Suspension-cultured BY-2 Cells Research Articles

Production of the SARS-CoV-2 Spike protein and its Receptor Binding Domain in plant cell suspension cultures

The COVID-19 pandemic, caused by the worldwide spread of SARS-CoV-2, has prompted the scientific community to rapidly develop efficient and specific diagnostics and therapeutics. A number of avenues have been explored, including the manufacture of COVID-related proteins to be used as reagents for diagnostics or treatment. The production of RBD and Spike proteins was previously achieved in eukaryotic cells, mainly mammalian cell cultures, while the production in microbial systems has been unsuccessful until now. Here we report the effective production of SARS-CoV-2 proteins in two plant model systems. We established transgenic tobacco BY-2 and Medicago truncatula A17 cell suspension cultures stably producing the full-length Spike and RBD recombinant proteins. For both proteins, various glycoforms were obtained, with higher yields in Medicago cultures than BY-2. This work highlights that RBD and Spike can be secreted into the culture medium, which will impact subsequent purification and downstream processing costs. Analysis of the culture media indicated the presence of the high molecular weight Spike protein of SARS-CoV-2. Although the production yields still need improvement to compete with mammalian systems, this is the first report showing that plant cell suspension cultures are able to produce the high molecular weight Spike protein. This finding strengthens the potential of plant cell cultures as production platforms for large complex proteins.

Plant-Derived Cell-Free Biofactories for the Production of Secondary Metabolites.

Cell-free expression systems enable the production of proteins and metabolites within a few hours or days. Removing the cellular context while maintaining the protein biosynthesis apparatus provides an open system that allows metabolic pathways to be installed and optimized by expressing different numbers and combinations of enzymes. This facilitates the synthesis of secondary metabolites that are difficult to produce in cell-based systems because they are toxic to the host cell or immediately converted into downstream products. Recently, we developed a cell-free lysate derived from tobacco BY-2 cell suspension cultures for the production of recombinant proteins. This system is remarkably productive, achieving yields of up to 3 mg/mL in a one-pot in vitro transcription–translation reaction and contains highly active energy and cofactor regeneration pathways. Here, we demonstrate for the first time that the BY-2 cell-free lysate also allows the efficient production of several classes of secondary metabolites. As case studies, we synthesized lycopene, indigoidine, betanin, and betaxanthins, which are useful in the food, cosmetic, textile, and pharmaceutical industries. Production was achieved by the co-expression of up to three metabolic enzymes. For all four products, we achieved medium to high yields. However, the yield of betanin (555 μg/mL) was outstanding, exceeding the level reported in yeast cells by a factor of more than 30. Our results show that the BY-2 cell-free lysate is suitable not only for the verification and optimization of metabolic pathways, but also for the efficient production of small to medium quantities of secondary metabolites.

Uranium(VI) toxicity in tobacco BY-2 cell suspension culture – A physiological study

For the first time, the physiological and cellular responses of Nicotiana tabacum (BY-2) cells to uranium (U) as an abiotic stressor were studied using a multi-analytic approach that combined biochemical analysis, thermodynamic modeling and spectroscopic studies. The goal of this investigation was to determine the U threshold toxicity in tobacco BY-2 cells, the influence of U on the homeostasis of micro-macro essential nutrients, as well as the effect of Fe starvation on U bioassociation in cultured BY-2 cells. Our findings demonstrated that U interferes with the homeostasis of essential elements. The interaction of U with BY-2 cells confirmed both time- and concentration-dependent kinetics. Under Fe deficiency, a reduced level of U was detected in the cells compared to Fe-sufficient conditions. Interestingly, blocking the Ca channels with gadolinium chloride caused a decrease in U concentration in the BY-2 cells. Spectroscopic studies evidenced changes in the U speciation in the culture media with increasing exposure time under both Fe-sufficient and deficient conditions, leading us to conclude that different stress response reactions are related to Fe metabolism. Moreover, it is suggested that U toxicity in BY-2 cells is highly dependent on the existence of other micro-macro elements as shown by negative synergistic effects of U and Fe on cell viability.

