Water absorption and dormancy-breaking requirements of physically

Seed Science Research, page 1 of 8
q Cambridge University Press 2012
doi:10.1017/S0960258512000013
Water absorption and dormancy-breaking requirements
of physically dormant seeds of Schizolobium parahyba
(Fabaceae – Caesalpinioideae)
Thaysi Ventura de Souza, Caroline Heinig Voltolini, Marisa Santos and
Maria Terezinha Silveira Paulilo*
Departamento de Botânica, Universidade Federal de Santa Catarina, Florianópolis 88040-900, Brazil
(Received 14 July 2011; accepted after revision 18 January 2012)
Abstract
Physical dormancy refers to seeds that are water
impermeable. Within the Fabaceae, the structure
associated with the breaking of dormancy is usually
the lens. This study verified the role of the lens in
physical dormancy of seeds of Schizolobium parahyba, a gap species of Fabaceae from the Atlantic
Forest of Brazil. The lens in S. parahyba seeds
appeared as a subtle depression near the hilum and
opposite the micropyle. After treatment of the seeds
with hot water, the lens detached from the coat.
Blocking water from contacting the lens inhibited water
absorption in hot-water-treated seeds. High constant
(308C) and alternating (20/308C) temperatures promoted the breaking of physical dormancy and
germination in non-scarified seeds. Maximum percentage of germination occurred earlier for seeds
incubated at 20/308C than for those incubated at
308C. Seeds with a blocked lens did not germinate at
alternating or high temperatures. This study suggests
that alternating temperatures are probably the cause
of physical dormancy break of seeds of S. parahyba in
gaps in the forest.
Keywords: Fabaceae, lens, physical dormancy, water
uptake
Introduction
For plants, it is important that seed germination occurs
in the right place and at the right time, and, for this
reason, most species have mechanisms that delay
germination, such as seed dormancy (Fenner and
Thompson, 2005). The definitions of dormancy in
*Correspondence
Email: [email protected]
seeds have been a source of controversy (Fenner and
Thompson, 2005; Finch-Savage and Leubner-Metzger,
2006). A definition of dormancy that has been
proposed recently is that dormancy is an innate seed
property determined by genetics that defines the
environmental conditions in which the seed is able to
germinate (Finch-Savage and Leubner-Metzger, 2006).
Five classes of seed dormancy are recognized, and one
of them is physical dormancy (Baskin and Baskin,
2004), which is caused by a seed (or fruit) coat that
prevents absorption of water (Morrison et al., 1998;
Baskin and Baskin, 2001; Smith et al., 2002).
Physical dormancy is known to occur in 17 families
of angiosperms, including the Fabaceae (Baskin and
Baskin, 2000; Funes and Venier, 2006), where it occurs in
many species. Water-impermeability of the coat (or in
some species the fruit coat) is caused by the presence of
one or more layers of elongated, lignified Malpighian
cells that are tightly packed together and impregnated
with water-repellant chemicals (Morrison et al., 1998;
Baskin and Baskin, 2001; Smith et al., 2002; Baskin,
2003). Under natural conditions, it has been suggested
that physical dormancy is not broken by seeds passing
through the digestive tracts of an animal or by cracks in
the coat caused by animals (Baskin and Baskin, 2001;
Fenner and Thompson, 2005). One characteristic that
suggests this hypothesis is correct is the presence of a
specialized anatomical region in physically dormant
seeds that develops an opening where water can enter
the seeds (Baskin and Baskin, 2001). Several types of
specialized structures (‘water gaps’) have been found in
12 of the 17 families that have physical dormancy; for
example, the carpellary micropyle in Anacardiaceae;
the bixoide chalazal plug in Bixaceae, Cistaceae,
Cochlospermaceae, Dipterocarpaceae and Sarcolaenaceae; the imbibition lid in Cannaceae; the chalazal plug
in Malvaceae; the lens and hilar slit in Fabaceae (Baskin
et al., 2000) and the micropyle-water gap complex in
Geraniaceae (Gama-Arachchige et al., 2011). However,
in some Fabaceae (subfamilies Caesalpinioideae and
2
T.V. de Souza et al.
