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This is to certify that the
thesis entitled
Mechanisms of Epicuticular Wax Deposition

In Leaves of Brassica oleracea

presented by

Lori Henderson Anton

has been accepted towards fulfillment
of the requirements for

Master's degree in Botany & Plant Pathology

awn/134,,

Major professor

 

 

Date III/"I‘l/q/

0-7639 MS U is an Affirmative Action/Equal Opportunin Institution

 

 

 

 

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chmS—pd

MECHANISMS OF EPICUTICULAR WAX DEPOSITION
IN LEAVES OF BRASSICA OLERACEA

 

BY

Lori Henderson Anton

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1991

67’?- 575/?

ABSTRACT

MECHANISMS OF EPICUTICULAR WAX DEPOSITION
IN BRASSICA OLERACEA LEAVES

BY

Lori Henderson Anton

The exact mechanism of epicuticular wax deposition is not
known. Wax has been hypothesized to travel through the
cuticle either by pores or by diffusion. In this study,
Broccoli (Brassica oleracea var. botrytis) leaf waxes were
labelled with 1“C-acetate and examined during synthesis and
deposition using microautoradiography. Waxes were viewed
with light microscopy and scanning electron microscopy
(SEM), including cryo-SEM. In microautoradiography, silver
grains resulting from radioactive decay of labelled waxes
seemed to be distributed randomly throughout the cuticle.
This random distribution supported the hypothesis of
diffusion or complex anastomosing pores as the mechanism of
epicuticular wax deposition. The hypothesis of wax

depositing pores running perpendicular to the leaf surface

was not supported.

For my husband, Bryan, and my family

iii

ACKNOWLEDGEMENTS

I would like to thank Dr. Frank Ewers, Dr. Raymond
Hammerschmidt, and Dr. Karen Klomparens for their advice
and research materials. I also thank the staff of the
Center for Electron Optics at Michigan State University for

their assistance.

iv

TABLE

List of Tables . . . . . .
List of Figures. . . . . .
Introduction . . . . . . .
Materials and Methods. . .
Results. . . . . . . . . .
Discussion . . . . . . . .

List of References . . . .

OF CONTENTS

LIST OF TABLES

Table 1. Silver grain density (grains/”ma, mean t
standard error) based on light microscopic
observations of 89m sections perpendicular to the

leaf surface 0 O O O O O O O O O O O I O O O O O 17

vi

LIST OF FIGURES

Figure 1. Light micrograph of 3L“C-acetate labelled
leaf showing silver grains in vascular tissue
area . . . . . . . . . . . . . . . . . . . . . .
Figure 2. Light micrograph of unlabelled leaf showing
absence of silver grains in vascular tissue
area . . . . . . . . . . . . . . . . . . . . . .
Figure 3. Light micrograph of 1“C-acetate labelled leaf
showing silver grain (arrow points to one
grain) distribution in cuticle (C) and

mesophyll (M). . . . . . . . . . . . . . . . . .
Figure 4. Light micrograph of unlabelled leaf showing
absence of silver grains in cuticle (C) and

mesophyll (M). . . . . . . . . . . . . . . . . .

Figure 5. Scanning electron micrograph of 14C—acetate

labelled leaf. Backscatter electron mode.

vii

19

19

19

19

21

Figure 6. Scanning electron micrograph of 14C-acetate

labelled leaf. Secondary electron mode. . . . . 21

Figure 7. Scanning electron micrograph of unlabelled leaf

treated with chloroform to remove waxes. . . . 21

Figure 8. Cryo-Scanning electron micrograph of

unlabelled leaf. . . . . . . . . . . . . . . . . 21

Figure 9. Diffusion hypothesis - random distribution of

silver grains. . . . . . . . . . . . . . . . . . 27

Figure 10. Anastomosing pores - complex distribution of

Silver grains 0 O O O O O O O O O O O O O O O O O 27

Figure 11. Pore hypothesis - orderly pattern of silver

grains in pores perpendicular to surface. . . . . 27

viii

INTRODUCTION

Epicuticular wax is synthesized in plant epidermal cells
and must travel through the epidermal cell membrane, cell
wall, and the cuticle to reach its place on the surface of
above ground plant parts. The mechanism of wax deposition,
however, is not widely agreed upon. Scientists have studied
wax deposition since 1871 when deBary (1871) suggested the
presence of pores through the cuticle as a pathway for the

