Accumulation Of Lipid Bodies Research Articles

Disruption of ultrastructure and cytoskeletal network is involved with biocontrol of damping-off pathogen Aphanomyces cochlioides by Lysobacter sp. strain SB-K88

The rhizoplane bacterium Lysobacter sp. SB-K88 suppresses damping-off disease in sugar beet and spinach caused by Aphanomyces cochlioides through characteristic plant colonization and antibiosis. Interaction of the pathogen and SB-K88 on potato dextrose agar medium revealed remarkable curly growth, excessive branching, and inhibition of mycelial growth of the pathogen. This study elucidated the mode of antagonism of SB-K88 by detecting changes in ultrastructure and organization of cytoskeletal filamentous actin (F-actin) network in the affected hyphae and zoospores of A. cochlioides. Transmission electron microscopy aided in visualization of significant ultrastructural alterations including invagination of membrane, disintegration or necrosis of cell wall, accumulation of excessive lipid bodies, electron-dense enlarged vacuoles, and degradation of cytoplasm in the SB-K88-induced excessively branched and curled hyphae. Confocal laser scanning microscopy confirmed the role of SB-K88 or its metabolites in severe disruption of the organization of F-actin networks in both hyphae and zoospores of A. cochlioides. An inhibitor of actin polymerization, latrunculin B, also exhibited similar disruption of F-actin organization in the hyphae and zoospores of A. cochlioides. These results suggest that the inhibitory effect of Lysobacter sp. SB-K88 on A. cochlioides is likely due to a combination of F-actin disruption and ultrastructural alterations in the zoospore and hyphal cells.

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Sphingolipids and their metabolites have been thought crucial for cell growth and cell cycle progression, membrane and protein trafficking, signal transduction, and formation of lipid rafts; however, recent studies in trypanosomes point to the dispensability of sphingolipids in some of these processes. In this study, we explore the requirements for de novo sphingolipid biosynthesis in the insect life cycle stage of the African trypanosome Trypanosoma brucei by inhibiting the enzyme serine palmitoyltransferase (SPT2) by using RNA interference or treatment with a potent SPT2 inhibitor myriocin. Mass spectrometry revealed that upon SPT2 inhibition, the parasites contained substantially reduced levels of inositolphosphorylceramide. Although phosphatidylcholine and cholesterol levels were increased to compensate for this loss, the cells were ultimately not viable. The most striking result of sphingolipid reduction in procyclic T. brucei was aberrant cytokinesis, characterized by incomplete cleavage-furrow formation, delayed kinetoplast segregation and emergence of cells with abnormal DNA content. Organelle replication continued despite sphingolipid depletion, indicating that sphingolipids act as second messengers regulating cellular proliferation and completion of cytokinesis. Distention of the mitochondrial membrane, formation of multilamellar structures within the mitochondrion and near the nucleus, accumulation of lipid bodies and, less commonly, disruption of the Golgi complex were observed after prolonged sphingolipid depletion. These findings suggest that some aspects of vesicular trafficking may be compromised. However, flagellar membrane targeting and the association of the flagellar membrane protein calflagin with detergent-resistant membranes were not affected, indicating that the vesicular trafficking defects were mild. Our studies indicate that sphingolipid biosynthesis is vital for cell cycle progression and cell survival, but not essential for the normal trafficking of flagellar membrane-associated proteins or lipid raft formation in procyclic T. brucei.

Developmental Changes in Endogenous Abscisic Acid Concentrations and Asymbiotic Seed Germination of a Terrestrial Orchid, Calanthe tricarinata Lindl.

Changes in endogenous abscisic acid (ABA) concentrations were investigated in developing seeds and the pretreated seeds of Calanthe tricarinata, a hard-to-germinate terrestrial orchid. ABA concentration was as low as 2.16 to 2.26 ng·mg−1 fresh weight at the proembryo stage [60 to 90 days after pollination (DAP)] and then continuously increased to 11.6 ng·mg−1 fresh weight at 210 DAP. Seed maturation was accompanied by a dramatic decrease in water content and a prominent accumulation of protein and lipid bodies within the embryo proper. The optimum time for asymbiotic seed germination was obtained from immature seeds at 150 DAP. At this stage, the embryo proper reached its maximum size, and the seedcoat became dehydrated and gradually shrunk into a thin layer. By 180 DAP, seed germination declined sharply as seed approached maturity. Mature seeds pretreated with ultrasound (45 min), 1% NaOCl (45 to 60 min), or 1N NaOH (45 min) were effective in improving the germination percentage and lowering seed ABA concentrations. Our results suggest that high concentrations of endogenous ABA in orchid seeds may play a critical role in arresting embryo growth and in preventing seed germination.