Mimosoideae) the lens is absent (Gunn, 1984, 1991) and
after treating some legume seeds to break physical
dormancy, cracks develop in the extrahilar region
or in the hilum that permit entrance of water into the
seeds (Hu et al., 2008, 2009).
Several artificial techniques are used to break physical
dormancy in seeds, including mechanical, thermal and
chemical scarification, enzymes, dry storage, percussion,
low temperatures, radiation and high atmospheric
pressures (Baskin and Baskin, 2001). Studies on seeds
with physical dormancy have contributed greatly to
our understanding of water gaps, the effects of various
factors (e.g. drying, heating, low temperatures and
alternating temperatures) in breaking physical dormancy under natural conditions, and the rate and path
of water entrance into seeds that have become permeable
(Baskin and Baskin, 2001). Under natural conditions, it is
known that temperature is an important environmental
factor for breaking physical dormancy in seeds (Baskin
and Baskin, 2001). Vázquez-Yanez and Orozco-Segovia
(1982) verified that the highly fluctuating temperature
that occurs in gaps, but not in forest understorey, breaks
physical dormancy in gap forest species.
Schizolobium parahyba (Fabaceae –Caesalpinioideae)
is a pioneer woody species from the Atlantic Forest of
Brazil that occurs mostly in gaps and along forest
borders, with physically dormant seeds and anemochoric seed dispersal (Carvalho, 2003). The impermeable seed coat of this species can be broken artificially
by boiling water or mechanical scarification (Cândido
et al., 1981; Freire et al., 2007; Matheus and Lopes, 2007).
The aim of this work was to study the seeds of
S. parahyba with the objectives of: (1) locating the water
gap in the seeds; (2) describing the anatomical
structure of the water gap; and (3) testing the effect
of alternating temperatures on breaking the physical
dormancy of the seeds.
Materials and methods
Seed collection
Seeds of S. parahyba, which remain enclosed in the
similar-shaped papery envelope of endocarp resembling
a wing, were collected from the ground soon after wind
dispersal, during spring, in a section of Atlantic Forest
located in the municipality of Florianopolis, Santa
Catarina, Brazil (278350 3600 S, 488350 6000 W). The endocarp
was removed, and the seeds were stored in plastic bottles
at room temperature until they were used.
1981; Matheus and Lopes, 2007), the seed coats were
made impermeable in four ways: (1) extrahilar region
blocked with paraffin; (2) hilar region blocked with
paraffin; (3) hilum blocked with Super Bonderw glue
(Henkel, Jundiai, Brazil); and (4) lens blocked with
Super Bonderw glue. A control group was of nondormant, non-blocked seeds. Twenty seeds were
utilized for each treatment. Seeds were placed in
transparent plastic boxes of 11 £ 11 £ 3.5 cm on two
layers of filter paper (Whatman No. 1, Whatman
International Ltd, Maidstone, England) with 10 ml of
distilled water. The boxes were stored at 208C with a
photoperiod of 12 h/12 h. Incubated seeds were
counted at intervals of 2 or 3 d for 19 d, during which
time germination was observed.
Analysis of seed coat features
The hilar regions of five intact and five thermally
scarified seeds were fixed in 2.5% glutaraldehyde
in a 0.1 M sodium phosphate buffer at pH 7.2 and
dehydrated in a graded ethanol series. Sections of
40 mm thickness were cut using a sliding microtome.
Histochemical tests were made utilizing Sudan IV for
suberin, cutin, oils and waxes; acid phloroglucinol
and iron chloride for lignin (Costa, 1982); and toluidine
blue for polychromatic reactions to lignin and cellulose
(O’Brien et al., 1965). Images were taken with a digital
camera connected to an optical microscope (Leica MPS
30 DMLS). For scanning electron microscopy (SEM)
analyses, the dehydrated pieces of five intact and five
scarified seeds were immersed in hexamethyldesilasane (HMDS) for 30 min, as a substitute for critical
point drying (Bozzola and Russell, 1991) and then
mounted on aluminium stubs and blocked with a
gold layer (40 nm thick). The pieces were viewed using
a Jeol JSM 6390 LV scanning electron microscope.
