Annals of Botany 92: 289±297, 2003
doi:10.1093/aob/mcg136, available online at www.aob.oupjournals.org
Diffusion Barriers of Tripartite Sporopollenin Microcapsules Prepared
from Pine Pollen
G . B O H N E 1 , E . RI C H TE R 1 , H . W O E H L E C K E 2 and R . EH W A L D 1 , *
1Institut of Biology, Humboldt-University Berlin, Invalidenstr. 42, 10115 Berlin, Germany and
2L.U.M. GmbH, Rudower Chaussee 29, 12489 Berlin, Germany
Received: 7 February 2003 Returned for revision: 8 April 2003 Accepted: 7 May 2003
Tripartite sporopollenin microcapsules prepared from pine pollen (Pinus sylvestris L. and Pinus nigra Arnold)
were analysed with respect to the permeability of the different strata of the exine which surround the gametophyte and form the sacci. The sexine at the surface of the sacci is highly permeable for polymer molecules and
latex particles with a diameter of up to 200 nm, whereas the nexine covering the gametophyte is impermeable
for dextran molecules, with a Stokes' radius >4 nm (Dextran T 70), and for the tetravalent anionic dye Evans
Blue (Stokes' radius = 1´3 nm). The central capsules obtained by dissolution of the sporoplasts showed strictly
membrane-controlled exchange of non-electrolytes, with half-equilibration times in the range of minutes (monosaccharides, oligosaccharides) to hours (dextran molecules with Stokes' radii up to 2´5 nm). The dependence of
the permeability coef®cients of the nexine for non-electrolytes on Stokes' radius or molecular weight shows that
the aqueous pores through the nexine are inhomogeneous with respect to their size, and that most pores are too
narrow for free diffusion of sugar molecules. To explain the barrier function of the nexine for Evans Blue, it is
assumed that at least the larger pores, which enable slow permeation of dextran molecules, contain negative
charges.
ã 2003 Annals of Botany Company
Key words: Sporopollenin, exine, pores, size exclusion, permeability, sugars, dextran, Evans Blue, chromatography,
pine, Pinus sylvestris L., Pinus nigra Arnold.
INTRODUCTION
Moss and fern spores as well as most pollen grains have an
envelope (sporoderm, exine) consisting of sporopollenin
(Zetsche and Vicari, 1931), a biopolymer that is highly
resistant to enzymatic breakdown and hydrolytic decomposition in strongly acid or alkaline media. Sporopollenin
cannot be considered a uniform macromolecule. Infra-red
spectroscopy and 13C NMR spectroscopy of sporopollenin
derived from pteridophyta and spermatophyta demonstrate
the presence of aliphatic, aromatic, hydroxyl, carbonyl/
carboxyl and ether functions in various proportions
(Wilmesmeier et al., 1993). Sporopollenin is produced
largely from acyl lipid and phenylpropanoid precursors
(Wiermann and Gubatz, 1992; Piffanelli et al., 1998; Ahlers
et al., 2000). The mechanisms of its synthesis and of its
consolidation are not yet understood. With respect to its
composition and precursors, sporopollenin and suberin
show similarities (compare Bernards, 2002), and the
biosynthetic capacity of embryophyta to produce suberin
and sporopollenin might have a common origin (Kroken
et al., 1996). However, in contrast to the suberin lamellae,
sporodermata and exines are generally situated outside of
the cellulose/pectin wall (intine). Exines can be partly or
completely solubilized in hot aminoethanol (Bailey, 1960;
Southworth, 1974), and the dissolved polymers re-aggregate
spontaneously when the solvent is exchanged against water
(Jungfermann et al., 1997; Thom et al., 1998). After partial
* For correspondence. E-mail [email protected]
degradation of the exine by different methods, including
oxidation and aminoethanol treatment, regular substructures
of 100±200 nm diameter appear in scanning and transmission electron microscopy (SEM and TEM) images of exines
and sporodermata (Southworth, 1986; Rowley, 1996). There
is still uncertainty regarding the detailed chemical structure
of sporopollenin as well as regarding the conformation and
cross-linking of this polymer.
