Download Epicuticular wax patterns on plants in and conditions Brassica in

Epicuticular wax patterns on Brassica plants in in vivo and in vitro conditions
1
2
2
Jagna Karcz, Tomasz Ksiazczyk, Jolanta Maluszynska
1
Laboratory of Scanning Electron Microscopy, University of Silesia, Jagiellonska 28, 40-032, Katowice, Poland
e-mail [email protected]
2
Department of Plant Anatomy and Cytology, University of Silesia, Jagiellonska 28, 40-032, Katowice, Poland
INTRODUCTION
MATERIAL AND METHODS
The cuticles of the majority of higher plants are covered by epicuticular waxes with
considerable ultrastructural and chemical diversity. They often cause a glaucous
appearance. Many of them are of great systematic significance (Barthlott et al., 1998).
Waxes are sensitive to a range of environmental variables and therefore they can be used
as an indicator of pollution levels. These projections are extruded or migrated from the
epidermal cells and are crystalline on the cuticle surface. Epicuticular waxes may be
present as an amorphous wax film or as a wax film covered with wax crystalloids (Fig. 1).
Chemically, these secretions consist of a very heterogenous mixture of lipophilic
substances (e.g. hydrocarbons, esters, aldehydes, ketons, alcohols, triterpenoides).
In Brassica species the differences in the epicuticular wax structure have been studied.
Macroscopically, waxes are visible on Brassica leaf and stem surfaces as a bluish-white
colored coating, called glaucousness or waxy bloom. The epicuticular wax morphology
is composed primarily of rods, tubes, vertical plates, and dendritic- or umbrella-like
structures. Although the epicuticular wax studies on cultivated brassicas were carried out
in vivo and in vitro, little is known about structural wax deposition in the rapid-cycling
Brassica (RCBr) plants, especially in relation to in vitro conditions.
The aim of the study was: (1) to compare the epicuticular wax structure of the RCBr and
cultivated Brassica during developmental stages in in vivo and in vitro conditions; (2) to
determine if in vitro culturing may suppress epicuticular wax production in RCBr plants,
what was reported in case of other cultivated species. In our study three cultivars of
Brassica species (Brassica campestris, B. oleracea, B. napus) and their derivatives RCBr
lines in vivo and in vitro conditions were analyzed. Better understanding of epicuticular
wax structure provides more information about use of this diagnostic feature in studies on
plant-environmental interactions.
EWF
EWC
in vivo conditions. Seeds of common Brassica campestris (“Goldball”), Brassica oleracea
(“Amager”) were obtained from Enterprise for Garden Seeds and Nursery (PNOS, Poland),
and Brassica napus (“Kana”) from Plant Breeding and Acclimatization Institute (Ma³yszyn,
Poland). Seeds of rapid-cycling Brassica campestris (base population 1-1), Brassica oleracea
(base population 3-1) and Brassica napus (base population 5-1) were obtained from the
Crucifer Genetics Cooperative (CrGC 1997 seed stock). Plants were grown in flowerpots at
constant temperature of 22°C ± 2°C under 16h/8h photoperiod regime.
in vitro conditions. Seeds of investigated common Brassica and rapid-cycling Brassica were
surface-sterilized for 30 minutes in the sterilizing buffer (3% (v/v) hydrogen peroxide, 97%
(v/v) ethanol and two drops of Tween 20 and subsequently rinsed 3 times with autoclaved
water. Seeds (30 per vessel) were aseptically germinated on ½-strenght MS medium (Murashige
and Skoog, 1962) without growth regulators, supplemented with 3% (w/v) sucrose and
solidified with Difco agar (0.8% w/v). The pH was adjusted to 5.8 with 1N NaOH before
autoclaving. All cultures were kept at constant temperature of 20°C ± 2°C under 16h/8h
photoperiod regime.
Scanning electron microscopy (SEM)
Samples of all Brassica genotypes were collected from the glossy and glaucous leaves and
stems after 1 and 3 months growth in in vivo and in vitro conditions. To avoid alteration of the
wax crystals, no fixation procedures were applied to the specimens (Neinhuis and Barthlott,
1997). Small pieces were cut from the same area of the leaf lamina and the stem internode,
affixed to an aluminium stubs with double sided adhesive tape, air-dried, and then sputteredcoated (Pelco SC-6 sputter coater) with a thin film of gold to improve the electrically
conducting properties of the leaf and stem surface. Three to four replicates of coated samples
were examined with a Tesla BS 340 scanning electron microscope. The following SEM settings
were chosen: Acc. V (accelerating voltage): 20kV; detector: SE (secondary electrons); WD
(working distance): 10-15 mm distance from the final lens. Micrographs were taken using
Fomapan 400/120 film.
epicuticular wax
(EW)
cutin + wax
Figure 1. Schematic cross-section through the outer parts of a
plant epidermis devoid of wax crystals: CP-cuticle proper, CL-
CP
IW cutin
wax
pectins
cellulose
cutin
wax
pectins
cellulose
CL
RESULTS AND DISCUSSION
Our study is the first report on the epicuticular wax content and structure of the rapidcycling Brassica (RCBr) plants in relation to in vivo and in vitro developmental stages.
