Tobacco BY-2 Research Articles

P-393 - Is autophagy involved in the response to oxidative stress of tobacco BY-2 cells overexpressing thioredoxin o1?

Autophagy is a catabolic process for cellular remodeling and macromolecular recycling of cytoplasmic components and organelles. Interestingly, autophagy appears as a key mechanism to cope with adverse conditions by removing damaged cellular components, although less information exists about this process in plants. In this sense, little is known about the possible role of redox regulation through thiol oxidoreductases thioredoxins (Trxs), specifically the mitochondrial/nuclear Trxo1. We have previously described a protective role of pea thioredoxin (PsTrxo1) overexpressed in TBY-2 cells treated with H2O2, thus increasing their viability via antioxidants, delaying cell death. In this work, we analyze the possible implication of autophagy as a protective mechanism operating in the PsTrxo1 transformed TBY-2 cells. For this, together with cell viability, different markers such as autophagy-related proteins ATGs are analyzed by Western blot, in parallel to autolysosomes and autophagosomes formation by fluorescence microscopy. The results have shown significant changes in ATG4 and ATG8 contents as well as presence of both lytic structures in H2O2-treated overexpressing PsTrxo1 cells, which suggest that Trxo1 may be also involved in the redox control of some autophagy components.

Grapevine fatty acid hydroperoxide lyase generates actin-disrupting volatiles and promotes defence-related cell death.

Fatty acid hydroperoxides can generate short-chained volatile aldehydes that may participate in plant defence. A grapevine hydroperoxide lyase (VvHPL1) clustering to the CYP74B class was functionally characterized with respect to a role in defence. In grapevine leaves, transcripts of this gene accumulated rapidly to high abundance in response to wounding. Cellular functions of VvHPL1 were investigated upon heterologous expression in tobacco BY-2 cells. A C-terminal green fluorescent protein (GFP) fusion of VvHPL1 was located in plastids. The overexpression lines were found to respond to salinity stress or the bacterial elicitor harpin by increasing cell death. This signal-dependent mortality response was mitigated either by addition of exogenous jasmonic acid or by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases. By feeding different substrates to recombinantly expressed enzyme, VvHPL1 could also be functionally classified as true 13-HPL. The cognate products generated by this 13-HPL were cis-3-hexenal and trans-2-hexenal. Using a GFP-tagged actin marker line, one of these isomeric products, cis-3-hexenal, was found specifically to elicit a rapid disintegration of actin filaments. This response was not only observed in the heterologous system (tobacco BY-2), but also in a grapevine cell strain expressing this marker, as well as in leaf discs from an actin marker grape used as a homologous system. These results are discussed in the context of a role for VvHPL1 in a lipoxygenase-dependent signalling pathway triggering cell death-related defence that bifurcates from jasmonate-dependent basal immunity.

Proteomic analysis of xylem vessel cell differentiation in VND7-inducible tobacco BY-2 cells by two-dimensional gel electrophoresis.

The xylem vessel is an essential structure for water conduction in vascular plants. Xylem vessel cells deposit thick secondary cell walls and undergo programmed cell death, to function as water-conducting elements. Since the discovery of the plant-specific NAC domain-type VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors, which function as master switches of xylem vessel cell differentiation in Arabidopsis, much has been learned about the transcriptional regulatory network of xylem vessel cell differentiation. However, little is known about proteome dynamics during xylem vessel cell differentiation. Here, we performed two-dimensional electrophoresis-based proteomic analysis of xylem vessel cell differentiation using a transgenic tobacco BY-2 cell line carrying the VND7-inducible system (BY-2/35S::VND7-VP16-GR), in which synchronous trans-differentiation into xylem vessel cells can be induced by the application of a glucocorticoid. Of the 47 spots revealed by gel electrophoresis, we successfully identified 40 proteins. Seventeen proteins, including several well-characterized proteins such as a cysteine protease and serine carboxypeptidase (involved in programmed cell death), were upregulated after 24 h of induction. However, previous transcriptomic analysis showed that only eight of these proteins are upregulated at the transcriptional level during xylem vessel cell differentiation in BY-2/35S::VND7-VP16-GR cells. These findings suggest that post-transcriptional regulation strongly affects proteomic dynamics during xylem vessel cell differentiation.

Silver ions increase plasma membrane permeability through modulation of intracellular calcium levels in tobacco BY-2 cells.

