Ubisch bodies and pollen ontogeny in Oxalis articulata Savigny
Sonia Rosenfeldt and Beatriz G. Galati
Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Buenos Aires, Argentina.
Address correspondence to: Lic. Sonia Rosenfeldt. Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, UBA. 4º piso, Pabellón II, Ciudad Universitaria. C1428EHA Buenos Aires. ARGENTINA. E-mail: soniar@bg.fcen.uba.ar / galati@bg.fcen.uba.ar
ABSTRACT: The correlation between the ontogeny of Ubisch bodies and pollen development in Oxalis articulata was studied with Transmission Electron Microscopy (TEM). The ultrastructural changes observed during the different stages of development in the tapetal cells are related to Ubisch bodies, sporopollenin and pollen-kitt formation. The pro-orbicules have the appearance of lipid globuli and their formation is related to the endoplasmic reticulum of rough type (ERr). The lipid globules or pro-orbicules disappear in the mature Ubisch bodies, and the places that they occupied remain free of contents or with pollen-kitt.
Keyword: Pollen. Ubisch bodies. Ontogeny. Utrastructure. Oxalis
Introduction
The genus Oxalis L. comprises about 800 cosmopolitan
species. Previous studies on microsporogenesis
and microgametogenesis in this family are meagre (Johri
et al., 1992). The majority of them have been carried
out in the genus Averrhoa and very few ones in the genus Oxalis (Herr, 1972; Guth and Weller 1986).
Ubisch (1927) observed granular bodies along the
inner walls of tapetal cells in Oxalis and she pointed
out the staining properties similarities of these bodies
to pollen exine.
These granular bodies were described by Ubisch
(op. cit.) as very showy small plates due their size and
form. In spite of this fact, ultrastructural studies of these
bodies have been performed in only one species of this
genus until this moment (Carniel, 1967).
The aim of this paper is to describe the ultrastructure
and development of Ubisch bodies and pollen grains
in Oxalis articulata Savigny in order to advance the understanding
of the orbicules morphology and function.
Material and Methods
The material of Oxalis articulata Savigny was collected
in FCEyN, Ciudad Universitaria, Nuñez, Buenos
Aires, Argentina.
Material was fixed in FAA (formalin, alcohol, acetic-
acid) for light microscopy studies. Sections were
made by means of standard microtome techniques. The
slides were stained in a safranin-fast green combination,
cresyl violet or toluidine blue and mounted in synthetic
resin (D'Ambrogio, 1986). The material was observed
with a Wild M20 microscope.
Material fixed in FAA was transferred to 100° acetone
and then air dried for scanning electron microscopy
(SEM) studies. The sputtering treatment was made
with gold-palladium for 3 min.
For transmission electron microscopy (TEM) studies,
the material was pre-fixed in 2.5% glutaraldehyde
in phosphate buffer (pH 7.2) for 2 h and post-fixed in
OsO4 at 2ºC in the same buffer for 3 h. Then the material
was dehydrated in ascending ethanol series and
embedded in Spurr's resin. Fine sections were made on
a Sorvall ultramicrotome, stained with uranyl acetate
and lead citrate (O'Brien and Mc Cully, 1981), observed
and photographed in a JEOL 100c TEM.
Results
We identified five stages of Ubisch bodies and pollen development in Oxalis articulata.
Stage 1: Microspore Mother Cells (MMC)
The anther is tetrasporangiate and its wall consists of: epidermis (ep), endothecium (en), one or two middle layers (ml) and a secretory type tapetum (t) (Figs. 1, 3).
FIGURES 1-4. MMC stage in O. articulata. Figs. 1-2. Light micrographs. Fig. 1. Transverse section of a developing anther showing the anther wall layers and the MMC. Scale bar: 10 μm. Fig. 2. MMC with callosic wall (c) and cytoplasmic connections (arrow). Scale bar: 5 μm. Figs. 3-4. Transmission electron micrographs. Fig. 3. Anther wall layers (ep: epidermis, en: endothecium, ml: middle layer, t: tapetum) and MMC. Scale bar: 2 μm. Fig. 4. Detail of tapetal cell cytoplasm with abundant endoplasmic reticulum of rough type (ERr). Scale bar: 500 nm.
The MMC are uninucleate and with few vacuolated
cytoplasm (Fig. 1). They have abundant endoplasmatic
reticulum of rough type (ERr), mitochondria, proplastids,
and dictyosomes (Fig. 3).
The tapetal cells cytoplasm has many free ribosomes,
ERr and proplastids (Fig. 3).
When callose walls envelop the MMC, conspicuous
cytoplasmic connections are observed (Fig. 2).
