Cell Walls Of Embryos Research Articles

Germline development and seed set of metallophyte Biscutella laevigata L. (Brassicaceae)

The costs of Biscutella laevigata adaptation, a facultative metallophyte, to an environment polluted with heavy metals were established by analyzing the differences in embryological processes between plants from two populations in Southern Poland (a mountain, in the Tatra Mountains and calamine, in Bolesław). Disturbances in male and female lineage development occurred in plants from both populations. The higher frequency of degenerations in plants from the calamine population could be interpreted as a strategy to save resources limited by the environment. The distribution of high-esterified homogalacturonan detected by LM20 antibody in the cell walls of embryos from the calamine population could be part of a resistance/defense system. The results from both populations indicate that B. laevigata has already developed adaptation/tolerance, enabling maintenance of the calamine population over time. Tolerant species could be an important source for revitalization and/or phytoremediation of polluted environments.

Zygotic embryo cell wall responses to drying in three gymnosperm species differing in seed desiccation sensitivity.

Plant cell walls (CWs) are dynamic in that they can change conformation during ontogeny and in response to various stresses. Though seeds are the main propagatory units of higher plants, little is known of the conformational responses of zygotic embryo CWs to drying. This study employed cryo-scanning electron microscopy to compare the effects of desiccation on zygotic embryo CW morphology across three gymnosperm species that were shown here to differ in seed desiccation sensitivity: Podocarpus henkelii (highly desiccation-sensitive), Podocarpus falcatus (moderately desiccation-sensitive), and Pinus elliottii (desiccation-tolerant). Fresh/imbibed (i.e. fresh Podocarpus at shedding and imbibed Pi. elliottii) embryos showed polyhedral cells with regular walls, typical of turgid cells with an intact plasmalemma. Upon desiccation to c. 0.05gg-1 (dry mass basis), CWs assumed an undulating conformation, the severity of which appeared to depend on the amount and type of dry matter accumulated. After desiccation, intercellular spaces between cortical cells in all species were comparably enlarged relative to those of fresh/imbibed embryos. After rehydration, meristematic and cotyledonary CWs of P. henkelii and meristematic CWs of P. falcatus remained slightly undulated, suggestive of plasmalemma and/or CW damage, while those of Pi. elliottii returned to their original conformation. Cell areas in dried-rehydrated P. henkelii root meristem and cotyledon were also significantly lower than those from fresh embryos, suggesting incomplete recovery, even though embryo water contents were comparable between the two states. Electrolyte leakage measurements suggest that the two desiccation-sensitive species incurred significant plasmalemma damage relative to the tolerant species upon desiccation, in agreement with the CW abnormalities observed in these species after rehydration. Immunocytochemistry studies revealed that of the four CW epitopes common to embryos of all three species, an increase in arabinan (LM6) upon desiccation and rehydration in desiccation-tolerant Pi. elliottii was the only difference, although this was not statistically significant. Seed desiccation sensitivity in species like P. henkelii and P. falcatus may therefore be partly based on the inability of the plasmalemma and consequently CWs of dried embryos to regain their original conformation following rehydration.

Ultrastructural Impact of Tobacco Rattle Virus on Tobacco and Pepper Ovary and Anther Tissues