To verify the presence of callose in the seeds, sections
of non-fixed samples of the hilar and extrahilar
regions of five intact seeds were immersed in 0.05%
aniline blue with a 0.1 M potassium phosphate buffer
at pH 8.3 (Ruzin, 1951). As a control, some sections were
immersed only in the potassium phosphate buffer. The
sections were observed using an Olympus BX41
microscope, with a mercury vapour lamp (HBO 100)
and a blue epifluorescence filter (UMWU2), at 330–
385 nm excitation and 420 nm emission wavelengths.
Images were taken with a Q-imaging digital camera
(3.3 mpixel QColor3C) and the software Q-captures
Pro 5.1 (Q Images, Surrey, British Columbia, Canada).
Location of the water entrance region
Effect of alternating temperatures on germination
and dormancy break
After artificially breaking dormancy of the seeds by
placing them in water at 988C for 1 min (Cândido et al.,
Seeds were immersed in 5% sodium hypochlorite for
5 min and then washed three times in distilled water.
Physical dormancy in seeds of Schizolobium
For some of the seeds, the region with the lens was
covered using Super Bondw glue, which made the
seeds impermeable. Then the seeds were placed in
transparent plastic boxes on a 5 cm autoclaved layer of
sand moistened with distilled water. The boxes were
stored at 208C, 308C and a 12 h/12 h alternating
temperature regime of 20/308C with a photoperiod of
12 h. Four boxes, each with 20 blocked seeds and
another four, each with 20 non-blocked seeds, were
used for each treatment. Germinated seeds were
counted at intervals of 2 or 3 d for 29 d. To verify the
effect of the temperature on the breaking of dormancy
of the seeds, three boxes with 20 seeds (of known mass)
were stored at 208C, 308C, and at a 12h/12h alternating
temperature of 20/308C with a daily photoperiod of
12 h. Every day the mass of the seeds was measured
until the beginning of germination. The mass of each
seed, after and before the incubation period, was used
to calculate the amount of absorbed water.
Data analysis
A completely randomized design was used in all
experiments. Arcsine-transformed germination data
were analysed using one-way ANOVA with the
software Statistica (Statsoft, 2001). Tukey’s tests were
performed to compare treatments.
Results
Location of the water entrance
Germination (%)
After 19 d of incubation, germination of scarified seeds
exposed to boiling water, as well as the germination of
scarified seeds with the blocked extrahilar region, was
about 80% (Fig. 1). Germination of scarified seeds with
the blocked hilar region (i.e. the hilum plus lens) and
100
90
80
70
60
50
40
30
20
10
0
Control
Extrahilar
Hilar
Hilum
Lens
3
with the blocked lens was only 1.0%. However, 50% of
the scarified seeds with only the hilum blocked
germinated (Fig. 1). The germination levels at the last
day of incubation were similar for seeds blocked in the
lens and in the hilar regions, but significantly different
for scarified seeds and scarified seeds blocked in the
extrahilar region and hilum (P # 0.05).
Analysis of seed coat features
In S. parahyba, the hilar region is near the wide end of
the seeds and consists of the hilum, micropyle and
lens, with the hilum positioned between the micropyle
and lens (Fig. 2a, b).
The seed coat consists of one layer of thick walled,
tightly packed, columnar palisade cells (macrosclereids or Malpighian cells) and sclerenchymatous tissue;
osteosclereids (‘hourglass cells’) are not present
(Fig. 2c – f). The seed coat is covered by a thin cuticle.
In front view, the macrosclereid cells have a hexagonal
shape (Fig. 2d). The palisade layer is thinner in the lens
region than in the rest of the coat (Fig. 2e). It is possible
to see a light line crossing the macrosclereids in the
palisade layer (Fig. 2e, f). Below the sclerenchymatous
tissue and above the endosperm is the tegmen, formed
by a layer of crushed cells with thin walls between two
cuticle layers, which reacted positively to Sudan IV
(not shown). The macrosclereids and the subjacent
sclerenchymatous tissue reacted negatively for lignin
when exposed to iron chloride, phloruglucinol and
toluidine blue. However, the macrosclereids reacted
positively for cellulose when exposed to toluidine
blue. The cuticle reacted positively to Sudan IV.