Silver ions increase plasma membrane permeability for water and small organic compounds through their stimulatory effect on plasma membrane calcium channels, with subsequent modulation of intracellular calcium levels and ion homeostasis. The action of silver ions at the plant plasma membrane is largely connected with the inhibition of ethylene signalling thanks to the ability of silver ion to replace the copper cofactor in the ethylene receptor. A link coupling the action of silver ions and cellular auxin efflux has been suggested earlier by their possible direct interaction with auxin efflux carriers or by influencing plasma membrane permeability. Using tobacco BY-2 cells, we demonstrate here that besides adramatic increase of efflux of synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthalene acetic acid (NAA), treatment with AgNO3 resulted in enhanced efflux of the cytokinin trans-zeatin (tZ) as well as the auxin structural analogues tryptophan (Trp) and benzoic acid (BA). The application of AgNO3 was accompanied by gradual water loss and plasmolysis. The observed effects were dependent on the availability of extracellular calcium ions (Ca2+) as shown by comparison of transport assays in Ca2+-rich and Ca2+-free buffers and upon treatment with inhibitors of plasma membrane Ca2+-permeable channels Al3+ and ruthenium red, both abolishing the effect of AgNO3. Confocal microscopy of Ca2+-sensitive fluorescence indicator Fluo-4FF, acetoxymethyl (AM) ester suggested that the extracellular Ca2+ availability is necessary to trigger the response to silver ions and that the intracellular Ca2+ pool alone is not sufficient for this effect. Altogether, our data suggest that in plant cells the effects of silver ions originate from the primal modification of the internal calcium levels, possibly by their interaction with Ca2+-permeable channels at the plasma membrane.

A peptidogalactomannan isolated from Cladosporium herbarum induces defense-related genes in BY-2 tobacco cells

Cladosporium herbarum is a plant pathogen associated with passion fruit scab and mild diseases in pea and soybean. In this study, a peptidogalactomannan (pGM) of C. herbarum mycelium was isolated and structurally characterized, and its role in plant-fungus interactions was evaluated. C. herbarum pGM is composed of carbohydrates (76%) and contains mannose, galactose and glucose as its main monosaccharides (molar ratio, 52:36:12). Methylation and 13C-nuclear magnetic resonance (13C-NMR) spectroscopy analysis have shown the presence of a main chain containing (1 → 6)-linked α-D-Manp residues, and β-D-Galf residues are present as (1 → 5)-interlinked side chains. β-Galactofuranose containing similar structures were characterized by our group in A. fumigatus, A. versicolor, A. flavus and C. resinae.Tobacco BY-2 cells were used as a model system to address the question of the role of C. herbarum pGM in cell viability and induction of the expression of plant defense-related genes. Native and partially acid hydrolyzed pGMs (lacking galactofuranosyl side-chain residues) were incubated with BY-2 cell suspensions at different concentrations. Cell viability drastically decreased after exposure to more than 400 μg ml−1 pGM; however no cell viability effect was observed after exposure to a partially acid hydrolyzed pGM. BY-2 cell contact with pGM strongly induce the expression of plant defense-related genes, such as phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX), as well as the pathogen-related PR-1a, PR-2 and PR-3 genes, suggesting that pGM activates defense responses in tobacco cells. Interestingly, contact with partially hydrolyzed pGM also induced defense-related gene expression at earlier times than native pGM. These results show that the side chains of the (1 → 5)-linked β-D-galactofuranosyl units from pGM play an important role in the first line fungus-plant interactions mediating plant responses against C. herbarum. In addition, it was observed that pGM and/or C. herbarum conidia are able to induced HR when in contact with tobacco leaves and in vitro plantlets roots, producing necrotic lesions and peroxidase and NO burst, respectively.

Role of cerium oxide nanoparticle-induced autophagy as a safeguard to exogenous H2O2-mediated DNA damage in tobacco BY-2 cells.

