Suspension-cultured Tobacco Cells Research Articles

Sound waves alter the viability of tobacco cells via changes in cytosolic calcium, membrane integrity, and cell wall composition.

The effect of sound waves (SWs) on plant cells can be considered as important as other mechanical stimuli like touch, wind, rain, and gravity, causing certain responses associated with the downstream signaling pathways on the whole plant. The objective of the present study was to elucidate the response of suspension-cultured tobacco cells (Nicotiana tabacum L. cv Burley 21) to SW at different intensities. The sinusoidal SW (1,000 Hz) was produced through a signal generator, amplified, and beamed to the one layer floating tobacco cells inside a soundproof chamber at intensities of 60, 75, and 90 dB at the plate level for 15, 30, 45, and 60 min. Calibration of the applied SW intensities, accuracy, and uniformity of SW was performed by a sound level meter, and the cells were treated. The effect of SW on tobacco cells was monitored by quantitation of cytosolic calcium, redox status, membrane integrity, wall components, and the activity of wall modifying enzymes. Cytosolic calcium ions increased as a function of sound intensity with a maximum level of 90 dB. Exposure to 90 dB was also accompanied by a significant increase of H2O2 and membrane lipid peroxidation rate but the reduction of total antioxidant and radical scavenging capacities. The increase of wall rigidity in these cells was attributed to an increase in wall-bound phenolic acids and lignin and the activities of phenylalanine ammonia-lyase and covalently bound peroxidase. In comparison, in 60- and 75 dB, radical scavenging capacity increased, and the activity of wall stiffening enzymes reduced, but cell viability showed no changes. The outcome of the current study reveals that the impact of SW on plant cells is started by an increase in cytosolic calcium. However, upon calcium signaling, downstream events, including alteration of H2O2 and cell redox status and the activities of wall modifying enzymes, determined the extent of SW effects on tobacco cells.

'Candidatus Liberibacter asiaticus'-Encoded BCP Peroxiredoxin Suppresses Lipopolysaccharide-Mediated Defense Signaling and Nitrosative Stress In Planta.

The lipopolysaccharides (LPS) of gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic 'Candidatus Liberibacter' spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on nonhost tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host orange (Citrus sinensis), confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione pool, callose deposition, and activation of the salicylic acid and azelaic acid (AzA) signaling networks. Transient expression of 'Ca. L. asiaticus' bacterioferritin comigratory protein (BCP) peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that 'Ca. L. asiaticus' BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. 'Ca. L. asiaticus' BCP peroxiredoxin (i) attenuates NO-mediated SAR signaling and (ii) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of 'Ca. L. asiaticus' acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Plant-Based Cell-Free Transcription and Translation of Recombinant Proteins.

Plant cell-free lysates contain all the cellular components of the protein biosynthesis machinery, providing an alternative to intact plant cells, tissues, and whole plants for the production of recombinant proteins. Cell-free lysates achieve rapid protein production (within hours or days) and allow the synthesis of proteins that are cytotoxic or unstable in living cells. The open nature of cell-free lysates and their homogeneous and reproducible performance is ideal for protein production, especially for screening applications, allowing the direct addition of nucleic acid templates encoding proteins of interest, as well as other components such as enzyme substrates, chaperones, artificial amino acids, or labeling molecules. Here we describe procedures for the production of recombinant proteins in the ALiCE (Almost Living Cell-free Expression) system, a lysate derived from tobacco cell suspension cultures that can be used to manufacture protein products for molecular and biochemical analysis as well as applications in the pharmaceutical industry.

Use of a novel R2R3-MYB transcriptional activator of anthocyanin biosynthesis as visual selection marker for rubber tree (Hevea brasiliensis) transformation

Hevea brasiliensis is presently the only commercial source of natural rubber. The use of anthocyanin production as a visual and non-destructive selection marker may be a viable strategy for increasing the transformation efficiency of this species. In this study, high performance liquid chromatography (HPLC) analysis confirmed that the cyanidin-type anthocyanin is a major determinant of red color in Hevea tissues. It was also found that the anthocyanin production in Hevea somatic embryos could be activated by methyl jasmonate (MeJA) treatment. By sequence alignment and phylogenetic analysis, a novel Hevea R2R3-MYB transcription factor (TF) was isolated and designated here as Hevea brasiliensis Anthocyanin 2 (HbAN2). The expressions of HbAN2 correlate well with the anthocyanin levels in Hevea tissues. The regulatory roles of HbAN2 in anthocyanin biosynthesis were functionally characterized in the native and heterologous systems. In tobacco suspension culture cells, the expression of HbAN2 alone or in combination with two HbbHLH genes activated the promoter of NtDFR, a key gene responsible for anthocyanin biosynthesis. In tobacco plants and Hevea embryos, overexpression of HbAN2 upregulated the late stage anthocyanin pathway genes, resulting in increased anthocyanin accumulation in the transformed parts of the explants, which were therefore easily distinguishable from the non transformed parts. Although transgenic Hevea shoots failed to regenerate into whole plants, these results suggest that HbAN2 is a regulator of anthocyanin biosynthesis and has the potential to be used as visual selection marker for Hevea transformation.

