The growth and differentiation of aortal smooth muscle cells after calcitriol treatment are associated with microtubule reorganisation - an in vitro study

The smooth muscle cells (SMCs) of the arterial media play a predominant role in functional and structural alterations of the arterial wall. The transition from the “contractile” to the “synthetic” phenotype appears to be an early critical event in the development of atherosclerotic disease. A number of observations suggest that 1,25(OH)2D3 (calcitriol) is of importance in maintaining normal cardiovascular function through its receptors in cardiac myocytes or aortal SMCs. The present study has focused on the microtubular (MT) network reorganisation after exposure to calcitriol. SMCs isolated by enzymatic digestion from the aortal media of neonatal rats were cultured on glass cover slips. 1 μM of 1,25(OH)2D3 was added to the culture medium every second day. The cytoskeletal features of SMCs after calcitriol were visualised by the immunofluorescence staining of α-tubulin. The alterations in α-tubulin expression and the distribution of microtubules related to the activities of the vascular smooth muscle cells, namely adhesion, migration, multilayer formation and cell division, were observed. A spindle shape, decreased cell adhesion, low expression of α-tubulin and a longitudinally arranged microtubular network manifested the high rate of SMC differentiation in the calcitriol-treated culture. A flat stellate morphology, high expression of α-tubulin and a radially distributed three-dimensional microtubular network were observed in the SMCs of the control culture. Destructive changes in the microtubular architecture which altered the cellular shape were evident in SMCs undergoing apoptosis. Cells with apoptotic features were more frequent in calcitriol-exposed culture. In contrast to the regular SMC divisions observed in the control culture, some of the mitotic cells exposed to calcitriol contained broader bipolar, multipolar or disordered spindles. These alterations in the SMCs’ microtubular cytoskeleton after calcitriol treatment were concomitant with changes in cell growth, differentiation and apoptosis, and may suggest a similarity to atherosclerotic plaque formation

A scanning electron microscopic study of phenotypic plasticity and surface structural changes of aortal smooth muscle cells in primary culture

Phenotypic modulation of smooth muscle cells (SMCs) from a contractile to a synthetic state characterised by active proliferation appears to be an early event in the pathogenesis of atherosclerosis. A similar transition occurs when SMCs are established in culture. In this study the phenotypic plasticity and surface structural changes of aortal smooth muscle cells during the transition from the contractile to the synthetic state and during maturation have been structurally assessed by scanning electron microscope (SEM). The experiments were performed on SMCs obtained from aorta of neonatal rats after enzymatic digestion and then cultured on glass cover slips. SEM observations revealed a three-dimensional appearance characteristic for different stages of SMCs. Intensively proliferating cells from monolayer region were large, polygonal in shape with lamellipodia and well spread. Long, uniform in diameter, finger-like microvilli were densely arranged on the surface of these cells. In the thickened region of culture, the cells were rather small, generally spindle-shaped, not well spread, with low density of short, bubble-like microvilli on the surface. Numerous plasma membrane structural alterations in apoptotic cells were observed by SEM: loss of cellular adhesion, smoothing, shrinkage and outpouching of membrane segments have been recognised as markers associated with the cell injury and death. It was concluded that scanning microscopy observations would allow a more complete understanding of SMCs and their changes in culture and atherosclerotic disease

Floral structure and pollen morphology are important characters in taxonomy of the genus Viola (Violaceae)

the pistil with stigma, stamen appendages (nectaries) and pollen heteromorphism are important diagnostic features in the genus ViolaL. The style characters were crucial in the very early classifications of this genus (Clausen 1927). We analyzed in details, using scanning electron microscopy (SEM), the microstructural characters of generative organs (style and stigma, stamens with nectaries) and pollen in representatives of three sections (Viola L., Melanium Ging., DischidiumGing.) occurring in Poland to get insights into the relatedness among far-related (different sections) and closely related (sub-sections within section) species. There is a great difference in stigma micromorphology between sections. In the section Violaflowers have style beaked at the apex, glabrous or covered by papillae and/or hairs, depending of subsection. Monotypic section Dischidiumwith one species V. biflora L. characterizes 2-lobed stigma. Cup-shaped stigma with the hole on the top and a lip below, covered with papillae and hairs on its outer surface occurs in pansies of the section Melanium. Pollen is highly heteromorphic (different pollen morphs, from three up to six apertures within one flower or even within one pollen sac) in the Melaniumsection and weakly heteromorphic mainly with three apertures in diploids of Viola and Dischidiumsections. This character is independent of the polyploidy in the Melaniumbut not in Violasection (Dajoz 1999). The flower micromorphological characters are also useful in reconstruction of closely related species origin. Based on stigma and nectaries features, two zinc violets are more similar to the alpineV. lutea, than to V. tricolor, indicated also as the ancestor (Kuta et al.2012)

