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Are unusual plasmodesmata in the embryo-suspensor restricted to species from the genus Sedum among Crassulaceae?
Flora 206 (2011) 684–690
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Flora journal homepage: www.elsevier.de/flora
Are unusual plasmodesmata in the embryo-suspensor restricted to species from the genus Sedum among Crassulaceae? Małgorzata Kozieradzka-Kiszkurno a,∗ , Bartosz Jan Płachno b , Jerzy Bohdanowicz a a b
Department of Plant Cytology and Embryology, University of Gda´ nsk, Kładki 24 St., 80-822 Gda´ nsk, Poland Department of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52 St., 31-044 Cracow, Poland
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Article history: Received 13 August 2010 Accepted 25 November 2010 Keywords: Crassulaceae Jovibarba Plasmodesmata Sempervivum Suspensor Ultrastructure
a b s t r a c t The Crassulaceae family comprises mainly herbaceous leaf succulents, some of which have an ornamental value. During embryogenesis, they produce a suspensor with a giant polyploid basal cell. It has recently been shown that in Sedum acre and S. hispanicum this cell has compound plasmodesmata with an unusual dome of electron-dense material associated on the cell’s side. These compound plasmodesmata differ from the typical ones occurring in other angiosperms. In this study, the hypothesis was tested that the unusual plasmodesmata in the embryo-suspensor are a feature not only restricted to species from the genus Sedum, but are also found in other Crassulaceae genera. Suspensors of example species from the genera Sempervivum and Jovibarba, which have vegetative morphologies quite different from Sedum and which are placed in the traditional classification into another subfamily, were first examined using an electron microscope. It was found that the unusual compound plasmodesmata in the suspensor are not only restricted to species from the genus Sedum but are also found in species from other Crassulaceae genera (Sempervivum arachnoideum and Jovibarba sobolifera). It should be noted that some ultrastructural features of compound plasmodesmata in the analyzed genera (e.g. the character of the wall with plasmodesmata, plasmodesmata diameter or occurrence of the electron-dense material) are different from the suspensor plasmodesmata recorded in species from the Sedum genus. We found that in Sempervivum arachnoideum the size of the plasmodesmata diameter varies according to the micropylar-chalazal axis of the embryo. This is the first report of variation in the diameter of the plasmodesmata within the embryo of angiosperms. Further study will be needed to show whether compound plasmodesmata occur in other Crassulaceae clades, whether they are a stable feature at the genus level in this family, and also whether they have evolved several times or only once in Crassulaceae. © 2011 Elsevier GmbH. All rights reserved.
Introduction The Crassulaceae family, with a world-wide distribution and comprising 900–1500 species, has evolved mainly herbaceous leaf succulents (Berger, 1930; Eggli, 2005; ‘t Hart and Bleij, 2003; van Ham and ‘t Hart, 1998). Molecular studies have shown that Crassulaceae are part of the Saxifragales clade (e.g. Chase et al., 1993; Morgan and Soltis, 1993) and this family most likely has a southern African origin (Mort et al., 2001). Recently several papers have dealt with the phylogeny of Crassulaceae (e.g. Mort et al., 2001; van Ham and ‘t Hart, 1998). The family is monophyletic; however, six subfamilies are traditionally recognized as being polyphyletic (van Ham and ‘t Hart, 1998) and also the large genus Sedum is highly polyphyletic with representatives spread throughout the Sedoideae clade (Mort et al., 2001). It is worth mentioning that the
∗ Corresponding author. E-mail address: [email protected] (M. Kozieradzka-Kiszkurno). 0367-2530/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2010.11.017
members of Crassulaceae have ornamental value and some are also used in medicine (Eggli, 2005). Since the beginning of the 20th century, the Crassulaceae family has attracted the interest of researchers because of its interesting embryogenesis – mainly with respect to its large polyploid suspensors with haustoria (Kozieradzka-Kiszkurno and Bohdanowicz, 2003, 2006, 2010; Kozieradzka-Kiszkurno et al., 2010; Mauritzon, 1933; Niculae and Barca, 2001; Souéges, 1927, 1936). For example in Sedum acre, the nucleus of the suspensor basal cell increases its nuclear DNA content and attains a maximum level as high as 1024C (Kozieradzka-Kiszkurno and Bohdanowicz, 2003); therefore it could be a good model for studies on polyploid cells. Recently Kozieradzka-Kiszkurno and Bohdanowicz (2010) described unusual, compound plasmodesmata which connect the giant suspensor basal cell to the first layer of the chalazal suspensor cells. These compound plasmodesmata are wider than typical plasmodesmata in angiosperms and are covered by a dome of an electron-dense material, which might be a kind of filter, which selects the flow of nutrients via suspensor’s cells.
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Table 1 Comparison of suspensor’s plasmodesmata between Sedum acre, S. hispanicum and two species from other genera. Sempervivum arachnoideum and Jovibarba sobolifera are treated together because we did not find any major differences between these genera. Characters
Sedum acre and S. hispanicum
Sempervivum arachnoideum and Jovibarba sobolifera
Occurrence
Wall between the basal cell and the first layer of the chalazal suspensor cells Without ingrowths
Wall between the basal cell and the first layer of the chalazal suspensor cells With ingrowths – transfer wall type Wall thickness decreases in the plasmodesmata region Compound
Type of the wall with plasmodesmata Thickness of the wall
Type of plasmodesmata Plasmodesmata diameter (nm) Character of an electron-dense material Behavior of ER
Fig. 1. Plants of Sempervivum arachnoideum L. (A) and Jovibarba sobolifera (Sims) Opiz (B) in natural habitats.