Microscopies multiharmoniques pour l’imagerie structurale de tissus intacts

One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies. In recent years, significant progress has been made in understanding the contrast mechanisms of these recent methodologies, and high-resolution imaging based on intrinsic sources of signal has been demonstrated in cells and tissues. Harmonic generation exhibits structural rather than chemical specificity and can be obtained from a variety of non-fluorescent samples. SHG is observed specifically in dense, non-centrosymmetric arrangements of polarizable molecules, such as collagen fibrils, myofilaments, and polarized microtubule bundles. SHG imaging is therefore emerging as a novel approach for studying processes such as the physiopathological remodelling of the collagen matrix and myofibrillogenesis in intact tissue. THG does not require a non-centrosymmetric system ; however no signal can be obtained from a homogeneous medium. THG imaging therefore provides maps of sub-micrometer heterogeneities (interfaces, inclusions) in unstained samples, and can be used as a general purpose structural imaging tool. Recent studies showed that this technique can be used to image embryo development in small organisms and to characterize the accumulation of large lipid bodies in specialized cells. SHG and THG microscopy both rely on femtosecond laser technology and are easily combined with two-photon microscopy.

Modification of Host Lipid Raft Proteome upon Hepatitis C Virus Replication

Hepatitis C virus (HCV) replication complex resides in detergent-insoluble subcellular domains or lipid rafts. We used two proteomics approaches to characterize the protein content of lipid rafts isolated from Huh7 cells and its modification upon HCV replication. Using two-dimensional electrophoresis and mass spectrometry, we identified approximately 100 protein spots in the isolated lipid rafts; among them, 39 were reproducibly modified in HCV replicon cell lines as compared with control cell lines. We also used stable isotope labeling by amino acids in cell culture (SILAC) combined with one-dimensional electrophoresis separation and mass spectrometry. Using this approach, we identified 1036 individual proteins based on peptides selected with at least 95% confidence; among them, 413 proteins were identified with at least two peptides. Quantification analysis identified 150 proteins modified by at least 2.5-fold (110 up-regulated and 40 down-regulated) in HCV-replicating cells compared with controls. Protein identifications and quantifications obtained by both proteomics approaches were largely concordant. Modulated proteins included a majority of proteins involved in vesicular and protein trafficking and in cell signaling. Remarkably for a large number of proteins, their up-regulation in lipid rafts of HCV replicon cells was due to their relocalization. By using small interfering RNAs directed to the modulated small GTPases Cdc42 and RhoA, we observed an increase in HCV replication, whereas reduction of syntaxin 7 expression resulted in decreased replication of HCV. Our findings indicate that protein subcellular relocalization occurs in HCV-containing cells that can directly affect HCV replication.

Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes

Cellular imaging techniques based on vibrational spectroscopy have become powerful tools in cell biology because the molecular composition of subcellular compartments can be visualized without the need for labeling. Using high-resolution, nonresonant confocal Raman microscopy on individual cells, we demonstrate here that lipid bodies (LBs) rich in arachidonate as revealed by their Raman spectra associate with latex bead-containing phagosomes in neutrophilic granulocytes. This finding was corroborated in macrophages and in PLB-985 cells, which can be induced to differentiate into neutrophil-like cells, by selective staining of LBs and visualization by confocal fluorescence microscopy. We further show that the accumulation of LBs near phagosomes is mediated at least in part by the flavohemoprotein gp91phox (in which "phox" is phagocyte oxidase), because different LB distributions around phagocytosed latex beads were observed in WT and gp91phox-deficient PLB-985 cells. gp91phox, which accumulates in the phagosomal membrane, is the catalytic subunit of the leukocyte NADPH oxidase, a critical enzyme in the innate immune response. Finally, time-lapse fluorescence microscopy experiments on neutrophils revealed that the LB-phagosome association is transient, similar to the "kiss-and-run" behavior displayed by endosomes involved in phagosome maturation. Because arachidonic acid (AA) has been shown to be involved in NADPH oxidase activation and phagosome maturation in neutrophils and macrophages, respectively, the findings reported here suggest that LBs may provide a reservoir of AA for local activation of these essential leukocyte functions.

