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Solution of sporopollenin and reaggregation of a sporopollenin-like material: A new approach in the sporopollenin research
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© 1997 by Gustav Fischer Verlag. Jena
Solution of sporopollenin and reaggregation of a sporopollenin-like material: A new approach in the sporopollenin research l 2 CHRISTA]UNGFERMANN , FRIEDHELM AHLERS!, MONIKA GROTE , SABINE GUBATZl, l l 3 l STEFAN STEUERNAGEL , INA THOM , GERD WETZELS , and ROLF WIERMANN 1
Institut fur Botanik. SchlolSganen 3. D-48149 Munster. Germany
2
Institut fur Medizinische Physik und Biophysik, Hufferstralk 68. D-48149 Miinster. Germany
3
Bruker Analytische MelStechnik GmbH. Silberstreifen. D-76287 Rheinsterten. Germany
Received January 2. 1997 . Accepted February 9, 1997
Summary
Exines from Typha angustifolia L. pollen were completely dissolved in 2-aminoethanol if the exine material was isolated and purified by an enzymatic procedure. Acetolysis or treatment of the exine material with H 3P04 before the dissolution process led to insolubility. A reaggregation of the dissolved material was possible when the solvent was removed and H 20 added. The initial exine material and the reaggregated material were compared by means of staining, fluorescence microscopy, SEM, resistance against acetolysis, DSC, FTIR and BC CP/MAS NMR spectroscopy. Both materials showed great similarities with only little differences in the FTIR and BC NMR spectra and in the DSC thermograms.
Key words: 2-Aminoethanol Reaggregation, Sporopollmin. Solubilisation. Sporopollmin-like materia/, Typha angustifolia L. Abbreviations: BC CP/MAS NMR = Cross-polarization and magic-angle spinning nuclear magnetic resonance; DSC = Differential scanning calorimetry; FTIR = Fourier-transform-infrared; SEM = Scanning-electron-microscopy; TLC = Thin layer chromatography. Introduction
Sporopollenin, an extremely resistant biopolymer is substantially involved in the formation of the outer walls of spores and pollen. It is known to be insoluble in common acids and in most organic solvents. Strongly oxidizing chemicals degrade the polymer; however, due to the harsh conditions, the resulting molecules do not give conclusive evidence about the chemical nature of sporopollenins. Thus the chemical analysis of sporopollenin has been limited to methods that can be applied to the solid state only. Some morphological evidence exists that plants themselves can hydrolyse sporopollenin during early stages of germination (Dickinson and Lewis, 1974; Ahokas, 1976). 2-Aminoethanol is an organic solvent that has been used in cytological studies of pollen exines and intines for several
f. Plant Physiol. w,£ 151. pp. 513-519 (J997)
years (Bailey, 1960; Southworth, 1974; Rowley, 1978; HeslopHarrison and Heslop-Harrison, 1982; Kedves and Rojik, 1989; for review see Southworth, 1990). Southworth reponed that the outer exine layer of some gymnosperm and angiosperm pollen dissolves in 2-aminoethanol whereas the inner exine layer does not. During this process the exines of Lilium. Fagus and Juniperus turned into a lattice-like structure of interconnected granules interspersed with angular unstained openings called polygons (Southworth, 1974; 1985 a, b). Since there are similarities between these latticelike structures and developing primeexines of several genera, 2-aminoethanol might detach exine components in a process reverse to that of exine synthesis (Southworth, 1986). Furthermore, colloidal mixtures may play an important role in spore wall development. Hemsley et al. (1992 b) described a colloidal crystal-like structure of sporopollenin in
514
C. JUNGFERMANN, F. AHLERS, M. GROTE, S. GUBATZ, S. STEUERNAGEL, I. THOM, G. WETZELS, and R. WIERMANN
the spore walls of Selaginella and fossil Erlansonisporites. They found that the central region of these walls consists of closely packed particles in a semi-crystalline arrangement. The hypothesis of a colloidal exine construction in lycophyte megaspores with its inherent aspect of self-assembly has also been discussed by Collinson et al. (1993) and Hemsley et al. (1996). The authors concluded that this hypothesis reduces the need to invoke direct biological intervention and that therefore genetic input during the organization may be minimal. As shown above, 2-aminoethanol was successfully used for cytological studies, but not yet for biochemical experiments. The present investigation demonstrates the complete solubility of sporopollenin from Typha angustifolia L. in 2-aminoethanol and the subsequent reaggregation of the dissolved material to a sporopollenin-like substance. The initial and the reaggregated material are compared by staining, fluorescence microscopy, SEM, resistance against chemical degradation, DSC, FTIR- and I3C CP/MAS NMR-analysis.
Materials and Methods
Plant material For the experiments pollen of Tjpha angustifolia L. was used, because it can be harvested in sufficient amounts. In addition, according to results of cytological analyses the exine of Tjpha pollen is soluble in hot 2-aminoethanol (Southworth, 1974). The pollen was collected at the shedding stage from plants growing in the Botanical Garden, MUnster, Germany. The exine material was obtained by sonification of the pollen (Branson Sonifier Cell Distributer B-12, Standard Microtip, sonification intensity: == 50W/cm2 , 10 min) and enriched by filtration through nylon-meshes (mesh 20~m).
In order to degrade the intine, three methods were applied: acetolysis (acetic anhydride: H 2S04 conc. 8: 2, 100 ·C, 10 min), treatment with phosphoric acid (80 %, 50 ·C, 10 days) or enzymatic hydrolysis using Mazerozym RIO and Cellulase Onozuka RIO (yakult, Nishinomiya, Japan), each 1 % dissolved in 0.1 sodium acetate buffer, pH 4.5, 30 ·C, 3 days). The resulting material was extracted using solvents with increasing polarity (CHCI3/MeOH 1: 1 v/v; diethylether, acetone, MeOH, ethylene glycol monoethyl ether, H 20), each for 24h. For BC CP/MAS NMR analysis the material was extracted with each solvent until absolute purification (controlled byTLC analysis). After the extraction procedure the material was lyophilized for 48 h and stored in a desiccator until examination (Fig.1A).