AbstractThe one‐third of plant viruses are seed transmitted, and this has significant economic consequences. Tobacco rattle virus (TRV), belonging to the genus Tobravirus and family Virgaviridae, has one of the widest host range of any known plant viruses. TRV infects vegetative organ and effects seed and pollen development that results in a decrease in crop yield. The mechanisms by which Tobravirus is transmissible to seeds are still poorly understood. The presence of the virus in pollen grains and inside ovaries is linked with seed transmission and can have effects on virus particles' transport during the pollination and fertilization process. This paper focuses on the significant impact of TRV on pepper and tobacco anthers and ultrastructure changes in ovaries. The presence of two types of TRV particles in ovary wall parenchyma and vascular tissues as well as in placenta cells was demonstrated via ultrastructural analysis. For the first time, the regular inclusion of virus particles was reported in both ovule integuments and nucellus parenchyma cells. Immunolocalization of TRV capsid proteins indicated the deposition of TRV CP epitope in ovary vascular bundles and in placenta cells. Moreover, the presence of virus particles was demonstrated inside pepper seeds in endothelium and integument parenchyma layers as well as on the embryo cell wall. Virus particles were found not only on the surface of pollen grains but also inside pepper pollen protoplasts in mature anthers. Also, this is the first time where TRV particles are reported in both differentiated endothecium cells and the remaining tapetum cells. Moreover, the detection of TRV capsid protein epitope in tobacco and pepper vascular anther tissues as well as in tapetum and endothecium cells was correlated with TRV distribution in infected anthers. Demonstrated analyses indicated that pollen grains and ovaries with ovules as well as could be a natural source of TRV transmission.

Embryo cell wall properties in relation to development and desiccation in the recalcitrant-seeded Encephalartos natalensis (Zamiaceae) Dyer and Verdoorn.

Plant cell walls are dynamic entities that may change with development, differ between plant species and tissue type and play an important role in responses to various stresses. In this regard, the present investigation employed immunocytochemistry to determine wall composition and possible changes during development of immature and mature embryos of the recalcitrant-seeded cycad Encephalartos natalensis. Fluorescent and gold markers, together with cryo-scanning and transmission electron microscopy (TEM) were also used to analyse potential changes in the cell walls of mature embryos upon desiccation. Immature cell walls were characterised by low- and high methyl-esterified epitopes of pectin, rhamnogalacturonan-associated arabinan, and the hemicellulose xyloglucan. Arabinogalactan protein recognised by the LM2 antibody, along with rhamnogalacturonan-associated galactan and the hemicellulose xylan, were not positively localised using immunological probes, suggesting that the cell walls of the embryo of E. natalensis do not possess these epitopes. Interestingly, mature embryos appeared to be identical to immature ones with respect to the cell wall components investigated, implying that these may not change during the protracted post-shedding embryogenesis of this species. Drying appeared to induce some degree of cell wall folding in mature embryos, although this was limited by the abundant amyloplasts, which filled the cytomatrical space. Folding, however, was correlated with relatively high levels of wall plasticisers typified by arabinose polymers. From the results of this study, it is proposed that the embryo cell walls of E. natalensis are constitutively prepared for the flexibility required during cell growth and expansion, which may also facilitate the moderate cell wall folding observed in mature embryos upon drying. This, together with the abundant occurrence of amyloplasts in the cytomatrix, may provide sufficient mechanical stabilisation if water is lost, even though the seeds of this species are highly desiccation-sensitive.

Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus

Arabinogalactan proteins (AGPs), present in cell walls, plasma membranes and extracellular secretions, are massively glycosylated hydroxyproline-rich proteins that play a key role in several plant developmental processes. After stress treatment, microspores cultured in vitro can reprogramme and change their gametophytic developmental pathways towards embryogenesis, thereby producing embryos which can further give rise to haploid and double haploid plants, important biotechnological tools in plant breeding. Microspore embryogenesis constitutes a convenient system for studying the mechanisms underlying cell reprogramming and embryo formation. In this work, the dynamics of both AGP presence and distribution were studied during pollen development and microspore embryogenesis in Brassica napus, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM13, JIM14, MAC207) and analysing the expression pattern of the BnAGP Sta 39-4 gene. Results showed the developmental regulation and defined localization of the studied AGP epitopes during the two microspore developmental pathways, revealing different distribution patterns for AGPs with different antigenic reactivity. AGPs recognized by JIM13, JIM14 and MAC207 antibodies were related to pollen maturation, whereas AGPs labelled by LM2 and LM6 were associated with embryo development. Interestingly, the AGPs labelled by JIM13 and JIM14 were induced with the change of microspore fate. Increases in the expression of the Sta 39-4 gene, JIM13 and JIM14 epitopes found specifically in 2-4 cell stage embryo cell walls, suggested that AGPs are early molecular markers of microspore embryogenesis. Later, LM2 and LM6 antigens increased progressively with embryo development and localized on cell walls and cytoplasmic spots, suggesting an active production and secretion of AGPs during in vitro embryo formation. These results give new insights into the involvement of AGPs as potential regulating/signalling molecules in microspore reprogramming and embryogenesis.

