Download Characteristic Thickened Cell Walls of the Bracts

Annals of Botany 102: 31 –37, 2008
doi:10.1093/aob/mcn058, available online at www.aob.oxfordjournals.org
Characteristic Thickened Cell Walls of the Bracts of the ‘Eternal Flower’
Helichrysum bracteatum
KU NI KO N IS HI KAWA 1 , HI ROA K I ITO 2, * , TAT SU YA AWAN O 3 , M U NE TA KA HO S O KAWA 1
and S U S U M U YAZ AWA 1
1
Vegetable and Ornamental Horticulture, Division of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Kyoto
606-8502, Japan, 2AJINOMOTO Integrative Research for Advanced Dieting, Graduate School of Agriculture, Kyoto
University, Oiwake-cho, Kitashirakawa, Kyoto 606-8502, Japan and 3Tree Cell Biology, Division of Forest and
Biomaterials Science, Kyoto University, Oiwake-cho, Kitashirakawa, Kyoto 606-8502, Japan
Received: 31 January 2008 Returned for revision: 20 February 2008 Accepted: 20 March 2008 Published electronically: 23 April 2008
† Background and Aims Helichrysum bracteatum is called an ‘eternal flower’ and has large, coloured, scarious
bracts. These maintain their aesthetic value without wilting or discoloration for many years. There have been no
research studies of cell death or cell morphology of the scarious bract, and hence the aim of this work was to elucidate these characteristics for the bract of H. bracteatum.
† Methods DAPI (4’6-diamidino-2-phenylindol dihydrochloride) staining and fluorescence microscopy were used
for observation of cell nuclei. Light microscopy (LM), transmission electron microscopy (TEM) and polarized
light microscopy were used for observation of cells, including cell wall morphology.
† Key Results Cell death occurred at the bract tip during the early stage of flower development. The cell wall was the
most prominent characteristic of H. bracteatum bract cells. Characteristic thickened secondary cell walls on the
inside of the primary cell walls were observed in both epidermal and inner cells. In addition, the walls of all
cells exhibited birefringence. Characteristic thickened secondary cell walls have orientated cellulose microfibrils
as well as general secondary cell walls of the tracheary elements. For comparison, these characters were not
observed in the petal and bract tissues of Chrysanthemum morifolium.
† Conclusions Bracts at anthesis are composed of dead cells. Helichrysum bracteatum bracts have characteristic
thickened secondary cell walls that have not been observed in the parenchyma of any other flowers or leaves.
The cells of the H. bracteatum bract differ from other tissues with secondary cell walls, suggesting that they
may be a new cell type.
Key words: Helichrysum bracteatum, scarious bract, secondary cell wall, primary cell wall, cell morphology,
birefringence, orientated cellulose microfibrils, cell death, DAPI, transmission electron microscopy, polarized light
microscopy.
IN TROD UCT IO N
Helichrysum bracteatum has compound flowers comprised
of many tubular flowers and scarious bracts (Everett,
1980). The scarious bracts are large and coloured like a
corolla. They maintain their aesthetic value without
wilting or discoloration for many years, even after
cutting. Helichrysum bracteatum is, therefore, suitable as
a dried flower. Many species in the Compositae family
Inurae tribe have such characteristic flowers, including
Ammobium alatum, Anaphalis margaritacea, Antennaria
dioica, Acroclinium roseum, H. bracteatum and
Rhodanthe manglesi. In the Cardueae tribe, Carlina
acaulis and Xeranthemum annuum have similarly characteristic flowers. The same can be said of Gomphrena globosa
and Gomphrena haageana in the Amaranthaceae family
and of Limonium sinuatum in the Plumbaginaceae family.
In all these plants, the scarious bracts or sepals of their
flowers are large and coloured like a corolla, similarly to
those in H. bracteatum.