Solubilisation procedure An aliquot of 5 mg purified exine material was suspended in 3 mL 2-aminoethano!. The solubilisation procedure was carried out under stirring at 100·C for 1 h. Complete dissolution of the exines was controlled by light and fluorescence microscopy.
Reaggregation procedure In order to remove the 2-aminoethanol two methods are possible: a) Dialysis against double distilled water (Millipore), until pH-neutrality in and outside of the dialysis-tubing (Sigma, MWCO 12500-14000). With reduction of the 2-aminoethanol-concentration the reaggregated material appears in the dialysis-tubing.
b) Extraction with ethyl acetate until a brown material appears. Mter addition of 1 mL H 20 a precipitate is visible. This precipitation is improved by low temperature or by addition of several drops of HC!. The precipitate (= reaggregated material, Fig. 1 D) is washed three times with 6 mL distilled water, lyophilized for 48 h and stored in a desiccator until examination.
Fluorescence microscopy The initial and reaggregated material were analysed by epifluorescence microscopy using a Leirz Diaplan (Leirz, Werzlar, Germany) with filter block L3 (450-490nm).
FTIR-spectroscopy The initial exine material and the reaggregated material were analysed spectroscopically with a Nicolet-5DXC-FTIR-Spectrometer equipped with the OMNIC 1.2-program (Nicolet, Offenbach, Germany). The analyses were carried out at the Institut fur Organische Chemie der WWU-MUnster. KBr-pellets were prepared from 300 mg KBr and 2 mg sample; spectra were obtained between 4000 cm -1 and 400 cm-r:-
13C CPlMAS NMR-spectroscopy The BC NMR analyses were carried out with a Bruker DSX 300 or DRX 400 spectrometer (for detail see Wilmesmeier et aI., 1993). The analyses were carried out by Bruker Analytische Megtechnik, Rheinstetten, Germany.
Electron microscopy For SEM investigation, the lyophilized initial exine material and the reaggregated material were put on glass slides without any adhesive, coated with a thin layer of gold by sputtering and examined at an acceleration voltage of 25 KV in a Hitachi S 450 scanning electron microscope. The SEM-analyses were performed at the Institut fur Medizinische Physik der WWU-MUnster.
DSC experiment The DSC experiments were performed using a Nerzsch DSC 200 instrument. The DSC thermograms were obtained in a dry nitrogen atmosphere with a heating rate of 5 ·Clmin. The DSC experiments were carried out at the Institut fur Physikalische Chemie der WWU-MUnster.
Results and discussion
Isolated sporopollenin from Typha angustifolia L. pollen is completely soluble in hot 2-aminoethanol if the intine was originally removed by enzymatic decomposition. Light-microscopy shows a homogeneous solution without any pellet (Fig. 1 C), thus demonstrating that the outer as well as the inner exine layer are completely soluble in this organic solvent (Fig.lB,C). Acetolysis and treatment with phosphoric acid, both conventional methods for degrading the intine, seem to alter the exine material in a way that leads to non solubility in 2-aminoethanol. This is not an effect of higher temperatures as shown by control experiments using the same temperature treatment. The introduction of acetyl groups by acetolysis
Sporopollenin and sporopollenin-like material
B
A
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Fig. 1 A-J: Comparison of the initial exine material and the reaggregated material from 7jpha angustifolia. (A) initial exine material, (B) exine material suspended in 2-aminoethanol; (C) homogeneous solution of the exine material after 1 h in hot 2-aminoethanol; (D) reaggregated material. Staining with aniline-blue of (E) the initial exine material and (F) the reaggregated material. Autofluorescence of (G) the initial material and (H) the reaggregated material. SEM of the surface (I) of the initial exine material and of the structures generated during reaggregation after solubilization of the initial purified exine material in 2-aminoethanol. E- H X 345, bars = 3011m; I x 3200, J x 1500, bars = 3/1ffi.
m
516
C. JUNGFERMANN, F. AHLERS, M. GROTE, S. GUBATZ, S. STEUERNAGEL, I. THOM, G. WETZELS, and R. WIERMANN
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and probably of phosphoric groups by H 3P04-treatment may prevent an attack of 2-aminoethanol at reactive sites in the sporopoUenin skeleton. So far, neither the exact nature of these sites nor the type of reaction between 2-aminoethanol and sporopoUenin are known in detail.
1000
500
Fig. 2: FTIR-spectra of the initial exine material (A) and the reaggregated material (8). Labeling of the bands was carried out following WIlmesmeier et al. (1993). X: 1660 em-I C =0 stretching vibrations and Y: 1545 cm-I N-H deformation vibrations, both are characteristic for N-mono-substituted amides (secondary amides). The original signal E at 1678 cm-I is considerably overlapped by the new absorption of the band X or is completely reduced. Z: 1062 em -I CoO stretching vibrations caused by primary alcohols.