Co-Localization of ß-1,3-Glucanases and Callose During Somatic Embryogenesis in Cichorium

During direct somatic embryogenesis in leaves of Cichorium hybrid clone '474', 38 kDa ß-1,3-glucanases are accumulated in the culture medium of the embryogenic hybrid to a higher level when compared with a non-embryogenic cultivar. In the same time, embryogenic cells were surrounded by a cell wall that was characterized by the presence of callose. This callosic deposition disappeared as embryos grew. Callose consisted of ß-1,3-glucan linkages and so represented a possible substrate for ß-1,3-glucanases. Using immunolocalization experiments, we demonstrated that from the three types of callose deposits observed during the culturing of Cichorium leaf explants, only the callose present in the walls surrounding reactivated cells seemed specifically related to somatic embryogenesis. Moreover, callose and the 38-kDa ß-1,3-glucanases were co-localized dispersed throughout the thick and swelled walls of reactivated cells and embryo cell walls. This suggests that callose and ß-1,3-glucanases are implicated in the process of somatic embryogenesis since they were always detected in or quite near embryogenic and embryo cell. This also suggested that ß-1,3-glucanases could be involved in the degradation of this callose.

Subdomains for transport via plasmodesmata corresponding to the apical–basal axis are established during Arabidopsis embryogenesis

The axial body pattern of Arabidopsis is determined during embryogenesis by auxin signaling and differential gene expression. Here we demonstrate that another pathway, cell-to-cell communication through plasmodesmata (PD), is regulated during apical-basal pattern formation. The SHOOT MERISTEMLESS (STM) promoter was used to drive expression in the shoot apical meristem (SAM) and a subset of cells at the base of the hypocotyl of 1x,2x, and 3x soluble green fluorescent proteins (sGFPs), and the P30 movement protein of Tobacco mosaic virus (TMV) translationally fused to 1x and 2x sGFP. In the early heart stage, 2x sGFP (54 kDa) moves throughout the whole embryo, whereas 3x sGFP (81 kDa) shows more restricted movement. As the embryo develops, PD apertures are down regulated to form local subdomains allowing transport of different sized tracers. For example, movement of 2x sGFP to the cotyledon, and 3x sGFP to root tips, becomes restricted. Subdomains of cell-to-cell transport align with the apical-basal embryo body axis and correspond to the shoot apex, cotyledons, hypocotyl, and root. Studies with P30-GFP fusions reinforce the distinction between embryonic symplastic subdomains. Although P30 targets embryo cell walls as puncta (diagnostic for functional localization of P30 to PD in adult plants), P30 cannot dilate embryonic PD to overcome the barriers for transport between symplastic subdomains, suggesting that specific boundaries separate symplastic subdomains of the embryo. Thus, cell-to-cell communication via plasmodesmata conveys positional information critical to establish the axial body pattern during embryogenesis in Arabidopsis.