While the water content of the petals of Chrysanthemum
morifolium ‘seikounoaki’ was found to be 88.5 %, that of
* For correspondence. E-mail [email protected]
the scarious bracts of H. bracteatum ‘Jumbo Yellow’ was
38.6 %, and that of the scarious sepals of L. sunuatum
‘Sundaeviolet’ was 21.2 %. Water contents of the leaves
of these species were 88.9 %, 91.0 % and 75.9 %, respectively. Thus, scarious tissues have a low water content,
while growing plant tissues typically contain 80 to 90 %
water. Wood is composed mostly of dead cells, and has a
low water content. For instance, the sapwood that functions
in transport in via the xylem contains 35 – 75 % water (Taiz
and Zeiger, 2002). The scarious bract of C. acaulis is composed of dead cells (Troll, 1957). These observations
suggest that H. bracteatum scarious bracts and
L. sunuatum scarious sepals are composed of dead cells.
However, to the best of our knowledge, no research
studies show this, nor are there reports of the cell morphology of scarious bracts and sepals.
We investigated whether the cells of the scarious
bracts of H. bracteatum ‘Monstrosa’ are dead or alive by
observing nuclei of such cells stained by DAPI
(4’6-diamidino-2-phenylindol dihydrochloride) under a fluorescence microscope, and examined the morphology of
scarious bract cells under a light microscope, a transmission
electron microscope and a polarized light microscope.
# The Author 2008. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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32
Nishikawa et al. — Characteristic Thickened Cell Walls of Floral Leaves
M AT E R I A L S A N D M E T H O D S
Plants of Helichrysum bracteatum ‘Monstrosa’ were cultivated in a plastic greenhouse at Kyoto University in
Japan. They were grown in pots containing the growing
medium Metro-Mix 360 (Sun Gro Horticulture Canada
Ltd, Seba Beach, Canada) under natural sunlight. The composition of this medium is peat moss, vermiculite, bark ash,
bark, dolomitic limestone and a wetting agent. The solid
fertilizer IBS1 (N : P : K ¼ 1 : 1 : 1; JA Group, Tokyo,
Japan) was applied. Flowers of these plant were used in
the following experiments.
DAPI staining and fluorescence microscopy
Seven stages of H. bracteatum flower development were
considered (Fig. 1A): stage 1, bud ,8.0 mm wide; stage 2,
bud 8.0 10 mm wide; stage 3, bud 10 12 mm wide;
stage 4, bud 12 14 mm wide, with its second layers of
bracts starting to open; stage 5: 4th 5th bracts of the
bud starting to open; stage 6, innermost bracts of the bud
starting to open; and stage 7, all bracts completely opened
(anthesis). The innermost bracts at each stage of flower
development, or bracts adjacent to tubular flowers, were
used in the following experiments, which were performed
according to Gladish et al. (2006). Bracts at each stage
were stained with 1 mg L – 1 DAPI (4’6-diamidino2-phenylindol dihydrochloride) in 10 mmol L – 1 Tris/HCl
buffer ( pH 7.4). The bracts were soaked in DAPI solution
in a vacuum pump in the dark overnight to completely
stain the nuclei of all cells of the bracts. Nuclei of the
bract cells were observed after excitation at 340 – 380 nm
under a fluorescence microscope (Olympus BX60).
Each bract was divided into four equal lengths, and
tip – 1/4 and 1/4 – 2/4 were used in this experiment
(Fig. 1B). Nuclei and epidermal cells were counted and
the proportion of nuclei to epidermal cells of each part
was determined. Three bracts were used at each stage.