The elimination of the intine by hydrolytic enzymes has been proven to be a very gentle method for purification of sporopoUenin (Gubatz et al., 1986; Schulze Osthoff and Wiermann, 1987). Since the method used for purification of sporopoUenin effects the degree of solubilisation, the evalua-
SporopoUenin and sporopoUenin-like material
FJg.3: l3C CP/MAS NMR spectra of the initial exine material isolated from Ijpha angustifolia pollen by using enzymes (A) and of a sparopoUenin-like material (8) reaggregated from a soluble sporopollenin fraction obtained from the same species.
tion of an additional method for the production of exine material under mild conditions, e.g. by using 4-methylmorpholine N-oxide monohydrate (MMNO. H 20) as described by Loewus et al. (1985), Baldi et al. (1987) , and Tarlyn et al. (1993) is of great interest. Up to now reaction products after treatment with 2-aminoethanol could not be determined. Our current experiments are focussed on the thorough analysis of the solubilised material. Reaggregation of a sporopollenin-like material is possible in the two ways described above. The reaggregated material has properties similar to the initial exine material (Fig. 1 E-H). Both are stainable with aniline blue, both show intensive autofluorescence and both withstand acetolysis. With regard to their reaction with 80 % H 3P04, however, the two materials differ. The reaggregated material dissolves, whereas the initial exine material is resistant. The FTIR- and I3C NMR-spectra obtained from both materials are presented in Figs. 2 and 3. The FTIR-spectra of both samples (2A, 2 B) have very similar absorption patterns, but show clearly different bands around the wave numbers: 834 cm -1, 1660 cm -I, 1545 cm -I and 1062 cm -I. The peak at 834 cm -I corresponds to the out-of-plane C- H vagging vibration in aromatics. The C =0 stretching frequency in the native material (E = 1678 cm -1) is very strong and typical for conjugated carbonyl groups of (X,~-unsaturated compounds. In the FTIR-spectra of the reaggregated material the C = 0 band appears at a lower wave number (X 1660cm-l ) and in weaker intensity than obversed for the native material. In additon, only in the spc:ctrum of the reaggregated material is a peak seen at 1545cm-1 (Y in Fig. 2B). This signal is characteristic for N-H deformation vibrations. Furthermore, the peak at about 1062 cm -I (Z in Fig. 2 B) is only detectable in
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the reaggregated material. It is due to the primary alcohol group of 2-aminoethanol bound as an amide. All other signals in the spectra show high coincidence. In summary, the comparison of the two FTIR-spectra gives reasons to believe that the 2-aminoethanol reacts with a carboxy group in the native material to build a secondary amide. This assumption is confirmed by the results of the elemental analysis shown in Table 1. As expected, the nitrogen content is very low in the initial exine material. In contrast, the reaggregated material contains a significantly higher amount of nitrogen. Comparable results were obtained by I3C NMR analyses. The spectra of the initial and the reaggregated exine material are very similar. The most remarkable differences appear in the region of the signal group between 60 and 70 ppm (see and/or Fig. 3, arrow), indicating modifications of C-N functions. These modifications are probably caused by the treatment with 2-aminoethanol. The DSC thermograms for both materials are displayed in Fig. 4. The thermograms are similar in shape and have a transition temperature in the vicinity of 30"C. Furthermore, the transition temperature of the two samples depends on the heating rate (second-order transition). This relation is typical for macromolecules and biopolymers (Mandelkern, 1983).
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Table 1: Results of the elemental analyses. Typha angustifolia L.
C[%]
0[%]
H[%]
N[%]
purified exine material reaggregated material .
63.48 63.83
27.89 23.42
8.54 8.80
1.23 3.95
(Typhaceae)
518
C. JUNGFERMANN, F. AHLERS, M. GROTE, S. GUBATZ, S. STEUERNAGEL,I. THOM, G. WETZELS, and R. WIERMANN
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Fig.4: DSC thermograms of the initial exine material isolated from Typha angustifolia pollen by using enzymes (A) and of a sporopol-
lenin-like material (B) reaggregated from a soluble sporopollenin fraction obtained from the same species.
DSC investigations of the cuticula isolated from leaves of Citrus aurantium L. leads to transition temperatures around 7°C (Reynhardt and Riederer, 1991). The results presented here give no indication of fundamental differences between the initial and the reaggregated material. We regard them as either sporopollenin or sporopollenin-like material. It is not assumed that the reaggregated material is completely identical with the native sporopollenin, but both exhibit very similar properties and structures. In this connection it should be remembered that spectra of sporopollenins of different systematic origin revealed farreaching similarities in characteristic signals, but that the contributions of single functional groups were found to differ (Hemsley et al., 1992 a, 1993; Wilmesmeier et al., 1993). Therefore, to our current state of knowledge, sporopollenin is considered to represent a group of closely related macromolecules. The SEM-micrographs of the reaggregated material do not show a uniform material, but more or less globular structures of different sizes (Fig. 1, I-J). The average diameter of these particles is 4 J.l.m. Structures as those seen in native reticulate exines were not expected after reaggregation because no matrix giving a construction pattern is present during this procedure. According to unpublished results the type of structure depends on the way in which the sporopollenin fraction is obtained and the reaggregation is initiated. Control experiments were carried out either without exine material or using cellulose instead of exine material. These experiments did not result in the formation of any structures. Therefore, in our reaggregation experiments the formation of characteristic structures is due to the input of the sporopollenin material. Results of FTIR- and 13C NMR-spectroscopic analyses (Guilford et al., 1988; Espelie et al., 1989; Hemsley et al., 1992 a; Wilmesmeier et al., 1993) as well as those of electron microscopic investigations (Rowley, 1978; Southworth, 1986; Kedves and Rojik, 1989) clearly show that sporopollenins are closely related biopolymers with different functional groups
occurring in varying amounts and different properties. This is in accordance with our observations and early cytological studies (Southworth, 1974, 1990) demonstrating that exines from other species dissolved very poorly or only partially in hot 2-aminoethanol. The results of our experiments support the hypothesis that under certain conditions sporopollenin is a self-assembling biopolymer, as assumed recently by several authors (Hemsley et al., 1992 b, 1996; Collinson et al., 1993). By our experiments it was shown that it is possible to dissolve sporopollenin and reaggregate a sporopollenin-like material. The data clearly demonstrate that the macromolecule reaggregates by itself without either the addition of any substance or the presence of a matrix. During the development of pollen exines simple physical processes seem to be involved too, besides other as yet unknown processes. Our new approach is a very promising starting-point for the study of the chemical composition and structural organisation of sporopollenin by methods that could not be applied up to now, as solubilized sporopollenin is much more accessible to analytical methods than the particle form. This will be shown by several experiments with hydrolytic enzymes (in preparation). Sporopollenin, solubilised by different methods and reaggregated under varying conditions, is especially useful for investigation of the factors that influence in-vitro reconstitution. Acknowledgements
The financial support by the «Deutsche Forschungsgemeinschaft» and the «Fonds der Chemischen Industrie» is gratefully acknowledged. The authors are indebted to Prof. Gerhard Erker (Institut fur Organische Chemie, Munster) for making the FTIR-spectrometer available. We also thank Prof. Hellmut Eckert and the DSC group (Institut fur Physikalische Chemie, Munster) for their great help.