Embryological characteristics could be very useful in taxonom´ atek, ˛ ical and phylogenetical investigations (e.g. Płachno and Swi 2008, 2009, 2010). In this study, we test the hypothesis that the unusual compound plasmodesmata in the suspensor are not only restricted to species from the genus Sedum but also for species from other Crassulaceae genera. Suspensors from species of the genera Sempervivum and Jovibarba, which are readily available to us, were examined first because they are elements of Polish flora and their vegetative morphologies are quite different from those of Sedum. Materials and methods Flowering plants of Sempervivum arachnoideum L. (Fig. 1A) were provided by a commercial business company (Andrzej and Łucja Hinz, http://kaktusiarnia.pl/index1.html). Flowering plants of Jovibarba sobolifera (Sims) Opiz (Fig. 1B) were collected from a natural population in Kokotek near Lubliniec in the south of Poland. Some additional plants were also obtained from The Botanic Garden of the Jagiellonian University, Cracow, Poland. The ovules at different stages of embryo development were fixed in 2.5% formaldehyde and 2.5% glutaraldehyde in 0.05 M cacodylate buffer (pH = 7.0) for 4 h at room temperature. The material was post-fixed in 1% osmium tetroxide in cacodylate buffer at 4 ◦ C overnight, rinsed in the same buffer with four changes, treated with 1% uranyl acetate in distilled water for 1 h, dehydrated with acetone and embedded in Spurr’s resin (Spurr, 1969). Serial ultrathin (60–100 nm) sections were cut with a diamond knife on a SORVALL MT 2B microtome, and then post-stained with saturated solution of uranyl acetate in 50% ethanol for 20 min and with 0.04% lead citrate for 2 min. Observations were made using a Philips CM 100 transmission electron microscope. For light microscopy, semithin sections (0.5–2 m) were stained with 0.05% toluidine blue 0 in 1% sodium tetraborate. To prepare the data for Tables 1 and 2, at least twenty plasmodesmata for each of the studied species, were mea-
Wall has uniform thickness Very compound – branched 30–90
30–75
More electron transparent
More electron dense
Profiles of ER adjacent to the electron-dense material
Profiles of ER adjacent to the electron-dense material
sured. Additionally, measurements of the plasmodesmata diameter in the Sedum acre suspensor were performed using material described by Kozieradzka-Kiszkurno and Bohdanowicz (2010). Results Both Sempervivum arachnoideum and Jovibarba sobolifera undergo the Caryophyllad type of embryonic development, and because we did not find any major differences between these genera we treated them together. A suspensor consists of the giant basal cell, which is pyriform in shape, and a few chalazal cells, which are connected with the embryo-proper (Fig. 2A, D). There is a difference in the density of the cytoplasm of the basal and chalazal cells of the suspensor (Fig. 2B, C, E, F). The cytoplasm of the basal cell is more transparent but is rich with mitochondria, profiles of endoplasmic reticulum, dictyosomes, microbodies, lipid droplets, vesicles differing in size and giant plastids (Fig. 2B). The plastids are bigger then mitochondria; they may contain a few internal membranous structures, numerous small tubules and starch grains. The wall surrounding the basal cell is thicker then Table 2 Comparison of plasmodesmata characters in the Sempervivum arachnoideum embryo. Wall(s)
Diameter of plasmodesmata (nm)
Electron-dense material in the plasmodesmata
Between the basal cell and the first layer of the chalazal suspensor cells Between the first and second or (third) layer of chalazal suspensor cells Longitudinal between the cells inside the first layer of chalazal suspensor Of embryo-proper cells
30–75
Present in the shape of a “cloud”
25–65
Present
25–50
Present
20–30
Absent
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Fig. 2. Structure of plasmodesmata of embryo-suspensor in Sempervivum arachnoideum L. (A–C) and Jovibarba sobolifera (Sims) Opiz (D–F); (A and D) semithin sections; (B, C, E and F) electron micrographs. (A) Section showing the large basal cell (BC) with the micropylar haustorium (MH), a few chalazal suspensor cells (CHS) and the globular embryo-proper (EP). The wall, separating the BC from the first layer of the CHS (framed) is perforated by unusual plasmodesmata (arrowheads); scale bar = 25 m. (B) Fragment of the wall (W) between the basal cell (BC) and the first layer of the chalazal suspensor cells. Note unusual plasmodesmata (rings); (P) plastid; (M) mitochondria, (WI) wall ingrowths; scale bar = 1 m. (C) Plasmodesma (PD) from (B) at a higher magnification; note an unusual electron-dense “cloud” associated with the plasmodesma; (M) mitochondrion; (W) wall; (WI) wall ingrowths; ER (black arrows); scale bar = 500 nm. (D) Section showing the large basal cell (BC), two layers of chalazal suspensor cells (CHS) and the heart stage embryo-proper (EP). The wall, separating the basal cell from the first layer of the chalazal suspensor cells (framed) is perforated by unusual plasmodesmata (arrowheads); scale bar = 50 m. (E) Part of the wall between the basal cell and the first layer of the chalazal suspensor cell (CHS). Note unusual plasmodesmata (rings); (N) nucleus; (M) mitochondria, (WI) wall ingrowths; scale bar = 2 m. (F) Compound plasmodesma (PD) from (E) at a higher magnification. (BC) basal cell; (CHS) chalazal suspensor cells; (M) mitochondria; (WI) wall ingrowths; ER (black arrows); scale bar = 500 nm.