wax o

The epicuticular wax covering of plants has important
protective functions. These include prevention of excessive
water loss (Martin & Juniper, 1970) and prevention of the
entrance of certain gases, including pollutants (Lendzian &
Kersteins, 1988). Waxes have also been shown to protect the
plant from infection by fungi (Blakeman, 1971) and other
pathogens, and insects (Martin & Juniper, 1970).
Commercially, wax morphology and chemistry are of interest
since they affect the wettability of plant surfaces and
therefore the absorption of pesticides and growth
regulators (Bukovac, Flore & Baker, 1979). Waxes exist in
various shapes and forms due to chemistry, environment,

plant organ age, genetics, and possibly the mechanism of

2
wax deposition (vonWettstein-Knowles, 1974, Baker, 1974).
The cuticle has been described as having two layers, the
cuticle proper and the cuticular layer (Martin 8 Juniper,
1970). The cuticle proper and the cuticular layer make up
the cuticular membrane, which is all the material between
the epicuticular wax and the pectinaceous layer adjacent to
the cell wall. The cuticle proper is the outer layer of the
cuticular membrane with a thickness of at least 19m and is
composed of layers of cutin interspersed with layers of
wax. The cuticular layer is the inner layer of the
cuticular membrane with a thickness of 1-10pm (Holloway,
1977) and is made of cutin, waxes, some cellulose, and
possibly a pectin-like substance. Some investigators
recognize a third cuticular layer of cellulose and pectin
(Miller, 1982). The terms primary and secondary cuticle
have also been used to describe the cuticle layers based on
development (Sargent, 1976a). The primary cuticle (perhaps
similar to the cuticle proper) develops while the epidermal
cells underneath are still expanding. The secondary cuticle
(perhaps similar to the cuticular layer) develops after the
epidermal cells have completed their expansion. The cuticle
develops centripetally with the newest cuticle, the
secondary cuticle, found closest to the cell wall (Sargent,

1976a).

The synthesis of both epicuticular waxes and cutin begins

with the production of fatty acids of 16 to 18 carbons.

3
Malonyl-CoA, produced by acetyl-CoA carboxylase from
acetyl-CoA and C02, is converted to malonyl-ACP by the
enzyme malonyl-CoAzACP transacylase. The malonyl-ACP
combines with acetyl-CoA with a loss of a C02 through the
enzyme "short chain" 3-ketoacyl-ACP synthase III. Fatty
acid intermediates from C. to C15 are produced with 3-
ketoacyl ACP synthase I through reduction and dehydration.
The above reactions continue in a cycle consisting of
combination of fatty acid intermediates with malonyl-ACP,
decarboxylation, reduction, and dehydration. The cycle is
completed seven times until the fatty acid is elongated to
16 carbons. The enzyme 3-ketoacyl ACP synthase II then
makes a final elongation of the fatty acid from 16 to 18
carbons (0hlrogge, Browse & Sommerville, 1991). The
biosynthesis of waxes and cutin follows different paths

from this point.

Cutin is composed of esters of C16 and C13 hydroxy-fatty
acids. After fatty acid synthesis, the C16 and C19 fatty
acids are converted to hydroxy-fatty acids by w-
hydroxylation. The cutin monomers are then transported
across the cell wall where esterification and final
modifications, such as crosslinking into a polymer form,

take place (Holloway, 1977, Kolattukudy, 1984).

Waxes differ from cutin in that waxes consist of a greater

variety of compounds, with many of the carbon chains much

4
longer than in cutin. Epicuticular waxes can contain
hydrocarbons, fatty acids, aldehydes, primary and secondary
alcohols, esters, ketones, and terpenoids. The carbon chain
lengths range from C12 to C72, including the esters (Baker,

1982). In Brassica oleracea var. capitata (cabbage),

 

hydrocarbons account for 36% of the leaf wax, with primary
and secondary alcohols contributing 20%, ketones 14%,
esters 13%, acids 9%, and ketols 1% (Purdy & Truter,
1963a). The C29 compounds dominate the wax of cabbage
leaves (Purdy & Truter, 1963b). More quantitative data on
wax chemistry exists for cabbage leaf wax than for other
varieties of B; oleracea, such as broccoli and cauliflower.
However, through thin layer and gas chromatography,
cabbage, broccoli, and cauliflower were shown to have
almost identical chemical compounds in their epicuticular

waxes (Kolattukudy, 1965).