The effect of cerium oxide nanoparticle (CeNP) in plants has elicited substantial controversy. While some investigators have reported that CeNP possesses antioxidant properties, others observed CeNP to induce reactive oxygen species (ROS). In spite of considerable research carried out on the effects of CeNP in metazoans, fundamental studies that can unveil its intracellular consequences linking ROS production, autophagy and DNA damage are lacking in plants. To elucidate the impact of CeNP within plant cells, tobacco BY-2 cells were treated with 10, 50 and 250 µg ml-1 CeNP (Ce10, Ce50 and Ce250), for 24 h. Results demonstrated concentration-dependent accumulation of Ca2+ and ROS at all CeNP treatment sets. However, significant DNA damage and alteration in antioxidant defence systems were noted prominently at Ce50 and Ce250. Moreover, Ce50 and Ce250 induced DNA damage, analysed by comet assay and DNA diffusion experiments, complied with the concomitant increase in ROS. Furthermore, to evaluate the antioxidant property of CeNP, treated cells were washed after 24 h (to minimise CeNP interference) and challenged with H2O2 for 3 h. Ce10 did not induce genotoxicity and H2O2 exposure to Ce10-treated cells showed lesser DNA breakage than cells treated with H2O2 only. Interestingly, Ce10 provided better protection over N-acetyl-L-cysteine against exogenous H2O2 in BY-2 cells. CeNP exposure to transgenic BY-2 cells expressing GFP-Atg8 fusion protein exhibited formation of autophagosomes at Ce10. Application of vacuolar protease inhibitor E-64c and fluorescent basic dye acridine orange, further demonstrated accumulation of particulate matters in the vacuole and occurrence of acidic compartments, the autophagolysosomes, respectively. BY-2 cells co-treated with CeNP and autophagy inhibitor 3-methyladenine exhibited increased DNA damage in Ce10 and cell death at all assessed treatment sets. Thus, current results substantiate an alternative autophagy-mediated, antioxidant and geno-protective role of CeNP, which will aid in deciphering novel phenomena of plant-nanoparticle interaction at cellular level.

A rice class-XIV kinesin enters the nucleus in response to cold

Higher plants possess a large number of kinesins, but lack the minus-end directed dynein motors. However, the kinesin class XIV has strongly expanded, and minus-end directed motors from this class may have taken over functions of cytoplasmic dyneins. In this study, we address the functional aspects of a novel rice homologue of the Arabidopsis class-XIV kinesins ATK1 and ATK5. Since a loss-of-function rice mutant of this kinesin is not viable, the function was studied in tobacco BY-2 as heterologous system. OsDLK-GFP stably expressed in BY-2 cells decorates cortical microtubules, but also can shift into the nucleus of interphase cells. Because of this peculiar localisation, we coined the name Dual Localisation Kinesin (DLK). The nuclear import of this protein is strongly and reversibly promoted in response to cold. During mitosis, OsDLK is repartitioned between spindle and phragmoplast. Motility assays in vitro using show that OsDLK can convey mutual sliding of microtubules and moves at a velocity comparable to other class-XIV kinesins. When tobacco cells overexpressing OsDLK are synchronised, they exhibit a delayed entry into metaphase, while the later phases of mitosis are accelerated. The data are discussed in relation to additional functions of this kinesin type, beyond their transport along microtubules.

78. タバコ培養細胞 BY-2 を用いた新規植物免疫活性化剤と抑制剤の探索と解析(口頭発表,植物化学調節学会第48回大会)

78. タバコ培養細胞 BY-2 を用いた新規植物免疫活性化剤と抑制剤の探索と解析(口頭発表,植物化学調節学会第48回大会)

Gene expression analysis, subcellular localization, and in planta antimicrobial activity of rice (Oryza sativa L.) defensin 7 and 8

Defensins are a group of plant antimicrobial peptides. In a previous study, it was reported that two recombinant rice (Oryza sativa L.) defensin (OsDEF) genes (OsDEF7 and OsDEF8) produced heterologously by bacteria inhibited the growth of several phytopathogen. Here, we analyzed gene expression patterns in Thai jasmine rice (O. sativa L. ssp. indica ‘KDML 105’) using quantitative reverse transcription-polymerase chain reaction and compared them with those in Japanese rice (O. sativa L. ssp. japonica ‘Nipponbare’). Although the cultivars exhibited similar gene expression patterns at the developmental stages examined, the expression levels differed between organs. Upon Xanthomonas oryzae pv. oryzae infection in the leaves, both OsDEFs were highly upregulated at 8 days post-infection, suggesting that they play a role in pathogen defense. Moreover, in silico analyses revealed that OsDEF expression levels were affected by drought, cold, imbibition, anoxia, and dehydration stress. Using green fluorescent protein (GFP) fusions, we found that both OsDEFs were in the extracellular compartment, confirming their functions against pathogen infection. However, when recombinant OsDEFs (without GFP) were produced in tobacco BY-2 cells or Nicotiana benthamiana leaves, they could not be detected in either the culture medium or the cells. Yet, N. benthamiana leaves infiltrated with OsDEF7 or OsDEF8 constructs exhibited in planta inhibitory activity against the phytopathogen Xanthomonas campestris pv. glycines, suggesting that recombinant OsDEFs were present. Additionally, when targeting them to the ER compartment, recombinant OsDEFs could be detected. Lower inhibitory activity was observed when recombinant OsDEFs were targeted to the ER. These results suggest that OsDEFs play a role in controlling plant diseases.

