ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox.

KEY MESSAGE: Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability. We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O 2 (·-) , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA-GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.

Foliar micro-morphology of Gasteria bicolor haw. (Asphodelaceae) from South Africa.

BACKGROUND: The succulent genus, Gasteria, which comprises 16 species, is endemic to South Africa and has its main centre of distribution in the Savanna Region of the Eastern Cape. Whereas G. bicolor has been investigated phyto-chemically and pharmacologically, not much data concerning the anatomical and micro-morphological features can be found in literature. MATERIALS AND METHODS: This study was undertaken, using light and scanning electron microscopy to obtain information on the micro-morphological features of this important medicinal plant to facilitate its identification and authentication. The elemental composition of the leaf was determined by energy dispersive X-ray spectroscopy (EDXS). RESULTS: The epidermal cells are either hexagonal or pentagonal in form, and are compactly arranged with undulate anti-clinal cell walls. The epidermal cell width was approximately 50 µm. Stomata apertures are elliptical and the upper epidermis of the leaf has paracytic stomata which are slightly raised above the epidermal surface with 4 to 5 subsidiary cells surrounding each stoma. Based on the EDXS microanalysis, the mineral crystals present at the level of the mesophyll of G. bicolor were probably mixtures of calcium oxalate, calcium sulphate and silica. CONCLUSION: The co-occurrence of aluminum suggests the potential role of the crystals in detoxification of aluminum and heavy metals, as reported previously.

Anatomical and biochemical studies of bicolored flower development in Muscari latifolium.

The inflorescence of the broad-leafed grape hyacinth, Muscari latifolium, shows an interesting, two-tone appearance with the upper flowers being pale blue and the lower ones purple. To elucidate the mechanism of the differential color development, anatomical research was carried out and a cytological study of the colored protoplasts in which the shapes of the cells accumulating anthocyanin were observed by scanning electron microscopy. Next, vacuolar pH was recorded using a pH meter with a micro combination pH electrode, and the sap's metal-ion content was measured by inductively coupled plasma mass spectrometry. The anthocyanin and co-pigment composition was determined by high-performance liquid chromatography (HPLC). Chemical analyses reveal that the difference in metal-ion content of the two parts was not great. The vacuolar pHs of the upper and lower flowers were 5.91 and 5.84, respectively, with the difference being nonsignificant. HPLC results indicate that the dihydroflavonol and flavonol contents are also very similar in the two sorts of flower. However, the upper flowers contained only delphinidin, whereas the lower flowers also contained cyanidin. The total anthocyanin content in the lower flowers was 4.36 mg g(-1), which is approximately seven times higher than in the upper flowers, while the delphinidin content is four times higher. Quantitative real-time PCR analysis established that the two-tone flower was a result of different expressions of the F3'5'H, F3'H and DFR genes, and these lead to different amounts of anthocyanin.

Ultrastructural studies on the sporogenous tissue and anther wall of Leucojum aestivum (amaryllidaceae) in different developmental stages.

In this study, ultrastructures of anther wall and sporogenous tissue of Leucojum aestivum were investigated during different developmental stages. Cytomictic channels were seen between pollen mother cells during prophase I. Polar distribution was described in the organelle content of pollen mother cells and microspores in early phases of microsporogenesis and also in pollen mitosis. Active secretion was observed in tapetal cells. Previous reports about developmental stages of male gametophyte were compared with the results of this study.

Calreticulin expression and localization in plant cells during pollen-pistil interactions.

In this report, the distributions of calreticulin (CRT) and its transcripts in Haemanthus pollen, pollen tubes, and somatic cells of the hollow pistil were studied. Immunoblot analysis of protein extracts from mature anthers, dry and germinated pollen, growing pollen tubes, and unpollinated/pollinated pistils revealed a strong expression of CRT. Both in vitro and in situ studies confirmed the presence of CRT mRNA and protein in pollen/pollen tubes and somatic cells of the pistil transmitting tract. The co-localization of these molecules in ER of these cells suggests that the rough ER is a site of CRT translation. In the pistil, accumulation of the protein in pollen tubes, transmitting tract epidermis (tte), and micropylar cells of the ovule (mc) was correlated with the increased level of exchangeable calcium. Therefore, CRT as a Ca(2+)-binding/buffering protein, may be involved in mechanism of regulation calcium homeostasis in these cells. The functional role of the protein in pollen-pistil interactions, apart from its postulated function in cellular Ca(2+) homeostasis, is discussed.

Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation.

To characterize the spatial and temporal occurrence of programmed cell death (PCD) in Lilium anther tissues, we used both microscopical and molecular markers of apoptosis for developmental stages from meiosis to pollen release. The first hallmarks of PCD include cell condensation and shrinkage of the cytoplasm, separation of chromatin into delineated masses, and DNA fragmentation in the tapetum as early as the premeiosis stage. PCD then extended to other anther sporophytic tissues, leading to anther dehiscence. Although the PCD clearly affected the endothecium and the epidermis, these two cell layers remained alive until anther dehiscence. In pollen, no sign of PCD was found until pollen mitosis I, after what apoptotic features developed progressively in the vegetative cell. In addition, DNA ladders were detected in all sporophytic tissues and cell types throughout pollen development, whereas in the male gametophyte DNA ladders were only detected during pollen maturation. Our data suggest that PCD is a progressive and active process affecting all the anther tissues, first being triggered in the tapetum.

Of plants and other pets: practical aspects of freeze-substitution and resin embedding.

Representative tissues from higher plants (e.g. developing pollen, somatic anther tissues from the monocotyledonous angiosperm Ledebouria) and mammalian cell cultures were successfully cryoimmobilized by means of high-pressure freezing. Various substitution and embedding protocols were then evaluated considering the preservation of ultrastructural details, membrane staining, immunolabelling properties, as well as reproducibility and ease of use. Two types of recipe proved to be highly suitable for most applications, regardless of type, developmental stage or physiological conditions of the cells: (i) the best choice for morphology is still osmium in acetone (optionally supplemented with uranyl acetate) followed by embedding in Epon and/or Araldite; (ii) feasible approaches for immunocytochemistry are freeze-substitution with ethanol containing uranyl acetate and formaldehyde, or with pure acetone (in the case of fixation-sensitive antigens), followed by embedding with LR-white acrylic resin; though being far from optimal, these combinations represent, in my opinion, an acceptable compromise between labelling intensity, section stability, structural preservation and health hazards. Notably, the patterns observed in Ledebouria were consistent with data obtained from a broad range of other specimens from all kingdoms (e.g. leaves and callus cultures from angiosperms, gymnosperm roots with their ectomycorrhizal fungi, mammalian cell cultures and eubacteria). Finally, a warning is given as to the extractive potentials of embedding resins (Spurr's mixture, LR-white, but also Epon) being sometimes the cause of unacceptable artefacts, both in plant and in mammalian cells prepared by cryoimmobilization and freeze-substitution.

Cytological evidence for preservation of mitochondrial and plastid DNA in the mature generative cells of Chlorophytum spp. (Liliaceae).

Following 4',6-diamidino-2-phenylindole staining of mature pollen grains of Chlorophytum comosum, fluorescence microscopy confirmed that cytoplasmic nucleoids (DNA aggregates) were present in the generative cells, which indicated the possibility of biparental cytoplasmic inheritance. Electron and immuno-electron microscopy showed that both plastids and mitochondria were present in the generative cells, and both organelles contained DNA. These results indicate that mitochondria and plastids of C. comosum have the potential for biparental inheritance. Similar results were obtained with mature pollen grains of C. chinense. Therefore, we conclude the coincident biparental inheritance for mitochondria and plastids in the members of the genus Chlorophytum.

Accumulation of plant small heat-stress proteins in storage organs.

Plant small heat-stress proteins (sHSPs) have been shown to be expressed not only after exposure to elevated temperatures, but also at particular developmental stages such as embryogenesis, microsporogenesis, and fruit maturation. This paper presents new data on the occurrence of sHSPs in vegetative tissues, their tissue-specific distribution, and cellular localization. We have found sHSPs in 1-year-old twigs of Acer platanoides L. and Sambucus nigra L. and in the liana Aristolochia macrophylla Lamk. exclusively in the winter months. In tendrils of Aristolochia, sHSPs were localized in vascular cambium cells. After budding, in spring, these proteins were no longer present. Furthermore, accumulation of sHSPs was demonstrated in tubers and bulbs of Allium cepa L., Amaryllis ( Hippeastrum hybridum hort.), Crocus albiflorus L., Hyacinthus orientalis L., Narcissus pseudonarcissus L., Tulipa gesneriana L., and Solanum tuberosum L. (potato). In potato tubers and bulb scales of Narcissus the stress proteins were localized in the central vacuoles of storage parenchyma cells. In order to obtain more information on a possible functional correlation between storage proteins and sHSPs, the accumulation of both types of protein in tobacco seeds during seed ripening and germination was monitored. The expression of sHSPs and globulins started simultaneously at about the 17th day after anthesis. During seed germination the sHSPs disappeared in parallel with the storage proteins. Furthermore, in embryos of transgenic tobacco plants, which do not contain any protein bodies or storage proteins, no sHSPs were found. Thus, the occurrence of sHSPs in perennial plant storage organs seems to be associated with the presence of storage proteins.