The upper portion of the macrosclereids, mainly
the light line, showed aniline blue-induced fluorescence, indicating the presence of callose (Fig. 3a).
In non-scarified seeds, the lens is a slight depression at
the side of the hilum and opposite the micropyle
(Fig. 3b). In thermally scarified seeds, the hilum and
micropyle do not show alterations, but in the lens
region a crack forms between the macrosclereids,
exposing the underlying tissue (Fig. 3c).
Effect of alternating temperatures on dormancy
break and germination
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19
Days of incubation
Figure 1. Germination curves for seeds of Schizolobium
parahyba that were thermally scarified (control) and with
extrahilar, hilum and lens regions blocked after scarification.
Germination at day 19 of incubation was not significantly
different for seeds with blocked lens and hilar regions, but
significantly different for scarified seeds and scarified seeds
with blocked extrahilar region and hilum (Tukey’s test,
P # 0.05). Bars indicate standard deviation.
Seeds incubated at alternating temperatures of
20/308C and 308C absorbed about 10 g of water during
3 d of incubation, while those at 208C did not absorb
water (Fig. 4). The amount of absorbed water on the
third day of incubation was similar for seeds incubated
at 20/308C and 308C (about 10 g) but significantly
different for seeds at 208C (P # 0.05).
Seeds incubated at alternating temperatures of
20/308C reached the maximum percentage of germination (about 80%) after 13 d of incubation, while those
T.V. de Souza et al.
4
(b)
(a)
hi
mi
le
mi
hi
le
400 µm
0.5 µm
(c)
(d)
mc
20 µm
5 µm
(e)
(f)
le
mc
200 µm
sc
mc
100 µm
sc
Figure 2. Optical micrographs (OM) and scanning electron micrographs (SEM) of the seed coat of Schizolobium parahyba: (a) front
view of the hilar region (SEM); (b) longitudinal section of the hilar region (OM); (c) extrahilar region of seed coat, showing
macrosclereids (SEM); (d) front view of macrosclereids (SEM); (e) longitudinal section of peripheral tissues of hilar region
showing palisade layer, subjacent sclerenchymatous tissue and shorter macrosclereids in the region of the lens; arrow points to
the light line (OM); (f) longitudinal section of lens region showing palisade layer and sclerenchymatous tissue; arrow points to
the light line (OM). le, lens; hi, hilum; mc, macrosclereids; mi, micropyle; sc, sclerenchymatous tissue.
incubated at a constant temperature of 308C reached
only 7% of germination in the same period. The
percentages of germination at 20/308C and 308C at the
last day of incubation were not significantly different,
but both were significantly different from germination
at 208C (P # 0.05) which was less than 2% (Fig. 5).
Seeds with blocked lens did not germinate.
Discussion
Location of the water entrance region
In physically dormant seeds, dormancy break involves
disrupting an impermeable seed (fruit) coat, thereby
creating an opening for water to enter (Baskin and
Baskin, 2001). However, the initial site where water
enters after physical dormancy is broken varies in the
Fabaceae (Hu et al., 2008; Valtueña et al., 2008). The
hilum and micropyle have been reported to allow water
entrance into seeds after physical dormancy is broken
(Hyde, 1954; Zeng et al., 2005; Hu et al., 2008), as well as
cracks in the cuticle of the seed coat (Morrison et al.,
1998; Hu et al., 2009). Water entrance in the region of the
lens has been reported for legume seeds by several
authors (Dell, 1980; Hanna, 1984; Van Staden et al., 1989;
Serrato-Valenti et al., 1995; Morrison et al., 1998; Baskin
et al., 2000; Burrows et al., 2009; Hu et al., 2009).