The influence of fie and met1 mutations and in vitro culture conditions on autonomous endosperm development in unfertilized ovules of Arabidopsis thaliana

In flowering plants, seeds are produced both sexually (double fertilization is required) and asexually via apomixis (meiotic reduction and egg fertilization are omitted). An apomictic-like pattern of endosperm development in planta is followed by fis mutants of sexual Arabidopsis thaliana. In our experiments in planta, autonomous endosperm (AE) developed in met1 mutants. Furthermore we obtained autonomous endosperm formation in vitro not only in unfertilized ovules of fie mutants but also in wild genotypes (Col-0, MET1/MET1, FIE/FIE) and met1 mutants. AE induction and development occurred in all genotypes on the each of the media used and in every trial. The frequency of AE was relatively high (51.2% ovaries) and genotype-dependent. AE induced in vitro represents a more advanced stage of development than AE induced in fie mutants in planta. This was manifested by a high number of nuclei surrounded by cytoplasm and organized in nuclear cytoplasmic domains (NCDs), nodule formation, division into characteristic regions, and cellularization. The high frequency of AE observed in homozygous met1 (met1/met1) mutants probably is due to accumulation of hypomethylation as an effect of the met1 mutation and the in vitro conditions. AE development was most advanced in FIE/fie mutants. We suggest that changes in the methylation of one or several genes in the DNA of Arabidopsis genotypes caused by in vitro conditions resulted in AE induction and/or further AE development

Floral structure and pollen morphology of two zinc violets (Viola lutea ssp. calaminaria and V. lutea ssp. westfalica) indicate their taxonomic affinity to Viola lutea

Two zinc violets, the yellow form of the Aachen–Liège area and the blue morph of Blankenrode in western Westphalia, have very restricted occurrence on heavy metal waste heaps. Their taxonomic affinities have not been finally resolved. The flower micromorphological analysis presented here indicates that both zinc violets are closely related to the alpine Viola lutea, in line with our earlier published molecular data, but not with the conclusions of other authors. The zinc violets are classed at the rank of subspecies as V. lutea: ssp. calaminaria for the yellow zinc violet and ssp. westfalica for its blue counterpart. Although the violets examined (V. lutea, V. lutea ssp. calaminaria, V. lutea ssp. westfalica) are closely related, there is no evidence that V. lutea ssp. westfalica is a descendent of V. tricolor. Here we provide the most detailed information on generative organ structure in the four violets studied

Distribution of microtubules during regular and disturbed microsporogenesis and pollen grain development in Gagea lutea (L.) Ker.-Gaw.

The microtubular cytoskeleton in dividing microsporocytes and developing pollen grains of Gagea lutea (L.) Ker.-Gaw.(Liliaceae) was investigated with a modified indirect immunofluorescence method. Meiotic and mitotic stages were identified by DAPI staining. The microtubular cytoskeleton was compared in plants originating from natural localities and others grown in the laboratory. In natural conditions, microsporocytes and pollen grains of wild early-spring Gagea lutea plants are subjected to abiotic factors including cold exposure and lack of water. The persistent influence of these factors can disturb microtubular cytoskeleton functioning. The following disturbances were observed in the course of microsporogenesis and pollen development: abnormal chromosome configurations in the metaphase of meiosis I; abnormally divided dyads with irregular, radial microtubule systems around the nuclei; the formation of differently sized microspores with irregular shapes, and irregular division; and the formation of pollen grains with vacuoles abnormal for their development stage. Similar kinds of disturbances were observed after 1.5 months of cold treatment (4 ^{\circ}C) and drying in the laboratory. These abiotic factors simulated in laboratory conditions caused more disturbances in the course of microsporogenesis and produced more frequent defective pollen grains than in the sample that had experienced cold and drying in natural conditions