The levels of peroxisomal catalase protein and activity modulate the onset of cell death in tobacco BY-2 cells via reactive oxygen species levels and autophagy.

In plant cells, peroxisomes participate in the metabolism of reactive oxygen species (ROS). One of the major regulators of cellular ROS levels - catalase (CAT) - occurs exclusively in peroxisomes. CAT activity is required for immunity-triggered autophagic programmed cell death (PCD). Autophagy has been recently demonstrated to represent a route for degradation of peroxisomes in plant cells. In the present study, the dynamics of the cellular peroxisome pool in tobacco BY-2 cell suspension cultures were used to analyse the effects of inhibition of basal autophagy with special attention to CAT activity. Numbers of peroxisomes per cell, levels of CAT protein and activity, cell viability, ROS levels and expression levels of genes encoding components of antioxidant system were analysed upon application of 3-methyladenine (3-MA), an inhibitor of autophagy, and/or aminotriazole (AT), an inhibitor of CAT. When applied separately, 3-MA and AT led to an increase in cell death, but this effect was attenuated by their simultaneous application. The obtained data suggest that both the levels of CAT protein in peroxisomes as well as CAT activity modulate the onset of cell death in tobacco BY-2 cells via ROS levels and autophagy.

In Vitro Formation of Plant RNA-Induced Silencing Complexes Using an Extract of Evacuolated Tobacco Protoplasts.

Small RNA-mediated gene silencing is involved in a variety of biological processes among many eukaryotic organisms. The silencing effector, generally referred to as RNA-induced silencing complex (RISC), comprises an ARGONAUTE (AGO) protein and a small single-stranded guide RNA in its core. RISCs recognize target genes containing sequences complementary to the guide RNA and repress their expression transcriptionally or posttranscriptionally. In vitro systems that recapitulate RISC assembly are useful not only to decipher the molecular mechanisms underlying the assembly process itself but also to dissect the downstream silencing pathways mediated by RISCs. Here, we describe a method for in vitro plant RISC assembly, which relies on an extract of evacuolated protoplasts derived from Nicotiana tabacum BY-2 suspension-cultured cells. In this extract, synthetic duplexes of small RNAs are incorporated into AGO proteins that are synthesized by in vitro translation, and then duplex unwinding and selective strand elimination result in formation of mature RISCs.

Primary Metabolism during Biosynthesis of Secondary Wall Polymers of Protoxylem Vessel Elements.

Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary wall deposition and programmed cell death. These processes are regulated by the VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco (Nicotiana tabacum) BY-2 suspension culture cells carrying an inducible VND7 system to liquid chromatography-mass spectrometry-based wide-target metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed dynamic changes in metabolites related to amino acid biosynthesis. The concentration of glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific amino acids accumulated, including the shikimate-related amino acids and the translocatable nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved the active up-regulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and phenylalanine biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation. Taken together, our results show that protoxylem vessel element differentiation is associated with changes in primary metabolism, which could facilitate the production of polysaccharides and lignin monomers and, thus, promote the formation of the secondary cell wall. Also, these metabolic shifts correlate with the active transcriptional regulation of specific enzyme genes. Therefore, our observations indicate that primary metabolism is actively regulated during protoxylem vessel element differentiation to alter the cell's metabolic activity for the biosynthesis of secondary wall polymers.