The seeds of S. parahyba lack a conspicuous lens, as
observed by Gunn (1991) for the subfamily Caesalpinioideae, and it is distinguished on the seed coat as a
subtle depression close to the hilum and opposite the
micropyle. Our experiments in which the hilar and
extrahilar regions of thermally scarified seeds were
blocked, and also the SEM images of the lens region
after breaking seed dormancy with boiling water,
showed that the lens is the only region involved in the
absorption of water in seeds of S. parahyba. In the study
where dormancy was broken by alternating temperature, the seeds that had blocked lenses did not
Physical dormancy in seeds of Schizolobium
Imbibition (g)
(a)
18
16
14
12
10
8
6
4
2
0
20°C
5
30°C
0
20/30°C
1
2
3
Days of incubation
*
Figure 4. Imbibition curves (water absorbed in grams) for
intact seeds of Schizolobium parahyba incubated at 208C, 308C
and 20/308C. Imbibition at the third day of incubation was
similar for seeds incubated at 308C and 20/308C, but
significantly different for seeds at 208C (Tukey’s test,
P # 0.05). Bars indicate standard deviation.
100 µm
(b)
hi
mi
le
did not affect the location where the water initially
entered, which was always through the lens. Unfortunately, there are no data on this subject in the literature
about the genus Schizolobium.
Anatomical structure of the seed coat
(c)
mi
hi
le
500 µm
Figure 3. Morpho-anatomical aspects of the seeds of
Schizolobium parahyba. (a) Longitudinal section of the tegument
stained with aniline blue. Arrow indicates the light line with
high fluorescence and upper portion of the macrosclereı́ds.
Asterisk indicates the sclerenchymatous tissue which did not
show fluorescence. (b) Front view of hilar region of a seed (not
thermally scarified). (c) Thermally scarified seed, showing
cracks (arrow) between the macrosclereids and subjacent tissue
of the lens. le, lens; hi, hilum; mi, micropyle.
germinate, indicating that the lens is broken by
alternating temperature. However, Hu et al. (2009)
obtained results that indicated that the primary site of
water entry, after the breaking of physical dormancy,
can vary for Vigna oblongifolia and that it depended on
the treatment (boiling water or sulphuric acid). For
Sesbania sesban, however, Hu et al. (2009) found that the
treatment method used to break physical dormancy
Our study showed that the seed coat of S. parahiba is
composed of a coat and tegmen. The coat originates from
the outer integument of the ovule and the tegmen from
the inner integument (Corner, 1951). It is considered an
exotestal seed because the main mechanical layer of the
coat lies in the outer epidermis of the outer integument
(Corner, 1951). As described for other species of Fabaceae
(Corner, 1951), the coat of S. parahyba consists of a layer of
palisade cells with thick walls, that are packed tightly
together, a light line and sclerenchymatous tissue.
However, the layer of osteosclereid cells (also called
‘hourglass cells’) that usually lies below the palisade
layer is not present. Smith et al. (2002) reported that
‘hourglass cells’ are not universally present in Fabaceae.
The light line lies just beneath the cuticle, as in
Glycine max (Harris, 1983; Ma et al., 2004), but in
S. parahyba, this line crosses the palisade layer in the
middle third of the macrosclereids, as in other species
Germination (%)
200 µm
100
90
80
70
60
50
40
30
20
10
0
20°C
30°C
20°C/30°C
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Days of incubation
Figure 5. Germination curves for intact seeds of Schizolobium
parahyba incubated at 208C, 308C and 20/308C. Germination
on day 29 of incubation was not significantly different for
seeds at 308C and 20/308C, but significantly different for
seeds at 208C (Tukey’s test, P # 0.05). Bars indicate standard
deviation.
6
T.V. de Souza et al.
of Fabaceae (Serrato-Valenti et al., 1995; Leython and
Jáuregui, 2008). The origin of the light line has been
discussed by many authors, and Kelly et al. (1992)
suggested that it is an optical phenomenon generated
by the juxtaposition of the inner cellulose of the
palisade cells and the outer suberized caps. For Pisum
sativum, Harris (1983) noted that the light line becomes
discernable with a light microscope, at the junction of
the cellulosic tips of the macrosclereids and the line of
the subcuticular layer, and may represent the suberin
caps. Martens et al. (1995) indicated that the light line
in Trifolium repens is caused by an alteration of cellulose
microfibrillar orientation in palisade cell walls. Ma et al.