Wax synthesis continues from fatty acid synthesis with
carbon chain elongation. Although the elongation part of
wax synthesis is not well understood, genetic studies have
helped to reveal the presence of elongase enzymes.
vonWettstein—Knowles (1982) used light, mutations, and
inhibitors to show that several different elongases exist.
Bianchi gt 2;; (1985) found that in maize two elongases are

active in wax synthesis.

Kolattukudy, Croteau, and Buckner (1976) proposed a series

5
of reactions that produce the wide variety of compounds
found in waxes. Long chain fatty acids, NADPH, and the
enzyme acyl-CoA reductase allow for the production of
aldehydes. The aldehydes and another enzyme, aldehyde
reductase, again use NADPH to produce primary alcohols. Wax
esters are then formed with acyl-CoAzalcohol transacylase
and the combination of long chain fatty acids and primary
alcohols. Hydrocarbons are produced from the
decarboxylation of fatty acids. Hydroxylation of the
hydrocarbons produces secondary alcohols and a further step

of oxidation yields ketones (Bianchi, 95 al., 1985).

The epicuticular waxes are made in the epidermal cells
(Cassagne & Lessire, 1975), but once they are synthesized
they must pass through the epidermal cell membrane, cell
wall, and the cuticle to the plant surface. Hypotheses of
wax depostion can be placed into two main groups: the pore

hypothesis and the diffusion hypothesis.

The pore hypothesis states that pores in the cuticle
provide a path for wax to move to the plant surface. Hall
and Donaldson (1962) reported pores in Brassica oleracea,

Trifolium repens, and Poa colensoi leaves using carbon

 

 

replicas. Hall (1967) presented further evidence for pores
from carbon replicas for several species including

cauliflower (Brassica oleracea L. var. botrytis L). Many

 

other investigators have identified pores in leaf (Fisher &

6
Bayer, 1972, Chafe & Wardrop, 1973, Wells & Franich, 1977,
Miller, 1985) and fruit (Miller, 1982, Miller, 1983)
cuticles using a variety of methods. Sargent (1976a,b)
observed pores only in the secondary cuticle for Libertia
elegans (Iridaceae) and suggested that the pores form due
to expansion of the secondary cuticle during growth. Since
the cuticle also often shows a lamellar form, the wax could
travel through lamellations as if through anastomosing
pores (Hallam, 1964). An additional hypothesis related to
pores concerns treatment of tissue with Gilson's solution.
Gilson's solution is reported to reveal certain areas in
the cuticle which, due to a difference in chemistry from
the rest of the cuticle, accumulate mercury. Davis (1971)
suggested the possibility of similar chemically distinct

areas for the passage of waxes.

In contrast to the pore hypothesis, the diffusion
hypothesis states that wax dissolved in a volatile solvent
diffuses through the cell wall and cuticle and crystallizes
on contact with air at the surface (Baker, 1982, Jeffree,
Baker & Holloway, 1975). Evidence for the diffusion
hypothesis comes from experiments where surface waxes
removed in solvents were recrystallized in 31359 to produce
the wax morphology seen in_yigg (Jeffree, Baker & Holloway,
1975). Interestingly, for several species including
Brassica, the original wax morphology was recrystallized

only when the different classes of wax chemicals were

7
delivered to the surface one at a time (Jeffree, Baker &

Holloway, 1975).

The mechanism of deposition of epicuticular wax is poorly
understood due to difficulties in the microscopic
observation of the wax as it is deposited. In the present
study, 1‘C-acetate and microautoradiography allowed for the

examination of Broccoli (Brassica oleracea var. botrytis)

 

leaf waxes during synthesis and deposition. Since chemical
solvents are not necessary for cryo-scanning electron
microscopy (cryo-SEM), this method was employed to examine
waxes in a more natural state. The cryo-SEM results were
compared to SEM results in which waxes were removed with
chloroform to reveal any anatomical features possibly

involved in deposition.

MATERIALS AND METHODS

APPLICATION OF 14C-ACETATE

Broccoli (Brassica oleracea L. var. capitata L.) plants

were grown from seed in a greenhouse.