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Fig. 3. Plasmodesmata in the chalazal suspensor cells in Sempervivum arachnoideum L. (A, C) and Jovibarba sobolifera (Sims) Opiz (B); (A) semithin section. (B and C) Electron micrographs. (A) Section showing fragment of the large basal cell (BC) and a few chalazal suspensor cells (CHS). Areas framed and numbered 1 and 2 are enlarged on B and C, respectively; scale bar = 10 m. (B) The wall (W) between the first and second layer of the chalazal suspensor cells. Plasmodesmata (ring), mitochondria (M), lipid bodies (L), dictyosomes (D), profiles of endoplasmic reticulum (ER), wall ingrowths (WI); scale bar = 1 m. (C) Longitudinal wall (W) between the cells inside the first layer of chalazal suspensor. Plasmodesmata (ring), mitochondrion (M), vacuole (V); scale bar = 500 nm.
the embryo-proper wall (Fig. 2A). There are no plasmodesmata in the outer walls of the whole embryo, but they are numerous in the inner walls of the suspensor (Fig. 3A–C) and the embryoproper (Fig. 4A–D). In ultrastructure, the chalazal suspensor cells (Fig. 3B, C) and the embryo-proper cells (Fig. 4B–D) are similar with the exception of the appearance of plasmodesmata. The wall separating the basal cell from the first layer of the chalazal suspensor cells forms the transfer wall ingrowths. Numerous mitochondria, spherical to ellipsoidal in shape, are present between the transfer wall ingrowths (Fig. 2B, C, E, F). This wall is perforated by compound plasmodesmata with unusual electron-dense material on the basal cell side, in a similar manned as was previously described in Sedum acre and S. hispanicum (Kozieradzka-Kiszkurno
and Bohdanowicz, 2010). However, there are some differences between the suspensor plasmodesmata of these two species of Sedum and those reported in this article (see Table 1 and Fig. 5A). In Sempervivum arachnoideum and Jovibarba sobolifera the wall thickness decreases in the plasmodesmata region on the basal cell side. These plasmodesmata can vary in the diameter to length relations. Their diameters are slightly extended from the side of the chalazal suspensor cells. The unusual electron-dense material has the shape of a “cloud” (Fig. 2B, C, E, F). In this “cloud” shadows can be observed (Figs. 2C, F and 5B). The shadows show continuity with the profiles of the endoplasmic reticulum. It should be highlighted that the plasmodesmata which occur between chalazal suspensor cells, also contain some electron-dense material
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Fig. 4. Plasmodesmata within the embryo-proper in Jovibarba sobolifera (Sims) Opiz (A–C) and Sempervivum arachnoideum L (D). (A) Light micrograph, (B–D) electron micrographs. (A) Semithin section showing fragment of the basal cell (BC), the chalazal suspensor cells (CHS) and the embryo-proper (EP); scale bar = 10 m. (B) Fragment of the embryo-proper cells (from area framed in A) showing the general distribution of the cellular organelles; (N) nucleus, (Nu) nucleolus, (L) lipid droplets. Note plasmodesmata (rings) in the walls (W) between the embryo-proper cells; scale bar = 2 m. (C) Plasmodesma (ring) from B at a higher magnification; scale bar = 500 nm. (D) Cross sectional views of plasmodesmata (PD) in the embryo-proper. Note the numerous microtubules (MT) close to the cell wall (W); scale bar = 200 m.
(Fig. 3B, C). However, they are smaller than the plasmodesmata described above – their diameter gradually diminishes according to the micropylar-chalazal axis of the embryo (see Table 2). Sedum acre and S. hispanicum both lack these kinds of plasmodesmata in the chalazal cells (Kozieradzka-Kiszkurno and Bohdanowicz, 2010).
Discussion Distribution of plasmodesmata among Crassulaceae From present findings it is clear that unusual compound plasmodesmata in the suspensor are a feature that is not only restricted
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arachnoideum and Jovibarba sobolifera) and the Acre clade (Sedum acre). It should be highlighted that there is no evidence supporting a speculation that specific suspensor plasmodesmata are a synapomorphic feature of the Crassulaceae family. It is not known if these plasmodesmata occur in the sister groups to Crassulaceae or in basal Crassulaceae species which may have primitive features related to the ancestors of this family. Future studies will show if specific suspensor plasmodesmata are a characteristic feature at the genus level in the Crassulaceae family. According to Niculae and Barca (2001) embryological characteristics obtained using the paraffin embedding method of Sempervivum and Jovibarba confirm the close evolutionary relationship between these two genera. Our ultrastructural data about the suspensor structure and plasmodesmata also confirm the close affinity of these genera; however, in the future more species should be examined in order to prove this. The functions of the plasmodesmata
Fig. 5. Models of plasmodesmata in the embryo-suspensor for the genus Sedum (S. acre and S. hispanicum) (A) and other genera: Sempervivum (S. arachnoideum L.) and Jovibarba (J. sobolifera (Sims) Opiz) (B). These models were created on the basis of numerous micrographs of plasmodesmata from electron microscopy. The models represent our interpretation of these micrographs. They highlight the continuity of the profiles of the endoplasmic reticulum with the shadows inside the electrondense material. (A) Unusual compound plasmodesma between the basal cell (BC) and the first layer of chalazal suspensor cells (CHS). From the basal cell (BC) side there is a dome of an electron-dense material visible (gray). In the dome, there are shadows observed (light green). (ER) endoplasmic reticulum (dark green), (W) wall. (B) Unusual compound plasmodesma between the basal cell (BC) and the first layer of chalazal suspensor cells CHS). An electron-dense material in the shape of a “cloud” (gray) associates with the plasmodesma on the basal cell side. In the “cloud” shadows (light green) can be observed. (ER) endoplasmic reticulum (dark green); (W) wall; (WI) wall ingrowths. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