Exogenous steroid hormones stimulate full development of autonomous endosperm in Arabidopsis thaliana

Most flowering plants, including important crops, require double fertilization to form an embryo and endosperm, which nourishes it. Independence from fertilization is a feature of apomictic plants that produce seeds, from which the plants that are clones of the mother plant arise. The phenomenon of apomixis occurs in some sexual plants under specific circumstances. Since the launch of a fertilization-independent mechanism is considered a useful tool for plant breeding, there have been efforts to artificially induce apomixis. We have been able to produce fertilization-independent endosperm in vitro in Arabidopsis over the last few years. This paper demonstrates the methods of improving the quality of the endosperm obtained using plant and mammalian steroid hormones. Additionally, it shows the study on the autonomous endosperm (AE) formation mechanism in vitro.This paper examines the effect of exogenous steroid hormones on unfertilized egg and central cell divisions in culture of unpollinated pistils of Arabidopsis Col-0 wild-type and fie-1 mutant. All media with hormones used (estrone, androsterone, progesterone, and epibrassinolide) stimulated central cell divisions and fertilization-independent endosperm development. The stages of AE development followed the pattern of Arabidopsis thaliana wild type after fertilization. Subsequent stages of AE were observed from 2-nuclear up to cellular with the most advanced occurring on medium with 24-epibrassinolide and progesterone. The significant influence of mammalian sex hormones on speed of AE development and differentiation was noticed. Using restriction analysis, the changes in methylation of FIE gene was established under in vitro condition. The authors of this paper showed that Arabidopsis thaliana has a high potency to fertilization-independent development

Extracellular matrix surface network is associated with non-morphogenic calli of Helianthus tuberosus cv. Albik produced from various explants

Helianthus tuberosus is economically important species. To improve characters of this energetic plant via genetic modification, production of callus tissue and plant regeneration are the first steps. A new, potentially energetic cultivar Albik was used in this study to test callus induction and regeneration. Callus was produced on leaves, petioles, apical meristems and stems from field-harvested plants but was totally non-morphogenic. Its induction started in the cortex and vascular bundles as confirmed by histological analysis. The surface of heterogeneous callus was partially covered with a membranous extracellular matrix surface network visible in scanning and transmission electron microscopies. The results clearly indicate that: (i) the morphogenic capacity of callus in topinambur is genotype dependent, (ii) cv. Albik of H. tuberosus proved recalcitrant in in vitro regeneration, and (iii) extracellular matrix surface network is not a morphogenic marker in this cultivar

Rab-dependent vesicular traffic affects female gametophyte development in Arabidopsis

Eukaryotic cells rely on the accuracy and efficiency of vesicular traffic. In plants, disturbances in vesicular trafficking are well studied in quickly dividing root meristem cells or polar growing root hairs and pollen tubes. The development of the female gametophyte, a unique haploid reproductive structure located in the ovule, has received far less attention in studies of vesicular transport. Key molecules providing the specificity of vesicle formation and its subsequent recognition and fusion with the acceptor membrane are Rab proteins. Rabs are anchored to membranes by covalently linked geranylgeranyl group(s) that are added by the Rab geranylgeranyl transferase (RGT) enzyme. Here we show that Arabidopsis plants carrying mutations in the gene encoding the β-subunit of RGT (rgtb1) exhibit severely disrupted female gametogenesis and this effect is of sporophytic origin. Mutations in rgtb1 lead to internalization of the PIN1 and PIN3 proteins from the basal membranes to vesicles in provascular cells of the funiculus. Decreased transport of auxin out of the ovule is accompanied by auxin accumulation in tissue surrounding the growing gametophyte. In addition, female gametophyte development arrests at the uni- or binuclear stage in a significant portion of the rgtb1 ovules. These observations suggest that communication between the sporophyte and the developing female gametophyte relies on Rab-dependent vesicular traffic of the PIN1 and PIN3 transporters and auxin efflux out of the ovule