On the Physiological Role of Anaerobically Synthesized Lipids in Oryza sativa Seedlings

The objective of this work was to elucidate a possible adaptive role of lipid biosynthesis and unsaturated fatty acids (FAs), esterified to lipids, as terminal acceptors of electrons, alternative to molecular oxygen, in the shoots of rice seedlings (Oryza sativa L.) under conditions of strict anoxia. Biosynthesis of lipids and their accumulation, as well as the reduction of double bonds in unsaturated FAs, were studied by electron microscopic observation of the accumulation of lipid bodies in the cytoplasm and by the biochemical analysis of FAs in shoot lipids before and after anaerobic incubation of the shoots. The experiments were carried out with intact coleoptiles after 5 and 8 days of anaerobic germination of seeds (primary anoxia) and with detached shoots, preliminarily grown in air and then subjected to anoxia in the presence of 2% glucose for 48 h (secondary anoxia). In these experiments, lipid bodies did not accumulate in the cytoplasm under anoxic conditions. Lipid bodies appeared only during 48-h anaerobic incubation of detached coleoptiles in the absence of exogenous glucose, when mitochondria degraded. There was no change either in the double bond index of FAs, or in the qualitative and quantitative composition of FAs during shoot anaerobic incubation. We conclude that neither lipids synthesized under anaerobic conditions nor esterified unsaturated FAs are involved in plant adaptation to anaerobiosis as terminal acceptors of electrons, alternative to molecular oxygen. Lipid biosynthesis under anoxic conditions, which was demonstrated for anoxia-tolerant seedlings of Oryza sativa and Echinochloa phyllopogon in experiments with radioactive precursors, 14C-acetate and 3H-glycerol, is only the manifestation of a turnover of saturated FAs and various classes of lipids, which stabilizes cell membranes under adverse conditions of strict anoxia.

Neutral lipid bodies in prokaryotes: recent insights into structure, formation, and relationship to eukaryotic lipid depots.

Many prokaryotes are able to accumulate large amounts of lipophilic compounds as inclusion bodies in the cytoplasm. Members of most genera synthesize polymeric lipids such as poly(3-hydroxybutyrate) (PHB) or other polyhydroxyalkanoates (PHAs) (125), whereas accumulation of triacylglycerols (TAGs) and wax esters (WEs) in intracellular lipid-bodies is a property of only a few prokaryotes (10). Like the formation of PHAs, TAG and WE biosynthesis is also promoted in response to stress imposed on the cells and during imbalanced growth, for example by nitrogen limitation, if an abundant carbon source is present at the same time. All these lipids act as storage compounds for energy and carbon needed for maintenance of metabolism and synthesis of cellular metabolites during starvation and in particular if growth resumes. Although neutral lipid metabolism in bacteria, especially WE biosynthesis, has attracted increasing biotechnological interest, there has so far been little interest in medical research in the formation of prokaryotic lipid bodies. However, recent findings suggest that lipid body formation and accumulation of TAGs play an important role in the metabolism of the pathogenic bacteria like Mycobacterium tuberculosis (37, 52). In contrast to the restricted occurrence of storage TAGs in prokaryotes, intracellular TAGs are widespread in many eukaryotes (34, 35, 40, 53, 65, 106, 112, 132, 133). In eukaryotes, storage lipids are also deposited as lipid bodies. Their structure and formation have recently been reviewed in detail by several authors, but additional information on prokaryotic lipid-bodies has been only superficial (98, 100, 151). In contrast, intracellular accumulation of WEs as a storage lipid is a great exception in eukaryotes and occurs only in jojoba (Simmondsia chinensis) (147), and PHAs are not at all synthesized as a storage compound in eukaryotes. The number of studies investigating the enzymatic and structural fundamentals of storage lipid metabolism in bacteria has increased drastically in the last years, providing the knowledge that storage lipids in bacteria have a similar important metabolic function like in eukaryotes. This review will focus on prokaryotic neutral lipid bodies in comparison with the currently discussed models for lipid body structure and their formation in eukaryotes and with PHA inclusions in prokaryotes.