The cytoplasm of the tapetal cells is very dense, with
a similar ultrastructure to that of the MMC (Fig. 4).
Stage 2: Tetrad formation
Cytokinesis is simultaneous resulting in tetrahedral microspore tetrads (Fig. 5). The middle lamella of the MMC is disintegrated but the primary wall is still present (Fig. 6). When the tetrad is formed, an incipient primexine is seen between the callosic wall and the plasmalemma (Fig. 6).
FIGURES 5-7. Transmission electron micrographs of stage 2 (Tetrad formation). Fig. 5. Microspores tetrad. Scale bar: 2 μm. Fig. 6. Microspore cytoplasm detail; callose (c), lipidic globule (lg). Scale bar: 500 nm. Fig. 7. Tapetal cell detail with cytoplasmic connections (arrow). Endoplasmic Reticulum of rough type (ERr), plastid (p). Scale bar: 500 nm.
The microspore cytoplasm shows high activity and
has many mitochondria. Plastids and large osmiophilic
globules are scattered in the cytoplasm (Fig. 6).
In this stage, the tapetal cells are binucleate and
more vacuolated. Their cytoplasm shows an ERr increase.
The endoplasmic reticulum cisternae are arranged
in dense and uniformly thick rows, parallel to
the cell wall (Fig. 7). There are also many proplastids,
mitochondria and small lipidic globules (Fig. 7).
The tapetal cell wall begins to dissolve and presents
a lax structure. Many cytoplasmic connections go
through the radial walls of these cells (Fig. 7).
Stage 3: Free young microspores
After the dissolution of the callose wall, the sporopollenin wall begins to form. Three different electron- dense layers form the pollen grain wall: ectexine with interrupted tectum, columellae and foot layer; endexine and intine still in formation (Figs. 8, 11).
FIGURES 8-11. Transmission electron micrographs of stage 3 (Free young microspores). Fig. 8. Portion of a microspore and tapetal cell with Ubisch bodies. Scale bar: 1 μm. Fig. 9. Ubisch body and tapetal cell cytoplasm details. Endoplasmic reticulum of rough type (ERr), plastid (p). Scale bar: 500 nm. Fig. 10. Ubisch body detail; lipidic globule (white arrowhead ). Note the fibrillar electron dense material (black arrow). Scale bar: 200 nm. Fig. 11. Young microspore. Scale bar: 1μm.
The microspores have a conspicuous nucleus and
the structure of their cytoplasm is similar to the previous
stage (Fig. 11).
The tapetal cells show a dense cytoplasm with abundant
ERr, numerous free ribosomes and well development
plastids. The ERr lost the parallel configuration
and its cisternae are extensive (Fig. 9).
Moderate electron-dense globules or pro-orbicules
appear outside the plasmalemma at the inner tangential
face of the tapetal cells (Figs. 8, 9). These globules are
observed on depressions or crypts of the tapetal cells
plasmalemma.
A fibrillar electron-dense material is observed inside
the anther locule (Figs. 8-10). This material is deposited
on the developing exine and on several globules
or prorbicules (Fig. 10). One to six globules are
assembled for sporopollenin deposition and they form
the young Ubisch bodies (Figs. 8-10).
Stage 4: Free microspores
The microspores have a conspicuous nucleus and their cytoplasm is limited to a parietal position due to the presence of a large vacuole (Fig. 12).
FIGURES 12-18. Stage 4 (Free microspores, figs.12,14,16) and stage 5 (Mature pollen grain, figs.13,15,17,18) in O. articulata. Figs. 12,13,18. Ligth micrographs. Figs. 14,15,16. Transmission electron micrographs. Fig. 12. Free microspores. Scale bar: 10 μm. Fig. 13. Mature pollen grains and Ubisch bodies on the inner face of endothecial cells (arrow). Scale bar: 10μm. Fig. 14. Microspore wall, Ubisch bodies and tapetal cells (tc). Scale bar: 1μm. Fig. 15. Pollen wall and cytoplasm of the vegetative cell. Scale bar: 1μm. Fig. 16. Ubisch body detail with tapetal cell rests (tc). Scale bar: 500nm. Fig. 17. Mature Ubisch bodies showing the places that occupied the lipidic globules (arrow). Scale bar: 1 μm. Fig. 18. Ubsich bodies in paradermal view. Scale bar: 7,5 μm.
Many mitochondria and large lipidic globules are
observed in the microspores cytoplasm (Fig. 14).
Tapetal cells start autolysis, and only rests of these
cells can be observed (Figs. 12, 14, 16).