Light microscopy (LM) and transmission electron
microscopy (TEM)
A bract of H. bracteatum ‘Monstrosa’ at stage 7 (anthesis)
was used. A petal and a bract of Chrysanthemum morifolium
‘Piato’ were also used for comparison. A razor blade was
used to hand-section 3-mm wide segments of each tissue,
which were fixed in 3 % glutaraldehyde in 0.05 mol L – 1
phosphate buffer ( pH 7.2) overnight at 4 8C. The segments
were rinsed six times in 0.05 mol L – 1 phosphate buffer for
10 min each. They were post-fixed in 2 % osmium tetroxide
in 0.05 mol L – 1 phosphate buffer for 2 h at room temperature, and were rinsed three times in the same buffer for
10 min each. The segments were dehydrated through a
graded ethanol series: 30 %, 50 %, 70 %, 80 %, 90 %, 95
%, 95 %, 95 %, 99.5 %, 99.5 % and 99.5 % for 10 min
each. The segments were then substituted four times
with propylene oxide for 10 min each and were embedded
in epon resin. Semi-thin sections were cut using an
ultramicrotome (Reichert-Jung) and were stained with
safranine and observed under a light microscope
(Olympus BX60). Ultra-thin sections were cut using an
ultramicrotome (Reichert-Jung). These were stained with
2 % aqueous uranyl acetate and Reynold’s lead citrate and
observed under a transmission electron microscope (JOEL
JEM-1220).
Polarized light microscopy
A bract of H. bracteatum ‘Monstrosa’ at stage 7
(anthesis) was used. A petal and a bract of C. morifolium
‘Piato’ were also used for comparison. A razor blade was
used to hand-section 3-mm wide segments of each tissue,
which were fixed in FAA [100 % ethanol : DW : formalin
: acetic acid ¼ 12 : 6 : 1 : 1 (v/v)] overnight at room temperature, and dehydrated through a graded ethanol series:
30 %, 50 %, 70 %, 80 %, 90 %, 90 %, 90 %, 99.5 % and
99.5 % for 60 min each. They were embedded in
Technovit 7100 resin (Heraeus Kuzer). Semi-thin sections
were cut using a rotary microtome (Leica RM2155), and
were observed under a light microscope (Olympus BX60),
a polarized light microscope (Olympus BHA-751P), and
the same polarlized light microscope with compensator.
Sections stained with safranine were observed under a
light microscope.
R E S U LT S
Nuclei in cells of bracts at each stage
A
1
B
2
3
4
5
6
7
Tip~1/4
1/4~2/4
F I G . 1. (A) Seven stages of H. bracteatum flower development. Stage 1,
bud ,8.0 mm-wide; stage 2, bud 8.0 –10 mm wide; stage 3, bud 10–
12 mm wide; stage 4, bud 12–14 mm wide, with its second layers of
bracts starting to open; stage 5: 4th –5th bracts of the bud starting to
open; stage 6, innermost bracts of the bud starting to open; stage 7, all
bracts are completely opened (anthesis). Scale bar ¼ 1 cm. (B) Division
of bract into four equal parts in terms of length, as labelled.
Fluorescence of many nuclei in individual cells was
observed at the bract tip at stage 1 (Fig. 2A); fewer nuclei
were observed at stages 2 and 3 (Fig. 2B, C) and most
had disappeared at stage 4 (Fig. 2D). In contrast, fluorescence of many nuclei was observed in bract bases at all
stages (data not shown). The ratio of nuclei to epidermal
cells in the parts tip – 1/4 and 1/4 –2/4 decreased with
stage advancement (Fig. 3). The ratio in the 1/4– 2/4 part
at stages 1 and 2 were over 1.0; the reason for this is that
the number of nuclei was determined in both the underlying
layers of the epidermis as well as in the epidermis
itself, whereas the cell number was only counted in the
latter. No nuclei were observed in the tip – 1/4 part at
stage 5; thus, cell death occurred at the bract tip at the
Nishikawa et al. — Characteristic Thickened Cell Walls of Floral Leaves
A
C
B
D
F I G . 2. DAPI-staining of the bract tip of H. bracteatum at (A) stage 1, (B)
stage 2, (C) stage 3, and (D) stage 4. A fluorescent dot in the bract cell
indicates the nucleus. Many nuclei of bract cells are observed at stage 1,
fewer at stages 2 and 3; most nuclei have disappeared by stage 4. Scale
bars ¼ 200 mm.
early stage of flower development. Most nuclei disappeared
in the upper half of the bract by stage 5 (before anthesis).
