Integument cell gelatinisation—the fate of the integumentary cells in Hieracium and Pilosella (Asteraceae)
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Members of the genera Hieracium and Pilosella are model plants that are used to study the mechanisms of apomixis. In order to have a proper understanding of apomixis, knowledge about the relationship between the maternal tissue and the gametophyte is needed. In the genus Pilosella, previous authors have described the specific process of the “liquefaction” of the integument cells that surround the embryo sac. However, these observations were based on data only at the light microscopy level. The main aim of our paper was to investigate the changes in the integument cells at the ultrastructural level in Pilosella officinarum and Hieracium alpinum. We found that the integument peri-endothelial zone in both species consisted of mucilage cells. The mucilage was deposited as a thick layer between the plasma membrane and the cell wall. The mucilage pushed the protoplast to the centre of the cell, and cytoplasmic bridges connected the protoplast to the plasmodesmata through the mucilage layers. Moreover, an elongation of the plasmodesmata was observed in the mucilage cells. The protoplasts had an irregular shape and were finally degenerated. After the cell wall breakdown of the mucilage cells, lysigenous cavities that were filled with mucilage were formed.
KeywordsApomixis Asteraceae Integument Lysigenous cavities Mucilage cells Ovule Plasmodesmata Ultrastructure Idioblasts
Members of the genera Hieracium L. and Pilosella Vaill. are important model plants for understanding the mechanisms of apomixis in angiosperms (e.g. Koltunow et al. 2011a,b; Tucker et al. 2012; Okada et al. 2013; Hand and Koltunow 2014; Hand et al. 2015; Shirasawa et al. 2015; Rabiger et al. 2016; Rotreklová and Krahulcová 2016).
According to Koltunow et al. (1998), the development of the embryo and endosperm in Hieracium aurantiacum L. [= Pilosella aurantiaca (L.) F. W. Schultz & Sch. Bip.], H. pilosella L. [= P. officinarum Vaill.] and P. piloselloides (Vill.) Soják [= H. piloselloides Vill.] coincide with the intensive “liquefaction” of the integument cells that surround the embryo sac. This process was observed in both sexual and apomictic plants and relied on changes in integument cell wall followed by integument cell liquefaction near the endothelium and finally the accumulation of carbohydrate-rich material. According to Koltunow et al. (1998), this material may serve a nutritive role and moreover, they suggested that the accumulation of a large pool of nutrients around the embryo sac might have helped the evolution of the apomictic trait within the genus. This suggestion about a nutritional function of these specific integumentary cells was accepted and repeated by other authors, e.g. Van Baarlen et al. (1999) wrote that the ovules of Hieracium and Taraxacum contain a protein-rich storage tissue, which nourishes the embryo and reduces the importance of the endosperm function. It was also suggested by these authors that the presence of this tissue might explain the evolution of autonomous embryo development in most of the Asteraceae apomicts. A similar suggestion was repeated in the case of Taraxacum and Chondrilla by Musiał et al. (2013) and later by Musiał and Kościńska-Pająk (2013). These integumentary cells were called integumentary “nutritive tissue” and its presence and ultrastructure in different members of Asteraceae was discussed by Kolczyk et al. (2014). Although data about the ultrastructure of the integument in both Hieracium and Pilosella are still lacking, progress has been made in the case of another apomictic genus, Taraxacum, which belongs to the same subfamily. Płachno et al. (2016) showed that the “nutritive tissue” (= peri-endothelial tissue) in the Taraxacum ovule consists of specialised mucilage cells. During the differentiation of these cells and the deposition of mucilage, the plasmodesmata become elongated and are associated with structures called “cytoplasmic bridges.”
It is well known that the plasmodesmata are plant cell communication channels that are crucial for controlling the intercellular transport of macromolecules such as mRNA, signals including proteins and transcriptional factors (e.g. Oparka 2004; Gursanscky et al. 2011; Hyun et al. 2011). Symplasmic isolation/communication between the ovular sporophytic tissues and the megagametophyte and later the embryo is necessary for successful development (e.g. Ingram 2010; Bencivenga et al. 2011; Marzec and Kurczynska 2008, 2014; Wróbel-Marek et al. 2017 and literature therein). Sporophytic ovule tissues also have an influence on apomixis, e.g. Tucker et al. (2012) showed that in Pilosella ovules, sporophytic information is potentiated by the growth of the funiculus and also that polar auxin transport influences ovule development, the initiation of apomixis and the progression of embryo sac. According to Okada et al. (2013), signalling molecules such as the kinases from the sporophytic ovule cells have an influence on the aposporous embryo sac formation in the apomictic Hieracium species. Thus, in order to properly understand the symplasmic isolation/communication in Pilosella and Hieracium ovules, basic knowledge about the ultrastructure of the sporophyte tissues is needed.