Cell Walls Are Remodeled to Alleviate nY2O3 Cytotoxicity by Elaborate Regulation of de Novo Synthesis and Vesicular Transport.

Yttrium oxide nanoparticles (nY2O3), one of the broadly used rare earth nanoparticles, can interact with plants and possibly cause plant health and environmental impacts, but the plant defense response particularly at the nanoparticle-cell interface is largely unknown. To elucidate this, Bright Yellow 2 (BY-2) tobacco (Nicotiana tabacum L.) suspension-cultured cells were exposed to 50 mg L-1 nY2O3 (30 nm) for 12 h. Although 42.2% of the nY2O3 remained outside of protoplasts, nY2O3 could still traverse the cell wall and was partially deposited inside the vacuole. In addition to growth inhibition, morphological and compositional changes in cell walls occurred. Together with a locally thickened (7-13-fold) cell wall, increased content (up to 58%) of pectin and reduction in (up to 29%) hemicellulose were observed. Transcriptome analysis revealed that genes involved in cell wall metabolism and remodeling were highly regulated in response to nY2O3 stress. Expression of genes for pectin synthesis and degradation was up- and down-regulated by 31-78% and 13-42%, respectively, and genes for xyloglucan and pectin modifications were up- and down-regulated by 82% and 81-92%, respectively. Interestingly, vesicle trafficking seemed to be activated, enabling the repair and defense against nY2O3 disturbance. Our findings indicate that, although nY2O3 generated toxicity on BY-2 cells, it is very likely that during the recovery process cell wall remodeling was initiated to gain resistance to nY2O3 stress, demonstrating the plant's cellular regulatory machinery regarding repair and adaptation to nanoparticles like nY2O3.

In vitro inhibition of shikimate hydroxycinnamoyltransferase by acibenzolar acid, the first metabolite of the plant defence inducer acibenzolar-S-methyl

Acibenzolar acid, the first metabolite formed in planta from the defence inducer acibenzolar-S-methyl (ASM), has been shown to be an inhibitor of the enzyme shikimate hydroxycinnamoyltransferase (HST), extracted from grapevine or tobacco cell suspension cultures. Using a purified recombinant Arabidopsis thaliana HST, the inhibition was found to be competitive, acibenzolar acid binding reversibly to the shikimate binding site of the HST:p-coumaroyl-CoA complex, with a Ki value of 250 μM. The other hydroxycinnamoyltransferases tested in the course of this study, using either hydroxypalmitic acid, putrescine, tyramine, or quinic acid as acyl acceptors were not, or only slightly, inhibited by acibenzolar acid. To understand the specificity of the interaction of acibenzolar acid with HST, we analyzed the structure-activity relationship of a series of benzoic or acibenzolar acid analogues, tested either as AtHST substrates or as inhibitors. This analysis confirmed previously published data on the substrate flexibility of HST and demonstrated that both the carboxyl group and the thiadiazole moiety of acibenzolar acid are playing an important role in the interaction with the shikimate binding site. Acibenzolar acid, which cannot form an ester bond with p-coumaric acid, was however a less potent inhibitor than protocatechuic or 3-hydroxybenzoic acids, which are used as acyl acceptors by HST. Our results show that the interaction of acibenzolar acid with HST, which is probably directly linked to the substrate promiscuity of HST, is unlikely to play a direct role in the defence-inducing properties of ASM in plants.

Nucleoside derivatives of 5-methylcytosine suppress 5-azacytidine-induced reactivation of a silent transgene in suspension-cultured tobacco cells.