Development of white spruce (Picea glauca(Moench.) Voss) somatic embryos during culture with abscisic acid and osmoticum, and their tolerance to drying and frozen storage

The limit of permeability of white spruce (Picea glauca [Moench.] Voss) somatic embryo cell walls to molecules was in the order of 30 Å. Polyethylene glycols (PEGs) and dextrans of molecular weights greater than 1000 and 6000, respectively, produced a nonpermeating (non-plasmolysing) water stress which improved embryo development. Somatic embryos converted to plantlets at frequencies of 76–84% following slow drying and storage at −20 °C for 1 year, which was similar to the 77% recorded for control somatic embryos slowly dried then germinated without freezing or storage. Culture for 7–8 weeks with medium containing abscisic acid, 3% sucrose, and 7.5% PEG 4000 yielded somatic embryos with five times the embryo storage lipid content recorded for zygotic embryos. During culture with PEG the moisture content of the somatic embryos decreased from 96% for immature suspension-cultured somatic embryos, to 47% for mature embryos. Somatic embryos cultured for 7–8 weeks survived rapid drying to 5% moisture content, and converted to plantlets at frequencies of 60–70%, but no somatic embryos survived rapid drying when cultured for only 4 weeks; however, slow drying did induce desiccation tolerance in 3-week cultured somatic embryos. Abscisic acid was important to maintain embryos in a developmental state, but ABA alone did not induce desiccation tolerance. In order to induce desiccation tolerance a water stress treatment was required. Tolerance of rapid drying coincided with moisture contents below 55%, which occurred after 5 weeks of culture in the presence of PEG 4000 and abscisic acid.

Analysis of embryo growth in mature fruits of celery (Apium graveolens)

A celery ‘seed’ is a schizocarpic fruit and consists largely of endosperm surrounded by a thin testa and a thick pericarp. Embryo growth in fruits of celery (Apium graveolens L. var. rapaceum [Miller] D. C.) cv. Monarch with low and high densities was studied during incubation in water and in polyethylene glycol (PEG). Embryo growth in water was characterized by both cell division and an increase in cell size, while embryo growth in PEG solutions solely occurred through cell division. Low density fruits differed from high density fruits in a higher cell division rate during incubation both in water and PEG; in water also the cell size of the embryo was larger. As a consequence, the low density fruits reached the embryo length of 1.5 mm, which was critical for radicle protrusion, already after 6.1 days in water and 1.7 days before the high density fruits. In ‐1.2 MPa PEG the embryos in both fractions reached their maximum length of 0.9 and 0.7 mm in low and high density fruits, respectively, after 13 days of incubation. When incubation for 17 days in PEG was followed by incubation in water, the embryo growth in both fractions was faster than without a pre‐incubation in PEG and also the cell size increased. The differences between the fractions persisted, however. It was concluded that embryo cell wall extensibility increased during PEG‐incubation. During incubation in water, low density fruits had a higher water content than high density fruits. It was concluded that this difference was caused by a lower pericarp pressure potential (Ψp, pericarp). When the pericarp was removed, the differences in water content disappeared, the cell size in both fractions became similar, and the difference in mean germination time was reduced. The difference in embryo growth rate of the two fruits during incubation both in water and in PEG could be eliminated by lowering the osmotic potential (Ψπ) of the incubation medium of low density fruits. It was concluded that the pericarp of celery fruits is an important factor in regulation of embryo growth.

Parental Stress and Prechilling Effects on Pennsylvania Smartweed (Polygonum pensylvanicum) Achenes

Pennsylvania smartweed (Polygonum pensylvanicum L.) achenes were harvested from plants growing either free from competition or in competition with corn (Zea mays L. ‘Pioneer 3780′) plants. Seeds were dormant when harvested. After 15 weeks of prechilling, 4 and 35% of the seeds germinated from plants with and without corn competition, respectively; after 30 weeks of prechilling, more than 92% of all seeds germinated. Scanning electron microscopy revealed that the carpel walls of achenes from plants with corn competition were porous with many channels. Carpel walls of achenes from plants without corn competition were without pores and channels. Transmission electron microscopy showed more lipid bodies in the embryo epidermal cells of seeds from plants with corn competition. Cell walls of embryos from non-prechilled seeds from plants with corn competition contained lipoidosomes that traversed cell walls. Lipoidosomes did not occur in cells of prechilled seeds.