The multilayered sporopollenin coatings of spores and
pollen grains show a fascinating diversity of structural
details and an often highly symmetrical pattern of sculptures
and (if present) pores or colpi. The shape, pore arrangement,
morphology and ultrastructure of sporodermata and exines
have proved informative to palaeobotanists and in systematics (e.g. Straka, 1975; Blackmore, 1990; Hesse, 1991;
Moore et al., 1991). However, with the exception of UVshielding (Rozema et al., 2001), the essential functions of
sporopollenin coats are not yet well understood. There are
severe de®cits in our knowledge on the physical properties
of sporopollenin, which might be signi®cant for physiological processes. It is likely that these properties are related
to the dispersal of spores and pollen grains, which requires
their transient exposure to the dry atmosphere. However, the
view that `one of the main protective functions' of the
sporoderm and exine `must be against excessive desiccation' (Heslop-Harrison, 1973), is not convincing, since
desiccation and rehydration of spores and pollen are usually
rapid processes and, in most cases, the poikilohydric
sporopolasts tolerate severe desiccation (e.g. Linskens,
1964).
Annals of Botany 92/2, ã Annals of Botany Company 2003; all rights reserved
290
Bohne et al. Ð Sporopollenin Microcapsules from Pine Pollen
Table 1. Composition of the dextran-probing solution (DPS)
Component
Manufacturer
Dextran T 70
Dextran T 20
Dextran 15
Dextran 8
Dextran 4
Dextran 4, hydrolysed for 24 hours²
Dextran 4, hydrolysed for 48 h²
a-Methyl-glucoside
Pharmacia (Uppsala, Sweden)
Pharmacia (Uppsala, Sweden)
Serva (Heidelberg,Germany)
Serva (Heidelberg,Germany)
Serva (Heidelberg,Germany)
Fluka, Buchs, Switzerland
Stokes' radius of
the most concentrated
size fraction * (nm)
Concentration in the
probing solution (mg ml±1)
4´56
3´99
3´61
2´46
1´99
1´50
1´24
0´38
0´900
0´975
1´125
3´000
0´750
0´750
1´050
0´525
* Obtained by SEC on a calibrated Superdex-75-R-column at the peak maximum.
0´06 M sulfuric acid, 80 °C.
²
Data on the permeability of sporodermata and aporate
exines for solutes of different size are informative with
respect to the dimensions of the water-®lled channels
through the polymer matrix. Such data might be relevant for
a better understanding of the physiological role of the
sporopollenin membranes and their eventual technical
application, too. Advantages of sporopollenin packing
particles and microcapsules puri®ed from pollen or spores
are their high resistance to extreme pH values, their
relatively uniform size and their mechanical stability. The
relatively high content of hydrophilic groups in sporopollenin may be used to anchor primers for the solid-phase
synthesis of biopolymers (Mackenzie and Shaw, 1980;
Adamson et al., 1983) or the immobilization of ligands for
chromatographic purposes (Pehlivan and Yildiz, 1994;
ErsoÈz et al., 1995; Vural et al., 1995; C
Ë engeloglu et al.,
1998).
Pine exines are available in large amounts and show
excellent mechanical rigidity and ®ltration properties for
®xed bed applications (Woehlecke et al., 2002). A detailed
description of the ultrastructure and ontogenesis of the pine
exine has been given by Rowley et al. (1999, 2000a, b). In
this study, we describe the exchange of sugars, lowmolecular-weight dyes, dextran molecules of different size
and submicrometer latex particles with tripartite microcapsules obtained from pine pollen by solubilization of the
sporoplasts.
MATERIALS AND METHODS
Dyes
The following dyes were used: carboxy¯uorescein (Molecular Probes Inc., Eugene, OR, USA); Evans Blue (Reanal,
Budapest, Hungary); FITC (¯uorescein isocyanate)-Dextran
(FD-250S, MR = 282 000 g mol±1; Sigma-Aldrich Chemie
GmbH, Steinheim, Germany); FITC-labelled latex beads
(Fluoresbrite Plain YG 0´2 micron microspheres,
é = 0´217 6 0´015 mm; Polyscience Europe, Eppelheim,
Germany) and polyacrylic acid 2100 sodium salt (Fluka
Chemie AG, Buchs, Switzerland)
Pine pollen and sporopollenin capsules
Protoplasts were removed from mature pine pollen by the
following preparation steps: (1) lipid extraction, (2) treatment with hot diluted acid, (3) treatments with hydrolytic
enzymes and (4) washings. Pollen grains were denatured
using 50 % ethanol, and lipids were extracted using hot
ethanol (60 °C) and dioxan (80 °C). Particles were then
incubated in a closed vessel containing diluted sulfuric acid
(30 g l±1) and sodium sul®te (3 g l±1) for 48 h at 90 °C. The
subsequent treatment with cell-wall lysing enzymes was
carried out as described by Jungfermann et al. (1997).