Acetate labelled with 14C was used in this experiment
because it is a major wax precursor. Malonyl-CoA, the
compound involved in the addition of carbons to growing
fatty acid chains, is produced from acetate by acetyl-CoA
carboxylase. For optimal leaf size determination, excised
leaves were given an application of 1“C-acetate (ZOpCi per
gram of leaf tissue) as described below and then dipped in
chloroform for 303 to remove the epicuticular wax. The
quantity of label in the wax dissolved in chloroform was
then counted for 5min with a Packard 1500 Tri-Carb
scintillation analyzer. Leaves measuring 99mmx62mm and
74mmx50mm accumulated the most label in the epicuticular
wax. Leaves of this size range were used in all further

14C-acetate applications.

Label was applied in a single dose of 4h or was applied in
a pulse-chase experiment for 4h followed by a 24h period
without label. Young leaves were cut from plants and placed

8

9
in the dark for 1h with their petioles immersed in water
(Kolattukudy, 1965). After 1h leaves were placed in vials
containing 2ml of water and zopci or lopCi 14C-acetate per
gram of leaf tissue. The leaves were kept under a
flourescent light with light intensity (fluence rate) of
8.9 pmol/m’s for 4h. For microautoradiography, leaves were
then prepared for microscopy or were removed from the 1“C-
acetate solution to cold acetate for 24h under the
fluorescent light for a pulse-chase experiment before

microscopic preparation.

Microautoradiography experiments were run twice with a
total of eight leaves given label and nine control leaves
without label. Six leaves labelled with 14C-acetate for
light microscopy ranged from 80 to 100mm in length and from
55 to 70mm in width. Four leaves ranging from 80 to 95mm in
length and 55 to 67mm in width were examined using the SEM.
Control leaves, which were given distilled water instead of
the radioactive solution, ranged from 72 to 108mm in length
and from 50 to 70mm in width for both light microscopy and

SEM microautoradiography.

Four leaves of a wider range of sizes, from 43 to 85mm in
length and from 28 to 72mm in width, were examined using
cryo-SEM. A leaf measuring 75mmx55mm was viewed with the
SEM after removal of waxes with chloroform as described

below.

10
For each leaf, small rectangles of tissue measuring 1mmx3mm
were cut from a smooth, glaucous area often containing
minor veins and fixed as described below. For scanning
electron microscopy, three rectangles per leaf were viewed
at magnifications from 480 to 3000x. For light microscopy
sections from two rectangles per leaf, for a total of 113
microscope slides, were examined at magnifications up to

1200K.

TISSUE PREPARATION

 

Light Microscopy

 

Labelled leaves were prepared for light microscopy and
sectioned either on the IEC (International Equipment
Company)-CTF Microtome-Cryostat or the Sorvall MT-2
ultramicrotome. For cryostat sections, tissue was placed
immediately in 0.2M phosphate buffer or was lightly fixed
in 2% glutaraldehyde in 0.1M phosphate buffer for 5min,
washed twice with 0.2M phosphate buffer at 5min each, and
stored in fresh buffer. Leaf tissue was frozen in Tissue-
Tek O.C.T. compound and sectioned at 8pm with a single edge
teflon-coated razor blade in the cryostat. The Tissue-Tek
was determined to be a possible source of chemography,
exposure in microautoradiography caused by chemicals or
substances other than radioactive decay, and therefore the
Tissue-Tek was washed from the sections with two changes of

distilled water. Sections were then air dried onto

11
microscope slides coated with a solution of 0.5g gelatin
and 0.059 chrome alum in 100ml distilled water to ensure
that sections adhered to the slides (Orr, G. L., Orr, N. &
Hollingworth, 1990). For ultramicrotome sections, tissue
was fixed in 4% glutaraldehyde in 0.1M phosphate buffer for
1 1/2h, rinsed three times in 0.2M phosphate buffer for
30min each, and dehydrated in an acetone series for 30min
at each step. Tissue was then embedded in Spurrs firm resin
and sectioned at 19m on the ultramicrotome using a glass
knife. Sections were dried onto uncoated slides on a slide

warmer .

Scanning Electron Microscopy

 

For SEM examination, leaf tissue was fixed with 4%
glutaraldehyde in 0.1M phosphate buffer for 3h, washed
twice with 0.2M phosphate buffer at 30min each, and
dehydrated in an ethanol series for 30min at each step. The
tissue was then dried with a Balzers critical point dryer
using C02 and mounted onto carbon planchets epoxied to

aluminum stubs.

