Micron 44 (2013) 312–316
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Micron
journal homepage: www.elsevier.com/locate/micron
The role of fibres and the hypodermis in Compositae melanin secretion
Orlando Cavalari De-Paula a,∗ , Juliana Marzinek b , Denise Maria Trombert Oliveira c ,
Silvia Rodrigues Machado d
a
Departamento de Botânica, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, PR, Brazil
Instituto de Biologia, Universidade Federal de Uberlândia, Uberlândia, MG, Brazil
Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
d
Departamento de Botânica, Instituto de Biociências, UNESP-Universidade Estadual Paulista, Botucatu, SP, Brazil
b
c
a r t i c l e
i n f o
Article history:
Received 5 May 2012
Received in revised form 6 August 2012
Accepted 7 August 2012
Keywords:
Asteraceae
Cypsela
Fruit
Phytomelan
Phytomelanin
Secretory structure
a b s t r a c t
Melanins are dark, insoluble pigments that are resistant to concentrated acids and bleaching by oxidising
agents. Phytomelanin (or phytomelan) is present in the seed coat of some Asparagales and in the fruits
of some Compositae. In Compositae fruits, melanin is deposited in the schizogenous spaces between the
hypodermis and underlying fibrous layer. Phytomelanin in Compositae is poorly understood, and there
are only speculations regarding the cells that produce the pigment and the cellular processes involved in
the secretion and polymerisation of phytomelanin. This report describes the cellular processes involved
in the secretion of phytomelanin in the pericarp of Praxelis diffusa, a species with a structure typical of
the family. The ovaries and fruits at different stages were fixed and processed according to the standard methods of studies of light microscopy and transmission electron microscopy. Hypodermal cells
have abundant rough endoplasmic reticulum and mitochondria, and the nuclei have chromatin that is
less dense than other cells. These characteristics are typical of cells that synthesise protein/amino acids
and suggest no carbohydrate secretion. The fibres, however, have a dense cytoplasm rich in the Golgi
bodies that are associated with vesicles and smooth endoplasmic reticulum, common characteristics of
carbohydrate secretory cells. Our results indicate that the hypodermal cells are not responsible for the
secretion of phytomelanin, as previously described in the literature; in contrast, this function is assigned
to the adjacent fibres, which have an organisation typical of cells that secrete carbohydrates.
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Melanins are dark, insoluble pigments that are resistant to
degradation by concentrated acids and bleaching by oxidising
agents (Nicolaus et al., 1964). These substances can be found
in plants, animals and microorganisms (Bell and Wheeler, 1986;
Kotob et al., 1995; Hill, 1997; Gibson and George, 1998; Prota
et al., 1998; Sarna and Swartz, 1998). In plants, a compound
known as phytomelanin (or phytomelan) is present in the seed
coat some taxa of Asparagales (Dahlgren and Clifford, 1982) and
the fruits of certain Compositae (Pandey and Singh, 1982; Pandey
et al., 1989; Marzinek et al., 2008, 2010; Marzinek and Oliveira,
2010).
Fruits with phytomelanin are found in approximately 5400
species in 460 genera, including 11 tribes, and form a monophyletic group called the Phytomelanin Cypsela Clade (PCC), with
∗ Corresponding author at: Universidade Federal do Paraná, Departamento de
Botânica, Setor de Ciências Biológicas, Centro Politécnico, Av Cel Francisco H dos
Santos, s/n, Jardim das Américas, CP 19031, CEP 81531-990, Curitiba, PR, Brazil.
Tel.: +55 34 91670707.
E-mail address: orlandocavalari@gmail.com (O.C. De-Paula).
0968-4328/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.micron.2012.08.003
neotropical representatives accounting for approximately one
quarter of the species of Compositae (Panero and Funk, 2008).
Beyond the PCC, the presence of phytomelanin was confirmed in
fruits of subtribe Sipolisiinae (Vernonieae; Loeuille, 2011). It is
believed that the adaptive value of phytomelanin is related to the
protection of the fruit against predation (Johnson and Beard, 1977).
However, it is possible that the occurrence of phytomelanin has
been underestimated in the family, as there are recent reports of
its presence in the vegetative organs of tribe Cardueae (Fritz and
Saukel, 2011).
