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Development of spinous exine in Nuphar japonicum De Candolle (Nymphaeaceae)
Reviewof Palaeobotanyand Palynology, 75 ( 1992): 317-322 Elsevier Science Publishers B.V., Amsterdam
317
Development of spinous exine in Nupharjaponicum De Candolle (Nymphaeaceae) Masamichi Takahashi
Department of Biology, Kagawa University, Takamatsu, 760 Japan (Received July 7, 1992; revised and accepted September 30, 1992)
ABSTRACT Takahashi, M., 1992. Development of spinous exine in Nupharjaponicum De Candolle (Nymphaeaceae). Rev. Palaeobot. Palynol., 75: 317-322. Pollen development in Nupharjaponicum De Candolle (Nymphaeaceae) was studied with transmission and scanning electron microscopy (TEM and SEM), with special attention to the formation of spinous exine. At the early tetrad stage, globular components firstly initiate on the plasma membrane within the callose wall. Before any other exinous components appear, the globules develop into elongating spines in the callose wall. The inner constitution of developing spines shows a nonhomogeneous substructure composed of fused fibrous units, ca. 5-7 nm in diameter. After the spines elongate in the callose wall, a protectum is formed on a weakly undulated plasma membrane, and then probacules and a foot layer are formed underneath the protectum, The spinous exine further develops and reaches to 6-8 ~tm high after dissolution of the callose wall. The present study suggests that the developmental process of the exinous spines in Nuphar is distinguished from that of supratectal spines which begin to form after dissolution of the callose wall. The different developmental sequences imply that the spines of pollen grains in Nuphar are not homologous with the supratectal spines.
Introduction The great diversity o f pollen m o r p h o l o g y has resulted in it receiving much attention in the field o f plant systematics and ontogeny. Some workers recently suggested that the development o f exine patterning is under the control o f the microspore plasma m e m b r a n e within the tetrad. F o r example in Caesalpinia (Leguminosae) and Bougainvillea (Nyctaginaceae), the exine pattern is determined by the invaginated plasma m e m b r a n e that takes a pattern corresponding to the mature exine o r n a m e n t a t i o n (Takahashi, 1989; Takahashi and Skvarla, 1991a). Some specific variation in exine m o r p h o l o g y results from a distinct or modified process o f exine formation, as reviewed by Takahashi and Skvarla (1991b). In Hibiscus, the spines on the exine begin to form by the deposition o f
lamellated sheets after dissolution o f the callose wall (Takahashi and Kouchi, 1988). Pollen grains o f Nuphar are m o n o c o l p a t e with long spines (Erdtman, 1972). Abadie et al. (1986-1987) suggested that tubular subunits are exceptionally well stained and discernible in the exine o f Nuphar, due possibly to a difference from pollen o f other plants in the a m o u n t o f sporopollenin or its level o f polymerization. In the present study, pollen wall formation is traced from initiation within the callose wall to the mature pollen stage in N. japonicum De Candolle using a combination of transmission and scanning electron microscopy with a freeze cleaved method. Special attention is given to the formation and substructure o f long spines o f the pollen grains.
Materials and methods
Correspondence to."Dr. M. Takahashi, Department of Biology, Faculty of Education, Kagawa University, Takamatsu 760, Japan. 0034-6667/92/$05.00
Fresh anthers at various developmental stages were obtained from plants o f Nuphar japonicum
,~) 1992 - - Elsevier Science Publishers B.V. All rights reserved.
M. I A K A H , \ S H I
PLATE
I
319
SPINOUS EXINE DEVELOPMENT IN NUPH,4R JAPONICUM
collected in Kagawa Pref., Japan. Fixation and observation methods for scanning and transmission electron microscopy (SEM and TEM) were conducted as in Takahashi and Kouchi (1988); 200 nm thick sections were studied with a Hitachi H-7100 TEM at an accelerating voltage of 125 kV, in an attempt to clarify the constitution of substructural units of developing spines. Results
Appearance of globular structure Immediately after meiotic cytokinesis the microspore plasma membrane is almost smooth without any conformational changes (Plate I, 1). Globular structures, 0.2 lam in diameter, firstly initiate to form on the plasma membrane within the callose wall (Plate I, 2). It was impossible to find any particular organella associated with the sites of globules in the cytoplasm. The globules consist of a spongy-like substructure composed of fibrous units (Plate I, 3). The globules develop into elongating spines in the callose wall before other exinous components appear (Plate I, 4). The elongating spines are 1.2-1.4 ~tm high, and include an electron translucent cavity at the base.