Particles were bathed at 37 °C for 1 d in a phosphate buffer
(pH 6´5) containing 2 g l±1 of a bovine pancreas enzyme
mixture supplied as `trypsin for cell culture' by Bernd
Belger (Klein-Machnow, Germany). They were subsequently washed on a glass ®lter in a sodium chloride
solution (10 g l±1) and then incubated for 1 d at room
temperature in a large volume of a sodium carbonate
solution (10 g l±1) containing sodium dodecyl sulfate (1 g
l±1) and sodium dithionite (1 g l±1). Finally, particles were
washed using a sodium chloride solution (10 g l±1) and water
until the extract was free of UV-absorbing substances
(280 nm). The sporopollenin microcapsules obtained were
stored as a suspension in 50 % ethanol.
Measuring sugar and dextran ef¯ux
A previously described system enabling the continuous
polarimetric analysis of the particle-free medium (Ehwald
et al., 1973; Fleischer and Ehwald, 1995) was used to record
the ef¯ux of sugars and Dextran T 70 (Pharmacia, Uppsala,
Sweden) from sporopollenin capsules that had been
presaturated with the sugar or dextran solution. A ¯ow of
water-saturated air (approx. 40 ml min±1) was used to agitate
the suspension and to drive circulation of the particle-free
liquid through a 2 ml bypass including the polarimeter cell.
The whole volume in the system was 8±9 ml, and the ¯ow
through the detecting bypass was 20±30 ml min±1. Using the
recording polarimeter 141 M (Perkin Elmer, Ueberlingen,
Germany) and an analogue/digital converter, the angle of
rotation was registered at a wavelength of 366 nm with an
absolute accuracy of 6 0´001°.
Bohne et al. Ð Sporopollenin Microcapsules from Pine Pollen
291
F I G . 1. Scheme of the sporopollenin strata of the pine exine. The two
outer layers of the ectexine are detached from the foot layer, thus
forming a saccus.
Sporopollenin capsules were saturated with the respective
sugar or dextran solution for 3 d, vacuum-®ltered on a nylon
sieve (20 mm pore size) for 30 s, and stored in a closed
vessel. Sampling was carried out by ®lling an acrylic-glass
tube (diameter 8 mm) with the ®ltered mass, which was then
weighed on an electronic balance. To start an ef¯ux course,
the mass was pushed into the measuring system by means of
a ®tting cylinder.
Partitioning of Dextran T 70
Sporopollenin capsules were saturated with a standard
buffer solution (10 mM sodium phosphate, pH 7, 100 mM
NaCl, and 1 g l±1 NaN3) and collected on a nylon ®lter
as described above. One-gram samples of the ®ltered
mass were mixed with 1 ml standard buffer containing
a-methylglucoside (16 g l±1) and Dextran T 70 (32 g l±1).
After the respective diffusion time, sporopollenin capsules
were sedimented by centrifugation. The original solution
and the supernatant were chromatographed on a Superdex
75 HR column (30 3 1 cm; Pharmacia) with a polarimetric
detector (Chiralyser; IBZ Messtechnik, Hanover, Germany)
to determine the ratio between sugar and dextran concentrations from the peak areas. Since the sugar partition space
in the ®ltered mass is 0´94 ml g±1, the partition space of
dextran in 1 g of the ®ltered mass of sporopollenin capsules
is equal to (1.94 rr0 ± 1) ml, where r is the sugar : dextran
ratio obtained in the supernatant and r¢ is the sugar : dextran
ratio of the original solution.
Exchange of the smaller dextran size fractions
Dextran exchange with the central capsule was analysed
using a previously described method based on size exclusion chromatography of a polydispersed dextran-probing
solution (DPS) in combination with the diffusion experiment (Woehlecke and Ehwald, 1995). The composition of
the DPS used in this study is given in Table 1. Sporopollenin
capsules were shaken in DPS for 10 d, collected by vacuum®ltration on a 20 mm nylon sieve, and drained for 30 s.