Chemical Composition and Fungicidal Activity of Commercial Essential Oils of Thymus vulgaris L.

The antifungal activity of commercial Thymus vulgaris oils on the development of mycelium of the the phytopathogenic fungi Fusariumsolani (Mart.) Sacc., Rhizoctoniasolani Kühn and Colletotrichumlindemuthianum (Sacc. et Magn.) Briosi et Cav. was studied. The gas chromatography data showed that the essential oils were rich in thymol (22–38%) and its biogenetic precursors γ-terpinene and p-cymene. The carvacrol content was relatively low in all the oils tested, ranging between 1% and 2%. The fungicidal activity of the oils was correlated with their thymol content. The principal chemical components of thyme oils were then tested using the same concentrations as in the thyme oils. Only the fungicidal activity of thymol was high and close to that of the oils with the same thymol content. The effects of the thyme oils and thymol on the hyphae cytomorphology of the studied fungi was an increased vacuolization of the cytoplasm and accumulation of lipid bodies, undulations of the plasmalemma, and alterations of the mitochondria and endoplasmic reticulum.

Biological effects due to weak magnetic field on plants

Throughout the evolution process, Earth’s magnetic field (MF, about 50 μT) was a natural component of the environment for living organisms. Biological objects, flying on planned long-term interplanetary missions, would experience much weaker magnetic fields, since galactic MF is known to be 0.1–1 nT. However, the role of weak magnetic fields and their influence on functioning of biological organisms are still insufficiently understood, and is actively studied. Numerous experiments with seedlings of different plant species placed in weak magnetic field have shown that the growth of their primary roots is inhibited during early germination stages in comparison with control. The proliferative activity and cell reproduction in meristem of plant roots are reduced in weak magnetic field. Cell reproductive cycle slows down due to the expansion of G 1 phase in many plant species (and of G 2 phase in flax and lentil roots), while other phases of cell cycle remain relatively stabile. In plant cells exposed to weak magnetic field, the functional activity of genome at early pre-replicate period is shown to decrease. Weak magnetic field causes intensification of protein synthesis and disintegration in plant roots. At ultrastructural level, changes in distribution of condensed chromatin and nucleolus compactization in nuclei, noticeable accumulation of lipid bodies, development of a lytic compartment (vacuoles, cytosegresomes and paramural bodies), and reduction of phytoferritin in plastids in meristem cells were observed in pea roots exposed to weak magnetic field. Mitochondria were found to be very sensitive to weak magnetic field: their size and relative volume in cells increase, matrix becomes electron-transparent, and cristae reduce. Cytochemical studies indicate that cells of plant roots exposed to weak magnetic field show Ca 2+ over-saturation in all organelles and in cytoplasm unlike the control ones. The data presented suggest that prolonged exposures of plants to weak magnetic field may cause different biological effects at the cellular, tissue and organ levels. They may be functionally related to systems that regulate plant metabolism including the intracellular Ca 2+ homeostasis. However, our understanding of very complex fundamental mechanisms and sites of interactions between weak magnetic fields and biological systems is still incomplete and still deserve strong research efforts.

Triacsin C inhibits the formation of 1H NMR-visible mobile lipids and lipid bodies in HuT 78 apoptotic cells

Nuclear magnetic resonance-visible mobile lipids (ML) have been reported to accumulate during cell apoptosis in vitro and in vivo. The biogenesis, biochemical nature and structure of these lipids are still under debate. In this study, a human lymphoblastoid cell line, HuT 78, was induced to apoptosis by exposure to anti-Fas monoclonal antibodies (α-Fas mAb) followed by incubation for different time intervals (1–24 h, hypodiploid cell fraction, H, varying from 1% to over 60%) either in the presence or in the absence of 5.0 μM Triacsin C (TRC), specific inhibitor of long-chain acyl-CoA synthetase (ACS). The increase of ML in apoptotic cells correlated linearly with H and was associated with: (a) accumulation of intracellular lipid bodies, detected by confocal laser scanning microscopy in lipophilic dye-stained cells; (b) increases, detected by thin-layer chromatography in total lipid extracts, in the relative abundance of triacylglycerides (TAG) and cholesteryl esters (CE), with corresponding decreases of phospholipids (PL). TRC completely abolished both ML and lipid body formation in anti-Fas-treated apoptotic cells, with concomitant reversion of TAG, CE and PL to control levels, but did not alter cell viability nor did it inhibit apoptosis. ML signals detected during anti-Fas-induced apoptosis therefore appear to originate from neutral lipids assembled in intracellular lipid bodies, synthesised from cellular acyl-CoA pools.

