Ewa U. Kurczyńska

Histological analysis of direct somatic embryogenesis in Arabidopsis thaliana (L.) Heynh

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected.

Distribution of lipid transfer protein 1 (LTP1) epitopes associated with morphogenic events during somatic embryogenesis of Arabidopsis thaliana

AbstractUsing immunocytochemical methods, at both the light and electron microscopic level, we have investigated the spatial and temporal distribution of lipid transfer protein 1 (LTP1) epitopes during the induction of somatic embryogenesis in explants of Arabidopsis thaliana. Immunofluorescence labelling demonstrated the presence of high levels of LTP1 epitopes within the proximal regions of the cotyledons (embryogenic regions) associated with particular morphogenetic events, including intense cell division activity, cotyledon swelling, cell loosening and callus formation. Precise analysis of the signal localization in protodermal and subprotodermal cells indicated that cells exhibiting features typical of embryogenic cells were strongly labelled, both in walls and the cytoplasm, while in the majority of meristematic-like cells no signal was observed. Staining with lipophilic dyes revealed a correlation between the distribution of LTP1 epitopes and lipid substances within the cell wall. Differences in label abundance and distribution between embryogenic and non-embryogenic regions of explants were studied in detail with the use of immunogold electron microscopy. The labelling was strongest in both the outer periclinal and anticlinal walls of the adaxial, protodermal cells of the proximal region of the cotyledon. The putative role(s) of lipid transfer proteins in the formation of lipid lamellae and in cell differentiation are discussed. Key message Occurrence of lipid transfer protein 1 epitopes in Arabidopsis explant cells accompanies changes in cell fate and may be correlated with the deposition of lipid substances in the cell walls.

Histology and symplasmic tracer distribution during development of barley androgenic embryos

The present study concerns three aspects of barley androgenesis: (1) the morphology and histology of the embryos during their development, (2) the time course of fluorescent symplasmic tracers’ distribution, and (3) the correlation between symplasmic communication and cell differentiation. The results indicate that barley embryos, which are developing via an androgenic pathway, resemble their zygotic counterparts with respect to their developmental stages, morphology and histology. Analysis of the distribution of the symplasmic tracers, HPTS, and uncaged fluorescein indicates the symplasmic isolation of (1) the protodermis from the underlying cells of the late globular stage onwards, and (2) the embryonic organs at the mature stage of development.

Cellular Markers for Somatic Embryogenesis

Somatic embryogenesis (SE) is a process in which somatic cells under special conditions develop into embryos and in the end into a plant. That is why SE is a good model system for studying the genetic, molecular, physiological, biochemical, histological and cellular mechanisms underlying not only somatic but also zygotic embryogenesis and the totipotency of plant cells. SE begins with a transition of somatic cells to an embryogenic state and it can be induced under certain in vitro conditions. The mechanisms which determine SE induction the transition of cells from the vegetative to the embryogenic state and the conditions underlying such changes are the main questions of developmental biology (for a review see: de Jong et al., 1993; von Arnold et al., 2002; Feher et al., 2003; Namasivayam, 2007; Yang & Zhang, 2010).

Rays, intrusive growth, and storied cambium in the inflorescence stems of Arabidopsis thaliana (L.) Heynh

Arabidopsis thaliana is a model plant used in analysis of different aspects of plant growth and development. Under suitable conditions, secondary growth takes place in the hypocotyl of Arabidopsis plants, a finding which helps in understanding many aspects of xylogenesis. However, not all developmental processes of secondary tissue can be studied here, as no secondary rays and intrusive growth have been detected in hypocotyl. However, results presented here concerning the secondary growth in inflorescence stems of Arabidopsis shows that both secondary rays and intrusive growth of cambial cells can be detected, and that, in the interfascicular regions, a storied cambium can be developed.

Spatio-temporal distribution and methyl-esterification of pectic epitopes provide evidence of developmental regulation of pectins during somatic embryogenesis in Arabidopsis thaliana

The aim of the present study was to describe the occurrence of three pectic epitopes, recognized by JIM7, LM19, and LM5 antibodies, during somatic (SE) and zygotic (ZE) embryogenesis in Arabidopsis thaliana. The epitopes recognized by JIM7 and LM19 antibodies showed different distributions during SE stages. Moreover, in the early stages of somatic embryo development, a cytoplasmic occurrence of LM19 epitope was detected. Distribution of a pectic epitope recognized by LM5 antibody corresponded to a vascular system differentiation pattern. Occurrence of LM5 epitope was the same in both zygotic and somatic embryos and often restricted to newly synthesized walls of two adjacent cells. These data suggest that both low and high methyl-esterified pectins (recognized by LM19 and JIM7 antibodies, respectively) are developmentally regulated during SE stages and (1→4)-β-D-galactan epitope (recognized by LM5 antibody) may play a role in cell cytokinesis.