References AHOKAS, H.: Evidence of a pollen esterase capable of hydrolyzing sporopollenin. Experientia 32, 175-176 (1976). BAILEY, I. W: Some useful techniques in the study and interpretation of pollen morphology. J. Arnold Arbor. Harv. Univ. 41, 141-151 (1960). BALDI, B. G., V. R. FRANCESCHI, and F. A. LoEWUS: Preparation and properties of pollen sporoplasts. Protoplasma 141, 47-55 (1987). COLLINSON, M. E., A. R. HEMSLEY, and W A. TAYLOR: Sporopollenin exhibiting colloidal organization in spore walls. Grana Suppl. 1,31-39 (1993). DICKINSON, H. G. and D. LEWIS: Changes in the pollen wall of Linum grandiflorum following compatible and incompatible inttaspecific pollinations. Ann. Bot. 38, 23-29 (1974). EsPELIE, K. E., F. A. LoEWUS, R. J. PUGMIRE, W R. WOOLFENDEN, B. G. BALDI, and P. H. GWEN: Structural analysis of Lilium Iongiflorum sporopollenin by l3C NMR spectroscopy. Phytochemistry 28, 751-753 (1989). GUBATZ, S., S. HERMINGHAUS, B. MEURER, D. STRACK, and R. WIERMANN: The location to hydroxycinnamic acid amides in the exine of Corylus pollen. Pollen et Spores XXVIIl, 347-354 (1986).
Sporopollenin and sporopollenin-like material GUILFORD, W. J., D. M. SCHNEIDER, J.l..ABOVlTZ, and S. J. OPELLA: High resolution solid state BC NMR spectroscopy of sporopollenins from different plant taxa. Plant Physiol. 86, 134-136 (1988). HEMSLEY, A. R., W. G. CHALONER, C. J. GROOMBRIDGE, and A. C. SCOTT: Carbon-13 solid state nuclear magnetic resonance of sporopollenins from modern and fossil plants. Ann. Bot. 69, 545549 (1992a). _ HEMSLEY, A. R., M. E. COLLINSON, and A. P. R. BRAIN: Colloidal crystal-like structure of sporopollenin in the megaspore walls of recent &laginella and similar fossil spores. Bot. J. Linnean Soc. J08, 307-320 (1992b). HEMSLEY, A. R., P. J. BARRIE, W. G . CHALONER, and A. C. SCOTT: The composition of sporopollenin and its use in living and fossil plant systematics. Grana, Suppl. 1, 2-11 (1993). HEMSLEY, A. R., P. D. JENKINS, M. E. COLLINSON, and B. VINCENT: Experimental modelling of exine self-assembly. Bot. J. Linnean Soc. 121, 177-187 (1996). HESWP-HARRISON, Y. and J. HESWP-HARRISON: The microfibrillar component of the pollen intine: some structural features. Ann. Bot. 50, 831-842 (1982). KEDVES, M. and I. ROJIK: Investigation of the biopolymer organisation of partially degraded exines with the fragmentation method. Acta BioI. Szeged. 35, 71-80 (1989). LoEWUS, F. A., B. G. BALDI, V. R. FRANCESCHI, L. D. MEINERT, and J. J. MCCOLLUM: Pollen sporoplasts: Dissolution of pollen walls. Plant Physiol. 78, 652-654 (1985). MANDELKERN, L.: An introduction to macromolecules. New York: Springer (1983).
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REYNHARDT, E. C. and M. RIEDERER: Structure and molecular dynamics of the cuticular wax from leaves of Citrus aurantium L. J. Phys. D: Appl. Phys. 24, 478-486 (1991). ROWLEY, J. R.: The origin, ontogeny, and evolution of the exine. In: BHARADWAJ, D. C. (ed.): IV Int. Palynol. Conf., Lucknow (1976-77) 1, pp. 126-136 (1978). SCHULZE OSTHOFF, K. and R. WIERMANN: Phenols as integrated compounds of sporopollenin from Pinus pollen. Plant Physiol. 131, 5-15 (1987). SOUTHWORTH, D.: Solubiliry of pollen exines. Amer. J. Bot. 61, 36-44 (1974). - Pollen exine substructure. I. Lilium Iongijlorum. Amer. J.Bot. 72, 1274-1283 (1985a). - Pollen exine substructure. II. Fagus sylvatica. Grana 24, 161-166 (1985 b) . - Substructural organization of pollen exines. In: BLACKMORE, S. and I. K. FERGUSON (eds.): Pollen and Spores: Form and Function, pp. 61-69. Academic Press, London (1986). - Exine biochemistry. In: BLACKMORE, S. and R. B. KNox (eds.): Microspores: Evolution and ontogeny, pp. 193-212. Academic Press (1990). TARLYN, N. M., V. R. FRANCESCHI, J. D.EVERARD, and F. A. LoEwus: Recovery of exine from mature pollens and spores. Plant Sci. 90, 219-224 (1993). WILMESMEIER, S., S. STEUERNAGEL, and R. WIERMANN: Comparative FIlR and BC CP/MAS NMR spectroscopic investigations on sporopollenin of different systematic origins. Z. Naturforsch. 48c, 697-701 (1993).
.,OU••ALOF.
PlIId ...