Despite its importance in the family, the processes involved in
forming the phytomelanin layer remain unclear; indeed, several
authors have debated for a century about which pericarp tissue is
responsible for its production, what the cellular mechanisms are
and how the polymerisation occurs. Hanausek (1912) suggested
that phytomelanin is formed by the modification of cell wall fibres.
Sárkány (1947) proposed that the layer of phytomelanin may be
produced by the hydration of the inner wall of the hypodermis,
whereas Knowles (1978) indicated that this layer would be formed
by lysis of the fibres. Rogers et al. (1982) suggested that the phytomelanin layer may be the result of the lysis of hypodermal cells
discharging their content into the fibres. De Vries (1948), Misra
(1964, 1972), Pandey and Singh (1982, 1983) and Pandey et al.
O.C. De-Paula et al. / Micron 44 (2013) 312–316
313
(1989) proposed that the layer phytomelanin is secreted by the
hypodermis. Pandey et al. (1989) also observed the occurrence of
endoplasmic reticulum (apparently smooth) in the hypodermis,
thus enhancing the secretory potential of hypodermal cells. According to certain authors, phytomelanin precursors are synthesised in
the hypodermis and polymerised by enzymatic activity in the cell
wall of the fibres. However, there are no reports of secretion in
the hypodermis or fibres, and the cellular processes involved in
melanin production in Compositae remain unknown.
Here, we describe the cellular processes involved in the production of phytomelanin in Compositae and clarify the role of the
hypodermis and fibres in the synthesis and deposition of this compound. In addition, we discuss the possible pathways of secretion,
as compared with other groups of plants that produce this pigment.
synthetic resin and observed using an Olympus BX41 light microscope.
For transmission electron microscopy (TEM), the samples were
fixed in glutaraldehyde (2.5% in 0.1 M phosphate buffer, pH 7.3)
for 24 h, post-fixed in osmium tetroxide (1% in 0.1 M phosphate
buffer, pH 7.3) incubated in uranyl acetate (0.5% aqueous solution), dehydrated through an acetonic series and embedded in
Araldite. Ultrathin sections (50 nm) were contrasted using a saturated solution of uranyl acetate and lead citrate (Reynolds, 1963)
and examined using a TEM Philips EM 100 microscope at 80 kV.
2. Material and methods
The outer ovarian epidermis is uniseriate, with cuboids cells.
The mesophyll features two regions: a hypodermis with two layers
(slightly elongated cells) and an inner region with four to five layers
(highly elongated cells). The inner epidermis is uniseriate, also with
highly elongated cells (Fig. 1a and b).
The early development of the phytomelanin layer is characterised by the formation of intercellular spaces between the
hypodermis and underlying layer (Fig. 1c), as evidenced by a
degraded middle lamella (Fig. 1d). The cytoplasm of cells of the
hypodermis and underlying layer is restricted to the periphery
(Fig. 1c–f) and contains Golgi bodies, mitochondria, plastids with
plastoglobulin (Fig. 1e), a large central vacuole (Fig. 1c–f) and a
nucleus with condensed chromatin (Fig. 1f).
Praxelis diffusa (Rich.) Pruski plants were collected in ruderal
areas in the region of Botucatu, São Paulo, Brazil (22◦ 53 11,4 S,
48◦ 26 07,8 W). A voucher specimen was deposited at the Herbarium BOTU (Holmgren et al., 1990) under accession number 25,555.
For light microscopy (LM), the ovary and fruit at different stages
were fixed in FAA 50 for 48 h (Johansen, 1940) and stored in 70%
ethanol (Jensen, 1962). The samples were dehydrated through an
ethanol series and embedded in methacrylate (Leica), according
to the manufacturer’s instructions. Transverse and longitudinal
sections (8 ␮m) were stained with toluidine blue (0.05% in 0.1 M
acetate buffer, pH 4.7) (O’Brien et al., 1964 modified), mounted in
3. Results
3.1. Flower buds
Fig. 1. Flower buds of P. diffusa during the early development of the schizogenous space where the phytomelanin layer will be deposited. (a and b light micrographs): (a) Ovary
transversal section; (b) Detail of the ovary wall in longitudinal section; (c–f TEM micrographs from transverse sections): (c) Overview of the ovary wall showing the epidermis,
two layers of hypodermis, a layer of undifferentiated fibres and the remaining undifferentiated mesophyll, note the beginning of the formation of the schizogenous space
(arrow); (d) Detail of the previous panel, showing the separation of the hypodermis and undifferentiated fibres; (e) Details of undifferentiated fibres showing the organelles;
(f) Detail of undifferentiated fibres showing the nucleus with condensed chromatin (ep: epidermis; fl: fibre layer; gb: Golgi bodies; hy: hypodermis; m: mitochondria; ml:
middle lamella; n: nucleus; ov: ovule; ow: ovary wall; p: plastid; v: vacuole).