DNA hypomethylation concomitant with the overproduction of ROS induced by naphthoquinone juglone on tobacco BY-2 suspension cells

Juglone, 5-hydroxy-1,4-naphthoquinone, is a naturally occurring plant secondary metabolite with allelopathic and cytotoxic properties. The cytotoxic effect of juglone and changes at the level of DNA methylation were investigated on a plant cell model, tobacco BY-2 cell suspension culture. We found that juglone in a concentration-dependent manner inhibits cell growth and causes creation of reactive oxygen species (ROS) that were visible mainly in nucleus, nucleolus, cytoplasm, and plasma membrane. Analysis of the changes in DNA methylation level using Luminometric Methylation Assay showed that juglone at the concentrations of 60 and 80μM causes hypomethylation of DNA. Decrease in DNA methylation was confirmed by the more sensitive technique, confocal immunofluorescence microscopy, already at 30μM, and was accompanied by significant overproduction of hydrogen peroxide, hydroxyl radicals, and superoxide anion radicals. The highest percentage of the signs of programed cell death, especially apoptic-like bodies that indicate DNA fragmentation was observed at 30μM of juglone. On the basis of results we suggest that overproduction of ROS is the main mechanism that causes hypomethylation of DNA, which may contribute to the initiation of the programmed cell death.

Plant peroxisomes are degraded by starvation-induced and constitutive autophagy in tobacco BY-2 suspension-cultured cells.

Very recently, autophagy has been recognized as an important degradation pathway for quality control of peroxisomes in Arabidopsis plants. To further characterize the role of autophagy in plant peroxisome degradation, we generated stable transgenic suspension-cultured cell lines of heterotrophic Nicotiana tabacum L. cv. Bright Yellow 2 expressing a peroxisome-targeted version of enhanced yellow fluorescent protein. Indeed, this cell line model system proved advantageous for detailed cytological analyses of autophagy stages and for quantification of cellular peroxisome pools under different culturing conditions and upon inhibitor applications. Complementary biochemical, cytological, and pharmacological analyses provided convincing evidence for peroxisome degradation by bulk autophagy during carbohydrate starvation. This degradation was slowed down by the inhibitor of autophagy, 3-methyladenine (3-MA), but the 3-MA effect ceased at advanced stages of starvation, indicating that another degradation mechanism for peroxisomes might have taken over. 3-MA also caused an increase particularly in peroxisomal proteins and cellular peroxisome numbers when applied under nutrient-rich conditions in the logarithmic growth phase, suggesting a high turnover rate for peroxisomes by basal autophagy under non-stress conditions. Together, our data demonstrate that a great fraction of the peroxisome pool is subject to extensive autophagy-mediated turnover under both nutrient starvation and optimal growth conditions. Our analyses of the cellular pool size of peroxisomes provide a new tool for quantitative investigations of the role of plant peroxisomes in reactive oxygen species metabolism.

Cloning of a cDNA encoding the Gly m Bd 28K precursor and its vacuole transport in tobacco BY2 suspension-cultured cells.

Gly m Bd 28K (Gm28K), a soybean allergen, is formed as a preproprotein consisting of a predicted signal peptide, Gm28K, and the 23-kDa peptide (Gm23K). Gm28K and Gm23K are found in the protein-storage vacuoles (PSVs) of developing soybean seeds. However, the complete structure of Gm28K has not yet been identified and its processing and transport to the vacuoles has never been clarified. In the present study, we elucidated the 5'-nucleotide sequence of cDNA encoding the Gm28K precursor and identified a putative signal peptide (SP) with 24 N-terminal amino acid residues. We expressed peptides from the Gm28K precursor as fusion proteins with enhanced green fluorescent protein (EGFP) in tobacco BY2 suspension-cultured cells. BY2 cells transformed by an expression vector for SP-EGFP-Gm28-Gm23K (SP-EGFP-Gm28-Gm23K/BY2 cells) and SP-Gm28-Gm23K-EGFP/BY2 cells produced the EGFP fused-Gm28K precursor, and the EGFP-fluorescence in their vacuoles were recorded. In the experiments with SP-EGFP/BY2 and SP-EGFP-Gm28K/BY2 cells, large amounts of the EGFP segments were secreted into the medium. On the other hand, the fluorescence of EGFP in SP-EGFP-Gm23K/BY2 cells was shown to accumulate only in the endoplasmic reticulum without secretion into the medium. These findings show that the SP signals the precursor to enter the lumen of the endoplasmic reticulum and that both the Gm28K and Gm23K components may be involved in the transport from the endoplasmic reticulum (ER) lumen via the Golgi to the vacuoles in a proprotein form.