Substructural units of developing spines The developing spines seem to be composed of fibrous units, based on the observation of 200 nm thick sections (Plate I, 5). The fibrous units, 5-7 ~tm thick, are irregularly oriented and fused. Further minute helical fibrils could be recognized in the fibrous units, although they are visible elsewhere, even in the non-structural parts of sections (Plate I, 6).
Formation of protectum and probacules While the spinous elements are elongating into the callose wall, protectum is formed on the protuberant sites of the invaginated plasma membrane (Plate I, 7). Subsequently, probacules are formed within the protectum above those regions of the plasma membrane (Plate I, 8). As protectum and probacules develop, they increase in electron density. The protectum and probacules are distinct from the developing spines in structure and in developmental process. The spines, 1.5 lam high, develop at the base and further elongate into the callose wall, including electron translucent spots in section (Plate I, 9).
Dissolution of the callose wall Tetrads include four microspores enveloped in the thick callose wall. The developing spines thrust into the callose wall (Plate II, 10). The dissolution of callose wall frees each microspore in the loculus (Plate II, 11). The free microspores enlarge and modify their shape. At the dissolution of callose wall, the spines are ca. 2.0 ~tm high with a granular surface (Plate II, 12). The endexine is formed by the accumulation of lamellations (Plate II, 13). Small spinules develop on the tectum (Plate II, 13). The intine appears within the exine, particularly thickened at the aperture region (Plate II, 14).
Mature pollen grains During the last stage of pollen development, mature spinous pollen grains are dispersed in the loculus (Plate II, 15). The spines are ca. 6-8 lam long. Thickness of the exine, except for the spines, is 0.6-0.7 ~tm (Plate II, 16). The pollen grains are
PLATEI Early exine developmentin Nuphar. 1. Plasma membrane of microspore immediatelyafter meiosis. Bar = 0.2 ~tm. 2. Globularbody presents on the plasma membrane. Bar = 0.1 ~m. 3. Inner structure of globule. Bar= 50 rim. 4. Elongatingspine into callose wall. Bar=0.5/am. 5. 200 nm thick section showing fused exine fibrous subunits. Bar= 10 nm. 6. Further minute helical elements observed in elsewhere. Bar = I0 nm. 7. Initiation of protectum. Bar = 0.3 ~m. 8. Protectum and probacula. Bar = 0.1 ~tm. 9. Developingspinous exine in callose wall. Bar = 0.5 ~tm.
320 P L A T E II
M. T A K A H A S H I
321
SPINOUS EXINE DEVELOPMENT IN NUPHAR JAPONICUM
spheroidal and monocolpate, 55-58 !am in the longest axis of equatorial diameter and 43-46 gm in polar axis. The furrow extends to the ends of the pollen grains. Long spines are also deposited on the furrow (Plate II, 17). Discussion
Previous pollen developmental studies indicated that the pattern in the mature exine corresponds to a pattern originally formed by plasma membrane (Takahashi, 1989; Takahashi and Skvarla, 1991a). The present study in Nuphar shows that the long spines are produced by a unique developmental sequence. The plasma membrane does not take a pattern corresponding to the long spines before the initiation of spines. The spine initiates to form as a globular structure before any other exinous elements appear on the plasma membrane within the callose wall. It is also unusual that the developing spines thrust into the callose wall. The long spine is a distinct and different element from the tectum which is formed later than the long spines. Although the plasma membrane determines the pattern of the tectum, it does not determine the pattern of the long spines in ,Nuphar. Some other studies suggested that supratectal elements develop on the rectum during free microspore stage, as reviewed by Takahashi and Skvarla (1991b). Pollen grains of Hibiscus have the similar long spines as Nuphar pollen. The developmental sequence of these spines is different. In Hibiscus pollen, the spines on the exine are formed by the deposition of lamellated sheets after dissolution of the callose wall. Therefore, pollen grains of these genera would probably share analogous characters of long spinous exines.