Importance of symplasmic communication in cell differentiation.

Symplasmic communication via plasmodesmata (PD) is part of the system of information exchange between plant cells. Molecules that pass through the PD include ions, some hormones, minerals, amino acids, and sugars but also proteins, transcription factors, and different classes of RNA, and as such PD can participate in the coordination of plant growth and development. This review summarizes the current literature on this subject and the role of PD in signal exchange, the importance of symplasmic communication and symplasmic domains in plant cell differentiation, and highlights the future prospective in the exploration of PD functions in plants. Moreover, this review also describes the potential use of barley root epidermis and non-zygotic embryogenesis in study of symplasmic communication during cell differentiation.

Spatial Distribution of Selected Chemical Cell Wall Components in the Embryogenic Callus of Brachypodium distachyon.

Brachypodium distachyon L. Beauv. (Brachypodium) is a species that has become an excellent model system for gaining a better understanding of various areas of grass biology and improving plant breeding. Although there are some studies of an in vitro Brachypodium culture including somatic embryogenesis, detailed knowledge of the composition of the main cell wall components in the embryogenic callus in this species is missing. Therefore, using the immunocytochemical approach, we targeted 17 different antigens of which five were against the arabinogalactan proteins (AGP), three were against extensins, six recognised pectic epitopes and two recognised hemicelluloses. These studies were complemented by histological and scanning electron microscopy (SEM) analyses. We revealed that the characteristic cell wall components of Brachypodium embryogenic calli are AGP epitopes that are recognised by the JIM16 and LM2 antibodies, an extensin epitope that is recognised by the JIM11 antibody and a pectic epitopes that is recognised by the LM6 antibody. Furthermore, we demonstrated that AGPs and pectins are the components of the extracellular matrix network in Brachypodium embryogenic culture. Additionally, SEM analysis demonstrated the presence of an extracellular matrix on the surface of the calli cells. In conclusion, the chemical compositions of the cell walls and ECMSN of Brachypodium callus show spatial differences that correlate with the embryogenic character of the cells. Thus, the distribution of pectins, AGPs and hemicelluloses can be used as molecular markers of embryogenic cells. The presented data extends the knowledge about the chemical composition of the embryogenic callus cells of Brachypodium.

Increased symplasmic permeability in barley root epidermal cells correlates with defects in root hair development

It is well known that the process of plant cell differentiation depends on the symplasmic isolation of cells. Before starting the differentiation programme, the individual cell or group of cells should restrict symplasmic communication with neighbouring cells. We tested the symplasmic communication between epidermal cells in the different root zones of parental barley plants Hordeum vulgare L., cv. ‘Karat’ with normal root hair development, and two root hairless mutants (rhl1.a and rhl1.b). The results clearly show that symplasmic communication was limited during root hair differentiation in the parental variety, whereas in both root hairless mutants epidermal cells were still symplasmically connected in the corresponding root zone. This paper is the first report on the role of symplasmic isolation in barley root cell differentiation, and additionally shows that a disturbance in the restriction of symplasmic communication is present in root hairless mutants.

Integument cell differentiation in dandelions (Taraxacum, Asteraceae, Lactuceae) with special attention paid to plasmodesmata

The aim of the paper is to determine what happens with plasmodesmata when mucilage is secreted into the periplasmic space in plant cells. Ultrastructural analysis of the periendothelial zone mucilage cells was performed on examples of the ovule tissues of several sexual and apomictic Taraxacum species. The cytoplasm of the periendothelial zone cells was dense, filled by numerous organelles and profiles of rough endoplasmic reticulum and active Golgi dictyosomes with vesicles that contained fibrillar material. At the beginning of the differentiation process of the periendothelial zone, the cells were connected by primary plasmodesmata. However, during the differentiation and the thickening of the cell walls (mucilage deposition), the plasmodesmata become elongated and associated with cytoplasmic bridges. The cytoplasmic bridges may connect the protoplast to the plasmodesmata through the mucilage layers in order to maintain cell-to-cell communication during the differentiation of the periendothelial zone cells.