The Golgi entry core compartment functions as a COPII-independent scaffold for ER-to-Golgi transport in plant cells.

Many questions remain about how the stacked structure of the Golgi is formed and maintained. In our previous study, we challenged this question using tobacco BY-2 cells and revealed that, upon Brefeldin A (BFA) treatment, previously undescribed small punctate structures containing a particular subset of cis-Golgi proteins are formed adjacent to the ER-exit sites and act as scaffolds for Golgi regeneration after BFA removal. In this study, we analyzed these structures further. The proteins that localize to these punctate structures originate from the cis-most cisternae. 3D time-lapse observations show that the trans-Golgi marker is transported through these structures during Golgi regeneration. These data indicate that the cis-most cisternae have a specialized region that receives cargo from the ER, which becomes obvious upon BFA treatment. Expression of a dominant mutant form of SAR1 does not affect the formation of the punctate structures. We propose to call these punctate structures the 'Golgi entry core compartment' (GECCO). They act as receivers for the rest of the Golgi materials and are formed independently of the COPII machinery.This article has an associated First Person interview with the first author of the paper.

High Level Production of δ-Guaiene, a Bicyclic Sesquiterpene Accumulated in Agarwood, by Co-expression of δ-Guaiene Synthase and Farnesyl Diphosphate Synthase Genes in Tobacco BY-2 Cells

Two genes encoding δ-guaiene synthase ( GS) and farnesyl diphosphate synthase ( FPS) involved in δ-guaiene biosynthesis in Aquilaria microcarpa were co-expressed in tobacco ( Nicotiana tabacum) BY-2 cells by Agrobacterium -mediated transformation. GC-MS analysis revealed that the transformed tobacco cells liberated δ-guaiene, and the compound was found in the headspace of the culture but not accumulated either in the medium or in the cells. Tobacco cells transformed by solely GS produced 0.2 mg δ-guaiene /L culture, however, concentration of the compound elevated to 0.6 – 5.9 mg/L when GS and FPS were co-expressed in the cells. The stirring efficiency of the cell suspension was improved by the reduction of the culture volume in the vials, and this resulted in an appreciable increase in δ-guaiene content to the level of 102 mg/L culture. Addition of mevalonolactone as the precursor markedly activated δ-guaiene production, and content of the compound elevated to more than 400 mg/L culture. These results strongly suggested that tobacco BY-2 is a suitable host to construct the bio-production system of sesquiterpene compounds, and co-expression of FPS and appropriate sesquiterpene synthase genes in the cells should be the good strategy to establish the highly productive platform of this class of compounds.

Phenotype-Based Screening of Small Molecules to Modify Plant Cell Walls Using BY-2 Cells.

The plant cell wall is an important and abundant biomass with great potential for use as a modern recyclable resource. For effective utilization of this cellulosic biomass, its ability to degrade efficiently is key point. With the aim of modifying the cell wall to allow easy decomposition, we used chemical biological technology to alter its structure. As a first step toward evaluating the chemicals in the cell wall we employed a phenotype-based approach using high-throughput screening. As the plant cell wall is essential in determining cell morphology, phenotype-based screening is particularly effective in identifying compounds that bring about alterations in the cell wall. For rapid and reproducible screening, tobacco BY-2 cell is an excellent system in which to observe cell morphology. In this chapter, we provide a detailed chemical biological methodology for studying cell morphology using tobacco BY-2 cells.

Ethyl Gallate Displays Elicitor Activities in Tobacco Plants.

Alkyl gallates showed elicitor activities on tobacco in both whole plants and cell suspensions. Methyl gallate (MG), ethyl gallate (EG), and propyl gallate (PG) infiltration into tobacco leaves induced hypersensitive reaction-like lesions and topical production of autofluorescent compounds revealed under UV light. When sprayed on tobacco plants at 5 mM, EG promoted upregulation of defense-related genes such as the antimicrobial PR1, β-1,3-glucanase PR2, Chitinase PR3, and osmotin PR5 target genes. Tobacco BY-2 cells challenged with EG underwent cell death in 48 h, which was significantly reduced in the presence of the protease inhibitor aprotinin. The three alkyl gallates all caused alkalinization of the BY-2 extracellular medium, whereas gallic acid did not trigger any pH variation. Using EGTA or LaCl3, we showed that Ca2+ mobilization occurred in BY-2 cells elicited with EG. Overall, our findings are the first evidence of alkyl gallate elicitor properties with early perception events on the plasma membrane, potential hypersensitive reactions, and PR-related downstream defense responses in tobacco.