There are several different views on the exine substructure, and the substructural constitution of exine still remains to be solved (Takahashi, 1993). Abadie et al. (1986-1987) proposed an exine model of tubular subunits, based on the FlynnRowley model (Flynn and Rowley, 1971) and observation with Nuphar variegatum. In the present study, the developing exine shows a spongy structure composed of fibrous units, 5-7 nm in diameter. The fibrous units are similar to the exine subunits proposed by Rowley and his co-workers (Abadie et al., 1986-1987, table 1, fig. 12b, c). A regular patterned arrangement of exine subunits was not recognized in the present study. The fibrous units seem to be irregularly fused. Abadie et al. (1986-1987) showed that the exine subunit, 10 nm in diameter, is composed of further minute helical elements of subunits. Although the same minute helical coils in the fibrous units as their exine subunits model could be recognized (Plate I, 5), it was not clear if these minute helical coils are the elements of exine subunits, because minute helical elements could be observed elsewhere, even in the non-structural regions of sections (Plate II, 6). Takahashi (1993), using the method of freeze cleaving and field emission electron microscopy, showed in Caesalpinia pollen that the initial protectum is composed of irregularly oriented fibrous threads, 5-7nm in diameter, on the protuberant sites of the invaginated plasma membrane at the early tetrad stage, and that the fibrous threads are gradually masked by globular materials during later developmental stages. The fibrous threads have been assumed to function as receptors and/or skeleton of the developing exine (Takahashi, 1993). Wehling et al. (1989) analyzed
PLATE 1I Later exine development and mature pollen grain. I0. Tetrad in which spines thrust into callose wall (arrows). Bar= 3/,tm. ! 1. Dissolution of callose wall. Bar = 5 p.m. 12. Developing spine and endexine during free microspore stage. Bar=0.5 p.m. 13. Developing exine during young pollen stage. Spinules on rectum (arrows). Bar= I p,m. 14. Cross-section of young pollen grain. Furrow (arrow). Bar = 3 pm. 15. Mature pollen grains in loculus. Bar= I00/.tin. 16. Completed spinous exine. Bar= 3 p.m. 17. Mature pollen grain, Bar= 10 jam.
322
sporopollenin from wings of Pinus pollen by pyrolysis mass spectrometry, and they showed that mass peaks, which are characteristic for pCoumaric acid, were dominant in the spectrum. They assumed that p-Coumaric acid is a genuine structure unit in the sporopollenin skeleton. It is suggested that a combination of chemical analytical study and ultrastructural approach will be needed to elucidate the structural condition of exine subunits.
Acknowledgement This study was supported in part by the Nissan Science Foundation.
References Abadie, M., Hideux, M. and Rowley, J.R., 1986-1987. Ultrastructural cytology of the anther. II. Proposal for a model
M. TAKAHASHI ofexine considering a dynamic connection between cytoskeleton, glycolemma and sporopollenin-synthesis. Ann. Sci. Nat. Bot. Paris, 13e set., 8: 1-16. Erdtman, G., 1972. Pollen Morphology and Plant Taxonomy-Angiosperms. Hafner, New York, NY, 553 pp. Flynn, J.J. and Rowley, J.R., 1971. Wall microtubules in pollen grains. Zeiss Inform,, 76:40 45. Takahashi, M., 1989. Pattern determination of the exine in Caesalpinia japonica (Leguminosae: Caesalpinioideae). Am. J. Bot., 76: 1615-1626. Takahashi, M., 1993. Exine initiation and substructure in Caesalpinia japonica (Leguminosae: Caesalpinioideae). Am. J. Bot.: in press. Takahashi, M. and Kouchi, J., 1988. Ontogenetic development of spinous exine in Hibiscus syriacus (Malvaceae). Am. J. Bot., 75:1549 1558. Takahashi, M. and Skvarla, J.J., 1991a. Exine pattern formation in Bougainvillia spectabilis (Nyctaginaceae). Am. J. Bot., 78: 1063-1069. Takahashi, M. and Skvarla, J.J., 1991b. Development of striate exine in lpomopsis rudbura (Polemoniaceae). Am, J. Bot., 78: 1724-1731. Wehling, K., Niester, Ch., Boon, J.J., Willemse, M.T.M. and Wiermann, R., 1989. p-Coumaric acid--a monomer in the sporopollenin skeleton. Planta, 179: 376-380.