Behavior of storage lipids during development and germination of olive ( Olea europaea L.) pollen.

The presence of abundant oil bodies in the mature olive pollen grain has led us to focus on the behavior of these lipid bodies during pollen development and in vitro pollen germination. The appearance, increase, and accumulation of lipid bodies have been determined by following the sequential development of the pollen grain. Semithin slices of anthers and pollen grains were stained with Sudan Black B in order to identify neutral lipids. Ultrastructural studies were also carried out. Our results show a notable increase in lipid bodies between the young-pollen-grain stage and the mature-pollen-grain stage. Substantial polarization of lipid bodies was observed after 1 or 2 h of pollen incubation in germination medium. During pollen tube growth, the lipid bodies are located near the germinative aperture after 3 h of incubation, as well as inside the pollen tube, thus suggesting that the lipid bodies move from the pollen grain to the pollen tube. After 7 h of germination the presence of lipid bodies inside the pollen tube is no longer substantial. Our results support the idea that lipid bodies are involved in pollen germination, stigma penetration, and pollen tube growth. These results are discussed in connection with their implications for the pollen germination process.

Effects of tebuconazole on morphology, structure, cell wall components and trichothecene production of Fusarium culmorum in vitro.

The effects of tebuconazole, a systemic fungicide, on the morphology, structure, cell wall components and toxin production of Fusarium culmorum were investigated in vitro. Treatment was by application of four filter paper strips (0.75 cm x 5.0 cm) soaked in 20 micrograms ml-1 fungicide placed around a point inoculum in Petri dishes. Mycelial growth was strongly inhibited by fungicide treatment. Scanning electron microscopic observations showed that the fungicide caused irregular swelling and excessive branching of hyphae. The morphological changes induced by the fungicide at the ultrastructural level included considerable thickening of the hyphal cell walls, excessive septation, the formation of the incomplete septa, extensive vacuolisation, accumulation of lipid bodies and progressing necrosis or degeneration of the hyphal cytoplasm. Non-membrane inclusion bodies were often detected in the hyphal cytoplasm. Furthermore, the formation of new hyphae (daughter hyphae) inside collapsed hyphal cells was common following treatment. The daughter hyphae also displayed severe alterations such as irregular thickening of the cell walls and necrosis of the cytoplasm. Using cytochemical techniques, the labelling densities of chitin and beta-1,3-glucan in the cell walls of the fungicide-treated hyphae were more pronounced than in those of the control hyphae. Moreover, immunogold labelling with antiserum against deoxynivalenol (DON) revealed that Fusarium toxin DON was localized in the cell walls, cytoplasm, mitochondria and vacuoles of the hyphae from the control and the fungicide treatment, but the labelling density in the fungicide-treated hyphae decreased dramatically compared with the control hyphae, indicating that tebuconazole reduced Fusarium toxin production of the fungus.

Ultrastructural and morphometric study of the Sertoli cell of the viscacha (Lagostomus maximus maximus) during the annual reproductive cycle.

