Małgorzata Kozieradzka-Kiszkurno

Morphological, histological and ultrastructural features of osmophores and nectary of Bulbophyllum wendlandianum (Kraenzl.) Dammer (B. section Cirrhopetalum Lindl., Bulbophyllinae Schltr., Orchidaceae)

The species from Bulbophyllinae are generally regarded as fly-pollinated: myophilous or sapromyophilous. The section Cirrhopetalum is characterized by umbellate inflorescence and elongated lateral sepals. The aim of the floral anatomical investigation (micromorphology, histochemistry, ultrastructure) in Bulbophyllum wendlandianum (section Cirrhopetalum) was the detection of secretory activity. The appendages of dorsal sepal and petals function as osmophores. The exudation is transported inside vesicles via granulocrine secretion. The cuticle stretches and forms swellings on the entire cell surface. Such swellings of cuticle on the appendages of dorsal sepal and petals were not previously described in Bulbophyllum species. The nectary is located in the central groove on the adaxial lip surface. It comprises epidermal epithelial cells and few subepidermal layers.

Studies on the ultrastructure of a three-spurred fumeauxiana form of Anacamptis pyramidalis

Floral spurs are regarded as features affecting pollinator behaviour. Anacamptis pyramidalis is regarded as a deceitful, non-rewarding orchid species. In the form fumeauxiana, additional spurs occur on the lateral sepals. In this study we analyse micromorphological and ultrastructural floral features and suggest the mechanism of deception in A. pyramidalis and A. pyramidalis f. fumeauxiana. In f. fumeauxiana, the adaxial surface of the lip, the lip calli, the tips of the lateral sepals, the abaxial and adaxial epidermises of the lip spur, and the lateral sepal spur have a secretory function. Numerous stomata were observed on the abaxial surfaces of spurs and sepals. The characteristic features of the ultrastructure of osmophore cells were noted: dense cytoplasm with numerous profiles of ER, mitochondria, plastids with plastoglobuli and tubular structures, a large nucleus, lipid droplets, and vesicles fusing with the plasmalemma. The similarity of the floral morphology and anatomy, the flowering period, and pollinators of A. pyramidalis, A. pyramidalis f. fumeauxiana and Gymnadeniaconopsea suggest a possible food-deceptive mechanism—imitation of nectar presence produced in the spurs of Gymnadenia.

New data about the suspensor of succulent angiosperms: Ultrastructure and cytochemical study of the embryo-suspensor of Sempervivum arachnoideum L. and Jovibarba sobolifera (Sims) Opiz.

The development of the suspensor in two species — Sempervivum arachnoideum and Jovibarba sobolifera — was investigated using cytochemical methods, light and electron microscopy. Cytological processes of differentiation in the embryo-suspensor were compared with the development of embryo-proper. The mature differentiated suspensor consists of a large basal cell and three to four chalazal cells. The basal cell produces haustorial branched invading ovular tissues. The walls of the haustorium and the micropylar part of the basal cell form the wall ingrowths typical for a transfer cells. The ingrowths also partially cover the lateral wall and the chalazal wall separating the basal cell from the other embryo cells. The dense cytoplasm filling the basal cell is rich in: numerous polysomes lying free or covering rough endoplasmic reticulum (RER), active dictyosomes, microtubules, bundles of microfilaments, microbodies, mitochondria, plastids and lipid droplets. Cytochemical tests (including proteins, insoluble polysaccharides and lipids are distributed in the suspensor during different stages of embryo development) showed the presence of high amounts of macromolecules in the suspensor cells, particularly during the globular and heart-shaped phases of embryo development. The protein bodies and lipid droplets are the main storage products in the cells of the embryo-proper. The results of Auramine 0 indicate that a cuticular material is present only on the surface walls of the embryo-proper, but is absent from the suspensor cell wall. The ultrastructural features and cytochemical tests indicate that in the two species — S. arachnoideum and J. sobolifera — the embryo-suspensor is mainly involved in the absorption and transport of metabolites from the ovular tissues to the developing embryo-proper.