(2004) reported that in G. max the light line is not
merely an optical phenomenon caused by chemical
modifications, but is a real structure formed where the
secondary walls are tightly appressed to one another.
Baskin and Baskin (2001) suggested that the light line
is due to differences in refraction of light by the top
and bottom portions of the macrosclereids, which
differ in chemical composition. In S. parahyba it was
possible that the light line originates at the junction of
the upper portion of the macrosclereids with callose
and the inner portions without callose.
The seed coat of Fabaceae contains several substances, including polysaccharides, lignin, proteins,
phenolic compounds, pigments, waxes, fats and
resinous matter, that protect the embryo or create a
barrier to water (Bewley and Black, 1994). In S. parahyba,
the cell wall of the macrosclereids is composed of
cellulose, as indicated by histochemical tests, but
suberin and lignin, which have been found in the seed
coats of legumes (Kelly et al., 1992), were not present. In
another species of subfamily Caesalpinioideae, Cassia
cathartica, Souza (1981) also found macrosclereids that
only had walls made of cellulose. The presence of
callose in the upper portion of the macrosclereid cells,
and especially in the light line, in S. parahyba has also
been observed in other Fabaceae species (SerratoValenti et al., 1993; Ma et al., 2004), and its function is
associated with the impermeability of the coat to water
(Bhalla and Slattery, 1984; Serrato-Valenti et al., 1993).
The present study showed that in the lens region
the macrosclereids were shorter than in the rest of the
tegument. This has been observed in other legume
species (Serrato-Valenti et al., 1995; Baskin et al., 2000),
and it was suggested that this site is physically the
weakest part of the seed coat and thus more easily
broken by treatments (Serrato-Valenti et al., 1995;
Baskin et al., 2000; Hu et al., 2009).
Effect of alternating temperatures on dormancy
break and germination
Laboratory experiments showed that the physical
dormancy of S. parahyba seeds was broken when the
seeds were exposed to alternating temperatures of
20/308C and a constant temperature of 308C. Germination of the seeds also occurred at these temperature
regimes. These data are consistent with previous
studies, which suggest that the two factors that
influence the breaking of physical dormancy of seeds
are a high constant temperature and fluctuating
temperature (Bewley and Black, 1994; Argel and
Paton, 1999; Baskin and Baskin, 2001). The alternating
temperature that breaks the physical dormancy of a
seed depends on the amplitude of the fluctuation
(Quinlivan, 1966). Seeds of Trifolium subterraneum
soften in response to temperatures that fluctuate
between 308C and 608C each day over a period of
several weeks or months, which are similar to
fluctuations that occur on open soils in Mediterranean
and tropical climates (Hagon, 1971; Quinlivan, 1971;
Taylor, 1981). Germination of species from tropical
coastal dunes increased when temperature fluctuations were greater than 208C and lasted for more than
45 d (Moreno-Casasola et al., 1994). In Thermopsis
lupinoides (Fabaceae), which grows on dunes in Japan,
alternating temperatures of 258C/358C promoted
breaking of physical dormancy (Kondo and Takahashi,
2004). In water-soaked seeds of Ipomoea lacunosa
(Convolvulaceae), dormancy was broken by an
alternating temperature of 35/208C or a constant
temperature of 358C (Jayasuriya et al., 2008). The
regimes of temperatures tested for S. parahyba occur in
gaps in the Atlantic rainforest, the natural environment where this species grows. Thus, we suggest that
temperature is probably the factor involved in breaking the physical dormancy of the seeds of this species
in natural habitats, as reported for Heliocarpus donnellsmithii, a gap tree species from Mexico and Costa Rica
(Vázquez-Yanes and Orozco-Segovia, 1982).
Acknowledgements
This study received financial support from Coordenação de Aperfeiçoamento do Ensino Superior (CAPES),
Brazil.
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