Diffusion barriers of tripartite sporopollenin microcapsules prepared from pine pollen

Abstract

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 > or =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 coefficients 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.

Fig. 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.

Fig. 2. Scanning electron micrographs of dehydrated capsules and cryo‐sections 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‐µm‐thick cryo‐slices were air‐dried from tetramethyl‐silane after dehydration in ethanol and sputter‐coated with 10 nm gold. Imaged using a Leica S360 scanning electron microscope (Leica, Cambridge, UK).

Fig. 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 fluorescent 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 ± 15 nm. A, Carboxyfluorescein, 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 µm, 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.

Fig. 4. Efflux of Dextran T 70 from liquid‐saturated sporopollenin capsules into water. At time zero, 304 mg filtered 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 µl of the dextran solution after a shift of registration. In this experiment the partition space of the dextran molecules in the filtered mass was 0·66 ml g^–1.

Fig. 5. Efflux of raffinose from liquid‐saturated exine capsules (Pinus sylvestris) into water. At time zero, 215 mg sporopollenin capsules filtered from a 150 mm raffinose solution were added to 8 ml of water in the measuring system. Arrow, Calibration with 0·2 ml of the 0·15 m raffinose solution. The partition space of raffinose in the filtered mass was 0·94 ml g^–1. Inset: Plot of the logarithm of the difference between the final 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 first‐order line to time zero was 0·28 ml g^–1.

Fig. 6. Size‐dependence of the exchange quotients of dextran size fractions with the central capsule at two different efflux 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 α‐methylglucoside peaks. The exchange rate represents the fractional equilibration between the central capsule and the medium. The three given levels of γ were used for the calculation of the permeability coefficients and corresponding Stokes’ radii (Table 5).

Fig. 7. Dependence of the permeability coefficient (P) of sugars and dextran size fractions on the Stokes’ radius (r_s). 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 γ (closed circles, 0·33; open circles, 0·5; closed squares, 0·66) after different diffusion periods (cf. Fig. 6). Closed triangles, Permeability values of α‐methyl‐d‐glucose and raffinose based on efflux kinetics (cf. Fig. 5).

Fig. 8. Product of the permeability coefficient and the Stokes’ radius as dependent on molecular size. Symbols as in Fig. 7.