Regulators of GTP-binding proteins cause morphological changes in the vacuole system of the filamentous fungus, Pisolithus tinctorius

Tubule formation is a widespread feature of the endomembrane system of eukaryotic cells, serving as an alternative to the better-known transport process of vesicular shuttling. In filamentous fungi, tubule formation by vacuoles is particularly pronounced, but little is known of its regulation. Using the hyphae of the basidiomycete Pisolithus tinctorius as our test system, we have investigated the effects of four drugs whose modulation, in animal cells, of the tubule/vesicle equilibrium is believed to be due to the altered activity of a GTP-binding protein (GTP gamma S, GDP beta S, aluminium fluoride, and Brefeldin A). In Pisolithus tinctorius, GTP gamma S, a non-hydrolysable form of GTP, strongly promoted vacuolar tubule formation in the tip cell and next four cells. The effects of GTP gamma S could be antagonised by pre-treatment of hyphae with GDP beta S, a non-phosphorylatable form of GDP. These results support the idea that a GTP-binding protein plays a regulatory role in vacuolar tubule formation. This could be a dynamin-like GTP-ase, since GTP gamma S-stimulated tubule formation has only been reported previously in cases where a dynamin is involved. Treatment with aluminium fluoride stimulated vacuolar tubule formation at a distance from the tip cell, but NaF controls indicated that this was not a GTP-binding-protein specific effect. Brefeldin A antagonised GTP gamma S, and inhibited tubule formation in the tip cell. Given that Brefeldin A also affects the ER and Golgi bodies of Pisolithus tinctorius, as shown previously, it is not clear yet whether the effects of Brefeldin A on the vacuole system are direct or indirect.     

Brefeldin A-induced increase of sphingomyelin synthesis. Assay for the action of the antibiotic in mammalian cells.

Brefeldin A leads to an increase of sphingomyelin in Chinese hamster ovary cells. The antibiotic is known to cause a dramatic morphological change of the endomembrane system in various mammalian cells resulting in a redistribution of Golgi resident proteins to the endoplasmic reticulum (Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., Berger, E. G., Hauri, H. P., Yuan, L. C., and Klausner, R. D. (1990) Cell 60, 821-836). A strict correlation was found between the brefeldin A-induced increase of sphingomyelin and the biochemical criteria that apply for this morphological change. From our data we conclude that the increase in sphingomyelin caused by brefeldin A reflects translocation of the enzyme sphingomyelin synthase from the Golgi apparatus to the endoplasmic reticulum. Using a radioactively labeled truncated ceramide this increase in sphingomyelin synthesis is easily detectable, and thus this method can serve as a convenient biochemical assay for the action of brefeldin A in mammalian cells.     

Histological and Ultrastructural Observation Reveals Significant Cellular Differences between Agrobacterium Transformed Embryogenic and Non-embryogenic Calli of Cotton

Over the past few decades genetic engineering has been applied to improve cotton breeding. Agrobacterium medicated transformation is nowadays widely used as an efficient approach to introduce exogenous genes into cotton for genetically modified organisms. However, it still needs to be improved for better transformation efficiency and higher embryogenic callus induction ratios. To research further the difference of mechanisms for morphogenesis between embryogenic callus and non-embryogenic callus, we carried out a systematical study on the histological and cellular ultrastructure of Agrobacterium transformed calli. Results showed that the embryogenic callus developed nodule-like structures, which were formed by small, tightly packed, hemispherical cells. The surface of some embryogenic callus was covered with a flbrilar-like structure named extracellular matrix. The cells of embryogenic calli had similar morphological characteristics. Organelles of embryogenic callus cells were located near the nucleus, and chloroplasts degraded to proplastid-like structures with some starch grains, in contrast, the non-embryogenic calli were covered by oval or sphere cells or small clusters of cells. It was observed that cells had vacuolation of cytoplasm and plastids with a well organized endomembrane system. This study aims to understand the mechanisms of embryogenic callus morphogenesis and to improve the efficiency of cotton transformation in future.     