Samples of the ®ltered mass (150 mg) were shaken with
450 ml standard buffer (see above) for different periods.
F I G . 2. Scanning electron micrographs of dehydrated capsules and cryosections of a pine exine (Pinus sylvestris). A, Residue of the central
capsule envelope with lateral sacci. B, Close-up of a saccus, showing the
surface layer and the honeycomb-shaped support structures. C, Image of
the surface of the sexine covering a saccus. D, Close-up of a central
capsule, showing the dense nexine at the luminal face and the alveolar
sexine at the external face. Capsules or 20-mm-thick cryo-slices were airdried from tetramethyl-silane after dehydration in ethanol and sputtercoated with 10 nm gold. Imaged using a Leica S360 scanning electron
microscope (Leica, Cambridge, UK).
Subsequently, the liquid was separated from the capsules by
centrifugation and fractionated by SEC on a size-calibrated
Superdex 75HR (30/1) column (Pharmacia). The elugraphs
were compared with that of the original DPS using a
computer program, as described previously (Woehlecke and
Ehwald, 1995; Dautzenberg et al., 1999).
292
Bohne et al. Ð Sporopollenin Microcapsules from Pine Pollen
TA B L E 2. The ratio between volume V and surface area A of the central capsules as determined from morphometric
estimates of their perimeters
Mean values with standard deviation and number of measured perimeters (n).
RESULTS AND DISCUSSION
Structure and size of the sporopollenin microcapsules
At the site of a saccus, two outer strata of the ectexine
(tectum and columellae) are separated from two inner layers
(endexine and foot layer of the ectexine), as illustrated
schematically in Fig. 1. The external membrane of the sacci
represents the part of the exine termed the sexine. The two
inner layers covering the lumen of the central capsule have
been termed nexine (Straka, 1975; Rowley et al., 2000b).
SEM images of the puri®ed microcapsules are shown in
Fig. 2. The external wall of the sacci consists of a thin
sporopollenin membrane (tectum), which covers a thick but
macroporous layer formed by honeycomb-shaped support
structures (columellae), which are open to the lumen
(Fig. 2A and B). The tectum is perforated by scattered
pores of submicrometer size (Fig. 2C). The lumen of the
central capsule is bordered by a continuous dense
sporopollenin envelope. Apart from the sacci, the two
layers representing the sexine are present on the surface of
the central capsule (Fig. 2D).
Since the ratio of volume (V) to surface area (A) of the
central capsule was required for the calculation of permeability coef®cients, the approximately ellipsoid central
capsules were analysed morphometrically using a slide
calliper and printed light microscope images of dense
suspensions of the sporopollenin microcapsules in water.
The two short perimeters were obtained either from the top
view or from the lateral view (Table 2).
Permeation of dyes, ¯uorescent polymers and particles
The diffusion of dyes, ¯uorescently labelled dextran, and
latex particles into the central capsule and the lateral sacci
was studied by ¯uorescence and transmission light microscopy (Fig. 3). Carboxy¯uorescein was excluded from the
empty central capsules for the ®rst minutes (not shown) but
F I G . 3. Partitioning of low-molecular-weight dyes, stained polymers and
particles within the tripartite sporopollenin microcapsules, and denatured
pollen grains (Pinus sylvestris). Images of the capsules or pollen grains
in the ¯uorescent or stained medium were produced using Leica CLSM
(Leica Laser-Technik GmbH, Heidelberg, Germany); the wavelength of
excitation was 488 nm, and of emission approx. 535 6 15 nm. A,
Carboxy¯uorescein, 0´01 g l±1, 3 h, CLSM-image. B, Evans Blue, 10 g
l±1, 3 h, transmission image. C, FITC-Dextran, mean molecular weight
282 kDa, 0´4 g l±1, 3 h, CLSM image. D, FITC-latex particles, nominal
size 0´2 mm, 0´26 mg l±1, 15 min, CLSM-image. Capsules (A, C, D) or
denatured pollen grains (B) were washed with de-ionized water and
dispersed in the stained solutions.