We also compared the microstructure of epicuticular wax layer on cultivated brassicas
with the waxes of RCBr plants for a better understanding of wax changes during plant
development. Moreover it was analyzed the wax deposition and structural arrangement
of the leaf tip (old epidermal cells), and the leaf base (young cells) from the adaxial and
abaxial leaf surfaces. A good correlation was found in both groups of investigated plants:
the number and size of crystal-like structures declined from the leaf tip towards the leaf
base.
The SEM analysis showed that epicuticular waxes on leaves and stems of RCBr plants
were less pronounced and more amorphous (e.g. RC B. napus) than those found on
cultivated Brassica plants, which exhibited a higher degree of crystalline wax. A similar
observation was reported for other crops and ornamental plants, e.g.Allium, Rosa (Sutter,
1985; Shepherd et al., 1995). For example, Maier and Post-Beittenmiller (1998)
compared waxes on Allium plants to reveal some broad correlations between wax
composition and structure in different growth conditions.
Although we observed quantitative differences in wax deposition, we also detected
structural modifications. In cultivated brassicas the predominant wax form were: plates,
rods, tubes and dendritic lattice-like crystals. The more irregular plates, fused short rods,
branching rods and small horizontal dendrites were particularly associated with waxes of
RCBr plants. There were also small amounts of tube-like crystals. Therefore this type of
wax structure can be useful in RCBr diagnosis. The present research also show a very
high micromorphological diversity of epicuticular wax crystalloids in Brassica species.
Plant material and growth conditions.
cuticle cuticular layer, EWF-epicuticular wax film, EWC-epicuticular
REFERENCES:
Barthlott W, Neinhuis CH, Cutler D, Ditsch F, Meusel I, Theisen I, and Wilhelmi H. 1998. Classification and
terminology of plant epicuticular waxes. Botanical Journal of the Linnean Society 126: 237-260.
Jeffree C. E. 1996. Structure and ontogeny of plant cuticles. In: Plant cuticles an integrated functional
approach. Ed. G. Kerstiens. BIOS Scientific Publishers Limited, Oxford, pp. 33-82.
Maier C. G-A, and Post-Beittenmiller D. 1998. Epicuticular wax on leek in in vitro developmental stages and
seedlings under varied growth conditions. Plant Science 134: 53-67.
Murashige T, and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures.
Physiol Plant 15: 473-497
Neinhuis C, and Barthlott W. 1997. Characterization and distribution of water-repellent, self-cleaning plant
surfaces.Annals of Botany 79: 667-677.
Shepherd T, Robertson G. W, Griffiths D. W,A. N. E, Birch and Duncan G. 1995. Phytochemistry 40 (2): 407-417.
Sutter E. G. Morphological, physical, and chemical characteristics of epicuticular wax on ornamental plants
regenerated in vitro. 1985. Annals of Botany 55: 321-329.
wax crystalloids, IW-intracuticular waxes (Jeffree, 1996).
TABLE 1. Superficial waxes in Brassica species
Pectinaceous layer
and middle lamella (P)
cell wall (CW)
Wax coating
Taxon
Visual description1)
Stem
Brassica campestris a
leaf
±
ultrastructure2)
Wax components
plates tubes rods filaments dendrites
±
+
+
+/=
+
-
Surface covered by a mixture of
transversely ridged tubes, irregular
plates, rods and filaments
superimposed upon tubes in
variable quantities
Brassica cultivars
B. campestris
(turnip)
genome AA
2n=2x=20
B. oleracea
(cabbage)
genome CC
2n=2x=18
B. napus
(winter oil seed rape)
genomes AACC
2n=4x=38
Brassica napus
(B. campestris x
B. oleracea)
++a,c
++a,c
+/=
+
+/-
+
+
Brassica oleracea c
++
+
+/=
+
+/=
+
+
RC Brassica campestris
RC Brassica napus
Types of epicuticular wax crystals
RC Brassica oleracea
__
__
__
__
Cylindrical upright tubes
Plates and short wax rods and long tapering wax rods Irregularly shaped rods
__
Filaments
__
Large interlocking dendrites
Scale bar 2 um
AA
__
4
__
2
5
±
++a,c
+/-
+/-
+/=
+
__
3
+/-
+/-
+/-
+/-
Wax syntopism of plates, rods,
branching rods, filaments and
dendrite - like wax crystals
.
Large areas of wax-free cuticle.