Are there symplastic connections between the endosperm and embryo in some angiosperms? : a lesson from the Crassulaceae family

It is believed that there is symplastic isolation between the embryo (new sporophyte) and the endosperm (maternal-parental origin tissue, which nourishes the embryo) in angiosperms. However, in embryological literature there are rare examples in which plasmodesmata between the embryo suspensor and endosperm cells have been recorded (three species from Fabaceae). This study was undertaken in order to test the hypothesis that plasmodesmata between the embryo suspensor and the endosperm are not so rare but also occur in other angiosperm families; in order to check this, we used the Crassulaceae family because embryogenesis in Crassulaceae has been studied extensively at an ultrastructure level recently and also we tread members of this family as model for suspensor physiology and function studies. These plasmodesmata even occurred between the basal cell of the two-celled proembryo and endosperm cells. The plasmodesmata were simple at this stage of development. During the development of the embryo proper and the suspensor, the structure of plasmodesmata changes. They were branched and connected with electron-dense material. Our results suggest that in Crassulaceae with plasmodesmata between the endosperm and suspensor, symplastic connectivity at this cell–cell boundary is still reduced or blocked at a very early stage of embryo development (before the globular stage). The occurrence of plasmodesmata between the embryo suspensor and endosperm cells suggests possible symplastic transport between these different organs, at least at a very early stage of embryo development. However, whether this transport actually occurs needs to be proven experimentally. A broader analysis of plants from various families would show whether the occurrence of plasmodesmata between the embryo suspensor and the endosperm are typical embryological characteristics and if this is useful in discussions about angiosperm systematic and evolution.

Embryogenesis in Sedum acre L.: structural and immunocytochemical aspects of suspensor development

The changes in the formation of both the actin and the microtubular cytoskeleton during the differentiation of the embryo-suspensor in Sedum acre were studied in comparison with the development of the embryo-proper. The presence and distribution of the cytoskeletal elements were examined ultrastructurally and with the light microscope using immunolabelling and rhodamine-phalloidin staining. At the globular stage of embryo development extensive array of actin filaments is present in the cytoplasm of basal cell, the microfilament bundles generally run parallel to the long axis of basal cell and pass in close to the nucleus. Microtubules form irregular bundles in the cytoplasm of the basal cell. A strongly fluorescent densely packed microtubules are present in the cytoplasmic layer adjacent to the wall separating the basal cell from the first layer of the chalazal suspensor cells. At the heart-stage of embryo development, in the basal cell, extremely dense arrays of actin materials are located near the micropylar and chalazal end of the cell. At this stage of basal cell formation, numerous actin filaments congregate around the nucleus. In the fully differentiated basal cell and micropylar haustorium, the tubulin cytoskeleton forms a dense prominent network composed of numerous cross-linked filaments. In the distal region of the basal cell, a distinct microtubular cytoskeleton with numerous microtubules is observed in the cytoplasmic layer adjacent to the wall, separating the basal cell from the first layer of the chalazal suspensor cells. The role of cytoskeleton during the development of the suspensor in S. acre is discussed.

Unusual electron-dense dome associates with compound plasmodesmata in the embryo-suspensor of genus Sedum (Crassulaceae)

Plasmodesmata ensure the continuity of cytoplasm between plant cells and play an important part in the intercellular communication and signal transduction. During the development of the suspensor of both Sedum acre L. and Sedum hispanicum L., changes in the ultrastructure of plasmodesmata and adjoining cytoplasm are observed. Numerous simple plasmodesmata are present in the inner wall of the two-celled embryo separating the basal cell from the apical cell. From the early-globular to the torpedo stage of embryo development, the part of the wall separating the basal cell from the first layer of the chalazal suspensor cells is perforated by unusual, compound plasmodesmata. The role and the sort of transport through these plasmodesmata are discussed.