labelled the central capsule lumen within some hours
(Fig. 3A). Evans Blue, a water-soluble and protein-staining
dye containing four sulfonic acid residues (960 Da, Stokes'
Bohne et al. Ð Sporopollenin Microcapsules from Pine Pollen
F I G . 4. Ef¯ux of Dextran T 70 from liquid-saturated sporopollenin
capsules into water. At time zero, 304 mg ®ltered sporopollenin capsules
(Pinus sylvestris) that had previously been equilibrated in a solution of
Dextran T 70 (30 g l±1) were added to 8 ml water in the measuring
system. Arrow: calibration with 200 ml of the dextran solution after a
shift of registration. In this experiment the partition space of the dextran
molecules in the ®ltered mass was 0´66 ml g±1.
TA B L E 3. Partition space of Dextran T 70 in the ®ltered
mass of sporopollenin capsules (Pinus nigra) in a long-term
experiment
Diffusion time (h)
Dextran partition space (ml g±1 fresh weight)
24
0´58
72
0´65
240
0´62
720
0´68
radius = 1´3 nm) did not enter the empty central capsule or
the denatured pollen grain within 24 h, but accumulated
rapidly in the denatured sporoplasts when the exine was
injured (Fig. 3B). FITC-labelled serum albumin (image not
shown) and FITC-Dextran with a mean molecular weight of
282 kDa (Fig. 3C) did not permeate into the empty central
capsule. The ¯uorescing dextran and even ¯uorescing latex
beads of a preparation with a mean diameter of 218 nm
entered the lumen of sacci rapidly (Fig. 3D), whereas 1 mm
beads did not (image not shown).
Access of solutes to the central capsule is obviously
controlled by the nexine, since the sexine is a highly
permeable micro®lter, at least where it builds the sacci. The
rapid equilibration of large polymer molecules and even
latex beads with the sacci requires comment, since SEM
images of the sexine (Fig. 2C) suggest a relatively low
density of submicrometer pores in the surface layer of the
sexine. In this respect, it has to be stressed that the
sporopollenin membrane is relatively thin at the surface
compared with the diameter of the sacci. Owing to the
diffusion resistance of the unstirred liquid within the
capsules, a small fraction of pores in the membrane area
will be suf®cient to prevent membrane control of diffusional
exchange, provided that the pores are distributed over the
whole surface.
A relatively high maximum pore size and the mechanically rigid structure of the sexine (Fig. 2B) might be a
necessary presupposition for the entrance of air into the
293
F I G . 5. Ef¯ux of raf®nose from liquid-saturated exine capsules (Pinus
sylvestris) into water. At time zero, 215 mg sporopollenin capsules
®ltered from a 150 mM raf®nose solution were added to 8 ml of water in
the measuring system. Arrow, Calibration with 0´2 ml of the 0´15 M
raf®nose solution. The partition space of raf®nose in the ®ltered mass
was 0´94 ml g±1. Inset: Plot of the logarithm of the difference between
the ®nal and actual angles of rotation on diffusion time. In this
experiment the volume of the slowly exchanging compartment (central
capsule) derived by extrapolation of the ®rst-order line to time zero was
0´28 ml g±1.
sacci at desiccation, since puri®ed sporopollenin has an
hydrophilic surface with a contact angle close to zero for
water (not shown here). When desiccation of the watersaturated cell wall capsules was observed under the light
microscope, no shrinkage of the sacci was found before air
entrance, whereas central capsules collapsed completely.
Since the product of r (pore radius) and Dp (the pressure
difference required to remove water from a hydrophilic
pore) is known to be 0´144 mm MPa, the critical cohesive
tension for the entrance of air into the sacci should be about
1 MPa for a maximum pore size of 200±300 nm.
Ef¯ux of Dextran T 70 and sugars from pre-equilibrated
sporopollenin microcapsules
The ef¯ux of Dextran T 70 from the ®ltered mass of the
sporopollenin capsules was too rapid to be resolved
kinetically using the applied method (Fig. 4). The ®nal
value was obtained within the 30 s that were needed for
convective mixing in the air-lift system. The corresponding
dextran partition space was 0´6±0´7 ml g±1 of the ®ltered
mass. Dextran T 70 did not equilibrate with the whole liquid
space of the ®ltered mass of sporopollenin capsules within
30 d (Table 3).