Identification and localization of a beta-COP-like protein involved in the morphodynamics of the plant Golgi apparatus

This paper examines the molecular machinery involved in membrane exchange within the plant endomembrane system. A study has been undertaken on beta-COP-like proteins in plant cells using M3A5, an antibody raised against the conserved sequence of mammalian beta-COP proteins. In mammalian cells, beta-COP proteins are part of a complex named the coatomer, which probably recruits some specific areas of the endomembrane system. Immunofluorescence analyses by confocal laser scanning microscopy showed that beta-COP-like proteins marked predominantly the plant Golgi apparatus. Other proteins known to be part of a potential machinery for COPI vesicle formation (gamma-COP, beta'-COP and Arf1 proteins) were immunolocalized on the same membraneous structures as beta-COP. Moreover, beta-COP and other COPI antibodies stained the cell plate in dividing cells. It is further shown that, in maize root cells, and in contrast to observations upon mammalian cells, the drug Brefeldin A (BFA) does not induce the release of beta-COP and Arf1 proteins from the Golgi membrane into the cytosol. These data clearly demonstrate that the antibody M3A5 is a valuable marker for studies on trafficking events in plant cells. They also report for the first time the location of COP components in plant tissue at the light level, especially on a model well known for secretion, i.e. the maize root cells. They also suggest that the membrane recruitment machinery may function in a plant-specific way.     

Extracellular matrix surface network of embryogenic units of friable maize callus contains arabinogalactan-proteins recognized by monoclonal antibody JIM4

Embryogenic units of friable maize callus are formed as globular or oblong packets of tightly associated meristematic cells. These units are surrounded by conspicuous cell walls visible in light microscopy after staining with basic fuchsin. Transmission electron microscopy revealed that embryogenic cells are rich in endoplasmic reticulum, polysomes and small protein bodies, and that the outermost layer of their cell walls is composed of fibrillar material. Electron microscopy has also shown that this material covers the surface of embryogenic cells as a distinct layer which we denote as extracellular matrix surface network (ECMSN). Employing histochemical staining with β-glucosyl Yariv phenylglycoside, we localized arabinogalactan-proteins (AGPs) to the outer cell walls of embryogenic units including ECMSN. The most prominent staining was found in cell-cell junction domains. Large non-embryogenic callus cells were not stained with this AGP-specific dye. Immunofluorescence and silver-enhanced immunogold labelling using monoclonal antibody JIM4 has shown that the ECMSN of embryogenic cells is equipped with JIM4 epitope, while non-embryogenic callus cells are devoid of this epitope. We propose that some specific AGPs of the ECMSN might be relevant for cell-cell adhesion and recognition of embryogenic cells during early embryogenic stages, and that the JIM4 antibody can serve as an early marker of embryogenic competence in maize callus culture. Received: 13 March 1998 / Revision received: 6 June 1998 / Accepted: 1 July 1998     

Reversible dissociation of the plant golgi apparatus by brefeldin A

Summary— The effects of the drug Brefeldin A, shown to block the translocation of proteins between the endoplasmic reticulum and the Golgi apparatus in animal cells, were studied on different plant cell systems. In suspension culture cells and root tissues, the Golgi aparatus was affected by Brefeldin A treatments resulting in distortion and dissociation of the Golgi stacks, coupled with appearance of numerous vesicles in the cytoplasm. This process was reversible. Therefore, Brefeldin A provides a powerful tool with which to study Golgi dynamics and function in plant as well as in animal cells.     

The effect of monensin on gametogenesis and zoosporogenesis in the aquatic fungus,Allomyces macrogynus

Summary Cytoplasmic cleavage in the gametangia and zoosporangia ofA. macrogynus was studied using monensin, an ionophore known to disrupt several endomembrane functions in plant and animal cells. Monensin interfered with normal gamete and zoospore formation in a dose dependent manner such that at a 20 M concentration very abnormal cells were released from the reproductive structures. It was evident that monensin's effect was most pronounced during the first 25 minutes of gametogenesis and parallels in time the onset and continuation of the cytoplasmic cleavage events. Observations using fluorescence and differential interference contrast microscopy indicated that the ionophore inhibited normal cytoplasmic cleavage resulting in the production of multinucleate cells, many of which had either no flagella or multiple flagella. Transmission electron microscopy showed that the monensin-treated gametangia had many large vacuoles which contained amorphous electron-opaque material. X-ray microprobe analysis demonstrated that the elemental composition of the large vacuoles was identical to that of the dense globular inclusions seen in untreated gametangia, and morphological analysis confirmed the relationship between these endomembrane structures. Thus this swollen endomembrane component probably is not involved in the cleavage process. Single endomembrane cisternae which were very common in untreated gametangia were seldom seen in monensin-treated preparations. Instead, many smaller electron-transparent vacuoles were observed. These swollen cisternae may both represent monensin-modified Golgi apparatus equivalents and/or play a critical role during the process of gametogenesis and zoosporogenesis inA. macrogynus.     