317
Development of spinous exine in Nupharjaponicum De Candolle (Nymphaeaceae) Masamichi Takahashi
Department of Biology, Kagawa University, Takamatsu, 760 Japan (Received July 7, 1992; revised and accepted September 30, 1992)
ABSTRACT Takahashi, M., 1992. Development of spinous exine in Nupharjaponicum De Candolle (Nymphaeaceae). Rev. Palaeobot. Palynol., 75: 317-322. Pollen development in Nupharjaponicum De Candolle (Nymphaeaceae) was studied with transmission and scanning electron microscopy (TEM and SEM), with special attention to the formation of spinous exine. At the early tetrad stage, globular components firstly initiate on the plasma membrane within the callose wall. Before any other exinous components appear, the globules develop into elongating spines in the callose wall. The inner constitution of developing spines shows a nonhomogeneous substructure composed of fused fibrous units, ca. 5-7 nm in diameter. After the spines elongate in the callose wall, a protectum is formed on a weakly undulated plasma membrane, and then probacules and a foot layer are formed underneath the protectum, The spinous exine further develops and reaches to 6-8 ~tm high after dissolution of the callose wall. The present study suggests that the developmental process of the exinous spines in Nuphar is distinguished from that of supratectal spines which begin to form after dissolution of the callose wall. The different developmental sequences imply that the spines of pollen grains in Nuphar are not homologous with the supratectal spines.
Introduction The great diversity o f pollen m o r p h o l o g y has resulted in it receiving much attention in the field o f plant systematics and ontogeny. Some workers recently suggested that the development o f exine patterning is under the control o f the microspore plasma m e m b r a n e within the tetrad. F o r example in Caesalpinia (Leguminosae) and Bougainvillea (Nyctaginaceae), the exine pattern is determined by the invaginated plasma m e m b r a n e that takes a pattern corresponding to the mature exine o r n a m e n t a t i o n (Takahashi, 1989; Takahashi and Skvarla, 1991a). Some specific variation in exine m o r p h o l o g y results from a distinct or modified process o f exine formation, as reviewed by Takahashi and Skvarla (1991b). In Hibiscus, the spines on the exine begin to form by the deposition o f
lamellated sheets after dissolution o f the callose wall (Takahashi and Kouchi, 1988). Pollen grains o f Nuphar are m o n o c o l p a t e with long spines (Erdtman, 1972). Abadie et al. (1986-1987) suggested that tubular subunits are exceptionally well stained and discernible in the exine o f Nuphar, due possibly to a difference from pollen o f other plants in the a m o u n t o f sporopollenin or its level o f polymerization. In the present study, pollen wall formation is traced from initiation within the callose wall to the mature pollen stage in N. japonicum De Candolle using a combination of transmission and scanning electron microscopy with a freeze cleaved method. Special attention is given to the formation and substructure o f long spines o f the pollen grains.
Materials and methods
Correspondence to."Dr. M. Takahashi, Department of Biology, Faculty of Education, Kagawa University, Takamatsu 760, Japan. 0034-6667/92/$05.00
Fresh anthers at various developmental stages were obtained from plants o f Nuphar japonicum
,~) 1992 - - Elsevier Science Publishers B.V. All rights reserved.
M. I A K A H , \ S H I
PLATE
I
319
SPINOUS EXINE DEVELOPMENT IN NUPH,4R JAPONICUM
collected in Kagawa Pref., Japan. Fixation and observation methods for scanning and transmission electron microscopy (SEM and TEM) were conducted as in Takahashi and Kouchi (1988); 200 nm thick sections were studied with a Hitachi H-7100 TEM at an accelerating voltage of 125 kV, in an attempt to clarify the constitution of substructural units of developing spines. Results
Appearance of globular structure Immediately after meiotic cytokinesis the microspore plasma membrane is almost smooth without any conformational changes (Plate I, 1). Globular structures, 0.2 lam in diameter, firstly initiate to form on the plasma membrane within the callose wall (Plate I, 2). It was impossible to find any particular organella associated with the sites of globules in the cytoplasm. The globules consist of a spongy-like substructure composed of fibrous units (Plate I, 3). The globules develop into elongating spines in the callose wall before other exinous components appear (Plate I, 4). The elongating spines are 1.2-1.4 ~tm high, and include an electron translucent cavity at the base.