It should be stressed that the selection of our research material is not accidental. There are amphimictic diploids and also apomictic polyploids (mitotic diplospory) among the genus Hieracium. However, in the genus Pilosella, both amphimictic taxa (diploids, sometimes tetra- and hexaploids) and poliploidal facultative apomicts (apospory) are known. Thus, we would like to compare if any differences occur in the integument structure of these genera.
The main aim of our paper was to investigate the changes in the integument cells that surround the embryo sac in Hieracium and Pilosella.
Another question is what happens to the plasmodesmata in these cells. Are the plasmodesmata in the ovule integumentary cells of Hieracium and Pilosella associated with the cytoplasmic bridges (the thin strands of cytoplasm) like in Taraxacum ovules?
We also wanted to investigate whether there is an accumulation of a large pool of nutrients (protein and lipid storage) in the peri-endothelial integument cells that surround the embryo sac.
Material and methods
Light and electron microscopy studies
The preparation of the samples for TEM followed the procedure used by Płachno and Świątek (2011) and Kozieradzka-Kiszkurno and Płachno (2012). Semithin sections were stained using aqueous methylene blue with azure II for general histology (Humphrey and Pittman, 1974) for 1–2 min (MB/AII) and examined using an Olympus BX60 microscope. The cytochemical tests included Aniline Blue Black (Jensen, 1962) for proteins and Sudan Black B for lipids (Bronner, 1975). The periodic acid-Schiff (PAS) reaction was used to visualise the total carbohydrates of insoluble polysaccharides (Wędzony 1996).
Ultrathin sections were cut on a Leica Ultracut UCT ultramicrotome. After contrasting with uranyl acetate and lead citrate, the sections were examined using a Hitachi H500 electron microscope at 75 kV in the Faculty of Biology and Environmental Protection, University of Silesia in Katowice and a Jeol JEM 100 SX; JEOL, Tokyo, Japan, at 80 kV in the Department of Cell Biology and Imaging, Institute of Zoology, Jagiellonian University in Kraków.
A breakdown of the cell wall between the adjacent mucilage cells occurred (Fig. 7b), after which lysigenous cavities that were filled with mucilage were formed (Figs. 1b, c and 2b). The protoplast of mucilage cells was finally degraded (Fig. 7c, d).
The cytochemical tests for the storage lipids and protein gave negative results in the case of the mucilage cells (not shown). However, we observed small lipid droplets in the cytoplasm of the mucilage cells using TEM (Supplementary material 1). There was positive staining after periodic-acid-Schiff reaction. The total carbohydrates of insoluble polysaccharides (including mucilage carbohydrates) stain pink to purplish red (Supplementary material 2).
We showed that the intensive “liquefaction” of the integument cells surrounding the embryo sac, which was previously described by Koltunow et al. (1998), was in the fact gelatinisation: an accumulation of the mucilage in the cells and later the formation of lysigenous cavities that were filled with mucilage. Koltunow et al. (1998) wrote that the material that was accumulated during the intensive changes of the integument cells surrounding the embryo sac was carbohydrate-rich (positive staining after periodic-acid-Schiff reaction). This also agreed with our observation that this material is mucilage. The ultrastructure of the mucilage in Hieracium and Pilosella is similar to the previously observed mucilage in the integument mucilage cells of other Asteraceae genera—Taraxacum, Onopordum, Solidago, Chondrilla and Bellis (Płachno et al. 2016; Kolczyk et al. 2014, 2016 and literature therein). However, our results disagree with Van Baarlen et al. (1999) who wrote that the ovules of Hieracium contain a protein-rich storage tissue, because we did not find protein bodies or protein storage vacuoles in the periendothelial tissue. However, during the gelatinisation of the periendothelial cells and the degeneration of their protoplasts, some nutrients might be released and transported to the female gametophyte. But, such a hypothesis requires experimental confirmation. Moreover, the mucilage in the ovules and seeds may have a different function and be storage for water like the mucilage in cacti cells (Nobel et al. 1992).