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Fig. 2. P. diffusa flowers at post-anthesis, at the end of the formation of the schizogenous space and the early lignification of the fibre layer. (a) Light micrograph of overview
of the pericarp in longitudinal section showing the hypodermis and fibres, note the schizogenous space formed (arrow); (b–f TEM micrographs from transverse sections):
(b) Detail of the outer pericarp, note the separation between the anticlinal walls of the innermost layer of the hypodermis; (c) Pericarp detail showing the hypodermis and
fibre layer, note that the separation between the hypodermis and fibre layer is partial; (d) Detail of a hypodermal cell showing the organisation of their organelles; (e) Detail
of a fibre, note that there is no lignification near the hypodermis (arrowhead); (f) Detail of the previous panel showing the organisation of the organelles (ex: exocarp; fl:
fibre layer; gb: Golgi bodies; hy: hypodermis; m: mitochondria; n: nucleus; p: plastid; se: seed; ss: schizogenous space; v: vacuole; ve: vesicle).
3.2. Flowers at anthesis
In addition to the separation of the periclinal cell walls between
the hypodermis and the underlying layer (now undifferentiated
fibres), a separation between the anticlinal cell walls of the inner
hypodermis was observed, thus the outermost hypodermis layer
also remains in contact with the schizogenous space (Fig. 2a–c).
The cytoplasm volume of the hypodermal cells and undifferentiated fibres is considerably increased (Fig. 2b and c): the nucleus
has chromatin that is less dense and a large nucleolus; and the
cytoplasm has plastids, mitochondria, Golgi bodies and small vacuoles (Fig. 2d). The fibre cell walls begin to thicken (Fig. 2e and f),
but this process does not occur homogeneously (Fig. 2e), and there
was no thickening in the projections formed between the fibres
and the hypodermis (Fig. 2e). A proliferation of Golgi bodies and its
associated vesicles was observed in the fibres (Fig. 2e and f).
The cytoplasm is restricted to the cell periphery (Fig. 3b, e and f) and
concentrated in the projection (Fig. 3e–f). In the fibre projections,
the cytoplasm is more abundant, and the Golgi apparatus and associated vesicles are more developed. All of the organelles display a
higher electron density (Fig. 3e and f) compared with the previous
stage (Fig. 2e and f).
There is a secretion of electron-dense and amorphous material (Fig. 3g) in the periplasmic space of the fibres (Fig. 3e
and f). This material traverses the cell wall (Fig. 3g and h) and is
deposited on the surface of the cell wall, forming plaques within the
schizogenous space that is initially adjacent to the wall of the fibres
(Fig. 3b) and is thereafter found in the cell walls of the hypodermis
(Fig. 3g–i).
4. Discussion
3.3. Young fruits (3 days after anthesis)
4.1. Is the hypodermis responsible for phytomelanin secretion in
Compositae fruits?
At the third stage of development, the phytomelanin layer is
characterised by the secretion and polymerisation of phytomelanin
(Fig. 3). The hypodermal cells have thin walls and electron dense
and abundant cytoplasm (Fig. 3a–d), with small central vacuoles,
few plastids (Fig. 3b), a developed rough endoplasmic reticulum
(RER) and mitochondria (Fig. 3c). The RER is dispersed throughout the cytoplasm, and the mitochondria present developed cristae
(Fig. 3c). The nucleus is spherical and has less dense chromatin
(Fig. 3d).
The fibres show thick electron-dense walls and a protoplast
organisation similar to the previous phase (compare with Fig. 2b).