Influence of boron deprivation on Hg stress-induced programmed cell death in suspension-cultured tobacco BY-2 cells

ABSTRACTSome agricultural regions in large areas deficient in Boron (B) also present a risk of contamination of soils with excess mercury (Hg). The purpose of this study was to explore whether Hg stress induces programmed cell death (PCD) in tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow-2) cells and the influence of B deprivation on mercury (Hg) stress-induced PCD. It was shown that the BY-2 cells exposed to 30 μM mercury chloride (HgCl2) underwent PCDand in the meantime, the expression changes of PCD-related genes were found. Meanwhile, Hg stress in combination with B deprivation can cause much higher proportion of cells to undergo PCD than Hg stress alone. Accompanied with PCD, the HgCl2-stressed cells under B deprivation accumulated more H2O2 and lipid peroxides than control cells. Our results suggest that HgCl2 can induce PCD in tobacco BY-2 cells, that B deficiency would enhance this effect, and oxidative damage is directly involved in the PCD process.

CONTROL OF FIRE BLIGHT INFECTIONS WITH SYNTHETIC PEPTIDES THAT ELICIT PLANT DEFENSE RESPONSES

Antimicrobial peptides (AMPs) are reliable compounds to develop strategies for integrated plant disease management. These peptides can act directly against the pathogens and/or induce plant defense responses. In this work, 33 sequences including linear and cyclic peptides, linear and cyclic peptidotriazoles, and cyclic lipopeptides were tested for induction of plant defense. A first screening was carried out with BY2 tobacco cell cultures by determining the alkalinization and production of hydrogen peroxide. Peptides BP13, BP100, BP143 and BPC200W were slightly positive in these assays and were tested by RT-qPCR for expression of 10 defense-related genes in tomato, which are representative of the jasmonic acid, salicylic acid and ethylene pathways. BP13 and BPC200W induced overexpression of several genes that are also overexpressed by reference plant defense peptide elicitors. Plant assays with potted pear plants in the greenhouse confirmed the effect of peptides BP13 and BPC200W in controlling Erwinia amylovora infections, putatively through plant defense induction.

Effect of acibenzolar-S-methyl phototransformation on its elicitation activity in tobacco cells

The plant activator acibenzolar-S-methyl (BTH) undergoes phototransformation when exposed to solar radiation. Here we investigated the changes in its elicitation properties on BY-2 tobacco cells at different stages of the photochemical reaction. Both pure BTH and formulated BTH were irradiated in controlled conditions to achieve different extents of conversion. Both pure BTH (900 μM) and Bion® (0.4 g.L−1) induced BY-2 cell death, but BTH photoconverted to an extent of 25 ± 3% lowered the cell death rate. A kinetic study of β-1,3-glucanase and chitinase activities was conducted on BY-2 extracellular medium. Exposure of tobacco cells to either pure BTH or Bion® resulted in a significant increase in the activities of both defense enzymes, which peaked 48 h after the treatment. The pathogenesis-related (PR) protein activities were quantified 48 h after elicitation for a range of phototransformed BTH solutions. The enzyme activities were reduced when BY-2 cells were treated with solutions in which BTH conversion was 22 ± 3%, 42 ± 3% and 100 ± 3%, but were not affected by the solution in which BTH was phototransformed at 60%, suggesting that some of the secondary photoproducts also exhibit eliciting properties. Solar irradiation of BTH thus impairs its elicitation properties, but this impairment depends strongly on the extent of phototransformation.

Arabinogalactan Proteins Are Involved in Salt-Adaptation and Vesicle Trafficking in Tobacco by-2 Cell Cultures.