to the genus Sedum but also occurs in species from other Crassulaceae genera. According to Berger (1930) Sedum belongs to the subfamily Sedoideae and Sempervivum to the subfamily Sempervivoideae. By using these classifications, we found that compound plasmodesmata occur in species from two different subfamilies in the Crassulaceae family. However, the classification based on the chloroplast gene matK sequence analysis (van Ham and ‘t Hart, 1998) and on analysis of CpDNA (Mort et al., 2001) differs from Berger’s classification. According to van Ham and ‘t Hart (1998) four of the six traditionally recognized subfamilies are considered to be polyphyletic, and, what is more, the giant genus Sedum is also polyphyletic (Mort et al., 2001). Thus, using the single most parsimonious Wagner tree of Crassulaceae of van Ham and ‘t Hart (1998) compound plasmodesmata were found to occur in species from three clades: their clade 4 (Sempervivum), clade 5 (Sedum hispanicum) and clade 7 (Sedum acre). Taking reference to another molecular work (Mort et al., 2001), compound plasmodesmata were found to occur in species from three clades: the Lecosedum clade (Sedum hispanicum), the Sempervivum clade (Sempervivum
Although plasmodesmata were discovered over a hundred years ago, to date surprisingly little is known about their structure, functions and biogenesis. This applies to both their transport of low molecular weight compounds such as carbohydrates, amino acids and others, less than about 1 kDa, and also to macromolecular compounds, e.g. proteins and nucleic acids. In light of contemporary data plasmodesmata perform not only the regulation of intercellular transport of low molecular compounds, but are also involved in the integration of physiological processes at the sub-cellular level especially those related to development and interactions with the environment (Kragler et al., 1998; Lucas et al., 1993, 2009; Robards and Lucas, 1990; van Bel and van Kesteren, 1999; Zambryski and Crawford, 2000). Plasmodesmata may be functional for transferring molecules which co-ordinate cellular processes during cell differentiation, for instance phytohormones such as gibberellins (Kwiatkowska, 1991; Lucas et al., 1993). The plasmodesmata connecting the suspensor cells and the embryo cells may serve to maintain an open channel of communication for the massive flow of different solutes between the suspensor and the embryo-proper. The unusual electron-dense dome or “cloud” associated with compound plasmodesmata in the embryo-suspensor in Crassulaceae might control this transfer of nutrients. However, in order to test this idea more experiments are needed. Nowhere in the literature were plasmodesmata found in suspensors like those which were recorded in Crassusulaceae. Therefore, in this paper we present two models of plasmodesmata in the embryo-suspensor for the selected representatives of the Crassulaceae (Sedum, Sempervivum and Jobolifera, see Fig. 5). The models highlight the continuity of the endoplasmic reticulum profiles with the shadows inside the electron-dense material. In Crassulaceae, the basal cell of the suspensor forms special haustorial protrusions, penetrating the ovular tissues, and is apparently the main source of nutritive substances for the embryo-proper. The micropylar part of the suspensor basal cell and the micropylar haustorium in Sedum are covered with wall ingrowths typical of transfer cells (Kozieradzka-Kiszkurno, 2003). It is assumed that the nutrients penetrate through the wall ingrowths, become transferred into the basal cell and then are transported along the suspensor to the embryo-proper. Transfer cells are specialized for the short-distance, active transport of solution across the plasmalemma, whose surface area is considerably extended by the formation of wall ingrowths (Gunning and Pate, 1969). Such wall ingrowths also occur in the suspensor cells of, e.g.: Phaseolus (Clutter and Sussex, 1968), Stellaria (Newcomb and Fowke, 1974), Tropaeolum (Nagl, 1976), Sedum acre and S. hispanicum (Kozieradzka-Kiszkurno and Bohdanowicz, 2006, 2010). We found that in Sempervivum arachnoideum the size of the plasmodesmata
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diameter varies according to the micropylar-chalazal axis of the embryo. No data are available about the size of plasmodesmata in different parts of an embryo in other angiosperm species; this makes it difficult to discuss the potential function or the role of this gradient in the diameter of plasmodesmata. In addition, it is too early to relate these plasmodesmata with specific features of Crassulacean adaptations to drought (e.g. the succulence) or the acidic metabolism of the Crassulaceae (CAM). In the Sedum species examined only simple plasmodesmata occurred in the two-celled proembryo (Kozieradzka-Kiszkurno and Bohdanowicz, 2010). Thus the compound plasmodesmata, which occur during later stages of suspensor development, appear to be modified primary plasmodesmata. Conclusion The compound plasmodesmata of Crassulaceae may perform the role of special channels for symplasmic transport. However, additional studies – experiments with fluorochromes – should be performed in order to test this hypothesis. Further studies will be needed to show whether compound plasmodesmata are a characteristic feature at genus level in the Crassulaceae family (we studied only single species from different genera), or whether they occur in other Crassulaceae clades and whether they have arisen multiple times or only once in this family. It is worth to test that eventually these plasmodesmata are an evolutionary adaptation facilitating and/or controlling the flow of the nutritional substances, that was specifically developed in Crassulaceae. Perhaps these unusual plasmodesmata do appear in a larger group of related families of Crassulaceae, but this has not yet been explored. Acknowledgements We cordially thank the director of the Botanic Garden of the Jagiellonian University Prof. Bogdan Zemanek, and the Supervisor of Botanic Garden (Chief Gardener), Maria Uzarowicz, for permission to use plants from the Garden collections. BJP gratefully acknowledges the support of an award from the Foundation for Polish Sciences (Start Programme).