Although the peri-endothelial cell ultrastructure and mucilage deposition that were results obtained in this study resemble those in Taraxacum (Płachno et al. 2016), there are some differences. In both Hieracium and Pilosella, the formation of lysigenous cavities occurs at the mature female gametophyte stage, while in Taraxacum, the peri-endothelial cells still retain individuality at the mature female gametophyte stage (see Fig. 1a in Płachno et al. 2016) and the formation of these cavities occurs later, during embryogenesis (see Fig. 1c, din Gawecki et al. 2017). Cooper and Brink (1949) also described the disintegration of the peri-endothelial cells in Taraxacum during embryogenesis.
The breakdown of the cell wall between the mucilage cells that was observed here has also been described in other plants that are not related to the Asteraceae, e.g. Hibiscus schizopetalus (Malvaceae) (Bakker and Gerritsen 1992) and Cinnamomum (Lauraceae) (Bakker et al. 1991). However, in H. schizopetalus, the local breakdown of the cell wall between the mucilage and neighbouring non-mucilage cells has also been observed many times (Bakker and Gerritsen 1992). In Cinnamomum mucilage cells, the local breakdown of the cell wall is not common due to the occurrence of a suberised cell wall layer (Bakker et al. 1991). A suberised wall layer does not occur in the mucilage cells of Hibiscus (Bakker and Gerritsen 1992), Taraxacum (Płachno et al. 2016) or in Hieracium and Pilosella.
Plasmodesmata in mucilage idioblasts
Unfortunately, there are only a few studies about the plasmodesmata in mucilage idioblasts. In the mucilage cells of C. verum and Annona muricata (Annonaceae), the plasmodesmata show a bulge on the idioblast side of the cell wall. These plasmodesmata become occluded by the mucilage, and according to Bakker and Baas (1993), symplasmic transport is presumably blocked. However, in the mucilage cells of C. burmanni, Bakker et al. (1991) noted that in a few cases, connections between the plasmodesma and the cytoplasmic strand that was embedded in the mucilage occurred. Our observation that the plasmodesmata in the mucilage cells are linked to the protoplast via cytoplasmic bridges in Hieracium and Pilosella are in agreement with a similar situation that was observed in Taraxacum mucilage cells (Płachno et al. 2016). Like in Taraxacum, our ultrastructural documentation here indicates that there was an elongation of the primary plasmodesmata that correlated with an increase in the thickness of the mucilage. The concept that the plasmodesmata may undergo elongation is not new and was proposed by Ehlers and Kollmann (1996). Glockmann and Kollmann (1996) also documented an elongation of the primary plasmodesmata that correlated with an increase in the thickness of the wall in the Strasburger cells of the needles of Metasequoia.
In order to maintain the intercellular communication between integument cells in Hieracium and Pilosella during mucilage deposition, the primary plasmodesmata have to be elongated. We propose here a model of elongation of primary plasmodesmata in the mucilage idioblasts: a part of cytoplasmic strand which is embedded in mucilage and has contact with the primary plasmodesma become increasingly constricted and develop into plasmodesmal strand. The enclosed ER cisternae inside this strand, which is connected with desmotubule, is transformed into the plasmodesmal desmotubule. This process is similar to elongation of primary plasmodesmata during the thickening growth of the cell walls proposed by Ehlers and Kollmann (1996, 2001); however, with one major difference, that in the mucilage idioblasts, cytoplasmic strands enclosing ER cisternae are in mucilage.
We showed that the liquefaction process of the integument cells surrounding the embryo sac in Hieracium and Pilosella was in the fact of gelatinisation: an accumulation of mucilage in the cells and later the formation of lysigenous cavities that are filled with mucilage.
The plasmodesmata in the mucilage cells are linked to the protoplast via cytoplasmic bridges, which suggests that they are functional. Our observation may indicate that there is an elongation of the primary plasmodesmata that is correlated with the mucilage deposition.
Because the mucilage cells of Hieracium and Pilosella lack storage proteins, the mucilage may perform the role of water storage or may be a source of carbohydrates for the gametophyte and embryo.
This study was funded by the National Science Centre, Poland. Contract grant number: DEC-2013/09/B/NZ8/03308. We dedicated our work to memory of Prof. Zygmunt Hejnowicz (1929–2016).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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