Correlation between mass transfer coefficient kLa and relevant operating parameters in cylindrical disposable shaken bioreactors on a bench-to-pilot scale

BackgroundAmong disposable bioreactor systems, cylindrical orbitally shaken bioreactors show important advantages. They provide a well-defined hydrodynamic flow combined with excellent mixing and oxygen transfer for mammalian and plant cell cultivations. Since there is no known universal correlation between the volumetric mass transfer coefficient for oxygen kLa and relevant operating parameters in such bioreactor systems, the aim of this current study is to experimentally determine a universal kLa correlation.ResultsA Respiration Activity Monitoring System (RAMOS) was used to measure kLa values in cylindrical disposable shaken bioreactors and Buckingham’s π-Theorem was applied to define a dimensionless equation for kLa. In this way, a scale- and volume-independent kLa correlation was developed and validated in bioreactors with volumes from 2 L to 200 L. The final correlation was used to calculate cultivation parameters at different scales to allow a sufficient oxygen supply of tobacco BY-2 cell suspension cultures.ConclusionThe resulting equation can be universally applied to calculate the mass transfer coefficient for any of seven relevant cultivation parameters such as the reactor diameter, the shaking frequency, the filling volume, the viscosity, the oxygen diffusion coefficient, the gravitational acceleration or the shaking diameter within an accuracy range of +/− 30%. To our knowledge, this is the first kLa correlation that has been defined and validated for the cited bioreactor system on a bench-to-pilot scale.

Biochemical and physiological characterization of fut4 and fut6 mutants defective in arabinogalactan-protein fucosylation in Arabidopsis

Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline-rich glycoproteins present in plant cell walls. AGPs are characterized by arabinose-/galactose-rich side chains, which define their interactive molecular surface. Fucose residues are found in some dicotyledon AGPs, and AGP fucosylation is developmentally regulated. We previously identified Arabidopsis thaliana FUT4 and FUT6 genes as AGP-specific fucosyltransferases (FUTs) based on their enzymatic activities when heterologously expressed in tobacco (Nicotiana tabacum) BY2 suspension-cultured cells. Here, the functions of FUT4 and FUT6 and the physiological roles of fucosylated AGPs were further investigated using Arabidopsis fut4, fut6, and fut4/fut6 mutant plants. All mutant plants showed no phenotypic differences compared to wild-type plants under physiological conditions, but showed reduced root growth in the presence of elevated NaCl. However, roots of wild-type and fut4 mutant plants contained terminal fucose epitopes, which were absent in fut6 and fut4/fut6 mutant plants as indicated by eel lectin staining. Monosaccharide analysis showed fucose was present in wild-type leaf and root AGPs, but absent in fut4 leaf AGPs and in fut4/fut6 double mutant leaf and root AGPs, indicating that FUT4 was required for fucosylation of leaf AGPs while both FUT4 and FUT6 contributed to fucosylation of root AGPs. Glycome profiling of cell wall fractions from mutant roots and leaves showed distinct glycome profiles compared to wild-type plants, indicating that fucosyl residues on AGPs may regulate intermolecular interactions between AGPs and other wall components. The current work exemplifies the possibilities of refinement of cell wall structures by manipulation of a single or a few cell wall biosynthetic genes.