Ubisch bodies and exine have the same high electron-
density (Fig. 14). The globules present in the Ubisch
bodies at the previous stage are no longer observed (Figs.
14, 16).
Lipidic substances are seen among the exine columellae
(Fig. 14).
Stage 5: Mature pollen grain
Pollen grains are two-celled when shed. The vegetative cell encloses the generative cell (Fig. 20).
FIGURES 19-24. Stage 5 (Mature pollen grain) in O. articulata. Figs. 19-21. Transmission electron micrographs. Fig. 19. Detail of vegetative cell cytoplasm. Endoplasmic reticulum of rough type (ERr), vegetative nucleus (vn). Scale bar: 1 μm. Fig. 20. Detail of generative cell; generative nucleus (gn). Membranous inclusions in the electron- translucent wall (arrow). Scale bar: 500nm. Fig. 21. Detail of two pollen grains with abundant pollenkitt (arrow). Note the amyloplasts (a) in the vegetative cell cytoplasm. Scale bar: 2 μm. Figs. 22-24. Scanning electron micrographs in Oxalis articulata. Fig. 22. Pollen grain. Scale bar: 10 μm. Fig. 23. Details of colpus and exine. Scale bar: 10 μm. Fig. 24. Detail of Ubisch bodies. Scale bar: 10 μm.
The generative cell acquires a fusiform aspect with
a lobulate outline (Fig. 20).
This cell has an electron-translucent wall with some
plasmodesmata and membranous inclusions (Fig. 20).
The generative cell cytoplasm is highly reduced when
compared to the large nucleus (Fig. 20).
The vegetative cell cytoplasm is very dense, filled
with numerous small vesicles, mitochondria, lipidic
globules, amyloplasts and ERr with extended cisternae
(Figs. 15, 19). The vegetative cell nucleus is lobed, for
this reason, small portions of it are seen in the fine sections
(Fig. 19).
The pollen grain wall and the Ubisch bodies are
fully developed (Figs. 13, 15, 17). The ectexine and
the Ubisch bodies sporopollenin is compressed and
shows less electron-density that in the previous stage
(Figs. 15, 17).
At this stage, abundant pollenkitt is present
among pollen wall columellae and connects different
grains in the anther locule (Figs. 15, 21).
The places occupied by the globules during
Ubisch bodies formation are reduced in size and are
observed as small perforations in fine sections. These
places are free of contents or occupied by pollen-kitt
(Figs. 16, 17).
These Ubisch bodies are seen as multiperforated
plates in paradermal view under light microscopy (Fig.
18) and as plates with dome shape in lateral view and
smooth surface under scanning electron microspoy
(Fig. 24).
Pollen grain morphology
The pollen grain is tricolpate, anguloaperturate, prolate-
spheroidal to subprolate in equatorial view, columellate,
reticulate, with granular lumina (Figs. 22, 23).
The ectexine is reduced to small granular fragments
in the colpus (Fig. 23).
Discussion
This is the first report about Ubisch bodies ontogeny
in relation with pollen development in the genus Oxalis.
The microsporogenesis and microgametogenesis in
O. articulata are similar to previous descriptions in other
species of the genus (Johri et al. 1992).
In this species the pollen grains are two-celled when
shed. According to Pargney (1978), O. corniculata pollen
grains are three celled at the shedding stage.
The ultraestructural changes observed during the
different stages of development in the tapetal cells are
related to Ubisch Bodies, sporopollenin and pollen-kitt
formation.
Tapetal development in Oxalis articulata is similar
to other Dicotyledons secretory tapetum descriptions,
however, tapetal cell walls loss occur very early, during
the tetrad stage. At this stage, there is a great amount of
ERr in the tapetal cell cytoplasm, and at the following
stage, the cisternae of ERr are very extensive. This fact
possibly marks the ERr involvement in the Ubisch bodies
synthesis. Similar origin was reported in Passiflora spp. (Amela Garcia et al., 2002), Jacaranda mimosifolia (Galati and Strittmatter, 1999), Ceiba insignis (Galati
and Rosenfeldt,1998), Prosopis julifora (Vijayaraghavan
and Chaudhry, 1993), Catharanthus roseus (EL-Ghazaly
and Nilsson, 1991), Anemarrhena asphodeloides (Chen et al., 1988), Lilium henryi (Herich and Lux, 1985),
Lavandula dentata (Suarez-Cervera and Seoane-Camba,
1986) and Allium cepa (Risueño et al., 1969).