Changes in cell wall polysaccharides of germinating barley grains

Abstract Decorticated barley grains were germinated at 25° for 6 days, until the endosperm reserves were nearly exhausted. The neutral monosaccharide components of the hydrolysates of the cell walls and gums from the embryo, aleurone layer and starchy endosperm and the endospermic starch were determined at daily intervals. The amount of embryo cell wall polysaccharide increased 40 times and glucose became the major component, followed in abundance by xylose and arabinose. The cell wall and gum polysaccharides of the aleurone layer (plus testa) and the starchy endosperm declined during germination and their compositions altered. The endospermic starch also decreased. In the early stages of germination the apparent composition of the cell walls of the aleurone layer and starchy endosperm depended upon how they had been prepared. After 6 days the cell walls and gums had provided a significant carbohydrate supply to the living tissues, equivalent to 18.5% of the endospermic polysaccharide degraded during growth, starch having provided the remaining 81.5%.

The composition and development of cell walls of Fucus embryos

Changes in the structure and composition of cell walls of Fucus gardneri Silva were related to embryo development. Results of histochemical treatments of walls from embryos of different ages were compared with those of differential extractions and electronmicroscopic examinations of isolated walls. At 24 h after fertilization alginic acid, fucoidin and cellulose were structural constituents of the embryo wall. The distribution of alginic acid and cellulose was uniform over the cell walls of embryos of any age, but fucoidin was concentrated at the rhizoid end of cell walls isolated from germinated embryos. Evidence is presented for the presence of sulfated xylogalactofucoglucuronan and β,1-3 glucan in the embryo cell-wall. Electron micrographs of untreated and differentially extracted cell walls showed that alginic acid comprised the fibrillar material and fucoidin comprised the amorphous material of the wall. Fucus eggs do not have cell walls and the zygotes began wall synthesis within 40 min after fertilization. During the first 2 h there was a rapid deposition of alginic acid. After 4 h the wall thickness increased linearly through 24 h. Embryos treated with cycloheximide ceased wall growth after 6 h. Wall isolated from 24-h-old cycloheximide-treated embryos resembled wall isolated from control embryos 2-4 h old.

EARLY DEVELOPMENT OF THE BARLEY EMBRYO: FINE STRUCTURE

The ultrastructure of early stages in embryogenesis in barley was examined. Post‐fertilization shrinkage does not occur. Plasmodesmata were not observed in cell walls of the zygote and outer cell walls of embryos. There is little evidence of cellular specialization in earliest embryonic stages, and planes of cell division tend to be irregular although a pattern of cell disposition characteristic of some grass embryos can be discerned. The embryo appears polarized after 2–3 division cycles, but no evolving of dorsiventrality occurs during this period. A basal supensor cell “anchors” the embryo during early embryogenesis, but by about five division cycles the embryo loses its attachment to the nucellus and is completely surrounded by endosperm. An increase in number of ribosomes and mitochondria takes place during early embryogenesis, and mitochondrial dimensions are reduced. A shift in vacuole distribution occurs.

Early Development of the Barley Embryo: Fine Structure

The ultrastructure of early stages in embryogenesis in barley was examined. Post-fertilization shrinkage does not occur. Plasmodesmata were not observed in cell walls of the zygote and outer cell walls of embryos. There is little evidence of cellular specialization in earliest embryonic stages, and planes of cell division tend to be irregular although a pattern of cell disposition characteristic of some grass embryos can be discerned. The embryo appears polarized after 2–3 division cycles, but no evolving of dorsiventrality occurs during this period. A basal supensor cell “anchors” the embryo during early embryogenesis, but by about five division cycles the embryo loses its attachment to the nucellus and is completely surrounded by endosperm. An increase in number of ribosomes and mitochondria takes place during early embryogenesis, and mitochondrial dimensions are reduced. A shift in vacuole distribution occurs.