1
Title: Visual perception of colourful petals reminds us of classical fragments
Sophia Rhizopoulou
1
, Apostolis Argiropoulos
1
, Emmanuel Spanakis
2
, Demetris
Gikas
1
, Nikos Alexandredes
1
, Danae Koukos
1
& Demetrios Anglos
2
1
National and Kapodistrian University of Athens, Faculty of Biology, Department of
Botany, Panepistimioupolis, Athens 157 84, Greece
2
Foundation of Research and Technology, Institute of Electronic Structure & Laser,
Heraklion 711 10 Crete, Greece
Text
Colour has attracted the interest and attention of many of the most gifted
intellects of all time. Ideas of early thinkers were not -and could not have been-
grasped on a scientific level without knowledge of a kind that lay far in the
future. One character that is being considered is the colourful surfaces of living
tissues, which could hardly have been visualized without a corresponding
reference to the microscale parallel. Millions of years before man made
manipulated synthetic structures, biological systems were using nanoscale
architecture to produce striking optical effects
1
. Here we show the
microsculpture of the adaxial surface of flower petals from the asphodel, the
Stork's-bill and the common poppy by using optical, scanning electron and
atomic force microscopy. Microsculpture has been studied in leaves
2, 3, 4, 5
and
pollen grains
6
of higher plants. To the best of our knowledge imaging and
nanoscale morphometry of petals has not been reported hitherto. Our findings
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on flower petals' microsculpture may be linked with aspects on colour revealed
from ancient literature.
Empedocles, Democritus, Plato, Aristotle, Theophrastus, Descartes, Hooke,
von Fraunhofer, Newton, Leonardo da Vinci (his notes reveal knowledge of
Theophrastus text on colours), Goethe (his books reveal knowledge of Leonardo's
ideas about colours), Hegel, Schopenhauer, Young, Maxwell, Helmholtz, Hering and
Schrödinger all have been intrigued by colour and have contributed to our knowledge
of it. Also, colour had always been an interesting subject for philosophers, although
their ideas could be neither confirmed nor disproved for nearly two thousand years.
Democritus (460370 B.C.) regarded colours as the visually perceived result of
various shapes, orderings, and positions of atoms and he conceived of colours as
quantities of energy (light) that reveals translucence as an incorporeal property
ranging from bright to dark. Plato (427347 B.C.) wrote that colours are generated
through the interaction of certain defined kinds of elemental particles and visual
organs
7, 8
. According to Aristotle (384322 B.C.) colour is a visible corporeal quality
9
.
The Pythagoreans named the corporeal surface colour; colour is either in the limits (of
bodies) or is the limit itself
9
. Theophrastus (370288 B.C.) was the first to come up
with a description of colour
10, 11
According to Zeno (332261 B.C.) colours constitute
the first determinant of the form of matter
11
, this extraordinary statement stands
furthermore isolated among the statements about colour and has received very little
attention
12, 13
. Newton (16421726) states that wavelength composition of a light
beam serves to define its colour, i.e. waves of length 400 to 450 nm are violet, 450 to
480 blue, 480 to 560 green, 560 to 590 nm yellow, 590 to 620 nm orange and 620 to
800 mm red. So pervasive is this doctrine in contemporary life that the existence of
other explanations of colour is considered a matter for history of science
11, 14, 15
. In the
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ongoing development of colour theory over centuries, Johann Wolfgang von Goethe
(1749-1832) rejuvenated classical, philosophical ideas on colour
16
.
In the plant kingdom, pigments of flowers can be exploited as sources for
natural pigments and structural patterns. It is likely that natural systems offer
technologically unrealised photonic structures and design protocols
2
, while modern
industry is conceived and nurtured largely by the demands for colour
17
.
Flowering plants known from antiquity
18
(presented by Theophrastus, named
by C. Linnaeus and collected by J. Sibthorp
18
) rely on a remarkable visual strategy in
order to attract pollinators. It is likely that colourful flowers have co-evolved with the
way pollinators see them
19, 20
, while floral colour has been used by pollinators as a
predictor of nutritional rewards
21, 22, 23
and warmth
24
.
We present
microscopic images of the adaxial surfaces of off-white petals (Fig.
1a) from Asphodelus ramosus L. (asphodel), of crimson petals (Fig. 1b) from
Erodium malacoides (L.) L' Hir. (Stork's-bill) and red petals (Fig. 1c) from Papaver
rhoeas L. (common poppy), consisting of two-three individual layers of cells,
approximately 230 m
thick
25, 26
.Cuticle folds of the surface of petals were observed
by using scanning electron (SEM) and atomic force microscopy (AFM). To perform
our work we relied on a high resolution tool in order to detect the first topographical
information of petal surfaces.
Images of the adaxial surface of petals from the asphodel flower were obtained
by using optical microscopy (Fig. 1a
1
), showing mature stomata. In SEM image,
nearly parallel cuticle folds were observed (Fig. 1a
2
). AFM imaging, over a 4 m
2
scan area reveals a periodicity of cuticle folds (Fig. 1a
3
) and a detailed surface relief
(Fig. 1a
4
). According to Theophrastus
10
what is smooth is white and all smooth
surfaces are brilliant
9
; but brilliant substances must also have open passages and be
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translucent
10
. Democritus calls white smooth and black rough and he refers to the
shape of the atomic figures
13
.