Sugars equilibrated with approximately the whole liquid
volume in the ®ltered mass (0´94 ml g±1), and leaked with a
biphasic kinetics (Fig. 5). The ®rst rapid phase (ef¯ux from
the sacci and interparticle space) could not be resolved, and
the slower phase (ef¯ux from the central capsule) showed
®rst order kinetics. Extrapolation of the semi-logarithmic
plot (inset of Fig. 5) to zero time allowed the separation of
the slowly exchanging volume, represented by the central
capsules (approx. 0´3 ml g±1 of the ®ltered mass), from the
rapidly exchanging volumes, represented by the sacci and
interparticle spaces (0´6±0´7 ml g±1). The half-life times of
294
Bohne et al. Ð Sporopollenin Microcapsules from Pine Pollen
TA B L E 4. Sugar ef¯ux from the preloaded central capsule
Species
Sugar
Pinus sylvestris a-Methyl-glucoside
Raf®nose
Pinus nigra
a-Methyl-glucoside
Partition space,Vp Rate coef®cient, Permeability coef®cient,
P 3 Mr0´5
Half-life time Half-life time
(ml g±1)*
k (s±1)*
P (mm s±1)*
(nm s±1 g0´5 mol0´5) measured t (s)* theor.t¢ (ms)²
0´29 6 0´02
0´30 6 0´01
0´34 6 0´01
0´0056 6 0´0021
0´0021 6 0´0001
0´0112 6 0´0021
0´028 6 0´0104
0´010 6 0´0004
0´062 6 0´0116
393
231
870
138
338
63
10´3
15´9
12´8
* Obtained by extrapolation of the ®rst-order line (Fig. 5) to zero time.
Calculated for diffusional exchange with a spherical unstirred water zone of the central capsules size. Mr, Sugar molecular weight.
²
TA B L E 5. Stokes' radii of dextran molecules coordinated to de®ned exchange rates after different diffusion times
Diffusion time (s)
Exchange rate
Rate constant (s±1 3 10±3)*
Permeability coef®cient (nm s±1)*
Stokes' radius (nm)²
1200
0´33
0´916
4´59
1´30
0´50
0´578
2´90
1´15
3600
0´66
0´338
1´69
1´05
0´33
0´305
1´53
1´83
0´50
0´193
0´97
1´58
10 800
0´66
0´113
0´56
1´35
0´33
0´102
0´51
2´20
0´50
0´064
0´32
1´95
32 400
0´66
0´038
0´19
1´60
0´33
0´034
0´17
2´95
0´50
0´021
0´11
2´50
0´66
0´013
0´06
2´13
Data obtained for central capsules of Pinus sylvestris L.
* Calculated from exchange rate and diffusion time, as explained in the Appendix.
² Read from graphs of the type shown in Fig. 6.
F I G . 6. Size-dependence of the exchange quotients of dextran size
fractions with the central capsule at two different ef¯ux times. Curves
were obtained using the preparation obtained from P. sylvestris pollen.
The exchange quotient q represents the quotient between the
concentration of a dextran size fraction in the original DPS and
the concentration of the same dextran size fraction in the medium of the
capsules. The Stokes' radii and the respective concentration values were
obtained by size exclusion chromatography on a column calibrated with
protein standards. q¢, Maximum value (impermeable fractions) obtained
from the peaks of Dextran T 70 at the void volume of the column; q¢¢,
minimum value (completely equilibrated fractions), obtained from the amethylglucoside peaks. The exchange rate represents the fractional
equilibration between the central capsule and the medium. The three
given levels of g were used for the calculation of the permeability
coef®cients and corresponding Stokes' radii (Table 5).
sugar exchange with the central capsule are three orders of
magnitude larger than half-life times expected for their
diffusional exchange with a spherical equivalent volume of
unstirred water (Table 4).
The strict membrane control and low permeability of the
sporopollenin membrane for sugars found here contrasts
with our previous ®ndings for primary plant cell walls.