Several authors have discussed the processes involved in the
production of phytomelanin in the fruits of Compositae, suggesting the occurrence of changes in the cell wall (Hanausek, 1912;
Sárkány, 1947), cell lysis, deposition of protoplasm (Rogers et al.,
1982) or secretion (Pandey et al., 1989). In our observations, the cell
wall (hypodermis and fibres) did not exhibit changes that indicate a
participation in the production of melanin during the development
of the fruit of P. diffusa, results that disagree with the assertions of
Hanausek (1912) and Sárkány (1947). Processes of cell lysis were
not observed in P. diffusa, refuting the reports by Rogers et al. (1982)
and Knowles (1978).
O.C. De-Paula et al. / Micron 44 (2013) 312–316
315
Fig. 3. P. diffusa fruits at three days post-anthesis during the early secretion of phytomelanin. (a) Light micrograph of cross section of the pericarp, note the layer of
phytomelanin (arrow); (b–i TEM micrographs from transverse sections): (b) Detail of the pericarp, note the layer of phytomelanin (arrow); (c) Detail of the previous panel
showing the organisation of the cytoplasm; (d) Detail of the hypodermal cell showing the nucleus with condensed chromatin; (e) Detail of the contact between the hypodermis
and fibre at the beginning of secretion (arrowhead); (f) Detail of the contact between the hypodermis and fibre, with a higher volume of secretion (arrowhead); (g) Detail of
a hypodermal cell showing the periplasmic space with phytomelanin (arrowhead) and the beginning of the polymerisation of the layer; (h) Hypodermal cell detail showing
the beginning of the formation of the phytomelanin layer; (i) Hypodermal cell with the phytomelanin layer polymerised (cw: cell wall; ex: exocarp; hy: hypodermis; fl: fibre
layer; gb: Golgi bodies; m: mitochondria; n: nucleus; p: plastid; pl: phytomelanin layer; rer: rough endoplasmic reticulum; ss: schizogenous space; ser: smooth endoplasmic
reticulum; v: vacuole; ve: vesicles).
The hypodermis is regarded by others as responsible for the
secretion of phytomelanin in Compositae (Misra, 1964, 1972;
Pandey and Singh, 1982, 1983; Pandey et al., 1989), however the only evidence of secretion, documented by Pandey
et al. (1989), was the presence of ER, possibly smooth. In the
present work, it was found that the hypodermis of P. diffusa
has rough ER but not smooth ER, as speculated by Pandey et al.
(1989).
Phytomelan is a brittle, coal-like substance (Dahlgren and
Clifford, 1982), chemically inert and has a carbon, hydrogen and
oxygen ratio of 3.7:2.1:1.0 respectively (Dafert and Miklauz, 1912).
According to Fahn (1979), the secretion of carbohydrates can be
performed by smooth ER, the Golgi apparatus or both. In the
hypodermis of P. diffusa, we observed a developed RER, many mitochondria and nuclei with less-dense chromatin, typical features of
cells actively synthesising a high level of proteins (Gunning and
Steer, 1996). There were no indications of secretion in the hypodermis of P. diffusa, and, therefore, we refute the hypothesis that
the hypodermis is responsible for phytomelanin secretion in Compositae.
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It is known that melanin consists of a single type of a high molecular weight substance formed by the enzymatic oxidation of the
precursor (Thomson, 1976). As hypodermis has cellular characteristics that indicate the synthesis of proteins/amino acids, it is
possible that the hypodermal cells are responsible for the production of the enzymes that oxidise the precursor of melanin. But, our
results do not provide sufficient evidence to support this hypothesis.
Differently from the hypodermis, the fibres demonstrate the
characteristics of cells that secrete carbohydrates, especially with
regard to the presence of a developed Golgi apparatus and associated vesicles. After the lignification of the fibre cell walls, there was
the further production of vesicles, having an electron-dense amorphous content that was restricted to the periphery of the vesicle,
which were secreted primarily at the junction of the fibres with
the hypodermis. Another argument against a role for the hypodermis in the secretion of phytomelanin is that the presence of this
pigment is not associated with the hypodermis in the roots and
rhizomes of some species of the Centaurea tribe (Cardueae; Fritz
and Saukel, 2011) and the fruits of Bishopalea, Heterocoma, Sipolisia
and Xerxes (Vernonieae; Loeuille, 2011). These observations reinforce the notion that the fibres are responsible for the secretion of
phytomelanin in P. diffusa.