Changes in the morphology of viscacha Sertoli cells were studied during the annual reproductive cycle. Sertoli cells exhibited marked nuclear and cytoplasmic changes. Seasonal variation in nuclear size and shape, chromatin texture, and nucleolus characteristics was observed. The seasonal patterns of the volume densities of the endoplasmic reticulum (ER), mitochondria, Golgi complex, dense bodies and lipid inclusions were distinct. Morphometric analysis revealed that the Golgi complex is the organelle most sensitive to seasonal change. It declined drastically in the regressed testes and its recovery was slow. The ER and mitochondria exhibited seasonal variations in their pattern and content, that was minimal during winter. In contrast, an accumulation of lipid and dense bodies, such as primary and secondary lysosomes, accompanied the spermatogenic arrest. The volume densities of both organelles were maximum during the restoration of spermatogenesis. The length and organization of the inter-Sertoli junctions also changed with the reproductive cycle. The Sertoli cell number per tubular cross section decreased significantly during the testicular regression, coincident with the presence of Sertoli cells with marked signs of involution. The degree of regression and recovery exhibited by the viscacha Sertoli cells was closely related to that shown by the associated germ cells. Therefore, seasonal endocrine fluctuations and local factors could be involved in the regulation of the morphological and functional characteristics of the viscacha Sertoli cells. These hormonal fluctuations are synchronized by the photoperiod through the pineal gland and its hormone, melatonin.

Ultrastructure and calcium balance in meristem cells of pea roots exposed to extremely low magnetic fields.

Investigations of low magnetic field (LMF) effects on biological systems have attracted attention of biologists due to planned space flights to other planets where the field intensity does not exceed 10(-5) Oe. Pea (Pisum sativum L.) seeds were grown in an environment of LMF 3 days. In meristem cells of roots exposed to LMF, one could observe such ultrastructural peculiarities as a noticeable accumulation of lipid bodies, development of a lytic compartment (vacuoles, cytosegresomes and paramural bodies), and reduction of phytoferritin in plastids. Mitochondria were the most sensitive organelle to LMF application. Their size and relative volume in cells increased, matrix was electron-transparent, and cristae reduced. Because of the significant role of calcium signalling in plant responses to different environmental factors, calcium participation in LMF effects was investigated using a pyroantimonate method to identify the localization of free calcium ions. The intensity of cytochemical reaction in root cells after LMF application was strong. The Ca2+ pyroantimonate deposits were observed both in all organelles and in a hyaloplasm of the cells. Data obtained suggest that the observed LMF effects on ultrastructure of root cells were due to disruptions in different metabolic systems including effects on Ca2+ homeostasis.

Activation of detergent-solubilized diacylglycerol acyltransferase by anionic phospholipids.

Diacylglycerol acyltransferase (DGAT), which catalyzes the final step in triacylglycerol (TG) biosynthesis, is crucial for lipid accumulation and formation of lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora. Since solubilization of DGAT in the lipid body fraction from this fungus with 0.5% Triton X-100 gave very low recovery of the activity, some activation factors for solubilized DGAT activity were investigated. Addition of phospholipids, especially anionic phospholipids such as phosphatidic acid and phosphatidylserine, to the assay mixture greatly increased DGAT activity. The activation by these phospholipids was most prominent when 0.2% Triton X-100 was added to the assay mixture. The effect of phosphatidic acid was reproduced using DGAT fraction obtained by 0.5 M KCl elution on Mono S column chromatography. The results provide new insight on activation of DGAT during TG accumulation as well as optimal DGAT assay conditions for solubilized fractions.

Effects of long-term open-air exposure to fluoride, nitrogen compounds and SO2 on visible symptoms, pollutant accumulation and ultrastructure of Scots pine and Norway spruce seedlings

Effects of SO2, aqueous fluoride (NaF) and a solution of nitrogen compounds (NH4NO3) on the visible symptoms, pollutant accumulation and ultrastructure of Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst.] seedlings were studied in an open-air experiment lasting for 3 consecutive years. Visible injury symptoms were most pronounced in combination exposures and whenever F was applied. Visible symptoms correlated well with needle pollutant concentrations. Exposure to NaF increased needle F contents particularly when F was applied with SO2 or NH4NO3. This suggests that a reduction in N or SO2 emissions, in F polluted areas, could improve the condition of conifers via decreased accumulation of phytotoxic F in the needles. Norway spruce needles accumulated 2–10 times as much S and F as those of Scots pine. Microscopic observations showed various changes in the needle mesophyll cell ultrastructure. In both species, exposure to SO2 increased significantly the amount of cytoplasmic vacuoles, suggesting detoxification of excess sulphate or low pH. F treatments resulted in a significant enlargement of plastoglobuli in Scots pine and a darkening of plastoglobuli in Norway spruce. All exposures enhanced the accumulation of lipid bodies. An increased portion of translucent plastoglobuli was most pronounced in N treatments. Many of the ultrastructural changes and visible symptoms appeared only as number of years exposed increased, indicating that long-term experiments are needed. Both visible symptoms and ultrastructural changes pointed to the more pronounced sensitivity of Norway spruce compared to Scots pine. Ultrastructural results mostly supported earlier qualitative observations of F, N and SO2 effects on needle mesophyll cell ultrastructure. However, no reduction of thylakoids in SO2 containing exposure or curling of thylakoids in F exposure could be detected in the present study.