Exchange of salts, sugars and even small colloids (Stokes'
radius up to 2 nm) with the empty plant cell wall capsules
(obtained from a plant cell suspension culture) is rapid
enough for ef®cient permeation chromatography (Ehwald
et al., 1992), which requires equilibration within less than
1 s. Although the nexine is a signi®cant diffusion barrier for
sugars, its permeability coef®cient for a monosaccharide
(Table 4) is high enough to enable a rate of sugar uptake by
the sporoplasts that satis®es the metabolic need. The
permeability coef®cients determined for a-methylglucoside
(Table 4) and the volume : surface area ratios of the central
capsule (Table 2) were used to calculate the stationary
concentration difference across the exine at an assumed high
rate of monosaccharide uptake (50 mmol per g fresh weight
and hour), which is twice the value found for maize root tips
at a glucose concentration of 200 mM (Ehwald et al., 1974).
The values obtained are in the millimolar range (see the
Appendix). On the other hand, the limitation of solute
diffusion by the exine found here is strong enough to reduce
hypo-osmotic shock and solute leakage when the air-dry
pollen grain is rehydrated. Furthermore, the diffusion
resistance of the sporopollenin membrane can facilitate
the maintenance of a speci®c pH and ion milieu close to the
plasma membrane of the male gametophyte. In its functional role as a relative, not absolute, diffusion barrier for
small solutes, the pine nexine resembles suberized and/or
cutinized cell walls and Casparian strips (Brauner, 1956;
Zimmermann et al., 2000; Hose et al., 2001). The permeability of the nexine for water, alcohols, salts and
ampholytes, as well as its barrier function for cation and
anion exchange, needs further study.
Exchange of smaller dextran size fractions with the central
capsule
Compared with plant cell wall capsules, which reach a
constant partition spectrum of the polydisperse dextran-
Bohne et al. Ð Sporopollenin Microcapsules from Pine Pollen
F I G . 7. Dependence of the permeability coef®cient (P) of sugars and
dextran size fractions on the Stokes' radius (rs). P is given on a
logarithmic scale to illustrate the range of values. Points with the same
symbol comprise data obtained at certain levels of the exchange rate g
(closed circles, 0´33; open circles, 0´5; closed squares, 0´66) after
different diffusion periods (cf. Fig. 6). Closed triangles, Permeability
values of a-methyl-D-glucose and raf®nose based on ef¯ux kinetics
(cf. Fig. 5).
probing solution within some minutes (Woehlecke and
Ehwald, 1995), the central capsule showed an extremely
slow exchange of the polymers. When sporopollenin
capsules were equilibrated with the polydisperse DPS and
then transferred to the buffer for ®xed diffusion periods, the
exchange ratio (q) and the exchange rate (g) of permeable
size fractions were obtained by the SEC method, as
explained in Fig. 6. Dextran molecules with Stokes' radii
between 1 and 2´5 nm showed intermediate values of these
parameters after some hours of diffusion (Table 4) but
reached the equilibrium values after 3 d (curves not shown).
Size dependence of permeability coef®cients
The Stokes' radii related to certain values of the exchange
rate g (0´33, 0´5 and 0´66) were read out from graphs of the
type shown in Fig. 6, and the permeability coef®cients of the
295
F I G . 8. Product of the permeability coef®cient and the Stokes' radius as
dependent on molecular size. Symbols as in Fig. 7.
central capsule envelope for these size fractions were
calculated as explained in the Appendix. Permeability
coef®cients (P) were obtained using the three values of g
®tted to the same curve (Fig. 7). Hence, in the size range
studied, exchange kinetics follows the ®rst-order model. An
inhomogeneity of the capsules with respect to the permeability coef®cients would lead to signi®cantly higher values
of P with the lowest value of g, but this is not evident.
Sporopollenin capsules prepared from Pinus sylvestris
showed a higher permeability for the larger dextran size
fractions and a lower permeability for the monosaccharide
than those prepared from Pinus nigra. Although this
difference was reproducible in the three parallel batches
analysed, it has to be stressed that all parallels were from
only one preparation each.