Epigenetic modifications, including DNA methylation, are involved in the regulatory mechanisms of gene expression in animals and plants. In this study, we investigated whether the action of 5-azacytidine (5-aza-Cd), which is a well-known DNA methylation inhibitor, in suspension-cultured tobacco cells is affected by treatment with nucleoside derivatives of 5-methylcytosine (5-mCs), namely 5-methylcytidine (5-mCd) and 5-methyl-2'-deoxycytidine (5-mdCd). In a tobacco cell line, 5-aza-Cd treatment reactivated an epigenetically silenced transgene containing the cauliflower mosaic virus 35S promoter fused to the β-glucuronidase coding region and the nopaline synthase polyadenylation signal. The reactivation was evident on the fifth day of treatment and was augmented during culture with application of 5-aza-Cd at every subcultivation. This treatment, provided only once in the initial culture, resulted in transient transgene reactivation, followed by attenuation of its activity. The reactivation induced by 5-aza-Cd was suppressed by concomitant treatment with either 5-mCd or 5-mdCd. These results suggest that the 5-mCs derivatives inhibit and/or reverse 5-aza-Cd-induced reactivation of a silent transgene in tobacco cells.

Subcellular Localization of Acyl-CoA: Lysophosphatidylethanolamine Acyltransferases (LPEATs) and the Effects of Knocking-Out and Overexpression of Their Genes on Autophagy Markers Level and Life Span of A. thaliana.

Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence.

Impact of phyto-hormone concentrations in optimizing cell suspension culture of flue-cured tobacco (Nicotiana tabaccum L.) cultivars

Impact of phyto-hormone concentrations in optimizing cell suspension culture of flue-cured tobacco (Nicotiana tabaccum L.) cultivars

Zearalenone‐malonyl‐glucosides as phase II metabolites in plant cell suspension cultures

AbstractBackground and objectivesConjugation of mycotoxins in the phase II metabolism of plants results in modified mycotoxins such as glucosides and malonyl‐glucosides. However, malonyl‐glucosides have not yet been completely elucidated for zearalenone (ZEN). Thus, the aim of this study was to produce and isolate malonyl‐glucosides of ZEN for an unambiguous identification by NMR spectroscopy.FindingsZearalenone was incubated in plant cell suspension cultures of wheat, soy, and tobacco, and phase II metabolites were analyzed by using LC‐DAD‐MS, ‐HRMS, and NMR spectroscopy. Four main metabolites of ZEN were detected in the cell extracts and identified as two glucosides (attached in positions 14 and 16) and their 6´‐malonyl derivatives.ConclusionsZearalenone‐malonyl‐glucosides should be incorporated in future analyses of modified mycotoxins because of their potential relevance for food and feed safety.Significance and noveltyFor the first time, the structures of the two malonyl‐glucosides of ZEN were unambiguously identified by NMR spectroscopy after preparative isolation as 14‐O‐(6’‐O‐malonyl‐β‐D‐glucopyranosyl)ZEN and 16‐O‐(6’‐O‐malonyl‐β‐D‐glucopyranosyl)ZEN.

Extracellular ATP affects cell viability, respiratory O2 uptake, and intracellular ATP production of tobacco cell suspension culture in response to hydrogen peroxide-induced oxidative stress

Extracellular adenosine 5′-triphosphate (ATP) is a well-known signalling molecule in plants and plays important roles during plant stress response. In the present work, the treatment of tobacco cell suspension culture with exogenous hydrogen peroxide (H2O2) caused the decreases of respiratory O2 uptake, intracellular ATP production, extracellular ATP level, and reduction of cell viability. Combining this observation with the finding that the oxidative phosphorylation uncoupler or the respiratory inhibitor not only decreased intracellular ATP production but also decreased the extracellular ATP level and cell viability, it is suggested that the decrease of extracellular ATP level and cell viability under H2O2 stress could be a result of the suppressed production of intracellular ATP. Treatment with ATP-degrading enzyme, apyrase, also caused the decreases of cell viability, respiratory O2 uptake, and intracellular ATP production. More importantly, addition of exogenous ATP alleviated the H2O2-induced decreases of cell viability, respiratory O2 uptake, and production of intracellular ATP. These results indicate that extracellular ATP could be an important effector in regulating the cell viability, respiratory O2 uptake, and intracellular ATP production of tobacco cell suspension culture under H2O2 stress.