Regeneration of roots, shoots and embryos: physiological, biochemical and molecular aspects

When the proper stimuli are given, somatic plant cells may form adventitious embryos, roots or shoots. The three pathways of regeneration show apparent similarities. They consist of three analogous phases: 1) dedifferentiation (during which the tissue becomes competent to respond to the organogenic/embryogenic stimulus), 2) induction (during which cells become determined to form either a root, a shoot or an embryo), and 3) realization (outgrowth to an organ or an embryo). The first phase may involve a period of callus growth (indirect regeneration), but often cells present in the explant become competent without cell division or without cell division at a large scale (direct regeneration). In an explant, only very few cells show the organogenic/embryogenic response. In direct regeneration, the three regenerative pathways start from cells in different tissues. This is most obvious when the different types of regeneration occur in the same explant. The hormonal trigger for the dedifferentiation phase is a general one, probably auxin. During the induction phase, each pathway requires specific hormonal triggers. During the realization phase, hormones should be absent or at low concentration. The successive steps in the regeneration process coincide with events on the molecular and biochemical levels, but so far no coherent picture has emerged. In particular during the early stages of regeneration, research on these levels is hampered by a technical problem, viz., the very low proportion of cells that participate in the process of regeneration. New methods may overcome this problem. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ilimaquinone inhibits gap-junctional communication prior to Golgi fragmentation and block in protein transport

Brefeldin A and ilimaquinone are compounds known to affect Golgi structure and function. In particular, the transport of proteins is blocked either at the level of exit from endoplasmic reticulum (brefeldin) or at cis-Golgi (ilimaquinone). Brefeldin caused a slow decrease in gap-junctional communication and a slow loss of all phosphorylated forms of connexin43 in hamster and rat fibroblasts, while ilimaquinone caused an abrupt decrease in gap-junctional communication and rapid loss of only the slowest migrating phosphorylated connexin43 band (P2). Ilimaquinone caused these effects prior to any significant Golgi fragmentation, especially in hamster fibroblasts. Concurrently, ilimaquinone minimally affected protein secretion, while brefeldin caused an instantaneous decrease. These results show that ilimaquinone inhibits gap-junctional communication in connexin43-expressing cells by a mechanism not dependent on Golgi fragmentation or block in protein transport.     

Reevaluating the effect of Brefeldin A (BFA) on ganglioside synthesis: the location of GM2 synthase cannot be deduced from the inhibition of GM2 synthesis by BFA.

Brefeldin A reversibly disassembles the Golgi complex, causing mixing of the Golgi cisternae with the ER while the trans Golgi network persists as part of a separate endosomal membrane system. Because of this compartmental separation, Brefeldin A treatment has been used to map the sub-Golgi locations of several Golgi enzymes including GM2 synthase. We previously proposed that GM2 synthase might be located in a distal portion of the Golgi complex which in the presence of Brefeldin A would be separated from the substrate ganglioside GM3 present in the mixed ER-Golgi membrane system. In the present study we show using GM2 synthase chimeras that GM2 synthesis was blocked by Brefeldin A when GM2 synthase was distributed throughout all Golgi subcompartments or even when it was restricted to the medial Golgi. Because these findings opposed our speculation regarding a distal location of this enzyme, we sought an alternative explanation for the inhibition of ganglioside synthesis by Brefeldin A. However, Brefeldin A did not degrade GM2 synthase, prevent its homodimerization, or inhibit its in vitro activity. Brefeldin A did result in the conversion of a portion of membrane bound GM2 synthase into a soluble form which has minimal capability to produce GM2 in whole cells. However, this conversion was not sufficient to explain the nearly total loss of GM2 production in intact cells in the presence of Brefeldin A. Nevertheless, the results of this study indicate that Brefeldin A-induced inhibition of ganglioside synthesis cannot be used to deduce the location of GM2 synthase.     