+
+
+/ -
+/=
+/-
+/-
A reduced density of tubular
waxes. Wax syntopism of
upright
rods,
platelets,
branching flat rods and
dendrites
2) Wax distribution patterns:
+ = wax structures are present
+/- = wax structures are reduced in frequency
+/= = wax structures are rare
- = wax structures are absent
__
6
__
10
__
8
11
__
__
__
9
__
__
12
__
__
__
__
Scale bar 2 um
1
4
CC
AACC
__
__
2
5
__
__
3
6
__
__
Figs 1 – 3. SEM images of leaf waxes.
Fig. 1. Differently oriented rods and plates on the adaxial leaf surface of RC Brassica campestris.
Fig. 2. Sparsely distributed fine filaments and small dendrites on the adaxial leaf surface of RC Brassica napus.
Fig. 3. Fine network of different wax particles on the adaxial leaf surface of RC Brassica oleracea. Scale bar: 5 µm
Figs 4 – 6. SEM images of stem waxes.
Fig. 4. Fused rods and plates on RC Brassica campestris.
Fig. 5. Wax syntopism. Thick truncated tubes and rods on RC Brassica napus.
Fig. 6. Close packed arrangement of plates, rods and dendrites on RC Brassica oleracea. Scale bar: 5 µm
in vitro conditions
Figures 7 – 9. SEM images of leaf waxes.
Fig. 7. Sparse distribution of plates and short rods on the adaxial leaf surface of Brassica campestris.
Fig. 8. Fine rods, tubes and small dendrites on the adaxial leaf surface of Brassica napus.
Fig. 9. Fine network of thin rods, filaments and dendrites on the adaxial leaf surface of Brassica oleracea. Scale bar: 5 µm
Figures 10 – 12. SEM images of stem waxes.
Fig. 10. Tapering wax rods and plates on Brassica campestris.
Fig. 11. Two types of wax layer. Epicuticular wax as a film on the stem surface and syntopism of wax crystals on Brassica napus.
Fig. 12. A reduced wax layer with fine filaments and rods on Brassica oleracea. Scale bar: 5 µm
__
Branching filaments and
Irregular plates and rods Branching flat filaments Small, horizontal dendrites
dendritic lattice-like crystals
Massive plates
AA
in vivo conditions
B. oleracea
genome CC
2n=2x=18
Types of epicuticular wax crystals
1) The visual description of the wax coating is based on the visually observed a general amount of wax
structures on the stem and leaf surfaces.
For the visual description of the wax coating the denotations:
+ = thick organized wax layer visible on stems and leaves;
± = thin organized wax layer visible on stems and leaves;
++a,c = wax pattern intermediate between the two putative parents
__
B. napus
genome AACC
2n=4x=38
Scattered
wax
particles.
Irregular plate -like and tube shaped structures, needles and
branching filaments
+/-
Figures 1 - 3. SEM images of leaf waxes.
Fig. 1. Sparse distribution of fine rods, tubes and plates on the adaxial leaf surface of Brassica campestris.
Fig. 2. Syntopism of rods, filaments and dendrites on the adaxial leaf surface of Brassica napus.
Fig. 3. Large interlocking dendrite-like crystals on the adaxial leaf surface of Brassica oleracea. Scale bar: 5µm
Figures 4 – 6. SEM images of stem waxes.
Fig. 4. Tapering wax rods and flat plates on Brassica campestris.
Fig. 5. Syntopism of filaments, rods and dendrite-like structures on Brassica napus.
Fig. 6. Dense coverage of rods, filaments and dendrites on Brassica oleracea. Scale bar: 5 µm
7
-
++a,c
+
Rapid-cycling Brassica (RCBr)
B. campestris
genome AA
2n=2x=20
Wax syntopism; close packed
arrangement of tubes, fuse
tubes, rods, branching
filaments and small dendrites
lying across tops of tubes
(varieties of B. oleracea can be
distinguished by the different
wax morphology)
CC
AACC
1
±
Syntopism of upright tubes and
transitional stages, rods,
branching filaments, plate -like
structures and flat, overlapping
dendritic platelets parallel to the
plant surface
7
10
__
__
8
11
__
__
9
12
__
__
Figs 7 – 9. SEM images of leaf waxes.
Fig. 7. Small, scattered, variously shaped rods and plates on the adaxial leaf surface of RC Brassica campestris.
Fig. 8. Short branched rods and small dendrites on the adaxial leaf surface of RC Brassica napus.
Fig. 9. Wax syntopism. A reduced wax coating on the adaxial leaf surface of RC Brassica oleracea. Scale bar: 5 µm
Figures 10 – 12. SEM images of stem waxes.
Fig. 10. Fine plates, short tubes and filaments on RC Brassica campestris.
Fig. 11. Two types of wax coating. Amorphous wax layer and differently lo cated wax crystals on RC Brassica napus.
Fig. 12. Horizontal and vertical rods and plates on RC Brassica oleracea. Scale bar: 5 µm