Adhesion and cell movement during pollination: cherchez la femme.

Pollination involves an interaction between the female tissues (stigma, style and ovary) and the male gametophyte or the pollen tube cell, which contains the sperm cells. Freezing methods now allow us to visualize the extracellular matrices that guide pollen tubes to the ovary. Adhesion of the pollen tube to these specialized extracellular matrices might be a mechanism of guidance and tube cell movement in the style. In lily, the stylar adhesion molecules are a pectin and a small, basic cysteine-rich protein, both of which are necessary to induce tube cell adhesion to an artificial, in vitro style matrix.

Developmental changes in the ultrastructure of the Sauromatum guttatum (Araceae) mitochondria.

Electron microscopic studies show that the electron density of the mitochondria of the thermogenic appendix of Sauromatum guttatum inflorescences changes during development. Two to 3 days before heat-production, the mitochondria accumulate osmiophilic, electron-dense material between the inner and outer membranes. During heat-production and the release of volatiles compounds the osmiophilic material disappears from the inter membrane space. Deposits with the same electron density are also found in the endoplasmic reticulum. It is concluded that the mitochondria may also have the capacity to accumulate that material in the inter membrane space.

Postharvest changes in white asparagus cell wall during refrigerated storage.

The postharvest changes of the white asparagus cell wall have been studied in relation to the toughening process along the length of a spear that was divided into three sections: apical, middle, and basal. Polysaccharides underwent significant turnover during storage. Uronic acid concentration decreased in all sections and in almost all polysaccharide fractions, while neutral sugars increased very slightly in the apical section, decreased in the middle, and increased in the basal one. Xylose, glucose, and galactose are the main neutral sugars implicated in the turnover; xylose accumulated in the hemicellulose fractions of the middle and basal sections, glucose decreased in the hemicellulose and cellulose fractions of the middle section and increased in the cellulose fraction of the basal one, and galactose disappeared mostly from the cellulose fractions of the three sections. Lignin increased most in the middle section and least in the basal one. No increase was detected in the apical one. No important increases of wall phenolics were detected in any part of the spear. The hardening process was limited only to the basal section.

Structure and DNA methylation pattern of partially heterochromatinised endosperm nuclei in Gagea lutea (Liliaceae).

Pentaploid endosperm nuclei in certain Gagea species exhibit large masses of sticky and dense chromatin, not observed in somatic nuclei. These heterochromatin masses most probably stem from the triploid chalasal polar nucleus of the embryo sac, thus representing an example of facultative heterochromatinisation in plants. In the present investigation, we studied the nuclei in Gagea lutea (L.) Ker-Gawl, endosperm tissue. The position of the heterochromatin in interphase nuclei was observed by confocal laser scanning microscopy (CLSM) and the DNA methylation status of the euchromatin and heterochromatin was analysed by immunolabelling with an antibody raised against 5-methylcytosine (anti-5-mC). In young endosperms, heterochromatin was relatively dispersed, occupying some peripheral and inner parts of the nuclei. In a later endosperm development, the nuclei became smaller and more pycnotic, and the heterochromatin masses were placed predominantly near the nuclear periphery. The distribution of anti-5-mC labelling on the heterochromatic regions was unequal: some parts appeared hypermethylated while other parts were, like the euchromatin, not labelled. During mitosis, the labelling intensity of all the chromosomes was approximately the same, thus indicating that there are no cytologically detectable methylation differences among the individual sets of chromosomes. However, differences in the anti-5-mC signal intensity along individual chromosomes were observed, resulting in banding patterns with highly positive bands apparently representing constitutive heterochromatic regions. From these results it is obvious that facultative heterochromatinisation, in contrast to constitutive heterochromatinisation, need not be strictly accompanied by a prominent DNA hypermethylation.