; V, oil cell vacuole. x 20,000.
Har=0.5 p m . Fig. 4 , Oil cell as in Fig. 2 showing variously shaped plastids ( P ) . G, Golgi body,
MV, rriultivesicularbody; N , nucleus, x 16,000. Bar=0.62 pm. Fig. 5.Oil cell inwhich the suberinwall
layer (SL) was formed. ER, Endoplasmic reticulum; G, Golgi body; MC,ordinary rnesophyll cell; V,
vacuole. x 32,000. Bar=O.31 pin.
 O I L CELLS IN LAURUS LEAVES 35

Figures 6-9. Fig. 6. Portion of an oil cell at a developmental stage similar to that of Fig. 5, showing a
multivesicular body (MVI outside the.plasmalemma. P, Plastid; SL, suberin wall layer. x 112,000.
Bar=0.09 pm. Fig. 7. Portion of an oil cell in which all three wall layers are seen: the inner cellulose
layer (IW), the outer cellulose layer (OW) and the lamellated suberin layer (SL). PI,
Plasmalemma. x 144,000. Bar=O.O7 fim. Fig. 8. A plasmodesma (Pd) in a wall protuberance of a
young oil cell. SL, suberin lamella. x 112,000. Bar= 0.09 pm. Fig. 9. Two plasmodesmata (Pd)seen in a
wall protuberance of a mature oil cell. IW, Inner cellulose wall layer; OW, outer cellulose wall layer;
SL, suberinlayer. x 67,000. Bar=O. 15pm.

3*
36 R. MARON AND A. FAHN

Figures 10- 1 I . Fig. 10. Part of an oil cell showing the thickened portion of the inner wall layer (TIW)
on the summit of which the cupule (Cu) occurs. OW, Outer cellulose wall layer; SL, suberin
layer. x 53,000. Bar=O.19 pm, Fig. 11. Portion of an old oil cell showing a thin layer of disorganized
cytoplasm and the large translucent spaceofthe oil drop (0). Cu, Cupule; M, neighbouringmesophyll
cell. x 20.000. Bar=0.5pm.

inner wall layer thicker than in the rest of the cell wall (Fig. 10). This thickened
portion of the inner wall layer, which forms a kind of cupule-base, exhibits a
specific texture. A thin region, facing the cytoplasm, is homogeneous, whereas
the rest of the thickened wall portion appears as a mosaic of electron opaque and
electron translucent material. On its summit the cupule develops. With the
development of the inner wall layer and cupule, the ground cytoplasm becomes
more electron opaque with only few small vacuoles and some osmiophilic
droplets present, the organelle membranes become indistinct and the large space
which develops contains the oil drop (Fig. 13). Inside this region portions of
foamy material can sometimes be seen.
 OIL CELLS IN LAURUS LEAVES 37

Figures 12-15. Fig. 12. Young oil cell in which only the outer cellulose wall layer and suberin layer
were formed. ER, Endoplasmic reticulum; G, Golgi body; M, mltochondnon; MV, multwesicular
body; P, p1astid.x 16,000. Bar=0.63 pm. Fig. 13. Mature oil cell with a very large space which
contained the oil drop (0). x 26,000. Bar=0.38 pm. Fig. 14. Section of an oil cell stained with
phosphotungstic acid-chromic acid, showing the plasmalemma (PI)lining the space where the oil
drop occurred (0).C, Cupule. x 30,000. Bar=0.33 pm. Fig. 15. Section of an oil cell treated with
periodic acid-hexamine-silver nitrate, showing reaction of cellulose wall layers and cupule. The
reaction of the cupule and the thickened portion of the inner wall layer is very strong. N o reaction is
seeninthesuberinlayer. x 24,000. Bar=0.41 pm.
38 R . MARON AND A. FAHN

With sections stained with phosphotungstic acid-chromic acid, it can clearly

be seen that the plasmalemma lines the space where the oil-drop occurs (Fig. 14).
The plasmalemma adheres to the inner surface of the cell wall, especially to the
portion bearing the cupule, and to the outer surface of the cupule. The
plasmalemma becomes detached from the inner surface of the cupule.
In the plasmodesmata (Fig. 9 ) it is difficult to distinguish the plasmalemma
and desmotubule. They seem to be at least partly occluded by electron opaque
material.
Stage 4 . The oil drop occupies most of the cell volume. The cytoplasm becomes
very electron opaque, spotted by electron translucent areas and organelles can no
longer be distinguished (Fig. 1 1).

Nature ofthe cupule

In sections stained with uranyl acetate and lead citrate the cupule appears
more or less similar to the inner cellulose wall layer and the innermost region of
the thickened wall portion (cupule base). The cupule fibrils are loosely and
randomly arranged. The cupule and the cell wall, except for the suberin layer,
react positively to periodic acid-hexamine-silver nitrate treatment (Fig. 15),
the reaction being strongest on the cupule.