© 1997 by Gustav Fischer Verlag. Jena
Solution of sporopollenin and reaggregation of a sporopollenin-like material: A new approach in the sporopollenin research l 2 CHRISTA]UNGFERMANN , FRIEDHELM AHLERS!, MONIKA GROTE , SABINE GUBATZl, l l 3 l STEFAN STEUERNAGEL , INA THOM , GERD WETZELS , and ROLF WIERMANN 1
Institut fur Botanik. SchlolSganen 3. D-48149 Munster. Germany
2
Institut fur Medizinische Physik und Biophysik, Hufferstralk 68. D-48149 Miinster. Germany
3
Bruker Analytische MelStechnik GmbH. Silberstreifen. D-76287 Rheinsterten. Germany
Received January 2. 1997 . Accepted February 9, 1997
Summary
Exines from Typha angustifolia L. pollen were completely dissolved in 2-aminoethanol if the exine material was isolated and purified by an enzymatic procedure. Acetolysis or treatment of the exine material with H 3P04 before the dissolution process led to insolubility. A reaggregation of the dissolved material was possible when the solvent was removed and H 20 added. The initial exine material and the reaggregated material were compared by means of staining, fluorescence microscopy, SEM, resistance against acetolysis, DSC, FTIR and BC CP/MAS NMR spectroscopy. Both materials showed great similarities with only little differences in the FTIR and BC NMR spectra and in the DSC thermograms.
Key words: 2-Aminoethanol Reaggregation, Sporopollmin. Solubilisation. Sporopollmin-like materia/, Typha angustifolia L. Abbreviations: BC CP/MAS NMR = Cross-polarization and magic-angle spinning nuclear magnetic resonance; DSC = Differential scanning calorimetry; FTIR = Fourier-transform-infrared; SEM = Scanning-electron-microscopy; TLC = Thin layer chromatography. Introduction
Sporopollenin, an extremely resistant biopolymer is substantially involved in the formation of the outer walls of spores and pollen. It is known to be insoluble in common acids and in most organic solvents. Strongly oxidizing chemicals degrade the polymer; however, due to the harsh conditions, the resulting molecules do not give conclusive evidence about the chemical nature of sporopollenins. Thus the chemical analysis of sporopollenin has been limited to methods that can be applied to the solid state only. Some morphological evidence exists that plants themselves can hydrolyse sporopollenin during early stages of germination (Dickinson and Lewis, 1974; Ahokas, 1976). 2-Aminoethanol is an organic solvent that has been used in cytological studies of pollen exines and intines for several
f. Plant Physiol. w,£ 151. pp. 513-519 (J997)
years (Bailey, 1960; Southworth, 1974; Rowley, 1978; HeslopHarrison and Heslop-Harrison, 1982; Kedves and Rojik, 1989; for review see Southworth, 1990). Southworth reponed that the outer exine layer of some gymnosperm and angiosperm pollen dissolves in 2-aminoethanol whereas the inner exine layer does not. During this process the exines of Lilium. Fagus and Juniperus turned into a lattice-like structure of interconnected granules interspersed with angular unstained openings called polygons (Southworth, 1974; 1985 a, b). Since there are similarities between these latticelike structures and developing primeexines of several genera, 2-aminoethanol might detach exine components in a process reverse to that of exine synthesis (Southworth, 1986). Furthermore, colloidal mixtures may play an important role in spore wall development. Hemsley et al. (1992 b) described a colloidal crystal-like structure of sporopollenin in
514
C. JUNGFERMANN, F. AHLERS, M. GROTE, S. GUBATZ, S. STEUERNAGEL, I. THOM, G. WETZELS, and R. WIERMANN
the spore walls of Selaginella and fossil Erlansonisporites. They found that the central region of these walls consists of closely packed particles in a semi-crystalline arrangement. The hypothesis of a colloidal exine construction in lycophyte megaspores with its inherent aspect of self-assembly has also been discussed by Collinson et al. (1993) and Hemsley et al. (1996). The authors concluded that this hypothesis reduces the need to invoke direct biological intervention and that therefore genetic input during the organization may be minimal. As shown above, 2-aminoethanol was successfully used for cytological studies, but not yet for biochemical experiments. The present investigation demonstrates the complete solubility of sporopollenin from Typha angustifolia L. in 2-aminoethanol and the subsequent reaggregation of the dissolved material to a sporopollenin-like substance. The initial and the reaggregated material are compared by staining, fluorescence microscopy, SEM, resistance against chemical degradation, DSC, FTIR- and I3C CP/MAS NMR-analysis.
Materials and Methods
Plant material For the experiments pollen of Tjpha angustifolia L. was used, because it can be harvested in sufficient amounts. In addition, according to results of cytological analyses the exine of Tjpha pollen is soluble in hot 2-aminoethanol (Southworth, 1974). The pollen was collected at the shedding stage from plants growing in the Botanical Garden, MUnster, Germany. The exine material was obtained by sonification of the pollen (Branson Sonifier Cell Distributer B-12, Standard Microtip, sonification intensity: == 50W/cm2 , 10 min) and enriched by filtration through nylon-meshes (mesh 20~m).
In order to degrade the intine, three methods were applied: acetolysis (acetic anhydride: H 2S04 conc. 8: 2, 100 ·C, 10 min), treatment with phosphoric acid (80 %, 50 ·C, 10 days) or enzymatic hydrolysis using Mazerozym RIO and Cellulase Onozuka RIO (yakult, Nishinomiya, Japan), each 1 % dissolved in 0.1 sodium acetate buffer, pH 4.5, 30 ·C, 3 days). The resulting material was extracted using solvents with increasing polarity (CHCI3/MeOH 1: 1 v/v; diethylether, acetone, MeOH, ethylene glycol monoethyl ether, H 20), each for 24h. For BC CP/MAS NMR analysis the material was extracted with each solvent until absolute purification (controlled byTLC analysis). After the extraction procedure the material was lyophilized for 48 h and stored in a desiccator until examination (Fig.1A).