Arabinogalactan proteins (AGPs) are a highly diverse family of glycoproteins that are commonly found in most plant species. However, little is known about the physiological and molecular mechanisms of their function. AGPs are involved in different biological processes such as cell differentiation, cell expansion, tissue development and somatic embryogenesis. AGPs are also involved in abiotic stress response such as salinity modulating cell wall expansion. In this study, we describe how salt-adaptation in tobacco BY-2 cell cultures induces important changes in arabinogalactan proteins distribution and contents. Using the immuno-dot blot technique with different anti-AGP antibodies (JIM13, JIM15, and others), we observed that AGPs were highly accumulated in the culture medium of salt-adapted tobacco cells, probably due to the action of phospholipases. We located these AGP epitopes using immunogold labeling in the cytoplasm associated to the endoplasmic reticulum, the golgi apparatus, and vesicles, plasma membrane and tonoplast. Our results show that salt-adaptation induced a significant reduction of the cytoplasm, plasma membrane and tonoplast content of these epitopes. Yariv reagent was added to the control and salt-adapted tobacco cell cultures, leading to cell death induction in control cells but not in salt-adapted cells. Ultrastructural and immunogold labeling revealed that cell death induced by Yariv reagent in control cells was due to the interaction of Yariv reagent with the AGPs linked to the plasma membranes. Finally, we propose a new function of AGPs as a possible sodium carrier through the mechanism of vesicle trafficking from the apoplast to the vacuoles in salt-adapted tobacco BY-2 cells. This mechanism may contribute to sodium homeostasis during salt-adaptation to high saline concentrations.

Dynamics of tobacco DNA topoisomerases II in cell cycle regulation: to manage topological constrains during replication, transcription and mitotic chromosome condensation and segregation.

The topoisomerase II expression varies as a function of cell proliferation. Maximal topoisomerase II expression was tightly coupled to S phase and G2/M phase via both transcriptional and post-transcriptional regulation. Investigation in meiosis using pollen mother cells also revealed that it is not the major component of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed. Synchronized tobacco BY-2 cell cultures were used to study the role of topoisomerase II in various stages of the cell cycle. Topoisomerase II transcript accumulation was observed during the S- and G2/M- phase of cell cycle. This biphasic expression pattern indicates the active requirement of topoisomerase II during these stages of the cell cycle. Through immuno-localization of topoisomerase II was observed diffusely throughout the nucleoplasm in interphase nuclei, whereas, the nucleolus region exhibited a more prominent immuno-positive staining that correlated with rRNA transcription, as shown by propidium iodide staining and BrUTP incorporation. The immuno-staining analysis also showed that topoisomerase II is the major component of mitotic chromosomes and remain attached to the chromosomes during cell division. The inhibition of topoisomerase II activity using specific inhibitors revealed quite dramatic effect on condensation of chromatin and chromosome individualization from prophase to metaphase transition. Partially condensed chromosomes were not arranged on metaphase plate and chromosomal perturbations were observed when advance to anaphase, suggesting the importance of topoisomerase II activity for proper chromosome condensation and segregation during mitosis. Contrary, topoisomerase II is not the major component of meiotic chromosomes, even though mitosis and meiosis share many processes, including the DNA replication, chromosome condensation and precisely regulated partitioning of chromosomes into daughter cells. Even if topoisomerase II is required for individualization and condensation of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed.

Unique Motifs and Length of Hairpin in Oleosin Target the Cytosolic Side of Endoplasmic Reticulum and Budding Lipid Droplet.

Plant cytosolic lipid droplets (LDs) are covered with a layer of phospholipids and oleosin and were extensively studied before those in mammals and yeast. Oleosin has short amphipathic N- and C-terminal peptides flanking a conserved 72-residue hydrophobic hairpin, which penetrates and stabilizes the LD Oleosin is synthesized on endoplasmic reticulum (ER) and extracts ER-budding LDs to cytosol. To delineate the mechanism of oleosin targeting ER-LD, we have expressed modified-oleosin genes in Physcomitrella patens for transient expression and tobacco (Nicotiana tabacum) BY2 cells for stable transformation. The results have identified oleosin motifs for targeting ER-LD and oleosin as the sole molecule responsible for budding-LD entering cytosol. Both the N-terminal and C-terminal peptides are not required for the targeting. The hairpin, including its entire length, initial N-portion residues, and hairpin-loop of three Pro and one Ser residues, as well as the absence of an N-terminal ER-targeting peptide, are necessary for oleosin targeting ER and moving onto budding LDs and extracting them to cytosol. In a reverse approach, eliminations of these necessities allow the modified oleosin to enter the ER lumen and extract budding LDs to the ER lumen. Modified oleosin with an added vacuole signal peptide transports the ER-luminal LDs to vacuoles. The overall findings define the mechanism of oleosin targeting ER-LDs and extracting budding LDs to the cytosol as well as reveal potential applications.

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.