DISCUSSION

The various developmental stages of the oil cells could not be correlated with
the ontogenetic stages of the leaves. The first oil cells were found in the third leaf
primordium. At this stage of leaf development, oil cells of various developmental
stages were already present. Therefore, an intrinsic parameter for the determina-
tion of the various developmental stages of the oil cells had to be chosen. The
parameter used was the sequence of deposition of the three cell-wall layers, i.e.
the outer cellulose layer, the suberin layer and the inner cellulose layer with the
cupule.
From the early developmental stages, the oil cells differed from the
neighbouring cells in their larger size, lower starch content, and in their plastids,
which contained only few thylakoids and rows of small electron translucent
vesicles at their periphery. The vesicles seemed to develop from invaginations of
the inner membrane of the plastid envelope.
The Golgi bodies developed and were most numerous at the early stage of oil
cell development. At this stage they were apparently involved in formation of the
outer cell-wall layer.
Multivesicular bodies outside the plasmalemma were observed, mainly at the
stage at which the suberin wall-layer was produced. This may perhaps indicate an
involvement in the deposition of'the suberin layer. Several stages in the deposition
of the lamellated suberin layer were observed. Thus suberin deposition does not
appear to be a brief intermission in cellulose deposition as was reported by
Wattendorf ( 1974)who studied the phellem cells of'dcacia senegal Willd.
Plasmodesmata occurred in special wall protuberances in the wall of the oil
cell. In the plasmodesmata of young oil cells it was easy to distinguish the
plasmalemma and desmotubuli. In mature oil cells they appeared to be at least
partly occluded by homogeneous electron opaque material. Because of the
 O I L CELLS IN LAURUS LEAVES 39

limited number of plasmodesmata in the oil cell waIl, it was difficult to conclude
whether the entire canal was occluded or only the cytoplasmic anuli, as was
observed by Evert, Eschrich & Heyser (1977) in plasmodesmata occurring
between mesophyll cells of Zea mays.
The occlusion of the plasmodesmata after the formation of the suberin wall
layer completely isolated the mature oil cell from the surrounding cells. Such
cells lose their vitality and can be compared to sealed, oil-containing vessels. The
vacuole attained its maximum volume towards the final stage of suberin
deposition. The space subsequently to be occupied by the oil drop had been
delimited by the time the deposition of the cell wall and cupule was complete.
The two main aims of the present investigation were to clarify the nature of the
cupule and to determine the location of the oil drop.
Amelunxen 8c Gronau (1969)and Tucker ( 1976)were unable to see a cupule in
Acorus calamus L. and Saururus cernuus L. respectively. However, in the oil cells of
most plants specific wall protuberances were observed (Berthold, 1886; Muller,
1905; Haberlandt, 1918; Lehmann, 1926; Leemann, 1928; Ziegler, 1960;
Holzner-Lendbradle, 1963; and others). In Laurus the wall protuberance, in the
shape of a cupule, was easily detectable. The strong reaction of the cupule to
periodic acid-hexamine-silver nitrate may suggest that it contained more free
aldehyde groups than the cellulose wall layers (cf. Pickett-Heaps, 1967). Berthold
(1886) and Lehmann (1926) also suggested that the cupule is of cellulose,
although Berthold added that the cupule is cutinized.
The thickened portion of the inner cellulose wall layer at the base of the cupule
consisted of an electron opaque matrix in which were embedded small electron
translucent areas, similar in appearance to the suberin layer. These areas appeared
to be in continuity with the suberin layer where they abutted on to this layer. It is
therefore suggested that the translucent areas contain suberin.
In the older literature, the oil drop is considered to be surrounded by an
envelo e. According to Berthold (1886)and Kisser ( 1926),this envelope consisted
Q
of cel ulose. Lehmann (1926) came to the conclusion, based on various
histochemical reactions and on experiments carried out on Laurus and Asarum,
that the envelope was a semipermeable plasmatic membrane, in which lipid,
perhaps suberin-like, substances occurred. Muller ( 1905) suggested that an oil-
containing vacuole, which is formed by fusion of smaller ones, joins the cell wall;
the vacuolar membrane producing the cupule and oil-drop envelope. Leemann
(1928) and Szentpetery et al. (19661, also came to the conclusion that the cupule
and oil-drop envelope were not cellulosic. Leemann suggested that the oil-drop
envelope and cupule consisted of the same phospholipid-like substance.
In the present study of electron microscope sections of the oil cells of Laurus
stained with phosphotungstic acid-chromic acid, it was clearly seen that the
plasmalemma surrounded the space which was previously occupied by the oil
drop. I t is therefore suggested that the oil is secreted to the outside of the
protoplast, accumulates at the place predetermined by the cupule, and forms the
oil drop.
A few osmiophilic droplets and small translucent vacuoles occurred in the oil
cells. It may possibly be that in the translucent vacuoles oil or its precursors were
present. The oil may have been extracted from them during preparation as was
the case with the oil drop itself. Several authors have suggested that the oil
precursors occurred as small droplets or in small vacuoles in the cytoplasm and