Solubilisation procedure An aliquot of 5 mg purified exine material was suspended in 3 mL 2-aminoethano!. The solubilisation procedure was carried out under stirring at 100·C for 1 h. Complete dissolution of the exines was controlled by light and fluorescence microscopy.
Reaggregation procedure In order to remove the 2-aminoethanol two methods are possible: a) Dialysis against double distilled water (Millipore), until pH-neutrality in and outside of the dialysis-tubing (Sigma, MWCO 12500-14000). With reduction of the 2-aminoethanol-concentration the reaggregated material appears in the dialysis-tubing.
b) Extraction with ethyl acetate until a brown material appears. Mter addition of 1 mL H 20 a precipitate is visible. This precipitation is improved by low temperature or by addition of several drops of HC!. The precipitate (= reaggregated material, Fig. 1 D) is washed three times with 6 mL distilled water, lyophilized for 48 h and stored in a desiccator until examination.
Fluorescence microscopy The initial and reaggregated material were analysed by epifluorescence microscopy using a Leirz Diaplan (Leirz, Werzlar, Germany) with filter block L3 (450-490nm).
FTIR-spectroscopy The initial exine material and the reaggregated material were analysed spectroscopically with a Nicolet-5DXC-FTIR-Spectrometer equipped with the OMNIC 1.2-program (Nicolet, Offenbach, Germany). The analyses were carried out at the Institut fur Organische Chemie der WWU-MUnster. KBr-pellets were prepared from 300 mg KBr and 2 mg sample; spectra were obtained between 4000 cm -1 and 400 cm-r:-
13C CPlMAS NMR-spectroscopy The BC NMR analyses were carried out with a Bruker DSX 300 or DRX 400 spectrometer (for detail see Wilmesmeier et aI., 1993). The analyses were carried out by Bruker Analytische Megtechnik, Rheinstetten, Germany.
Electron microscopy For SEM investigation, the lyophilized initial exine material and the reaggregated material were put on glass slides without any adhesive, coated with a thin layer of gold by sputtering and examined at an acceleration voltage of 25 KV in a Hitachi S 450 scanning electron microscope. The SEM-analyses were performed at the Institut fur Medizinische Physik der WWU-MUnster.
DSC experiment The DSC experiments were performed using a Nerzsch DSC 200 instrument. The DSC thermograms were obtained in a dry nitrogen atmosphere with a heating rate of 5 ·Clmin. The DSC experiments were carried out at the Institut fur Physikalische Chemie der WWU-MUnster.
Results and discussion
Isolated sporopollenin from Typha angustifolia L. pollen is completely soluble in hot 2-aminoethanol if the intine was originally removed by enzymatic decomposition. Light-microscopy shows a homogeneous solution without any pellet (Fig. 1 C), thus demonstrating that the outer as well as the inner exine layer are completely soluble in this organic solvent (Fig.lB,C). Acetolysis and treatment with phosphoric acid, both conventional methods for degrading the intine, seem to alter the exine material in a way that leads to non solubility in 2-aminoethanol. This is not an effect of higher temperatures as shown by control experiments using the same temperature treatment. The introduction of acetyl groups by acetolysis
Sporopollenin and sporopollenin-like material
B
A
c
515
o
H
G
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)
....... ~ .....
J
.
':..J ~ ~~
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Fig. 1 A-J: Comparison of the initial exine material and the reaggregated material from 7jpha angustifolia. (A) initial exine material, (B) exine material suspended in 2-aminoethanol; (C) homogeneous solution of the exine material after 1 h in hot 2-aminoethanol; (D) reaggregated material. Staining with aniline-blue of (E) the initial exine material and (F) the reaggregated material. Autofluorescence of (G) the initial material and (H) the reaggregated material. SEM of the surface (I) of the initial exine material and of the structures generated during reaggregation after solubilization of the initial purified exine material in 2-aminoethanol. E- H X 345, bars = 3011m; I x 3200, J x 1500, bars = 3/1ffi.
m
516
C. JUNGFERMANN, F. AHLERS, M. GROTE, S. GUBATZ, S. STEUERNAGEL, I. THOM, G. WETZELS, and R. WIERMANN
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4000
2000
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4000
3500
3000
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2000
1500
WAVENUMBER [em-I]
and probably of phosphoric groups by H 3P04-treatment may prevent an attack of 2-aminoethanol at reactive sites in the sporopoUenin skeleton. So far, neither the exact nature of these sites nor the type of reaction between 2-aminoethanol and sporopoUenin are known in detail.
1000
500
Fig. 2: FTIR-spectra of the initial exine material (A) and the reaggregated material (8). Labeling of the bands was carried out following WIlmesmeier et al. (1993). X: 1660 em-I C =0 stretching vibrations and Y: 1545 cm-I N-H deformation vibrations, both are characteristic for N-mono-substituted amides (secondary amides). The original signal E at 1678 cm-I is considerably overlapped by the new absorption of the band X or is completely reduced. Z: 1062 em -I CoO stretching vibrations caused by primary alcohols.