Cellular Adaptation from a Salt Tolerant Cell Line of Pisum sativum

Structural changes in tolerant calli that occur during salt adaptation of Pisum sativum calli are described. Ultrastructural studies, made in the sensitive and adapted calli of Pisum sativum , revealed that salt-tolerant calli showed several changes when compared with sensitive calli. Cells from sensitive calli were characterized by having large vacuoles, whereas salt-tolerant calli showed reduced vacuoles. There were changes in the appearance of plastids and in the accumulation of lipid bodies in cells of salt-tolerant calli. Cells with incomplete cytokinesis and anormal walls were seen in salt-tolerant calli. In these cells peroxidase is found mainly in the cell wall and in the intercellular space.

Effects of Iodoacetamide on the Fine Structure of Azuki Bean Protoplast and its Fusion Product.

Effects of iodoacetamide (IOA) on azuki bean protoplasts and their fusion products were examined with emphasis on thc fine structure. Protoplasts were isolated enzymatically from cell suspension culture 4∼6 days after subculture. Protoplasts were then treated with IOA before culturing. Protoplasts were also fused with untreated protoplasts of mung bean leaf. Untreated and IOA-treated protoplasts of azuki bean and their fusion products were treated for viability, cell wall formation test and electron microscopy. Viability of IOA-treated protoplasts was extremely reduced after 3 days in culture. Cell wall regeneration was not detected. Furthermore, no cell division was observed in these protoplasts. Ultrastructurally, IOA-treated protoplasts performed apparent peculiarity with an overformation of rough endoplasmic reticulum (rER) and formation of circular Golgi complexes. This was followed by accumulation of lipid bodies within the cytoplasm. After 3 days in culture, protoplasts exhibited abundant accumulation of electron dense particles. Some of them showed collapse as indicated by shrinkage of their organelles and cytoplasm. In contrast to these protoplasts, untreated protoplasts had a performance as meristematic cell and cell division firstly observed after 3 days in culture. The effects of IOA, such as overformation of rER and circular Golgi bodies, were not found in the fusion product with untreated protoplasts. Otherwise, swelling of thylakoid in chloroplast was detected. The heterokaryons were able to maintain their survival for several days.

Transcripts encoding an oleosin and a dormancy‐related protein are present in both the aleurone layer and the embryo of developing barley (Hordeum vulgare L.) seeds

In cereal seeds, the aleurone layer and the embryo share several characteristics, including synthesis and accumulation of lipid bodies, desiccation tolerance and dormancy. A number of Balem transcripts present in both the barley aleurone layer and the embryo have been cloned by differential screening of a cDNA library from aleurone layers of immature barley grains. The Balem clones constitute two subgroups, one for which the transcripts are detectable in aleurone layers and embryos of developing seeds only (B23D and B15C), and another for which transcripts are present also in germinating embryos and in maternal tissues (B12D, B14E and B31E). Sequence analysis identified B23D and B15C as the barley homologues of the 18 kDa oleosin of maize embryos (72% amino acid identity) and the dormancy-associated transcript pBS128 from Bromus secalinus (95% identity), respectively. In situ hybridization experiments demonstrate that in the embryo, the B23D transcript is mainly present in the scutellum, whereas the B15C transcript is predominantly present in shoot and root apices. Using anther-derived embryos and embryogenic cell suspensions, it is demonstrated that the B23D and B15C transcripts can be used as molecular markers for somatic embryogenesis. The functions of the transcripts in the second Balem subgroup remain unknown. Further studies on the Balem transcripts may shed light on the molecular basis for the extensive similarities between the embryo and the aleurone layer of the endosperm in the grass family.