Substructural units of developing spines The developing spines seem to be composed of fibrous units, based on the observation of 200 nm thick sections (Plate I, 5). The fibrous units, 5-7 ~tm thick, are irregularly oriented and fused. Further minute helical fibrils could be recognized in the fibrous units, although they are visible elsewhere, even in the non-structural parts of sections (Plate I, 6).
Formation of protectum and probacules While the spinous elements are elongating into the callose wall, protectum is formed on the protuberant sites of the invaginated plasma membrane (Plate I, 7). Subsequently, probacules are formed within the protectum above those regions of the plasma membrane (Plate I, 8). As protectum and probacules develop, they increase in electron density. The protectum and probacules are distinct from the developing spines in structure and in developmental process. The spines, 1.5 lam high, develop at the base and further elongate into the callose wall, including electron translucent spots in section (Plate I, 9).
Dissolution of the callose wall Tetrads include four microspores enveloped in the thick callose wall. The developing spines thrust into the callose wall (Plate II, 10). The dissolution of callose wall frees each microspore in the loculus (Plate II, 11). The free microspores enlarge and modify their shape. At the dissolution of callose wall, the spines are ca. 2.0 ~tm high with a granular surface (Plate II, 12). The endexine is formed by the accumulation of lamellations (Plate II, 13). Small spinules develop on the tectum (Plate II, 13). The intine appears within the exine, particularly thickened at the aperture region (Plate II, 14).
Mature pollen grains During the last stage of pollen development, mature spinous pollen grains are dispersed in the loculus (Plate II, 15). The spines are ca. 6-8 lam long. Thickness of the exine, except for the spines, is 0.6-0.7 ~tm (Plate II, 16). The pollen grains are
PLATEI Early exine developmentin Nuphar. 1. Plasma membrane of microspore immediatelyafter meiosis. Bar = 0.2 ~tm. 2. Globularbody presents on the plasma membrane. Bar = 0.1 ~m. 3. Inner structure of globule. Bar= 50 rim. 4. Elongatingspine into callose wall. Bar=0.5/am. 5. 200 nm thick section showing fused exine fibrous subunits. Bar= 10 nm. 6. Further minute helical elements observed in elsewhere. Bar = I0 nm. 7. Initiation of protectum. Bar = 0.3 ~m. 8. Protectum and probacula. Bar = 0.1 ~tm. 9. Developingspinous exine in callose wall. Bar = 0.5 ~tm.
320 P L A T E II
M. T A K A H A S H I
321
SPINOUS EXINE DEVELOPMENT IN NUPHAR JAPONICUM
spheroidal and monocolpate, 55-58 !am in the longest axis of equatorial diameter and 43-46 gm in polar axis. The furrow extends to the ends of the pollen grains. Long spines are also deposited on the furrow (Plate II, 17). Discussion
Previous pollen developmental studies indicated that the pattern in the mature exine corresponds to a pattern originally formed by plasma membrane (Takahashi, 1989; Takahashi and Skvarla, 1991a). The present study in Nuphar shows that the long spines are produced by a unique developmental sequence. The plasma membrane does not take a pattern corresponding to the long spines before the initiation of spines. The spine initiates to form as a globular structure before any other exinous elements appear on the plasma membrane within the callose wall. It is also unusual that the developing spines thrust into the callose wall. The long spine is a distinct and different element from the tectum which is formed later than the long spines. Although the plasma membrane determines the pattern of the tectum, it does not determine the pattern of the long spines in ,Nuphar. Some other studies suggested that supratectal elements develop on the rectum during free microspore stage, as reviewed by Takahashi and Skvarla (1991b). Pollen grains of Hibiscus have the similar long spines as Nuphar pollen. The developmental sequence of these spines is different. In Hibiscus pollen, the spines on the exine are formed by the deposition of lamellated sheets after dissolution of the callose wall. Therefore, pollen grains of these genera would probably share analogous characters of long spinous exines.