4.2. Comparison of phytomelanin secretion between Asparagales
and Compositae
The synthesis of phytomelanin in Asparagales is characterised
by three phases: the secretion of callose, the degradation of callose
and the deposition of phytomelanin (Wittich and Graven, 1995,
1998). The deposition of callose is performed by vesicles of the Golgi
apparatus, adding an internal reinforcement to the cellulosic wall of
the exotesta, as reported in Gasteria verrucosa (Wittich and Graven,
1995). In Compositae, it has not been reported that callose and phytomelanin are deposited outside of the fibre and hypodermis cell
walls.
In the second phase, the callose is degraded, possibly producing the precursors of phytomelanin in Asparagales (Wittich and
Graven, 1995, 1998). The data presented here for Compositae do
not indicate where the melanin precursors are produced. Concomitant with the degradation of callose in Gasteria verrucosa, Wittich
and Graven (1995, 1998) observed large amounts of RER that contained an osmiophilic material, interpreted as phenolic substances,
occasionally occupying the periplasmic space. In Praxelis diffusa,
the precursors are processed and secreted by the fibres into the
schizogenous space between the fibres and hypodermis.
The data obtained in this study permitted the conclusion that
the hypodermal cells do not have cellular apparatus for secreting
carbohydrates, so they cannot be responsible for secretion of phytomelanin as speculation in the literature. In contrast, this function
is assigned to the adjacent fibres, which have a typical organisation
of cells that secrete carbohydrates.
Acknowledgements
We thank the Centro de Microscopia Eletrônica (IB, UNESP Botucatu) for help in processing the samples for TEM. DMT Oliveira and
SR Machado thank Conselho Nacional de Desenvolvimento Científico e Tecnológico for research fellowships.
References
Bell, A.A., Wheeler, M.H., 1986. Biosynthesis and functions of fungal melanins.
Annual Review of Phytopathology 24, 411–451.
Dafert, F.W., Miklauz, R.v.S., 1912. Untersuchungen über die kohleähnliche Masse
der Kompositen (Chemischer Teil) Denkschriften der Östereichischen Akademie
der Wissenschaften. Mathematisch-Naturwissenschaftliche Klasse 87,
143–152.
Dahlgren, R.M.T., Clifford, H.T., 1982. The Monocotyledons. A Comparative Study.
Academic Press, New York.
De Vries, M., 1948. The Formation of Phytomelan in T. patula and Some Other Compositae. Universiteit Leiden, Leiden, p. 75.
Fahn, A., 1979. Secretory Tissues in Plants. Academic Press, London.
Fritz, E., Saukel, J., 2011. Secretory structures of subterranean organs of some species
of the Cardueae and their diagnostic value. Acta Biologica Cracoviensia. Series:
Botanica 53, 62–72.
Gibson, L.F., George, A.M., 1998. Melanin and novel melanin precursors from
Aeromonas media. FEMS Microbiology Letters 169, 261–268.
Gunning, B.E.S., Steer, M.W., 1996. Plant Cell Biology: Structure and Function. Jones
and Bartlett Publishers, London.
Hanausek, T.F., 1912. Untersuchungen über die kohleähnliche Masse der Kompositen (Botanischer Teilmit 3 Tafeln). Denkschriften der Östereichischen Akademie
der Wissenschaften. Mathematisch-Naturwissenschaftliche Klasse 87,
93–142.
Hill, H.Z., 1997. The photobiology of melanin. Photochemistry and Photobiology 65,
471.
Holmgren, P.K., Holmgren, N.H., Barnett, L.C., 1990. Index Herbariorum. Part I: The
Herbaria of the World. International Association for Plant Taxonomy, New York.
Jensen, W.A., 1962. Botanical Histochemistry: Principles and Practice. W.H. Freeman,
San Francisco.
Johansen, D.A., 1940. Plant Microtechnique. McGraw-Hill, New York.
Johnson, A.L., Beard, B.H., 1977. Sunflower moth damage and inheritance of the
phytomelanin layer in sunflower achenes. Crop Science 17, 369–372.
Knowles, P.E., 1978. Morphology and anatomy. In: Carter, J.F. (Ed.), Sunflower Science and Technology. ASA, Madison, pp. 55–88.
Kotob, S.I., Coon, S.L., Quintero, E.J., Weiner, R.M., 1995. Homogentisic acid is
the primary precursor of melanin syntesis in Vibrio cholerae, a Hyphomonas
strain, and Shewanella colwelliana. Applied and Environmental Microbiology 61,
1620–1622.