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Are unusual plasmodesmata in the embryo-suspensor restricted to species from the genus Sedum among Crassulaceae? Małgorzata Kozieradzka-Kiszkurno a,∗ , Bartosz Jan Płachno b , Jerzy Bohdanowicz a a b
Department of Plant Cytology and Embryology, University of Gda´ nsk, Kładki 24 St., 80-822 Gda´ nsk, Poland Department of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52 St., 31-044 Cracow, Poland
a r t i c l e
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Article history: Received 13 August 2010 Accepted 25 November 2010 Keywords: Crassulaceae Jovibarba Plasmodesmata Sempervivum Suspensor Ultrastructure
a b s t r a c t The Crassulaceae family comprises mainly herbaceous leaf succulents, some of which have an ornamental value. During embryogenesis, they produce a suspensor with a giant polyploid basal cell. It has recently been shown that in Sedum acre and S. hispanicum this cell has compound plasmodesmata with an unusual dome of electron-dense material associated on the cell’s side. These compound plasmodesmata differ from the typical ones occurring in other angiosperms. In this study, the hypothesis was tested that the unusual plasmodesmata in the embryo-suspensor are a feature not only restricted to species from the genus Sedum, but are also found in other Crassulaceae genera. Suspensors of example species from the genera Sempervivum and Jovibarba, which have vegetative morphologies quite different from Sedum and which are placed in the traditional classification into another subfamily, were first examined using an electron microscope. It was found that the unusual compound plasmodesmata in the suspensor are not only restricted to species from the genus Sedum but are also found in species from other Crassulaceae genera (Sempervivum arachnoideum and Jovibarba sobolifera). It should be noted that some ultrastructural features of compound plasmodesmata in the analyzed genera (e.g. the character of the wall with plasmodesmata, plasmodesmata diameter or occurrence of the electron-dense material) are different from the suspensor plasmodesmata recorded in species from the Sedum genus. We found that in Sempervivum arachnoideum the size of the plasmodesmata diameter varies according to the micropylar-chalazal axis of the embryo. This is the first report of variation in the diameter of the plasmodesmata within the embryo of angiosperms. Further study will be needed to show whether compound plasmodesmata occur in other Crassulaceae clades, whether they are a stable feature at the genus level in this family, and also whether they have evolved several times or only once in Crassulaceae. © 2011 Elsevier GmbH. All rights reserved.
Introduction The Crassulaceae family, with a world-wide distribution and comprising 900–1500 species, has evolved mainly herbaceous leaf succulents (Berger, 1930; Eggli, 2005; ‘t Hart and Bleij, 2003; van Ham and ‘t Hart, 1998). Molecular studies have shown that Crassulaceae are part of the Saxifragales clade (e.g. Chase et al., 1993; Morgan and Soltis, 1993) and this family most likely has a southern African origin (Mort et al., 2001). Recently several papers have dealt with the phylogeny of Crassulaceae (e.g. Mort et al., 2001; van Ham and ‘t Hart, 1998). The family is monophyletic; however, six subfamilies are traditionally recognized as being polyphyletic (van Ham and ‘t Hart, 1998) and also the large genus Sedum is highly polyphyletic with representatives spread throughout the Sedoideae clade (Mort et al., 2001). It is worth mentioning that the
∗ Corresponding author. E-mail address: [email protected] (M. Kozieradzka-Kiszkurno). 0367-2530/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2010.11.017
members of Crassulaceae have ornamental value and some are also used in medicine (Eggli, 2005). Since the beginning of the 20th century, the Crassulaceae family has attracted the interest of researchers because of its interesting embryogenesis – mainly with respect to its large polyploid suspensors with haustoria (Kozieradzka-Kiszkurno and Bohdanowicz, 2003, 2006, 2010; Kozieradzka-Kiszkurno et al., 2010; Mauritzon, 1933; Niculae and Barca, 2001; Souéges, 1927, 1936). For example in Sedum acre, the nucleus of the suspensor basal cell increases its nuclear DNA content and attains a maximum level as high as 1024C (Kozieradzka-Kiszkurno and Bohdanowicz, 2003); therefore it could be a good model for studies on polyploid cells. Recently Kozieradzka-Kiszkurno and Bohdanowicz (2010) described unusual, compound plasmodesmata which connect the giant suspensor basal cell to the first layer of the chalazal suspensor cells. These compound plasmodesmata are wider than typical plasmodesmata in angiosperms and are covered by a dome of an electron-dense material, which might be a kind of filter, which selects the flow of nutrients via suspensor’s cells.
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Table 1 Comparison of suspensor’s plasmodesmata between Sedum acre, S. hispanicum and two species from other genera. Sempervivum arachnoideum and Jovibarba sobolifera are treated together because we did not find any major differences between these genera. Characters
Sedum acre and S. hispanicum
Sempervivum arachnoideum and Jovibarba sobolifera
Occurrence
Wall between the basal cell and the first layer of the chalazal suspensor cells Without ingrowths
Wall between the basal cell and the first layer of the chalazal suspensor cells With ingrowths – transfer wall type Wall thickness decreases in the plasmodesmata region Compound
Type of the wall with plasmodesmata Thickness of the wall
Type of plasmodesmata Plasmodesmata diameter (nm) Character of an electron-dense material Behavior of ER
Fig. 1. Plants of Sempervivum arachnoideum L. (A) and Jovibarba sobolifera (Sims) Opiz (B) in natural habitats.