There are several different views on the exine substructure, and the substructural constitution of exine still remains to be solved (Takahashi, 1993). Abadie et al. (1986-1987) proposed an exine model of tubular subunits, based on the FlynnRowley model (Flynn and Rowley, 1971) and observation with Nuphar variegatum. In the present study, the developing exine shows a spongy structure composed of fibrous units, 5-7 nm in diameter. The fibrous units are similar to the exine subunits proposed by Rowley and his co-workers (Abadie et al., 1986-1987, table 1, fig. 12b, c). A regular patterned arrangement of exine subunits was not recognized in the present study. The fibrous units seem to be irregularly fused. Abadie et al. (1986-1987) showed that the exine subunit, 10 nm in diameter, is composed of further minute helical elements of subunits. Although the same minute helical coils in the fibrous units as their exine subunits model could be recognized (Plate I, 5), it was not clear if these minute helical coils are the elements of exine subunits, because minute helical elements could be observed elsewhere, even in the non-structural regions of sections (Plate II, 6). Takahashi (1993), using the method of freeze cleaving and field emission electron microscopy, showed in Caesalpinia pollen that the initial protectum is composed of irregularly oriented fibrous threads, 5-7nm in diameter, on the protuberant sites of the invaginated plasma membrane at the early tetrad stage, and that the fibrous threads are gradually masked by globular materials during later developmental stages. The fibrous threads have been assumed to function as receptors and/or skeleton of the developing exine (Takahashi, 1993). Wehling et al. (1989) analyzed
PLATE 1I Later exine development and mature pollen grain. I0. Tetrad in which spines thrust into callose wall (arrows). Bar= 3/,tm. ! 1. Dissolution of callose wall. Bar = 5 p.m. 12. Developing spine and endexine during free microspore stage. Bar=0.5 p.m. 13. Developing exine during young pollen stage. Spinules on rectum (arrows). Bar= I p,m. 14. Cross-section of young pollen grain. Furrow (arrow). Bar = 3 pm. 15. Mature pollen grains in loculus. Bar= I00/.tin. 16. Completed spinous exine. Bar= 3 p.m. 17. Mature pollen grain, Bar= 10 jam.
322
sporopollenin from wings of Pinus pollen by pyrolysis mass spectrometry, and they showed that mass peaks, which are characteristic for pCoumaric acid, were dominant in the spectrum. They assumed that p-Coumaric acid is a genuine structure unit in the sporopollenin skeleton. It is suggested that a combination of chemical analytical study and ultrastructural approach will be needed to elucidate the structural condition of exine subunits.
Acknowledgement This study was supported in part by the Nissan Science Foundation.
References Abadie, M., Hideux, M. and Rowley, J.R., 1986-1987. Ultrastructural cytology of the anther. II. Proposal for a model
M. TAKAHASHI ofexine considering a dynamic connection between cytoskeleton, glycolemma and sporopollenin-synthesis. Ann. Sci. Nat. Bot. Paris, 13e set., 8: 1-16. Erdtman, G., 1972. Pollen Morphology and Plant Taxonomy-Angiosperms. Hafner, New York, NY, 553 pp. Flynn, J.J. and Rowley, J.R., 1971. Wall microtubules in pollen grains. Zeiss Inform,, 76:40 45. Takahashi, M., 1989. Pattern determination of the exine in Caesalpinia japonica (Leguminosae: Caesalpinioideae). Am. J. Bot., 76: 1615-1626. Takahashi, M., 1993. Exine initiation and substructure in Caesalpinia japonica (Leguminosae: Caesalpinioideae). Am. J. Bot.: in press. Takahashi, M. and Kouchi, J., 1988. Ontogenetic development of spinous exine in Hibiscus syriacus (Malvaceae). Am. J. Bot., 75:1549 1558. Takahashi, M. and Skvarla, J.J., 1991a. Exine pattern formation in Bougainvillia spectabilis (Nyctaginaceae). Am. J. Bot., 78: 1063-1069. Takahashi, M. and Skvarla, J.J., 1991b. Development of striate exine in lpomopsis rudbura (Polemoniaceae). Am, J. Bot., 78: 1724-1731. Wehling, K., Niester, Ch., Boon, J.J., Willemse, M.T.M. and Wiermann, R., 1989. p-Coumaric acid--a monomer in the sporopollenin skeleton. Planta, 179: 376-380.