Since the diffusion coef®cient of small molecules in
water is inversely proportional
to the square root of the
p
molecular weight … M r †, and the diffusion coef®cient of
polymers correlates with the reciprocal of the Stokes' radius
(rs±1), it is useful to consider the products of the permeability coef®cient (P) of the central capsule membrane
(nexine) with the respective size parameters. P 3 rs rises
sharply as the molecular size of the dextran molecules
296
Bohne et al. Ð Sporopollenin Microcapsules from Pine Pollen
p
decreases (Fig. 8) and the product of P and M r of the
trisaccharide raf®nose was only half the value found for amethylglucose (Table 4). Hence, size-exclusion effects are
relevant for permeability in the whole range of permeand
sizes studied, and sugar permeation was not rate-controlled
by unhindered diffusion within the unstirred liquid of the
capsule lumen. The volume fraction of the large pores,
which enable dextran permeation up to a size of more than
2 nm, seems to be too small to control the sugar
permeability. It is obvious that the pore size distribution
in the nexine is not homogeneous. At present, it is unclear
whether the polymer-conducting diffusion pathways are
open in the physiological system. Their existence might be
physiologically signi®cant in pollen ontogenesis. However,
in the ripe pollen grain these pathways might be ®lled with
carbohydrate or lipids. In this case, even small digestive
enzymes such as trypsin (Stokes' radius = 2´4 nm) might be
excluded ef®ciently. The ®nding that dextrans with a
Stokes' radius of up to 2´5 nm equilibrated slowly with
the capsules, whereas Evans Blue (Stokes' radius = 1´3 nm)
was excluded, points to the presence of ®xed negative
charges in the pores. Due to electrical barrier effects, the
size exclusion limit of cation exchanger membranes can be
much smaller for polyanions than for non-electrolytes (e.g.
JaÈschke et al., 1992).
CONCLUSIONS
Results show that the permeability properties of the pine
nexine and sexine are very different. The nexine is an
ultra®lter membrane, which is impermeable to most
proteins. Its permeability to sugars is low compared with
that of a primary cell wall, but is nevertheless suf®cient for
the nutrition of the protoplast. The puri®ed sexine at the
external surface of the sacci is a micro®lter that is highly
permeable for even large polymer molecules and diffusible
sub-micrometer particles. With respect to applications for
chromatography and immobilization, the most interesting
properties of the sacci are rapid polymer exchange, a large
and well accessible inner sporopollenin surface, and a high
mechanical rigidity. The slow exchange of the central
capsule for polymers and polyvalent anions might be of
interest for controlled release applications.
ACKNOWLEDGEMENTS
We are grateful to Dr S. Rogaschewski for enabling and
supporting the work with the Leica SEM. The study was
supported by German Federal Ministry Economics (project
`ChromatographietraÈger' No 1279/00).
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APPENDIX
Permeability coef®cients
Permeability coef®cients, P, of the central capsule envelope
for the sugars and the dextran size fractions were calculated
for the case of membrane-controlled exchange (®rst-order
kinetics), where P can be derived from the rate constant k,
the capsule volume V, and capsule surface area A.
P = k V/A
Volume/surface ratio of the central capsules
An estimate of V/A of the central capsule was obtained
from mean values of the three perimeters of the ellipsoid
capsule a, b and c (Table 2) as:
V
ˆ
A
p
3 
abc
:
3
Rate constants
Rate constants, k, for sugar exchange were read directly
from semi-logarithmic plots shown in Fig. 5. Rate constants
of dextran exchange were calculated from the exchange rate
g (Fig. 6) and the exchange time, t, as:
k ˆ ln
t
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Half time of sugar exchange without membrane control
(Table 4)
p
3 
A sphere with radius r ˆ abc has approximately the
same volume as the ellipsoid central capsules with perimeters a, b and c. The half-saturation time, t*, of sugar
exchange with a spherical zone of unstirred liquid with this
radius (Table 3) was determined from a solution of the
2
second Ficks' law given by Crank (1957) as t* = 0.0305 rD ,
where D is the diffusion coef®cient of sugar in water.
Concentration difference across the nexine required for a
relatively high rate of monosaccharide uptake by the male
gametophyte
The stationary concentration difference, DC across the
external diffusion barrier (nexine) of the gametophyte
caused by a constant rate of monosaccharide uptake can
be obtained as:
DC ˆ
uV
PA
where P is the permeability coef®cient of the nexine for a
monosaccharide (Table 3), V/A is the volume/surface ratio
of the gametophyte (Table 2), and u is the volume-based
uptake rate. For an uptake rate of 50 mmol cm±3 h±1, the
concentration difference DC over the nexine is 2´48 mM for
the preparation of P. sylvestris and 1´25 mM for the
preparation of P. nigra.