Toxicity of aluminium oxide nanoparticles demonstrated using a BY-2 plant cell suspension culture model

Aluminium oxide nanoparticles have been applied in many branches of industry. They are also used in personal care products, such as cosmetics. Because of these uses, their impact on the environment must be considered and investigated. Almost nothing is known about the effects of aluminium oxide nanoparticles on plants at the cellular level; the objective of this work was thus to study the effects of nanoparticles on the plant cell model tobacco BY-2 cell suspension culture, which serves as a model comparable with the HeLa cells used for animal cell studies. We observed the impact of these nanoparticles at different levels. The inhibitory effect on growth was observed in both time- and concentration-dependent studies. In addition, the ability of the nanoparticles to generate reactive oxygen (hydrogen peroxide, superoxide anion radical) and nitrogen species (nitric oxide) has been established. The principal part of the work was focused on the ability of aluminium oxide nanoparticles to induce the processes of programmed cell death. Changes observed in the permeability of the plasma membrane are connected with the effects of the reactive oxygen species and lipid peroxidation. In addition, the loss of mitochondrial potential, the enhancement of the caspase-like activity and the fragmentation of DNA determined in both time- and concentration dependent studies are closely connected with the execution of the programmed cell death. Our results indicate the ability of aluminium oxide nanoparticles to induce programmed cell death in plant cells and may explain the toxic effect of these nanoparticles on plants.

Effect of magnetic nanoparticles on tobacco BY-2 cell suspension culture.

Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles), electronics (high-resolution imaging, logical circuits on the molecular level) and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases) or imaging (contrast agents). Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs) and modified magnetic nanoparticles (MNPs) on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis—total protein content, thiols—reduced (GSH) and oxidized (GSSG) glutathione, phytochelatins PC2-5, glutathione S-transferase (GST) activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension culture. The obtained results are discussed, especially in connection with already published data. Possible mechanisms of NPs’ and MNPs’ toxicity are introduced.

Effect of fluoranthene on plant cell model: Tobacco BY-2 suspension culture

Polycyclic aromatic hydrocarbons (PAHs) belong to the group of the most important pollutants of the living environment, which are present in air, soils, freshwater, seawater and sediments. They have very substantial effects on all living organisms including plants and animals. Plants represent important point in PAHs food chain entry. Despite the fact that PAHs influence on animals is intensively studied, effect on plants is almost unknown. In our study, action of polycyclic aromatic hydrocarbon fluoranthene on a plant cell experimental model – tobacco BY-2 cell suspension culture – was studied. BY-2 cells were exposed to the fluoranthene in concentration range from 0 to 1000μM, duration of treatment was 120h, respectively 5 days. Samples were collected in the strictly defined time intervals of 24h. Exposure of the BY-2 cells led to significant changes in viability, changes in autofluorescence due to accumulation of fluoranthene in lipophilic cell compartments, especially biomembranes, and production of reactive oxygen species, which resulted in damage of biomembranes and disruption of their semipermeability and initiation of process of programmed cell death. Obtained results bring new knowledge about phytotoxicity of fluoranthene and mechanism of its action.

Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells

Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells.

Role of α-tocopherol in cellular signaling: α-tocopherol inhibits stress-induced mitogen-activated protein kinase activation

Tocopherols belong to the plant-derived poly phenolic compounds known for antioxidant functions in plants and animals. Activation of mitogen-activated protein kinases (MAPK) is a common reaction of plant cells in defense-related signal transduction pathways. We report a novel non-antioxidant function of α-tocopherol in higher plants linking the physiological role of tocopherol with stress signalling pathways. Pre-incubation of a low concentration of 50 μM α-tocopherol negatively interferes with MAPK activation in elicitor-treated tobacco BY2 suspension culture cells and wounded tobacco leaves, whereas pre-incubated BY2 cells with α-tocopherol phosphate did not show the inhibitory effect on stimuli-induced MAPK activation. The decreased MAPK activity was neither due to a direct inhibitory effect of α-tocopherol nor due to the induction of an inhibitory or inactivating activity directly affecting MAPK activity. The data support that the target of α-tocopherol negatively regulates an upstream component of the signaling pathways that leads to stress dependent MAPK activation.

Cell proliferation, cell shape, and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March–April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.