The elimination of the intine by hydrolytic enzymes has been proven to be a very gentle method for purification of sporopoUenin (Gubatz et al., 1986; Schulze Osthoff and Wiermann, 1987). Since the method used for purification of sporopoUenin effects the degree of solubilisation, the evalua-
SporopoUenin and sporopoUenin-like material
FJg.3: l3C CP/MAS NMR spectra of the initial exine material isolated from Ijpha angustifolia pollen by using enzymes (A) and of a sparopoUenin-like material (8) reaggregated from a soluble sporopollenin fraction obtained from the same species.
tion of an additional method for the production of exine material under mild conditions, e.g. by using 4-methylmorpholine N-oxide monohydrate (MMNO. H 20) as described by Loewus et al. (1985), Baldi et al. (1987) , and Tarlyn et al. (1993) is of great interest. Up to now reaction products after treatment with 2-aminoethanol could not be determined. Our current experiments are focussed on the thorough analysis of the solubilised material. Reaggregation of a sporopollenin-like material is possible in the two ways described above. The reaggregated material has properties similar to the initial exine material (Fig. 1 E-H). Both are stainable with aniline blue, both show intensive autofluorescence and both withstand acetolysis. With regard to their reaction with 80 % H 3P04, however, the two materials differ. The reaggregated material dissolves, whereas the initial exine material is resistant. The FTIR- and I3C NMR-spectra obtained from both materials are presented in Figs. 2 and 3. The FTIR-spectra of both samples (2A, 2 B) have very similar absorption patterns, but show clearly different bands around the wave numbers: 834 cm -1, 1660 cm -I, 1545 cm -I and 1062 cm -I. The peak at 834 cm -I corresponds to the out-of-plane C- H vagging vibration in aromatics. The C =0 stretching frequency in the native material (E = 1678 cm -1) is very strong and typical for conjugated carbonyl groups of (X,~-unsaturated compounds. In the FTIR-spectra of the reaggregated material the C = 0 band appears at a lower wave number (X 1660cm-l ) and in weaker intensity than obversed for the native material. In additon, only in the spc:ctrum of the reaggregated material is a peak seen at 1545cm-1 (Y in Fig. 2B). This signal is characteristic for N-H deformation vibrations. Furthermore, the peak at about 1062 cm -I (Z in Fig. 2 B) is only detectable in
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0
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the reaggregated material. It is due to the primary alcohol group of 2-aminoethanol bound as an amide. All other signals in the spectra show high coincidence. In summary, the comparison of the two FTIR-spectra gives reasons to believe that the 2-aminoethanol reacts with a carboxy group in the native material to build a secondary amide. This assumption is confirmed by the results of the elemental analysis shown in Table 1. As expected, the nitrogen content is very low in the initial exine material. In contrast, the reaggregated material contains a significantly higher amount of nitrogen. Comparable results were obtained by I3C NMR analyses. The spectra of the initial and the reaggregated exine material are very similar. The most remarkable differences appear in the region of the signal group between 60 and 70 ppm (see and/or Fig. 3, arrow), indicating modifications of C-N functions. These modifications are probably caused by the treatment with 2-aminoethanol. The DSC thermograms for both materials are displayed in Fig. 4. The thermograms are similar in shape and have a transition temperature in the vicinity of 30"C. Furthermore, the transition temperature of the two samples depends on the heating rate (second-order transition). This relation is typical for macromolecules and biopolymers (Mandelkern, 1983).
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Table 1: Results of the elemental analyses. Typha angustifolia L.
C[%]
0[%]
H[%]
N[%]
purified exine material reaggregated material .
63.48 63.83
27.89 23.42
8.54 8.80
1.23 3.95
(Typhaceae)
518
C. JUNGFERMANN, F. AHLERS, M. GROTE, S. GUBATZ, S. STEUERNAGEL,I. THOM, G. WETZELS, and R. WIERMANN
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Fig.4: DSC thermograms of the initial exine material isolated from Typha angustifolia pollen by using enzymes (A) and of a sporopol-
lenin-like material (B) reaggregated from a soluble sporopollenin fraction obtained from the same species.
DSC investigations of the cuticula isolated from leaves of Citrus aurantium L. leads to transition temperatures around 7°C (Reynhardt and Riederer, 1991). The results presented here give no indication of fundamental differences between the initial and the reaggregated material. We regard them as either sporopollenin or sporopollenin-like material. It is not assumed that the reaggregated material is completely identical with the native sporopollenin, but both exhibit very similar properties and structures. In this connection it should be remembered that spectra of sporopollenins of different systematic origin revealed farreaching similarities in characteristic signals, but that the contributions of single functional groups were found to differ (Hemsley et al., 1992 a, 1993; Wilmesmeier et al., 1993). Therefore, to our current state of knowledge, sporopollenin is considered to represent a group of closely related macromolecules. The SEM-micrographs of the reaggregated material do not show a uniform material, but more or less globular structures of different sizes (Fig. 1, I-J). The average diameter of these particles is 4 J.l.m. Structures as those seen in native reticulate exines were not expected after reaggregation because no matrix giving a construction pattern is present during this procedure. According to unpublished results the type of structure depends on the way in which the sporopollenin fraction is obtained and the reaggregation is initiated. Control experiments were carried out either without exine material or using cellulose instead of exine material. These experiments did not result in the formation of any structures. Therefore, in our reaggregation experiments the formation of characteristic structures is due to the input of the sporopollenin material. Results of FTIR- and 13C NMR-spectroscopic analyses (Guilford et al., 1988; Espelie et al., 1989; Hemsley et al., 1992 a; Wilmesmeier et al., 1993) as well as those of electron microscopic investigations (Rowley, 1978; Southworth, 1986; Kedves and Rojik, 1989) clearly show that sporopollenins are closely related biopolymers with different functional groups
occurring in varying amounts and different properties. This is in accordance with our observations and early cytological studies (Southworth, 1974, 1990) demonstrating that exines from other species dissolved very poorly or only partially in hot 2-aminoethanol. The results of our experiments support the hypothesis that under certain conditions sporopollenin is a self-assembling biopolymer, as assumed recently by several authors (Hemsley et al., 1992 b, 1996; Collinson et al., 1993). By our experiments it was shown that it is possible to dissolve sporopollenin and reaggregate a sporopollenin-like material. The data clearly demonstrate that the macromolecule reaggregates by itself without either the addition of any substance or the presence of a matrix. During the development of pollen exines simple physical processes seem to be involved too, besides other as yet unknown processes. Our new approach is a very promising starting-point for the study of the chemical composition and structural organisation of sporopollenin by methods that could not be applied up to now, as solubilized sporopollenin is much more accessible to analytical methods than the particle form. This will be shown by several experiments with hydrolytic enzymes (in preparation). Sporopollenin, solubilised by different methods and reaggregated under varying conditions, is especially useful for investigation of the factors that influence in-vitro reconstitution. Acknowledgements
The financial support by the «Deutsche Forschungsgemeinschaft» and the «Fonds der Chemischen Industrie» is gratefully acknowledged. The authors are indebted to Prof. Gerhard Erker (Institut fur Organische Chemie, Munster) for making the FTIR-spectrometer available. We also thank Prof. Hellmut Eckert and the DSC group (Institut fur Physikalische Chemie, Munster) for their great help.