Embryological characteristics could be very useful in taxonom´ atek, ˛ ical and phylogenetical investigations (e.g. Płachno and Swi 2008, 2009, 2010). In this study, we test the hypothesis that the unusual compound plasmodesmata in the suspensor are not only restricted to species from the genus Sedum but also for species from other Crassulaceae genera. Suspensors from species of the genera Sempervivum and Jovibarba, which are readily available to us, were examined first because they are elements of Polish flora and their vegetative morphologies are quite different from those of Sedum. Materials and methods Flowering plants of Sempervivum arachnoideum L. (Fig. 1A) were provided by a commercial business company (Andrzej and Łucja Hinz, http://kaktusiarnia.pl/index1.html). Flowering plants of Jovibarba sobolifera (Sims) Opiz (Fig. 1B) were collected from a natural population in Kokotek near Lubliniec in the south of Poland. Some additional plants were also obtained from The Botanic Garden of the Jagiellonian University, Cracow, Poland. The ovules at different stages of embryo development were fixed in 2.5% formaldehyde and 2.5% glutaraldehyde in 0.05 M cacodylate buffer (pH = 7.0) for 4 h at room temperature. The material was post-fixed in 1% osmium tetroxide in cacodylate buffer at 4 ◦ C overnight, rinsed in the same buffer with four changes, treated with 1% uranyl acetate in distilled water for 1 h, dehydrated with acetone and embedded in Spurr’s resin (Spurr, 1969). Serial ultrathin (60–100 nm) sections were cut with a diamond knife on a SORVALL MT 2B microtome, and then post-stained with saturated solution of uranyl acetate in 50% ethanol for 20 min and with 0.04% lead citrate for 2 min. Observations were made using a Philips CM 100 transmission electron microscope. For light microscopy, semithin sections (0.5–2 m) were stained with 0.05% toluidine blue 0 in 1% sodium tetraborate. To prepare the data for Tables 1 and 2, at least twenty plasmodesmata for each of the studied species, were mea-
Wall has uniform thickness Very compound – branched 30–90
30–75
More electron transparent
More electron dense
Profiles of ER adjacent to the electron-dense material
Profiles of ER adjacent to the electron-dense material
sured. Additionally, measurements of the plasmodesmata diameter in the Sedum acre suspensor were performed using material described by Kozieradzka-Kiszkurno and Bohdanowicz (2010). Results Both Sempervivum arachnoideum and Jovibarba sobolifera undergo the Caryophyllad type of embryonic development, and because we did not find any major differences between these genera we treated them together. A suspensor consists of the giant basal cell, which is pyriform in shape, and a few chalazal cells, which are connected with the embryo-proper (Fig. 2A, D). There is a difference in the density of the cytoplasm of the basal and chalazal cells of the suspensor (Fig. 2B, C, E, F). The cytoplasm of the basal cell is more transparent but is rich with mitochondria, profiles of endoplasmic reticulum, dictyosomes, microbodies, lipid droplets, vesicles differing in size and giant plastids (Fig. 2B). The plastids are bigger then mitochondria; they may contain a few internal membranous structures, numerous small tubules and starch grains. The wall surrounding the basal cell is thicker then Table 2 Comparison of plasmodesmata characters in the Sempervivum arachnoideum embryo. Wall(s)
Diameter of plasmodesmata (nm)
Electron-dense material in the plasmodesmata
Between the basal cell and the first layer of the chalazal suspensor cells Between the first and second or (third) layer of chalazal suspensor cells Longitudinal between the cells inside the first layer of chalazal suspensor Of embryo-proper cells
30–75
Present in the shape of a “cloud”
25–65
Present
25–50
Present
20–30
Absent
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Fig. 2. Structure of plasmodesmata of embryo-suspensor in Sempervivum arachnoideum L. (A–C) and Jovibarba sobolifera (Sims) Opiz (D–F); (A and D) semithin sections; (B, C, E and F) electron micrographs. (A) Section showing the large basal cell (BC) with the micropylar haustorium (MH), a few chalazal suspensor cells (CHS) and the globular embryo-proper (EP). The wall, separating the BC from the first layer of the CHS (framed) is perforated by unusual plasmodesmata (arrowheads); scale bar = 25 m. (B) Fragment of the wall (W) between the basal cell (BC) and the first layer of the chalazal suspensor cells. Note unusual plasmodesmata (rings); (P) plastid; (M) mitochondria, (WI) wall ingrowths; scale bar = 1 m. (C) Plasmodesma (PD) from (B) at a higher magnification; note an unusual electron-dense “cloud” associated with the plasmodesma; (M) mitochondrion; (W) wall; (WI) wall ingrowths; ER (black arrows); scale bar = 500 nm. (D) Section showing the large basal cell (BC), two layers of chalazal suspensor cells (CHS) and the heart stage embryo-proper (EP). The wall, separating the basal cell from the first layer of the chalazal suspensor cells (framed) is perforated by unusual plasmodesmata (arrowheads); scale bar = 50 m. (E) Part of the wall between the basal cell and the first layer of the chalazal suspensor cell (CHS). Note unusual plasmodesmata (rings); (N) nucleus; (M) mitochondria, (WI) wall ingrowths; scale bar = 2 m. (F) Compound plasmodesma (PD) from (E) at a higher magnification. (BC) basal cell; (CHS) chalazal suspensor cells; (M) mitochondria; (WI) wall ingrowths; ER (black arrows); scale bar = 500 nm.