Epidermal cells of crimson Erodium flower petals display thickenings of the
cell wall at their base in a radial distribution (Fig. 1b
1
) and the rounded cells have a
conical-papillate shape (Fig. 1b
2
) that resembles images obtained by AFM (Fig. 1b
3
)
and the surface relief is shown in Figure 1b
4
. According to Theophrastus crimson
comes from white, red and black
10
; for thus it makes an appearance delightful to the
senses, while its brilliance and lustre testify to the presence of white
13, 15
. Aristotle
proposed purple as the strongest colour-energy after light itself, while warmth is
associated with red
27
. In the nanoworld, a particle with a diameter of about 100 nm
appears purple-pink, but the colour shifts to red for particles with diameters of around
20 nm
28
.
The adaxial surface of petals of Papaver (Fig. 1c) viewed by optical
microscope possesses oblong, red, epidermal cells with anticlinal undular walls (Fig.
1c
1
). Cuticle folds of petals form an irregular pattern, as is apparent in SEM (Fig. 1c
2
),
in AFM imaging (Fig. 1c
3
) and from the surface relief (Fig. 1c
4
). According to
Theophrastus red is composed of atoms whose form is the same as, but larger than,
that of things which are warm
10
.
Colour reflects the state of the object to which it belongs and it depends on a
relationship between light and the corporeal quality of the matter. However, "true"
and "pure" colours are never seen
27, 29
. It also seems likely that the perception of
colour has been influenced by qualities and temperaments being associated with
colours. In addition, colour can be seen on a surface, as a spirited thing and the word
to describe it was often fittingly applied as an adjective meaning something related to
the colour itself. On the other hand, colourful plant pigments concentrated in vacuoles
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and plastids are dependent on a mixture of elements in the cells, and in the
environment (e.g. pH, heat and moisture). Therefore, it might be an oversimplification
that a wavelength of light determines a colour.
Methods Summary
Fully expanded, turgid petals were harvested from flowers of Asphodelus ramosus
spring and then were carefully transferred to the laboratory. Fresh tissue samples were
cut and the surface sections were directly examined with a Zeiss Axioplan II
microscope (Zeiss, Oberkochen, Germany) equipped with a camera, using Kodac
colour 400 film. Petal samples were carefully cut in 24 mm
2
pieces and fixed in 3 %
glutaraldehyde in phosphate buffer at pH 7 at room temperature for 2 h. The tissues
were then postfixed in 1 % OsO
4
in the same buffer at 4 °C for 4 hours and
dehydrated in a graded acetone series. The dehydrated tissue samples were critical
point dried, mounted with a double adhesive tape on stubs, sputter coated with gold
30
and observed with a scanning electron microscope (JEOL 6300 SEM, Japan); SEM
pictures were digitally recorded. Floral tissues, in their natural state, are very sensitive
to the mechanical pressures of atomic force microscopy (AFM); thus, a mild method
of fixation was performed on petals by using glutaraldehyde, paraformaldehyde and
osmium tetroxide. Segments (4 mm
2
) were cut from the adaxial petal surface and
were immersed in a solution of 2 % glutaraldehyde plus 2% paraformaldehyde, in 0.1
M sodium cacodylate buffer (SCB), at pH 7.2, for 2.5 h, at room temperature. The
plant material was washed three times by immersion in SCB for 5 min, and postfixed
in 2 % OsO
4
for 5 h at 4°C. It was then washed three times in SCB for 30 min (each
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6
time) at room temperature. The tissues, immersed in the buffer solution, remained at
4°C until they were to be studied. In order to quantify surface profiles of petals from
topographic images, the stained tissues were screened in tap mapping atomic force
microscope (TM-AFM, Multimode SPM, Veeco, USA) and various parameters were
analysed and processed by using the software package Nanoscope III (Veeco, USA).
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Figure Legend
Figure 1 Morphometry of flower petal surface (open square indicates the area of
the petal adaxial surface obtained by using microscopes). a. Asphodelus ramosus' off-
white surface obtained by optical microscope (a
1
), SEM (a
2
) and TM-AFM (a
3
), with
profile view of the line section (a
4
). b. Erodium malacoides' crimson surface obtained
by optical microscope (b
1
), SEM (b
2
) and TM-AFM (b
3
) image, with profile view of
the line section (b
4
). c. Papaver rhoeas' red surface obtained by optical microscope
(c
1
), SEM (c
2
) and TM-AFM (c
3
), with profile view of the line section (c
4
).
Acknowledgements. This work was carried out as part of the Pythagoras (70/3/8036)
programme that is co-funded by the European Social Fund and National Resources
(EPEAEK II).
Author contributions. S.R., A.A. and D.A. designed the research. A.A., E.S. and
N.A. performed the experiments. S.R., A.A., E.S., D.G., D.K. and D.A. analysed the
data. S.R., A.A., D.K. and D.A. contribute to the preparation of the manuscript. S.R.
and D.K. wrote the paper.
Nature Precedings : hdl:10101/npre.2008.1523.1 : Posted 16 Jan 2008
a
b
c
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