Autoradiography Preparation and Processing

 

To prevent chemography, all slides and stubs with leaf
tissue were given a very light coat (approximately 10s at
40 amps) of carbon in a Varian Vacuum Evaporator VE 10. The
leaf tissue was then coated with liquified Kodak NTBB

emulsion using an eyedropper for an even coat. Slides and

12
stubs were stored at 4C in light tight boxes with
desiccant. The tissue was allowed to expose the emulsion
for 3 to 25 days, with a 6 day exposure as optimum.
Microautoradiograms were developed with full strength Kodak
D-19 for 2min, washed in distilled water for 105, fixed
with Kodak fixer for 5min, and washed three times with
distilled water for 3min each. Emulsion coating and
developing was performed in the dark with a Wratten #1 red

filter.

MICROSCOPY

 

For light microscopy, slides were either viewed without a
coverslip or with a coverslip applied with glycerol for oil
immersion. Some slides were stained with 0.05% toluidine
blue in phoshate buffer, pH 4.4 for Ss. Leaf tissue was
examined and photographed on a Zeiss Photomicroscope III or
Leitz Laborlux 12 with a Wild (Heerbrugg)

Microphotoautomat.

For SEM, stubs were coated with carbon on an Emitech K450
carbon coater before viewing. This carbon coat served the
same function as the gold coat usually given to SEM
samples, but carbon was used so as not to interfere with
imaging of the silver deposits that resulted from
radioactive decay of labelled waxes. The specimens were

viewed on the JEOL JSM-35CF SEM using both the secondary

13

electron (SE) mode and the backscatter electron (BSE) mode.

CHLOROFORM CLEARING

 

Unlabelled leaf samples treated with chloroform to remove
epicuticular wax were examined with the SEM for a
comparison to the cryo-SEM specimens. Fixation of the leaf
tissue began with 4% glutaraldehyde in 0.1M phosphate
buffer for 3h, a 30min 0.2M phosphate buffer wash, and
dehydration with an acetone series at 30min for each step.
Samples were then placed in a 1:1 mixture of 100% acetone
and chloroform for 20min followed by 20min in 100%
chloroform alone. The samples were placed in 1:1 acetone-
chloroform again for 20min and 100% acetone for 30min
before critical point drying. Samples were mounted on
aluminum stubs and coated with 30nm gold using an Emscope

SC 500 gold coater.

CRYO-SCANNING ELECTRON MICROSCOPY

 

Unlabelled broccoli leaf samples were examined using the
cryo-SEM SP2000 Emscope on the JEOL 35CF SEM. The leaf
tissue was applied to a shrouded holder with a mixture of
Dixon graphite powder #620 and Tissue-Tek. The specimen was
plunged into liquid nitrogen for quick freezing and then
was slowly warmed to -65C to etch away the water normally

found on biological specimens. After etching, the sample

14
was sputter coated with gold in the cryo-SEM unit and

examined at -150C.

RESULTS

Based on scintillation counts of the six leaves tested, the
largest leaf incorporated the greatest amount of label in
its waxes with 5.1dpm/mm2 and the third largest leaf
followed with 0.7dpm/mm’. The largest leaf incorporated
0.59% of the total radioactivity fed to the leaf and the
third largest leaf incorporated 0.047% of the total
radioactivity. Based on these analyses, leaves in the range
of 99mmx62mm to 74mmx50mm were chosen for labelling

experiments.

The density of silver grains corresponding to radioactive
decay was calculated for the cuticle and the adjacent
mesophyll (Table 1). Silver grains were counted in the
field of view of the oil immersion objective in the light
microscope, a magnification of 1200x. Background counts of
silver grains, taken from the slide in the field of View
adjacent to the cuticle, were subtracted from the silver
grain counts for the mesophyll and cuticle. The average
density of silver grains was greatest in the abaxial

cuticle, followed by the adaxial cuticle and mesophyll.

With light microscopy, silver grains were observed in the
vascular tissue, mesophyll, epidermis, and cuticle of the

15

16
broccoli leaves (Figures 1-4). A distinct pattern of
silver grains was not detected in any of the leaf tissues
including the cuticle. Instead, the silver grains appeared
to be randomly distributed. The pulse-chase experiment
revealed the same pattern of silver grains as single dose

applications of radioactivity.

The SE and BSE modes allowed examination of silver grains
at the electron microscope level. Since the BSE mode
detects atomic number differences, silver grains appeared
as bright dots (Figure 5) that could be seen very clearly.
The location of silver grains was also determined quickly
by scanning at low magnifications using the BSE mode.
Clusters of silver grains were approximately 35nm in
diameter. The SE (Figure 6) and BSE modes showed an
apparently random distribution of silver grains similar to
that seen with microautoradiography at the light microscope

level.