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Fig. 3. Plasmodesmata in the chalazal suspensor cells in Sempervivum arachnoideum L. (A, C) and Jovibarba sobolifera (Sims) Opiz (B); (A) semithin section. (B and C) Electron micrographs. (A) Section showing fragment of the large basal cell (BC) and a few chalazal suspensor cells (CHS). Areas framed and numbered 1 and 2 are enlarged on B and C, respectively; scale bar = 10 m. (B) The wall (W) between the first and second layer of the chalazal suspensor cells. Plasmodesmata (ring), mitochondria (M), lipid bodies (L), dictyosomes (D), profiles of endoplasmic reticulum (ER), wall ingrowths (WI); scale bar = 1 m. (C) Longitudinal wall (W) between the cells inside the first layer of chalazal suspensor. Plasmodesmata (ring), mitochondrion (M), vacuole (V); scale bar = 500 nm.
the embryo-proper wall (Fig. 2A). There are no plasmodesmata in the outer walls of the whole embryo, but they are numerous in the inner walls of the suspensor (Fig. 3A–C) and the embryoproper (Fig. 4A–D). In ultrastructure, the chalazal suspensor cells (Fig. 3B, C) and the embryo-proper cells (Fig. 4B–D) are similar with the exception of the appearance of plasmodesmata. The wall separating the basal cell from the first layer of the chalazal suspensor cells forms the transfer wall ingrowths. Numerous mitochondria, spherical to ellipsoidal in shape, are present between the transfer wall ingrowths (Fig. 2B, C, E, F). This wall is perforated by compound plasmodesmata with unusual electron-dense material on the basal cell side, in a similar manned as was previously described in Sedum acre and S. hispanicum (Kozieradzka-Kiszkurno
and Bohdanowicz, 2010). However, there are some differences between the suspensor plasmodesmata of these two species of Sedum and those reported in this article (see Table 1 and Fig. 5A). In Sempervivum arachnoideum and Jovibarba sobolifera the wall thickness decreases in the plasmodesmata region on the basal cell side. These plasmodesmata can vary in the diameter to length relations. Their diameters are slightly extended from the side of the chalazal suspensor cells. The unusual electron-dense material has the shape of a “cloud” (Fig. 2B, C, E, F). In this “cloud” shadows can be observed (Figs. 2C, F and 5B). The shadows show continuity with the profiles of the endoplasmic reticulum. It should be highlighted that the plasmodesmata which occur between chalazal suspensor cells, also contain some electron-dense material
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Fig. 4. Plasmodesmata within the embryo-proper in Jovibarba sobolifera (Sims) Opiz (A–C) and Sempervivum arachnoideum L (D). (A) Light micrograph, (B–D) electron micrographs. (A) Semithin section showing fragment of the basal cell (BC), the chalazal suspensor cells (CHS) and the embryo-proper (EP); scale bar = 10 m. (B) Fragment of the embryo-proper cells (from area framed in A) showing the general distribution of the cellular organelles; (N) nucleus, (Nu) nucleolus, (L) lipid droplets. Note plasmodesmata (rings) in the walls (W) between the embryo-proper cells; scale bar = 2 m. (C) Plasmodesma (ring) from B at a higher magnification; scale bar = 500 nm. (D) Cross sectional views of plasmodesmata (PD) in the embryo-proper. Note the numerous microtubules (MT) close to the cell wall (W); scale bar = 200 m.
(Fig. 3B, C). However, they are smaller than the plasmodesmata described above – their diameter gradually diminishes according to the micropylar-chalazal axis of the embryo (see Table 2). Sedum acre and S. hispanicum both lack these kinds of plasmodesmata in the chalazal cells (Kozieradzka-Kiszkurno and Bohdanowicz, 2010).
Discussion Distribution of plasmodesmata among Crassulaceae From present findings it is clear that unusual compound plasmodesmata in the suspensor are a feature that is not only restricted
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arachnoideum and Jovibarba sobolifera) and the Acre clade (Sedum acre). It should be highlighted that there is no evidence supporting a speculation that specific suspensor plasmodesmata are a synapomorphic feature of the Crassulaceae family. It is not known if these plasmodesmata occur in the sister groups to Crassulaceae or in basal Crassulaceae species which may have primitive features related to the ancestors of this family. Future studies will show if specific suspensor plasmodesmata are a characteristic feature at the genus level in the Crassulaceae family. According to Niculae and Barca (2001) embryological characteristics obtained using the paraffin embedding method of Sempervivum and Jovibarba confirm the close evolutionary relationship between these two genera. Our ultrastructural data about the suspensor structure and plasmodesmata also confirm the close affinity of these genera; however, in the future more species should be examined in order to prove this. The functions of the plasmodesmata
Fig. 5. Models of plasmodesmata in the embryo-suspensor for the genus Sedum (S. acre and S. hispanicum) (A) and other genera: Sempervivum (S. arachnoideum L.) and Jovibarba (J. sobolifera (Sims) Opiz) (B). These models were created on the basis of numerous micrographs of plasmodesmata from electron microscopy. The models represent our interpretation of these micrographs. They highlight the continuity of the profiles of the endoplasmic reticulum with the shadows inside the electrondense material. (A) Unusual compound plasmodesma between the basal cell (BC) and the first layer of chalazal suspensor cells (CHS). From the basal cell (BC) side there is a dome of an electron-dense material visible (gray). In the dome, there are shadows observed (light green). (ER) endoplasmic reticulum (dark green), (W) wall. (B) Unusual compound plasmodesma between the basal cell (BC) and the first layer of chalazal suspensor cells CHS). An electron-dense material in the shape of a “cloud” (gray) associates with the plasmodesma on the basal cell side. In the “cloud” shadows (light green) can be observed. (ER) endoplasmic reticulum (dark green); (W) wall; (WI) wall ingrowths. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