The localization of NADPH oxidase and reactive oxygen species in in vitro-cultured Mesembryanthemum crystallinum L. hypocotyls discloses their differing roles in rhizogenesis

This work demonstrated how reactive oxygen species (ROS) are involved in the regulation of rhizogenesis from hypocotyls of Mesembryanthemum crystallinum L. cultured on a medium containing 1-naphthaleneacetic acid (NAA). The increase of NADPH oxidase activity was correlated with an increase of hydrogen peroxide (H2O2) content and induction of mitotic activity in vascular cylinder cells, leading to root formation from cultured hypocotyls. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, inhibited H2O2 production and blocked rhizogenesis. Ultrastructural studies revealed differences in H2O2 localization between the vascular cylinder cells and cortex parenchyma cells of cultured explants. We suggest that NADPH oxidase is responsible for H2O2 level regulation in vascular cylinder cells, while peroxidase (POD) participates in H2O2 level regulation in cortex cells. Blue formazan (NBT) precipitates indicating superoxide radical (O2•−) accumulation were localized within the vascular cylinder cells during the early stages of rhizogenesis and at the tip of root primordia, as well as in the distal and middle parts of newly formed organs. 3,3′-diaminobenzidine (DAB) staining of H2O2 was more intense in vascular bundle cells and in cortex cells. In newly formed roots, H2O2 was localized in vascular tissue. Adding DPI to the medium led to a decrease in the intensity of NBT and DAB staining in cultured explants. Accumulation of O2•− was then limited to epidermis cells, while H2O2 was accumulated only in vascular tissue. These results indicate that O2•− is engaged in processes of rhizogenesis induction involving division of competent cells, while H2O2 is engaged in developmental processes mainly involving cell growth.

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

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.

The F-actin cytoskeleton in syncytia from non-clonal progenitor cells

The actin cytoskeleton of plant syncytia (a multinucleate cell arising through fusion) is poorly known: to date, there have only been reports about F-actin organization in plant syncytia induced by parasitic nematodes. To broaden knowledge regarding this issue, we analyzed F-actin organization in special heterokaryotic Utricularia syncytia, which arise from maternal sporophytic tissues and endosperm haustoria. In contrast to plant syncytia induced by parasitic nematodes, the syncytia of Utricularia have an extensive F-actin network. Abundant F-actin cytoskeleton occurs both in the region where cell walls are digested and the protoplast of nutritive tissue cells fuse with the syncytium and also near a giant amoeboid in the shape nuclei in the central part of the syncytium. An explanation for the presence of an extensive F-actin network and especially F-actin bundles in the syncytia is probably that it is involved in the movement of nuclei and other organelles and also the transport of nutrients in these physiological activity organs which are necessary for the development of embryos in these unique carnivorous plants. We observed that in Utricularia nutritive tissue cells, actin forms a randomly arranged network of F-actin, and later in syncytium, two patterns of F-actin were observed, one characteristic for nutritive cells and second—actin bundles—characteristic for haustoria and suspensors, thus syncytia inherit their F-actin patterns from their progenitors.

Floral structure of two species of Bulbophyllum section Cirrhopetalum Lindl.: B. weberi Ames and B. cumingii (Lindl.) Rchb. f. (Bulbophyllinae Schltr., Orchidaceae)

Flowers of Bulbophyllum weberi and B. cumingii are characterized by fly-pollinated features. The secretory activity was described in dorsal sepals in both species (putative osmophores), petals in B. weberi (possible osmophores) and adaxial surface of lips in both species. In the cells of dorsal sepals and petals of B. weberi proteins, dihydroxyphenols, lipids and starch grains were detected, in lateral sepals—lipids. Whereas in dorsal sepal of B. cumingii only lipids and starch grains were noted, in lateral sepals—proteins and dihydroxyphenols and in petals—proteins and starch grains. The lips in both species differed histochemically and ultrastructurally. The epidermal cells of lip groove in B. weberi contained lipids, proteins, starch grains in cytoplasm, dihydroxyphenols in vacuoles and pectic acids/mucilage on surface. Whereas in B. cumingii—few lipids, starch grains, no proteins, no dihydroxyphenols and no mucilage were noted. Ultrastructurally, in B. weberi, the secretory material was present on surface and vesicles building into plasmalemma, while in B. cumingii—cell wall ingrowths and microchannels in cuticle. The osmiophilic irregular materials and globular, osmiophilic globules in B. weberi are probably tannin-like materials. For the first time, we described the cell wall ingrowths in Bulbophyllum species: in lip of B. cumingii and petals of B. weberi.