Rab-H_1b is essential for trafficking of cellulose synthase and for hypocotyl growth in Arabidopsis thaliana

Cell-wall deposition of cellulose microfibrils is essential for plant growth and development. In plant cells,cellulose synthesis is accomplished by cellulose synthase complexes located in the plasma membrane. Trafficking of the complex between endomembrane compartments and the plasma membrane is vital for cellulose biosynthesis;however, the mechanism for this process is not well understood. We here report that, in Arabidopsis thaliana,Rab-H_1b, a Golgi-localized small GTPase, participates in the trafficking of CELLULOSE SYNTHASE 6(CESA6) to the plasma membrane. Loss of Rab-H_1b function resulted in altered distribution and motility of CESA6 in the plasma membrane and reduced cellulose content. Seedlings with this defect exhibited short, fragile etiolated hypocotyls.Exocytosis of CESA6 was impaired in rab-h1 b cells, and endocytosis in mutant cells was significantly reduced as well. We further observed accumulation of vesicles around an abnormal Golgi apparatus having an increased number of cisternae in rab-h1 b cells, suggesting a defect in cisternal homeostasis caused by Rab-H_1b loss function. Our findings link Rab GTPases to cellulose biosynthesis, during hypocotyl growth, and suggest Rab-H_1b is crucial for modulating the trafficking of cellulose synthase complexes between endomembrane compartments and the plasma membrane and for maintaining Golgi organization and morphology.e     

Mitotic partitioning of endosomes and lysosomes

BACKGROUND: Some of the mechanisms underlying cell division and partitioning of the cellular components into the daughter cells are well known. Within the endomembrane system, there is a general cessation of membrane traffic, including endocytosis and endosome fusion, at the onset of mitosis. However, the fate of endosomes and lysosomes during mitosis has been less well studied. RESULTS: Using video and confocal microscopy of living cells, we show here that endosomes and lysosomes remain intact and separate during mitosis. The segregation into daughter cells takes place by coordinated movements, and during cytokinesis, these organelles accumulate in the vicinity of the microtubule organization center. However, partitioning into daughter cells is not more accurate than a calculated stochastic distribution, despite the apparent order to the process. CONCLUSION: We conclude that partitioning of endosomes and lysosomes is an ordered, yet imprecise, process, and that the organelle copy number is maintained by the daughter cells.     

Brefeldin A-induced disassembly of the Golgi apparatus is followed by disruption of the endoplasmic reticulum in plant cells

Brefeldin A (BFA), a fungal fatty acid derivative, is a potentagent for disrupting the Golgi apparatus in plant and animalcells. We have examined its action using marker antibodies whichrecognize an epitope in the plant Golgi apparatus (JIM 84),and for proteins held in the endoplasmic reticulum by the HDELER-retention signal (2E7), in combination with double immunolabelling.In maize root cells, disruption of the ER occurs after breakdownof the Golgi apparatus is initiated. The redistribution of theGolgi is shown to be predominantly separate from that of theER, and as with the Golgi, the action of BFA on the ER is alsoreversible. The mode of action of BFA on the ER and Golgi ofplant cells is compared with that described for animal cells. Key words: Zea mays L, Brefeldin A, plant cells, endoplasmic reticulum, Golgi apparatus     

Cloning of cDNA encoding NtEPc, a marker protein for the embryogenic dedifferentiation of immature tobacco pollen grains cultured in vitro

We partially purified three Nicotiana tabacum L. embryogenic pollen-abundant phosphoproteins (NtEPa to c) which appeared in the cells undergoing a dedifferentiation process from immature pollen grains to embryogenic cells, caused by glutamine-deficiency in vitro. All the NtEPs had a highly conserved N-terminal amino acid sequence. Using degenerate oligonucleotide probes designed from the amino acid sequences, the cDNA for NtEPc was isolated from a cDNA library of pollen cultured in glutamine-free medium The cDNA sequence showed moderate homology with several type-1 copper-binding glycoproteins and with a kind of early nodulin though its function could not be predicted. Expression analysis revealed that the level of mRNA for NtEPc was high during the dedifferentiation and also in the very early period of pollen embryogenesis but it was low in the developmental process of microspores/pollen in anthers, in the in vitro maturation process and both in the stational and logarithmic growth phases of tobacco BY-2 cells. Furthermore, an acidic medium pH, which promoted the induction of dedifferentiation increased the level of mRNA for NtEPc, whereas the presence of 6-benzylaminopurine, which inhibited it, decreased the level. These results suggest that the expression of NtEPc gene is correlated with the dedifferentiation but not with pollen development or cell division.     