Leaves treated with chloroform to remove epicuticular waxes
revealed the cuticle, epidermal cells, and stomates (Figure
7). Cutin is not soluble in chloroform and therefore was
still present over the epidermis. Small particles measuring
approximately 11.5nm were visible on the cuticle (Figure

7).

Two types of wax morphology, filaments and plates, were

17
observed on the broccoli leaf with cryo-SEM (Figure 8).
Leaves showed thin filaments and plates of wax of varying
sizes. The wax plates appeared to be composed of filaments

fused together.

TABLE 1. Silver grain density (grains/”ma, mean 1 standard
error) based on light microscopic observations of 8pm
sections perpendicular to the leaf surface. Densities
reflect subtraction of background; negative density

indicates a high background level.

Cuticle Mesophyll
Abaxial Surface 0.0143 3 0.0004 0.00957 3 0.0002
Adaxial Surface 0.00465 g 0.0005 -0.00136 1 0.0001

Area Sampled 0.0131 1 0.0004 0.00410 * 0.00009

FIGURE 1.

FIGURE 2.

FIGURE 3.

FIGURE 4.

18
Light micrograph of 14C-acetate labelled leaf
showing silver grains in vascular tissue

area. Bar = 20pm.

Light micrograph of unlabelled leaf showing
absence of silver grains in vascular tissue

area. Bar = 209m.

Light micrograph of 14C-acetate labelled leaf
showing silver grain (arrow points to one
grain) distribution in cuticle (C) and

mesophyll (M). Bar = 20pm.

Light micrograph of unlabelled leaf showing
absence of silver grains in cuticle (C) and

mesophyll (M). Bar = 20pm.

19

 

Figure 5.

Figure 6.

Figure 7.

Figure 8.

20
Scanning electron micrograph of 14C-acetate
labelled leaf. Backscatter electron mode.
Bright dots are silver grains which correspond
to labelled waxes. Arrow indicates identical

area at arrow in Figure 6. Bar = Sum.

Scanning electron micrograph of 14C-acetate
labelled leaf. Secondary electron mode. Same
area of experimental leaf as Figure 5. Bar =

5pm.

Scanning electron micrograph of labelled leaf
treated with chloroform to remove waxes. Stomate

(8). Bar = sum.

Cryo-Scanning electron micrograph of
unlabelled leaf. Stomate (S), wax filament
(F), wax plate of fused filaments (P). Bar =

5pm.

21

 

DISCUSSION

The random silver grain distribution observed in the
cuticle, similar to the seemingly random distribution in
the mesophyll and vascular tissue, supports the diffusion
hypothesis (Figure 9). Alternatively, a complex system of
anastomosing pores whose paths cross many times may be
hidden within the grain distribution observed (Figure 10).
An orderly pattern of silver grains that might suggest
pores running perpendicular to the leaf surface through the
cuticle (Hall, 1962, Fisher & Bayer, 1972) was not observed

(Figure 11).

Small particles seen on chloroform treated leaves were the
only anatomical features observed on the cuticle surface.
It is possible that the particles could be obscuring very
small pores. These particles, much smaller than the silver
grains seen with SEM microautoradiography, were measured at
11.5nm, well within the reported size range of pores. In

leaves of Brassica oleracea and Trifolium repens, pores

 

 

were measured at 38 to 45nm (Hall, 1962). Smaller pores of
2 to 3.2nm were seen with the transmission electron

microscope in Plantago major leaves (Fisher & Bayer, 1972).

 

In apple fruit cuticles, larger pores, or macropores, of
500 to 2500nm were reported (Miller, 1982).

22

23
Other investigations have added to the understanding of wax
deposition. Hallam (1982) suggested the presence of a
protein carrier to move the wax across the cell membrane of
the epidermis. This protein would surround the wax with a
hydrophilic area outward and hydrophobic area inward next
to the wax, allowing the wax to cross the cell membrane.
The form of the wax as it moves to the surface is an
important consideration as well. The wax could exist as
precursors through most of the cuticle which then undergo
final chemical changes before reaching the surface
(Sargent, 1976a). In contrast, the wax could be cOmpletely
synthesized in the epidermal protoplast and travel as a

chemically finished product.