to the genus Sedum but also occurs in species from other Crassulaceae genera. According to Berger (1930) Sedum belongs to the subfamily Sedoideae and Sempervivum to the subfamily Sempervivoideae. By using these classifications, we found that compound plasmodesmata occur in species from two different subfamilies in the Crassulaceae family. However, the classification based on the chloroplast gene matK sequence analysis (van Ham and ‘t Hart, 1998) and on analysis of CpDNA (Mort et al., 2001) differs from Berger’s classification. According to van Ham and ‘t Hart (1998) four of the six traditionally recognized subfamilies are considered to be polyphyletic, and, what is more, the giant genus Sedum is also polyphyletic (Mort et al., 2001). Thus, using the single most parsimonious Wagner tree of Crassulaceae of van Ham and ‘t Hart (1998) compound plasmodesmata were found to occur in species from three clades: their clade 4 (Sempervivum), clade 5 (Sedum hispanicum) and clade 7 (Sedum acre). Taking reference to another molecular work (Mort et al., 2001), compound plasmodesmata were found to occur in species from three clades: the Lecosedum clade (Sedum hispanicum), the Sempervivum clade (Sempervivum
Although plasmodesmata were discovered over a hundred years ago, to date surprisingly little is known about their structure, functions and biogenesis. This applies to both their transport of low molecular weight compounds such as carbohydrates, amino acids and others, less than about 1 kDa, and also to macromolecular compounds, e.g. proteins and nucleic acids. In light of contemporary data plasmodesmata perform not only the regulation of intercellular transport of low molecular compounds, but are also involved in the integration of physiological processes at the sub-cellular level especially those related to development and interactions with the environment (Kragler et al., 1998; Lucas et al., 1993, 2009; Robards and Lucas, 1990; van Bel and van Kesteren, 1999; Zambryski and Crawford, 2000). Plasmodesmata may be functional for transferring molecules which co-ordinate cellular processes during cell differentiation, for instance phytohormones such as gibberellins (Kwiatkowska, 1991; Lucas et al., 1993). The plasmodesmata connecting the suspensor cells and the embryo cells may serve to maintain an open channel of communication for the massive flow of different solutes between the suspensor and the embryo-proper. The unusual electron-dense dome or “cloud” associated with compound plasmodesmata in the embryo-suspensor in Crassulaceae might control this transfer of nutrients. However, in order to test this idea more experiments are needed. Nowhere in the literature were plasmodesmata found in suspensors like those which were recorded in Crassusulaceae. Therefore, in this paper we present two models of plasmodesmata in the embryo-suspensor for the selected representatives of the Crassulaceae (Sedum, Sempervivum and Jobolifera, see Fig. 5). The models highlight the continuity of the endoplasmic reticulum profiles with the shadows inside the electron-dense material. In Crassulaceae, the basal cell of the suspensor forms special haustorial protrusions, penetrating the ovular tissues, and is apparently the main source of nutritive substances for the embryo-proper. The micropylar part of the suspensor basal cell and the micropylar haustorium in Sedum are covered with wall ingrowths typical of transfer cells (Kozieradzka-Kiszkurno, 2003). It is assumed that the nutrients penetrate through the wall ingrowths, become transferred into the basal cell and then are transported along the suspensor to the embryo-proper. Transfer cells are specialized for the short-distance, active transport of solution across the plasmalemma, whose surface area is considerably extended by the formation of wall ingrowths (Gunning and Pate, 1969). Such wall ingrowths also occur in the suspensor cells of, e.g.: Phaseolus (Clutter and Sussex, 1968), Stellaria (Newcomb and Fowke, 1974), Tropaeolum (Nagl, 1976), Sedum acre and S. hispanicum (Kozieradzka-Kiszkurno and Bohdanowicz, 2006, 2010). We found that in Sempervivum arachnoideum the size of the plasmodesmata
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diameter varies according to the micropylar-chalazal axis of the embryo. No data are available about the size of plasmodesmata in different parts of an embryo in other angiosperm species; this makes it difficult to discuss the potential function or the role of this gradient in the diameter of plasmodesmata. In addition, it is too early to relate these plasmodesmata with specific features of Crassulacean adaptations to drought (e.g. the succulence) or the acidic metabolism of the Crassulaceae (CAM). In the Sedum species examined only simple plasmodesmata occurred in the two-celled proembryo (Kozieradzka-Kiszkurno and Bohdanowicz, 2010). Thus the compound plasmodesmata, which occur during later stages of suspensor development, appear to be modified primary plasmodesmata. Conclusion The compound plasmodesmata of Crassulaceae may perform the role of special channels for symplasmic transport. However, additional studies – experiments with fluorochromes – should be performed in order to test this hypothesis. Further studies will be needed to show whether compound plasmodesmata are a characteristic feature at genus level in the Crassulaceae family (we studied only single species from different genera), or whether they occur in other Crassulaceae clades and whether they have arisen multiple times or only once in this family. It is worth to test that eventually these plasmodesmata are an evolutionary adaptation facilitating and/or controlling the flow of the nutritional substances, that was specifically developed in Crassulaceae. Perhaps these unusual plasmodesmata do appear in a larger group of related families of Crassulaceae, but this has not yet been explored. Acknowledgements We cordially thank the director of the Botanic Garden of the Jagiellonian University Prof. Bogdan Zemanek, and the Supervisor of Botanic Garden (Chief Gardener), Maria Uzarowicz, for permission to use plants from the Garden collections. BJP gratefully acknowledges the support of an award from the Foundation for Polish Sciences (Start Programme).
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