At the free young microspores stage, numerous
globuli appear outside the plasmalemma at the inner
tangencial face of the tapetal cells. Due to their electron-
density, these globuli seem to be constituted by
lipidic substances and may be consider as pro-orbicules.
Similar observations and interpretations were made by
Clement and Audran (1993 a,b) and Heslop-Harrison
and Dickinson (1969) in Lilium; Christensen et al.
(1972) in Sorgum bicolor and Steer (1977) in Avena.
Pro-orbicules are observed on depressions or crypts
of the tapetal cells plasmalemma. Rowley and Walles
(1987) and Steer (1977) found similar crypts and thought
that they constituted a site of lipid deposition during
pro-orbicule formation.
A fibrillar substance with the same electron density
as sporopollenin accumulates on the globuli after
these bodies are outside the plasmalemma of the tapetal
cells. For this reason, the accretion of sporopollenin on
them appears extracellular. These observations are in
concordance with the opinion of Amela Garcia et al.
(2002), Galati and Strittmatter (1999), Galati and
Rosenfeldt (1998), Clement and Audran (1993 a,b),
Christensen et al. (1972), and Echlin (1971).
According to Carniel (1967), the Ubisch bodies of
O. pubescens are developed before tapetal cell wall total
desintegration. On the contrary, in O. corniculata the tapetal cells wall begins to dissolve at the tetrade
stage, and these walls are totally degraded when the
sporopollenin accumulates on the globuli at the young
microspore stage.
The lipid globules or pro-orbicules disappear in the
mature Ubisch bodies, and the places that they occupied
remain free of contains or with pollen-kitt.
These observations were also reported for O. pubescence (Carniel, 1967).
In the young pollen grain, the vegetative cell goes
through a growth period, characterized by the synthesis
of new cytoplasm and an increase in the number of organelles,
as it has been reported for the majority of the
ultrastructurally studied species (Raghavan, 1997).
Many small vesicles are scattered in the cytoplasm of
this cell. According to Fisher et al. (1968) these vesicles
derive from dictyosomes and according to Noguchi
(1990), vesicles of this type could fuse to form vacuoles
in the emerging pollen tube. Amela García et al.
(2002) thought that the small vesicles could constitute
a membrane reservoir for the initial fast growth of the
pollen tube, assuring its anchoring on the stigma.
The generative cell of Oxalis articulata presents
some evaginations that protrude into the cytoplasm of
the vegetative cell. Tiwari (1994) found that the wall
processes arise from all faces of the generative cell and
protrude into the cytoplasm of vegetative cell. According
to Theunis et al. (1985) the ends of Rhododendron
laetum generative cell narrow into long evaginations.
We can speculate that O.articulata generative cell
morphology could support the transport of metabolites
from the vegetative cell.
The pollen morphology coincides with the general
description for the genus (Erdtman, 1952).
According to Wang and Chen (2001) O. acetocella pollen grains are tetracolpate and rarely tri or penta
colpate. However, we have not observed pollen grains
4-or 5-colpate in O articulata, only 3-colpate.
Acknowledgements
We thank Lic. Lara Strittmatter for reviewing the English. This work was supported by Grant Nº 2514 from CONICET, Argentine.
References
1. Amela Garcia MT, Galati BG, Anton AM (2002). Microsporogenesis,
microgametogenesis and pollen morphology of Passiflora
spp. (Passifloraceae). Bot J Linn Soc 139: 383-394.
2. Carniel K (1967). Licht- und electromenmikroskopische
Untersuchung der UbischKörperentwicklung in der gattung Oxalis. Österr Bot Z 114:490-501.
3. Christensen JE, Horner HT Jr, Lersten NR (1972). Pollen wall and
tapetal orbicular wall development in Sorghum bicolor
(Gramineae). Amer J Bot 59: 43-58.
4. Clément C, Audran JC (1993a). Cytochemical and ultrastructural
evolution of orbicules in Lilium. Pl Syst Evol (Suppl.) 7: 63-
74.
5. Clément C, Audran JC (1993b). Orbicule wall surface characteristics
in Lilium (Liliaceae). An ultrastructural and cytochemical
approach. Grana 32: 348-353.
6. Chen ZK, Wang FH, Zhou F (1988). On the origin, development
and ultrastructure of the orbicules and pollenkit in the tapetum
of Anemarrhena asphodeloides (Liliaceae). Grana 27:
273-282.
7. D'Ambrogio A (1986). Manual de Técnicas en Histología Vegetal.
Ed. Hemisferio Sur S.A. Buenos Aires. Argentina. pp. 1-
83.
8. Echlin P (1971). The role of the tapetum during microsporogenesis
of angiosperms. In: J. Heslop-Harrison (ed.), Pollen development
and physiology. Butterworths, London. pp. 41-61.