Systematic analysis of specific and nonspecific auxin effects on endocytosis and trafficking

The phytohormone auxin and its directional transport through tissues are intensively studied. However, a mechanistic understanding of auxin-mediated feedback on endocytosis and polar distribution of PIN auxin transporters remains limited due to contradictory observations and interpretations. Here, we used state-of-the-art methods to reexamine the auxin effects on PIN endocytic trafficking. We used high auxin concentrations or longer treatments versus lower concentrations and shorter treatments of natural indole-3-acetic acid (IAA) and synthetic naphthalene acetic acid (NAA) auxins to distinguish between specific and nonspecific effects. Longer treatments of both auxins interfere with Brefeldin A-mediated intracellular PIN2 accumulation and also with general aggregation of endomembrane compartments. NAA treatment decreased the internalization of the endocytic tracer dye, FM4-64; however, NAA treatment also affected the number, distribution, and compartment identity of the early endosome/trans-Golgi network, rendering the FM4-64 endocytic assays at high NAA concentrations unreliable. To circumvent these nonspecific effects of NAA and IAA affecting the endomembrane system, we opted for alternative approaches visualizing the endocytic events directly at the plasma membrane (PM). Using total internal reflection fluorescence microscopy, we saw no significant effects of IAA or NAA treatments on the incidence and dynamics of clathrin foci, implying that these treatments do not affect the overall endocytosis rate. However, both NAA and IAA at low concentrations rapidly and specifically promoted endocytosis of photo-converted PIN2 from the PM. These analyses identify a specific effect of NAA and IAA on PIN2 endocytosis, thus, contributing to its polarity maintenance and furthermore illustrate that high auxin levels have nonspecific effects on trafficking and endomembrane compartments.

Natural and synthetic auxins affect various aspects of the endomembrane system at high concentrations, but promote clathrin-mediated endocytosis of the PIN2 auxin transporter at low concentrations.     

The Role of auxin,pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa

Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 microM) or high (10 microM) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mM) in the presence of 1 microM 2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 microM 2,4-D-containing culture medium by 10 mM 2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.     

石刁柏胚性细胞诱导过程中的内源激素和多胺含量变化

用高效液相色谱法分析石刁柏愈伤组织胚性细胞诱导过程中不同时期内源激素和多胺含量的结果表明,在胚性细胞诱导过程中,Put和GA3一直呈上升趋势,胚性细胞出现时,IAA、Put和GA3含量都达到最高水平,显示高含量的IAA以及高比例的Pu“（Spd＋Spm）可能有利于胚性细胞的形成。     

High conversion frequency of germinated somatic embryos of Siberian ginseng (Eleutherococcus senticosus Maxim) using a bubble column bioreactor

Embryogenic callus was obtained from young petiole, stem, and root explants of 4-year-old Siberian ginseng plants on medium supplemented with 2,4-dichlorophenoxyacetic acid. Embryogenic callus differentiated into somatic embryos (SEs), most of which could germinate but developed abnormally. Friable embryogenic callus was induced mainly from the root regions of germinated primary SEs or regenerated plantlets on plant growth regulator-free medium. Histological studies showed that the embryogenic callus initiated from the subepidermal cells of young roots. The bioreactor system was more efficient than suspension culture regarding the number and growth of SEs, although a similar amount of embryonic tissue was used. An average of 115,370 germinated SEs developed from an initial 400?mg of embryogenic callus, and 64.7?% of germinated SEs converted into plantlets after a 4-weeks culture on agar medium. During the bioreactor culture process, secondary SEs were induced directly from SEs at various stages, a phenomenon that rarely occurred in suspension culture. These secondary SEs developed quickly and germinated during the bioreactor culture process. Proline content and peroxidase and catalase activities of SEs cultured in bioreactors were higher than in SEs cultured in suspension culture.     

4-Hydroxybenzyl alcohol accumulates in suspension-cell cultures and inhibits somatic embryogenesis in carrot

Somatic embryogenesis in carrot ( Daucus carota L.) is strongly inhibited by certain factors that accumulate in culture medium of high-density cultures of embryogenic cells. We previously identified 4-hydroxybenzyl alcohol (4HBA) as one of the inhibitory factors. In this study, we analyzed the accumulation pattern of 4HBA in the cultures of carrot suspension cells. When somatic embryogenesis was induced by culturing embryogenic cells in phytohormone-free Murashige and Skoog medium at various initial cell densities, 4HBA accumulated in the culture medium. The concentration of 4HBA in high cell density cultures was higher than in low cell density cultures. The accumulation of 4HBA in high cell density cultures was rapid during the early days of culture. This rapid accumulation of 4HBA in high cell density cultures might result in the strong inhibition of somatic embryogenesis. The production of 4HBA decreased as the somatic embryos developed. In addition, embryogenic cells released larger amount of 4HBA into the culture medium compared with non-embryogenic cells. These results suggest that the production of 4HBA is both related to embryogenic competence and developmentally regulated during somatic embryogenesis.