Cell morphology and characteristic secondary cell wall
Spongy parenchyma was observed in petal and bract
tissues of C. morifolium (Fig. 4B, C), but not in
H. bracteatum bract tissue (Fig. 4A). Cells of
H. bracteatum were closely arranged and smaller than
those of C. morifolium.
A large vacuole was observed at the centre of cells of the
petal and bract tissues of C. morifolium (Fig. 4F – I), with
1·8
Ratio of nuclei to epidermal cells
1·6
Tip–1/4
1/4–2/4
1·4
1·2
1·0
0·8
0·6
0·4
0·2
0
Stage 1
Stage 2
Stage 3
Stage 4
Stage 5
Stage of H. bracteatum flower development
F I G . 3. Ratio of nuclei to epidermal cells in two parts (tip– 1/4 and
1/4– 2/4) of the H. bracteatum bract (see Fig. 1). Bars represent the
mean value + s.e. of three independent experiments.
33
cytoplasm surrounding the vacuole. Some organelles,
such as the nucleus and chloroplasts, were observed in the
cytoplasm. On the other hand, no organelles were observed
in the cells of H. bracteatum bract tissue (Fig. 4D, E). The
cell walls were the most prominent characteristic of cells of
the H. bracteatum bract. The primary cell walls form the
outermost layer of cells in all tissues of the two species.
Only primary cell walls were observed in the cells of the
petal and bract of C. morifolium (Fig. 4F– I), whereas
characteristic thickened secondary cell walls on the inside
of the primary walls were observed in both epidermal and
inner cells of the H. bracteatum bract (Fig. 4D, E). The
outer periclinal walls of all epidermal cells were thickened
primary cell walls in petal and bract tissue of C. morifolium
(Fig. 4F, H). On the other hand, there were two layers, comprised of a thin primary cell wall and a thickened secondary
cell wall, in all epidermal cells in the bract tissue of
H. bracteatum (Fig. 4D). A cuticle layer was observed on
the outside of the primary cell walls in all the tissues of
the two species that were examined. Only flat primary
cell walls of all inner cells, except for tracheary elements,
were observed in the petals and bracts of C. morifolium
(Fig. 4G, I), whereas in bract tissue of H. bracteatum, secondary cell walls on the inside of flat primary cell walls of
all the inner cells had irregular thickening as lobes
(Fig. 4E).
Birefringent properties of cell walls
The general secondary cell wall in tracheary elements
(tracheid and vessel) or fibres has a highly birefringent
property that can be observed under a polarized light microscope (Leney, 1981; Lev-Yadun, 1997; Jang, 1998;
Bergander et al., 2002; Donaldson and Xu, 2005;
Thygesen and Hoffmeyer, 2005; Lev-Yadun et al., 2005).
This is because cellulose, which is the main component
of the secondary cell wall, has crystalline properties resulting from the orderly arrangement of cellulose molecules in
microfibrils (Smith et al., 1998). The orientation of cellulose microfibrils is neatly aligned parallel to each other in
secondary cell walls (Taiz and Zeiger, 2002), so that birefringence is generated when viewed under polarized light
(Evert, 2006). The primary cell wall cannot be observed
under a polarized light microscope because it has a rather
random arrangement of microfibrils.