References AHOKAS, H.: Evidence of a pollen esterase capable of hydrolyzing sporopollenin. Experientia 32, 175-176 (1976). BAILEY, I. W: Some useful techniques in the study and interpretation of pollen morphology. J. Arnold Arbor. Harv. Univ. 41, 141-151 (1960). BALDI, B. G., V. R. FRANCESCHI, and F. A. LoEWUS: Preparation and properties of pollen sporoplasts. Protoplasma 141, 47-55 (1987). COLLINSON, M. E., A. R. HEMSLEY, and W A. TAYLOR: Sporopollenin exhibiting colloidal organization in spore walls. Grana Suppl. 1,31-39 (1993). DICKINSON, H. G. and D. LEWIS: Changes in the pollen wall of Linum grandiflorum following compatible and incompatible inttaspecific pollinations. Ann. Bot. 38, 23-29 (1974). EsPELIE, K. E., F. A. LoEWUS, R. J. PUGMIRE, W R. WOOLFENDEN, B. G. BALDI, and P. H. GWEN: Structural analysis of Lilium Iongiflorum sporopollenin by l3C NMR spectroscopy. Phytochemistry 28, 751-753 (1989). GUBATZ, S., S. HERMINGHAUS, B. MEURER, D. STRACK, and R. WIERMANN: The location to hydroxycinnamic acid amides in the exine of Corylus pollen. Pollen et Spores XXVIIl, 347-354 (1986).
Sporopollenin and sporopollenin-like material GUILFORD, W. J., D. M. SCHNEIDER, J.l..ABOVlTZ, and S. J. OPELLA: High resolution solid state BC NMR spectroscopy of sporopollenins from different plant taxa. Plant Physiol. 86, 134-136 (1988). HEMSLEY, A. R., W. G. CHALONER, C. J. GROOMBRIDGE, and A. C. SCOTT: Carbon-13 solid state nuclear magnetic resonance of sporopollenins from modern and fossil plants. Ann. Bot. 69, 545549 (1992a). _ HEMSLEY, A. R., M. E. COLLINSON, and A. P. R. BRAIN: Colloidal crystal-like structure of sporopollenin in the megaspore walls of recent &laginella and similar fossil spores. Bot. J. Linnean Soc. J08, 307-320 (1992b). HEMSLEY, A. R., P. J. BARRIE, W. G . CHALONER, and A. C. SCOTT: The composition of sporopollenin and its use in living and fossil plant systematics. Grana, Suppl. 1, 2-11 (1993). HEMSLEY, A. R., P. D. JENKINS, M. E. COLLINSON, and B. VINCENT: Experimental modelling of exine self-assembly. Bot. J. Linnean Soc. 121, 177-187 (1996). HESWP-HARRISON, Y. and J. HESWP-HARRISON: The microfibrillar component of the pollen intine: some structural features. Ann. Bot. 50, 831-842 (1982). KEDVES, M. and I. ROJIK: Investigation of the biopolymer organisation of partially degraded exines with the fragmentation method. Acta BioI. Szeged. 35, 71-80 (1989). LoEWUS, F. A., B. G. BALDI, V. R. FRANCESCHI, L. D. MEINERT, and J. J. MCCOLLUM: Pollen sporoplasts: Dissolution of pollen walls. Plant Physiol. 78, 652-654 (1985). MANDELKERN, L.: An introduction to macromolecules. New York: Springer (1983).
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REYNHARDT, E. C. and M. RIEDERER: Structure and molecular dynamics of the cuticular wax from leaves of Citrus aurantium L. J. Phys. D: Appl. Phys. 24, 478-486 (1991). ROWLEY, J. R.: The origin, ontogeny, and evolution of the exine. In: BHARADWAJ, D. C. (ed.): IV Int. Palynol. Conf., Lucknow (1976-77) 1, pp. 126-136 (1978). SCHULZE OSTHOFF, K. and R. WIERMANN: Phenols as integrated compounds of sporopollenin from Pinus pollen. Plant Physiol. 131, 5-15 (1987). SOUTHWORTH, D.: Solubiliry of pollen exines. Amer. J. Bot. 61, 36-44 (1974). - Pollen exine substructure. I. Lilium Iongijlorum. Amer. J.Bot. 72, 1274-1283 (1985a). - Pollen exine substructure. II. Fagus sylvatica. Grana 24, 161-166 (1985 b) . - Substructural organization of pollen exines. In: BLACKMORE, S. and I. K. FERGUSON (eds.): Pollen and Spores: Form and Function, pp. 61-69. Academic Press, London (1986). - Exine biochemistry. In: BLACKMORE, S. and R. B. KNox (eds.): Microspores: Evolution and ontogeny, pp. 193-212. Academic Press (1990). TARLYN, N. M., V. R. FRANCESCHI, J. D.EVERARD, and F. A. LoEwus: Recovery of exine from mature pollens and spores. Plant Sci. 90, 219-224 (1993). WILMESMEIER, S., S. STEUERNAGEL, and R. WIERMANN: Comparative FIlR and BC CP/MAS NMR spectroscopic investigations on sporopollenin of different systematic origins. Z. Naturforsch. 48c, 697-701 (1993).