Microinjecting FM4–64 validates it as a marker of the endocytic pathway in plants

The amphiphilic dye FM4-64 is used to investigate endocytosis and vesicle trafficking in living eukaryotic cells. The standing hypothesis is that it is inserted into the outer leaflet of the plasma membrane and, from there, is passed on to intracellular membrane compartments by endocytosis. We tested this hypothesis by microinjecting FM4-64 into the cytoplasm and vacuole of Nicotiana tabacum BY-2 suspension culture cells and Tradescantia virginiana stamen hair cells. We found that the dye did not label any membranes when injected into the cytoplasm, but clearly labelled the tonoplast when injected directly into the vacuole. However, because the dye is pH-sensitive, the fluorescence intensity between the plasma membrane and tonoplast varied. We conclude that FM4-64 is a specific marker for the endocytic pathway. Nevertheless, little is known about the molecular interactions of FM4-64 with these particular phospholipid membrane leaflets. We, therefore, appeal for biochemical research to determine which membrane lipids FM4-64 interacts with.

Cadmium and zinc-mediated oxidative burst in tobacco BY-2 cell suspension cultures

Generation of reactive oxygen species (ROS) constitutes an important first reaction under many stress conditions in plants. We demonstrate that Nicotiana tabacum L. cv. Bright Yellow 2 (TBY-2) cells in suspension cultures, generate superoxide radical and hydrogen peroxide upon treatment with cadmium and zinc. Addition of catalase and N,N-diethyldithiocarbamate (DDC) decreased the level of H2O2, whereas superoxide dismutase (SOD) induced a slight increase of the H2O2 production. The effects of catalase, DDC and SOD on the heavy metal-induced ROS production indicate that it occurs outside of the cells, and that at least part of the hydrogen peroxide is produced by dismutation of the superoxide radical (O2·−). The effect of pretreatment of the cell cultures with commonly used mammalian NADPH oxidase inhibitors was also tested. Strong inhibitions of cadmium and zinc-mediated ROS production were obtained with the flavoprotein inhibitors—diphenylene iodonium (DPI) and quinacrine and with an inhibitor of b-type cytochromes—imidazol. Membrane permeable-N-ethyl maleimide (NEM) and iodoacetate, and membrane non-permeable thiol reagents—para-chloromercuribenzoic acid (pCMBS) also inhibited the ROS production. These results suggested that the enzyme responsible for cadmium and zinc-induced ROS production in tobacco cells contains a flavocytochrome. They also show the importance of intra- and extracellular thiol groups in the observed stress reaction. The induction of ROS production with heavy metals showed properties comparable to the elicitor-induced oxidative burst in other plant cells.

The tobacco Ntann12 gene, encoding an annexin, is induced upon Rhodoccocus fascians infection and during leafy gall development

SUMMARY Annexins are calcium-binding proteins that have been associated in plants with different biological processes such as responses to abiotic stress and early nodulation stages. Until now, the implication of annexins during plant-pathogen interactions has not been reported. Here, a novel plant annexin gene induced in tobacco BY-2 cell suspension cultures infected with the phytopathogenic bacterium Rhodococcus fascians (strain D188) has been identified. Expression of this gene, called Ntann12, is also induced, but to a lower extent, by a strain (D188-5) that is unable to induce leafy gall formation. This gene was also induced in BY-2 cells infected with Pseudomonas syringae but not in cells infected with Agrobacterium tumefaciens or Escherichia coli. Ntann12 expression was also found to be stimulated by abiotic stress, including NaCl and abscissic acid, confirming a putative role in stress signal transduction pathways. In addition, promoter-GUS analyses using homozygous transgenic tobacco seedlings showed that the developmentally controlled expression of Ntann12 is altered upon R. fascians infection. Finally, up-regulation of Ntann12 during leafy gall ontogenesis was confirmed by RT-qPCR. Discussion is focused on the potential role of Ntann12 in biotic and abiotic stress responses and in plant development, both processes that may involve Ca(2+)-dependent signalling.