Tracheary elements exhibited birefringence in petal and
bract tissues of C. morifolium (Fig. 5E, H), because they
have a secondary cell wall. The outer periclinal wall of epidermal cells also exhibited birefringence in C. morifolium
bract tissue (Fig. 5H); although it is a primary cell wall,
it consists of many layers (Fig. 4H) and so it exhibited birefringence. Observation under a polarized light microscope
with a compensator showed the orientation of cellulose
(Fig. 5C, F, I). In the figure, blue and yellow interference
colours are vertical; blue interference colour shows that
the orientation of cellulose is parallel to the x-axis, whilst
yellow interference colour shows that the orientation of cellulose is parallel to the z-axis. Tracheary elements in petal
and bract tissues of C. morifolium had two vertical orientations of cellulose (Fig. 5F, I). However, the cell walls of
34
Nishikawa et al. — Characteristic Thickened Cell Walls of Floral Leaves
A
B
C
200 m m
D
F
H
CU
CU
PW
CU
PW
PW
V
CYT
E
CP
V
G
CYT
I
PW
CYT
V
PW
PW
V
CYT
F I G . 4. (A– C) Light micrographs in transverse section (adaxial uppermost) with safranine stain: (A) bract of H. bracteatum; (B) petal of C. moriforium;
(C) bract of C. moriforium. Scale bars: (A) ¼ 100 mm; (B, C) ¼ 200 mm. (D–I) Electron micrographs in transverse section: (D, F, H) epidermal cells,
corresponding to the areas circled in (A–C); (E, G, I) inner cells, corresponding to the areas indicated by the squares in (A– C). (D, E) The bract of
H. bracteatum; (F, G) the petal of C. moriforium; (H, I) the bract of C. moriforium. Abbreviations: CP, chloroplast; CU, cuticle layer; CYT, cytoplasm;
PW, primary cell wall; V, vacuole; arrowhead, middle lamella (intercellular layer); $, characteristic thickened secondary cell wall. Scale bars: (D– H) ¼
4 mm; I ¼ 8 mm.
parenchyma cells, except for those of tracheary elements
and the outer periclinal walls, exhibited no birefringence
in C. morifolium petal and bract tissues. On the other
hand, cell walls of all the cells exhibited birefringence in
H. bracteatum bract tissue (Fig. 5B). They had the same
orientation of cellulose as tracheary elements in
C. morifolium petal and bract tissues (Fig. 5C). Moreover,
the orientation of cellulose of the outer periclinal walls
was parallel to the x-axis, not parallel to the z-axis in the
H. bracteatum bract (Fig. 5C).
DISCUSSION
Most nuclei disappeared in the upper half of the bract of
H. bracteatum before anthesis, as observed by DAPI staining and fluorescence microscopy (Figs 2, 3); moreover, no
organelles were observed in the cells of the H. bracteatum
bract (Fig. 4D, E). For these reasons, it is shown that the
bracts at anthesis are composed of dead cells.
Characteristic thickened secondary cell walls on the inside
of the primary cell wall were observed in all cells of
the bract tissue of H. bracteatum by TEM (Fig. 4D, E).
Nishikawa et al. — Characteristic Thickened Cell Walls of Floral Leaves
A
D
G
B
E
H
C
F
I
35
x
z
F I G . 5. Polarized light micrograph in transverse section. (A– C) Bract of H. bracteatum; (D–F) petal of C. moriforium; (G– I) bract of C. moriforium.
(A, D, G) Light micrograph with safranine stain; (B, E, H) normal polarized light micrographs; (C, F, I) polarized light micrographs with compensator.
Vertical directions of polarized waves are indicated by x and z. Scale bars ¼ 50 mm.
The walls of all the cells of H. bracteatum bract tissue
exhibited birefringence, as determined by polarized light
microscopy (Fig. 5B, C); the birefringence arises from the
characteristic thickened secondary cell wall. It is therefore
suggested that these walls of H. bracteatum bract cells
have orientated cellulose microfibrils, as do the secondary
cell walls of the tracheary elements of petal and bract
tissues of C. morifolium. The birefringence at the outer
periclinal walls of the epidermal cells of C. morifolium
bract tissue (Fig. 4H, I) may be from many layers of
primary cell walls. In summary, the bract of
H. bracteatum is composed of dead cells, which have
characteristic thickened secondary cell walls. These secondary cell walls have orientated cellulose microfibrils.
Cell walls of plants are classified into two types, primary
and secondary. Growing cells, which have a viscoelastic
property and can expand, form primary cell walls.