Loeuille, B.F.P., 2011. Towards a phylogenetic classification of Lychnophorinae
(Asteraceae: Vernonieae). Instituto de Biociências, Departamento de Botânica,
Universidade de São Paulo, São Paulo, p. 432.
Marzinek, J., De-Paula, O.C., Oliveira, D.M.T., 2008. Cypsela or achene? Refining terminology by considering anatomical and historical factors. Revista Brasileira de
Botânica 31, 549–553.
Marzinek, J., De-Paula, O.C., Oliveira, D.M.T., 2010. The ribs of Eupatorieae (Asteraceae): of wide taxonomic value or reliable characters only among certain
groups? Plant Systematics and Evolution 285, 127–130.
Marzinek, J., Oliveira, D.M.T., 2010. Structure and ontogeny of the pericarp of six
Eupatorieae (Asteraceae) with ecological and taxonomic considerations. Anais
da Academia Brasileira de Ciências 82, 279–291.
Misra, S., 1964. Floral morphology of the family Compositae. 2. Development of the
seed and fruit in Flaveria repanda. Botanical Magazine Tokyo 77, 290–296.
Misra, S., 1972. Floral morphology of the family Compositae. IV. Tribe
Vernonieae–Vernonia anthelmintica. Botanical Magazine Tokyo 85, 187–199.
Nicolaus, R.A., Piatelli, M., Fattorusso, E., 1964. The structure of melanins and
melanogenesis: IV. On some natural melanins. Tetrahedron 20, 1163–1172.
O’Brien, T.P., Feder, N., Mccully, M.E., 1964. Polychromatic staining of plant cell walls
by toluidine blue. Protoplasma 59, 368–373.
Pandey, A.K., Singh, R.P., 1982. Development and structure of seeds and fruits
in Compositae: Coreopsis species. Journal of the Indian Botanical Society 61,
417–425.
Pandey, A.K., Singh, R.P., 1983. Development and structure of seeds and fruits in Compositae: tribe Eupatorieae. Journal of the Indian Botanical Society 62, 276–281.
Pandey, A.K., Lee, W.W., Sack, F.D., Stuessy, T.F., 1989. Development of the phytomelanin layer in fruits of Ageratum conyzoides (Compositae). American Journal of
Botany 75, 739–746.
Panero, J.L., Funk, V.A., 2008. The value of sampling anomalous taxa in phylogenetic studies: major clades of Asteraceae revealed. Molecular Phylogenetics and
Evolution 47, 757–782.
Prota, G., D’Ischia, M., Napolitano, A., 1998. The chemistry of melanins and related
metabolites. In: Nordlund, J.J., Boissy, R.E., Hearing, V.J., King, R.A., Ortonne, J.P.
(Eds.), The Pigmentary System. Oxford University Press, Oxford, pp. 307–332.
Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain
in electron microscopy. Journal of Cell Biology 17, 208–212.
Rogers, C.E., Stafford, R.E., Kreitner, G.L., 1982. Phytomelanin development and role
in hybrid resistance to Homoeosoma electellum (Hulst.) larvae. In: Proceedings
of the 10th International Sunflower Conference, Surfers Paradise, Australia, pp.
138–141.
Sárkány, S., 1947. A napraforgó nemesitése a fitomelán kérdé. Agrártudományi
Szemle 1, 97–103.
Sarna, T., Swartz, H.M., 1998. The physical properties of melanins. In: Nordlund, J.J.,
Boissy, R.E., Hearing, V.J., King, R.A., Ortonne, J.P. (Eds.), The Pigmentary System.
Oxford University Press, Oxford, pp. 333–357.
Thomson, R.H., 1976. Miscellaneous pigments. In: Goodwin, T.W. (Ed.), Chemistry
and Biochemistry of Plant Pigments. Academic Press, London, pp. 597–623.
Wittich, P.E., Graven, P., 1995. Histochemical study of the development of the phytomelan layer in the seed coat of Gasteria verrucosa (Mill.) H. Duval. Protoplasma
187, 72–78.
Wittich, P.E., Graven, P., 1998. Callose deposition and breakdown, followed by
phytomelan synthesis in the seed coat of Gasteria verrucosa (Mill.) H. Duval.
Protoplasma 201, 221–230.