Are extracellular matrix surface network components involved in signalling and protective function?

Abstract

Endosperm is an interesting model for in vitro experiments, because of its unique origin, development and ploidy level. Here we used Actinidia deliciosa endosperm-derived callus to investigate morphology, histology and chemistry of extracellular matrix (ECM) structures in morphogenically stable tissue from long - term culture. SEM and TEM analysis showed that ECM is a heterogenous layer which consists of amorphous, dark – staining material, osmiophilic granules and reticulated fibres outside the outer callus cell wall. This structure may serve as a structural marker of morphogenic competence in endosperm – derived callus, because of its presence on the surface of callus forming morphogenic domains and its disappearance during organ growth. Based on immunolabelling, histochemistry, solvent and enzyme treatments, we suggest that pectins and lipids are components of the ECM layer. These results might indicate protective, water retention and/or cell communication functions for this ECM layer.

Addendum to: Popielarska-Konieczna M, Kozieradzka-Kiszkurno M, Świerczyńska J, Góralski G, Ślesak H, Bohdanowicz J. Ultrastructure and histochemical analysis of extracellular matrix surface network in kiwifruit endosperm-derived callus culture. Plant Cell Rep 2008; 27:1137-45.

In several plants cultured in vitro, SEM analysis revealed that induction of morphogenesis is linked to the appearance of a fibrillar network referred to as the extracellular matrix surface network (ECMSN).1 Šamaj et al.,2,3 reported, that the ECMSN plays an important morphoregulatory role during somatic embryogenesis and organogenesis, implying an active role in plant morphogenesis. According to Bobák et al.,4 the chemical composition and structural arrangement of the ECMSN on the cell surface indicate that it may play a fundamental role in cell-to-cell recognition and interaction, cell division and differentiation, and also in generation and maintenance of some traits in plant cell populations. There is not a large body of data confirming the occurrence of ECM during in vitro organogenesis.5,6 Most data concern the formation, structure and chemical composition and function of ECM in somatic and androgenic embryogenesis (reviewed in refs. 6–10). In previous histological and SEM analysis of morphogenic endosperm-derived callus of A. deliciosa, we described the presence of a membranous layer covering the callus surface, which we termed extracellular matrix (ECM).11 The present experiments employed SEM, TEM and histochemical analysis to study the structure and composition of material coating the callus surface.

SEM revealed the presence of heterogeneous material covering the callus surface. A smooth membranous layer coated some parts of the callus surface (Fig. 1A), while other regions were covered by fibrillar structures and granules of mucilage-like secretion forming a network at site of cell-cell adhesion. Similar structures were observed during in vitro induction of somatic embryogenesis, androgenesis and organogenesis in different species (reviewed in refs. 7–13).

TEM showed a layer composed of amorphous material with fibrillar and spherical components covering morphogenic cell clusters. The cell wall of non-embryogenic and senescent cells was also covered with ECM, but it differed in appearance: the layer was granular, with amorphous deposits. According to Rumyansteva et al.,14 loss of embryogenic competence was preceded by modification of the ECM structure from fibrillar to glue-like.

Callus induction was observed after 2–3 weeks of culture (Fig. 1B). Membraneous and fibrillar structures were visible on the very small callus surface by SEM (Fig. 1C and D), though the first signs of organogenesis were noted 4–6 weeks later. TEM observations of the first steps of callogenesis also showed fine, amorphous ECM on the cell surface (data not shown). This might suggest that the extracellular matrix described above probably has a protective rather than signalling function.

SEM of root tips appearing during culture showed that root cap cells were enveloped by a specific, partially damaged layer (Fig. 1E and F) similar in appearance to membraneous ECM on the callus surface. It is commonly known, that terminal part of the root tip is composed of pectins and performs a protective function, but signalling molecules were also detected on the root tip. In maize roots in vivo, Abeysekera and McCully15 found ECM occurring as a pellicle covering young meristematic epidermal cells, which contained arabinogalactan proteins.

Solvent treatment and histochemical staining with Sudan Black B suggested that some lipophilic substances are part of the ECM in kiwifruit. Damage to the membranous layer observed by SEM after solvent treatment might be the results of removal of lipid components from the ECM. Moreover, TEM of the surface layer revealed globular deposits similar in electron density and appearance to lipid bodies. Apart from Actinidia, lipophilic components of the ECM have been reported only in Triticum.10 The probable presence of lipids in the examined ECM may be connected with protective and signalling function, especially in oxygen stress conditions.16 Pectinase digestion caused only partial disappearance of the ECM, exposing granular remnants on the cell surface and filaments forming thick fibrils at sites of cell adhesion, visible by SEM. Cellulose-binding fluorescent dye ruled out cellulose polymers as an element of ECM structure. Additionally, we identified a callose component in morphogenic callus. Deposition of polysaccharides around embryogenic cells has been suggested by Šamaj et al.,3 as an early structural marker for these cells.

During these experiments we detected JIM5-labelled low-methylesterified pectins in intercellular spaces and on the surface layer. A comparison of our results with those obtained in Triticum6 and Zea3 supports Šamaj et al.,3 assumption that regulation of pectin localization within the ECM differs between monocotyledonous and dicotyledonous.

Our results suggest the possible role of the extracellular material during kiwifruit regeneration. Pectins serve as a reservoir of signalling molecules and for water retention. Consequently, ECM may be involved in integration and recognition of morphogenic cells within multicellular callus domains, and a protective function cannot be excluded.

Abbreviations

ECM	=	extracellular matrix
ECMSN	=	extracellular matrix surface network
SEM	=	scanning electron microscopy
TEM	=	transmission electron microscopy

Figures and Tables

Figure 1 SEM images of kiwifruit endosperm-derived callus after callogenesis induction, long-term culture and organogenesis. (A) Layer composed of membranous, partially damaged (arrows) structure enveloping callus surface after long-term culture. (B–D) Callus induction after 3 weeks of culture. Remnants of seed coat (sc) and cells of non-morphogenic callus (c) are observed (B). However, some part of the callus surface is covered with fibrillar (arrowheads) and membranous extracellular matrix (ecm) with holes (arrows) (C and D). (E and F) Rhizogenesis after 6 weeks of culture. Root with root cap (rc) and root hairs (rh) is emerging from callus domain (d). Partly damaged (arrows) membranous material covering cells of root tip (F). Bars represent 50 µm for (D); 100 µm for (C and F); 200 µm for (A and B); and 500 µm for (E).

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