Secondary cell walls are formed on the inside of primary
cell walls after cell growth has ceased. They function in
mechanical support in plants. They contain a higher
36
Nishikawa et al. — Characteristic Thickened Cell Walls of Floral Leaves
proportion of cellulose than primary cell walls, and the
orientation of cellulose microfibrils may be more neatly
aligned parallel to each other than in primary cell walls
(Taiz and Zeiger, 2002). Due to this property, only secondary cell walls exhibit birefringence. Moreover, cells with
secondary cell walls are often dead. Are the characteristic
secondary cell walls of the H. bracteatum bract the same
as general secondary cell walls? We compared cells of
the bract of H. bracteatum with taxonomical cells with
general secondary cell walls.
Plant cells are classified into many cell types. According
to the classification of Raven et al. (2005), cells with secondary cell walls are classified into three types: specialized
parenchyma cells, sclerenchyma cells, and cells of
tracheary elements. Sclerenchyma consists of fibres and
sclereids, whilst tracheary elements consist of vessels and
tracheids.
Leaves and petals contain a high amount water, and are
composed mostly of parenchyma cells with primary cell
walls and living cytoplasms. The cells of petal and bract
tissues of C. morifolium had only a primary cell wall
(Fig. 4F – I) and no birefringent properties (Fig. 5E, F, H,
I). ‘Transfer cells’ are specialized parenchyma cells with
secondary cell walls. Morphologically two categories of
cell wall ingrowths can be recognized for most transfer
cells; reticulate and flange (Pate and Gunning, 1972;
Talbot et al., 2002). The shape of the secondary cell
walls of the H. bracteatum bract is similar to that of transfer
cells; however, cells of the H. bracteatum bract differ from
transfer cells in their function, their non-living state and
their location. Lobes of the secondary cell walls of transfer
cells are for the transfer of solutes over short distances
(Gunning and Pate, 1969; Gunning, 1977; McDonald
et al., 1996; Harrington et al., 1997), and hence these
cells are not dead. The locations of transfer cells are potential sites of intensive short-distance solute transfer, for
example xylem, phloem and tissues of reproductive and
glandular structures (Gunning et al., 1970; Rost and
Lersten, 1970; Diane et al., 2002; Pate and Gunning, 1972).
Cells of the H. bracteatum bract are similar to sclerenchyma cells in function. The principal function of sclerenchyma cells is mechanical support, and these cells have
secondary cell walls. However, cells of the H. bracteatum
bract differ from fibres, which are a kind of sclerenchyma
cell, in the location and the shape of secondary cell walls.
The locations of fibres are the xylem, phloem, hypodermis,
cortex and central cylinder (Evert, 2006), and secondary
cell walls of fibre cells form a flat, thickened layer (Evert,
2006). Another type of sclerenchyma cell, sclereids, also
have various types; leaves are a rich source of sclereids
(Foster, 1955, 1956; Esau, 1977; Karabourniotis et al.,
1994; Karabourniotis, 1998). Cells of the H. bracteatum
bract also differ from sclereids in their overall shape, the
shape of the secondary cell walls, their non-living state
and their location. The sclereids of leaves have numerous
simple pits, are often alive at maturity, and are located in
particular parts of leaves, for example the ends of small
veins, patches, epidermis and intercellular spaces.
Cells of the H. bracteatum bract are similar to those of
tracheary elements in their non-living state and the shape
of the secondary cell walls. Cells of tracheary elements
are dead at maturity and their secondary cell walls have
irregular thickeness and form some lobes (Esau and
Charvat, 1978; Burgess and Linstead, 1984; Groover
et al., 1997; Karlsson et al., 2005). However, cells of the
H. bracteatum bract differ from tracheary elements in
their function; the principal function of tracheary elements
is in water conduction.
In conclusion, cells of the H. bracteatum bract differ
from those of other tissues whose characterized cells have
secondary cell walls, and they may be a new cell type.
All cells of the H. bracteatum bract have characteristic
thickened secondary cell walls that have not been reported
in the parenchyma of any other flowers or leaves. Cells of
the H. bracteatum bract are therefore an interesting
subject for further research on differentiation and
development.
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