9. EL-Ghazaly G, Nilsson S (1991). Development of tapetum and
orbicules of Catharanthus roseus (Apocynaceae). In: S.
Blackmore & S.H.Barnes (eds.), Pollen and spores. Systematics
Association Special Vol. 44. Clarendon Press, Oxford.
Pp. 317-329.
10. Erdtman G (1952). Pollen morphology and plant taxonomy:
Waltham Massachusset, USA. The Chronica Botanica.
11. Fisher DB, Jensen WA, Ashton ME (1968). Histochemical studies
of pollen: storage pockets in the endoplasmic reticulum (ER).
Histochemie 13: 169-182.
12. Galati BG, Rosenfeldt S (1998). The pollen development in Ceiba
insignis (Kunth) Gibbs & Semir ex Chorisia speciosa St. Hil.
(Bombacaceae). Phytomorphology 48(2): 121-129.
13. Galati BG, Strittmatter LI (1999). Correlation between pollen development
and Ubisch bodies ontogeny in Jacaranda
mimosifolia (Bignoniaceae). Beitr Biol Pflanz 71: 249-260.
14. Guth CJ, Weller SG (1986). Pollination, fertilization and ovule abortion
in Oxalis magnifica. Am J Bot 73: 246-257.
15. Herich R, Lux A (1985). Lytic activity of Übisch bodies (orbicles).
Cytologia 50: 563-569.
16. Herr JM Jr (1972). An extended investigation on the megagametophyte
in Oxalis corniculata L. In: Murty YS; Johori BM;
Mohan Ram HY;Varghese TM. Advances in plant morphology.
Sarita Prkasham. Meerut (India). pp 92-101.
17. Heslop- Harrison J, Dickinson HG (1969). Time Relationships of Sporopollenin Synthesis Associated with Tapetum and Microspores
in Lilium. Planta 84: 199-214.
18. Johri BM, Ambegaokar KB, Srivastava PS (1992). Comparative
embryology of angiosperms. Vol. 1. Springer-Verlag. Berlín
Heidelberg.
19. Noguchi T (1990). Consumption of lipid granules and formation
of vacuoles in the pollen tube of Tradescantia reflexa.
Protoplasma 156: 19-28.
20. O'Brien TP, McCully ME (1981). The study of plant structure. Principles
and selected methods. Termarcarphi Pty. Ltd.,
Melbourne. Australia.
21. Pargney JC (1978). Étude ultrastructurale de la gamétogenèse mâle
dans une espèce à floraison cléistogame: Oxalis corniculata,
suivie de quelques considérations générales sur la cléistogamie.
Can J Bot 56: 1262-1269.
22. Raghavan V (1997). Molecular Embryology of Flowering Plants.
Cambridge. University Press. pp: 690.
23. Risueño MC, Giménez-Martín G, López-Sáez JF, García MIR (1969). Origin and development of sporopollenin bodies. Protoplasma
67: 361-374.
24. Rowley JR, Walles B (1987). Origin and structure of Ubisch bodies
in Pinus Sylvestris. Acta Societatis Botanicorum Poloniae 56(2):
215-227.
25. Suárez-Cervera M, Seoane-Camba JA (1986). Ontogénese des grains
de pollen de Lavandula dentata L. et évolution des cellules
tapétales. Pollen et Spores 28: 5-28.
26. Steer MW (1977). Differentiation of the tapetum in Avena. I. The
cell surface. J Cell Sci 25: 125-138.
27. Theunis CH, Conchie CA, Knox RB (1985). Three dimensional reconstruction
of the generative cell and its wall connection in
mature bicellular pollen of Rhododendron. Micron Mic Acta
16: 225-231.
28. Tiwari S (1994). An intermediate -voltage electron microscopic study
of freezer -substituted generative cell in pear (Pyrus communis
L.): features with relevance to cell- cell communication between
the cells of a germinating pollen. Sex Plant Reprod 7:
177-186.
29. Ubisch G (1927). Zur Entewicklungsgeschichte der Antheren. Planta
3: 490-495.
30. Vijayaraghavan MR, Chaudhry B (1993). Structure and development
of orbicules in the tapetum of Prosopis juliflora (Leguminosae,
Mimosoideae). Phytomorphology 43: 41-48.
31. Wang Y-F, Chen S-H (2001). Pollen Flora of Yuenyang Lake Nature
Preserve, Taiwan (II). Taiwania 46: 167-191.
Received on November 24, 2004.
Accepted on May 16, 2005.