Many differences have been reported between epicuticular
waxes on abaxial and adaxial leaf surfaces. The adaxial
surface often has a greater density of waxes than the
abaxial surface (Davis, 1971, Baker 8 Hunt, 1981). In the
present study, the abaxial surface had a greater silver
grain density. Wax morphology may also differ between

abaxial and adaxial surfaces (Davis, 1971).

Cryo-SEM is a valuable method for viewing epicuticular
waxes. The dehydration steps often used in fixation for SEM
involve solvents that partially or completely dissolve
plant waxes. Since specimens are prepared with freezing in

cryo-SEM instead of fixatives and solvents, the

24
epicuticular waxes are preserved to a more natural state.
Filaments and wax plates composed of fused filaments were
observed in broccoli leaves in this study. Other reports of

Brassica oleracea wax morphology include hollow tubes, with

 

some situated perpendicular to the surface (Baker 5
Parsons, 1981, Baker, 1982). Wax plates similar to those
seen in this study were observed on brussel sprout (B;
oleracea var. gemmifera) leaves (Baker, 1982). It is
possible that epicuticular wax morphology may be a useful
taxonomic tool (Hallam, 1964). Future studies could use
cryo-SEM to examine waxes of related species since this
technique minimizes artifacts and damage to the

epicuticular waxes.

Because of the similar biochemistry of cutin and wax, the
1‘C-acetate used in this study could have labelled cutin as
well as waxes. The epicuticular waxes did incorporate the
1“C-acetate since radioactivity was detected in waxes
extracted with chloroform. To distinguish between silver
grains associated with labelled cutin or labelled wax,
microautoradiography at the transmission electron
microscope level could be considered. The primary cuticle,
formed during epidermal cell expansion, was most likely
fully formed when the label was applied. Cutin synthesis,
if still occuring, would be contributing only to the
secondary cuticle. Therefore, silver grains from the

secondary cuticle, the inner layer, may be due to both

25
labelled wax and cutin, but silver grains from the primary
cuticle, the outer layer, would be from labelled

epicuticular waxes.

The subcellular location of synthesis has not been
precisely determined. Evidence from differential
centrifugation suggests that different stages of wax
synthesis may occur in the endoplasmic reticulum membrane
(Kolattukudy, 1967, Bianchi gt git, 1985), in the cell
membrane, or in the cell wall (vonWettstein-Knowles, 1982).
A popular hypothesis states that fatty acid synthesis
occurs in the plastids and elongation occurs in the

endoplasmic reticulum (Ohlrogge gt al., 1991).

Pulse-chase experiments performed to detect the order of
wax deposition showed results comparable to single dose
experiments. Since silver grains were seen in the vascular
tissue, the label was not completely incorporated into
waxes in the time period of the 24h chase. A very short

pulse may give more favorable results.

Atomic force microscopy presents a possible means of
examining epicuticular wax deposition. In atomic force
microscopy, a probe rests directly on the surface of a
specimen. Movement of the probe across the specimen allows
the detection of surface features at the molecular level

(Pool, 1990). Atomic force microscopy could aid in the

26
location of anatomical features, such as pores, through

which wax may be deposited.

In conclusion, microautoradiography suggests epicuticular
wax deposition in broccoli leaves occurs either by
diffusion of wax through the cuticle or by travel through
narrow and complex anastomosing pores. Pores running
perpendicular to the leaf surface were not observed. Cryo-
SEM and microautoradiography are approaches that could have
many applications in wax studies because they avoid sample
preparation methods that can dissolve waxes and allow for

observation of waxes as they are synthesized.

27

 

Epicuticular Wax

. . Cuticle
with silver grains

. (.)

ES Epidermis

 

E Mesophyll

Figure 9. DIFFUSION HYPOTHESIS - random distribution of
silver grains

 

Epicuticular Wax

x x x < x . > > ~ + + o o ’ =.

< < < x . x x > ~ ~ + + o ’ = . Cuticle

< < . . . x ~ > o o o + ' ' ' ' = . with silver grains

. < . ~ x ~ > o , ' + + = = = . (. x < ~ > o + ' =)
< ~ ~ x x > o , , = = + + .

.1 Epidermis

§ Mesophyll

 

Figure 10. ANASTOMOSING PORES - complex distribution of
silver grains

 

Epicuticular Wax

Cuticle
with silver grains

(.)

Epidermis

A? Mesophyll

 

Figure 11. PORE HYPOTHESIS - Orderly pattern of silver
grains in pores perpendicular to surface

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31

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