Cell viability in the cadmium-stressed cell suspension cultures of tobacco is regulated by extracellular ATP, possibly by a reactive oxygen species-associated mechanism

Cell viability in the cadmium-stressed cell suspension cultures of tobacco is regulated by extracellular ATP, possibly by a reactive oxygen species-associated mechanism

Stronger cellulose microfibril network structure through the expression of cellulose-binding modules in plant primary cell walls

Cellulose-binding modules (CBMs) are non-catalytic domains typically occurring in glycoside hydrolases. Their specific interaction with diverse polysaccharides assists hydrolysis by the catalytic subunits. In this work, we have exploited the interactions between a CBM from family 3 (CBM3) and cell wall polysaccharides to alter the structure and mechanical properties of cellulose microfibrils from BY-2 tobacco cell suspension cultures. A CBM3 from Clostridium thermocellum was overexpressed in the cells using Agrobacterium-mediated transformation. Water suspensions of cellulose microfibrils were prepared by the removal of the non-cellulosic components of the primary cell walls, followed by mild disintegration using sonication. The morphology of the microfibrils was characterized by transmission electron microscopy and atomic force microscopy. These cellulose microfibrils were further hydrolyzed with 64 wt% sulfuric acid to produce cellulose nanocrystals (CNCs). The average length of CNCs prepared from the CBM3-transformed cells was 201 nm, higher than that from the wild-type cells (122 nm). In addition, the mechanical properties and deformation mechanism of nanopapers prepared from suspensions of cellulose microfibrils were investigated. The nanopapers obtained from the CBM3-transformed cells exhibited enhanced tensile strength and work of fracture, 40% and 128% higher than those prepared from wild-type tobacco cells, respectively.Graphical abstract

Using FM Dyes to Study Endomembranes and Their Dynamics in Plants and Cell Suspensions.

FM (Fei-Mao) styryl dyes are commonly used for the fluorescence imaging of plasma membrane (PM) and endocytosis in vivo. Thanks to their amphiphilic character, these dyes are incorporated in the outer leaflet of the PM lipid bilayer and emit fluorescence in its hydrophobic environment. The endocytic pathway of FM dye uptake starts with rapid PM staining and continues in PM invaginations and membrane vesicles during endocytosis, followed by staining of trans-Golgi network (TGN) and ending in tonoplast (vacuolar membrane). FM dyes do not stain endoplasmic reticulum and nuclear membrane. The time-lapse fluorescence microscopy could track endocytic vesicles and characterize the rate of endocytosis in vivo. On the other hand, fixable FM dyes (FX) can be used for the visualization of particular steps in the FM dye uptake in situ. Staining with FM dyes and subsequent microscopic observations could be performed on both tissue and cellular level. Here, we describe simple procedures for the effective FM dye staining and destaining in root tip of Arabidopsis thaliana seedlings and suspension-cultured tobacco cells.

A new monoclonal antibody against rhamnogalacturonan II and its application to immunocytochemical detection of rhamnogalacturonan II in Arabidopsis roots.

B: boron; B-RG-II: borate-RG-II complex; ELISA: enzyme-linked immunosorbent assay; IgG: immunoglobulin G; mBSA: methylated bovine serum albumin; PGA: polygalacturonic acid; PLL: poly-l-lysine; RG-I: rhamnogalacturonan I; RG-II: rhamnogalacturonan II.

On the mechanism of the cell cycle control of suspension-cultured tobacco cells after exposure to static magnetic field

One of the main sites of the magnetic fields influence on living cells is the cell cycle. The intensity of this influence however, varies depending on the cell type and the duration of the treatment. Suspension of cultured tobacco cells (Nicotiana tabacum cv. Barley 21) were synchronized via sucrose starvation at their stationary growth phase. The cells were then exposed to 0.2 m T SMF up to 24 h. The progression of different cell cycle phases was monitored through flow cytometry in a time course manner. Expression of cell cycle controlling genes and amounts of certain signaling molecules were measured as well. Exposure to SMF delayed G1.S transition which was accompanied by decrease of cyclin-dependent kinases A (CDK A) and D-type cyclin, but an increase in the adenylyl cyclase (AC), transcription factor E2F, retinoblastoma protein (Rbp), and CDK-inhibitor protein 21 (p21) transcript accumulation. Exposure to SMF also increased the contents of nitric oxide (NO), hydrogen peroxide (H2O2), and salicylic acid (SA), compared to the control group. The results suggest a signaling pathway triggered by SMF starting from accumulation of NO and H2O2 followed by downstream events including the increase of cyclic nucleotides and subsequent decrease of both CDKA and CycD.

Toxicity impacts of chemically and biologically synthesized CuO nanoparticles on cell suspension cultures of Nicotiana tabacum

Nanotechnology has quite a lot of applications in various fields of industrial sectors like food and agriculture. Although nanotechnology can improve the quality of life, its possible associated risks should be assessed. Here copper oxide nanoparticles (CuO NPs) were synthesized by chemical (polymer pyrolysis) and biological (green) methods with an average size of 30 and 44 nm, respectively. Afterwards, a cell biology approach was applied to evaluate the toxic effects of chemically and biologically synthesized CuO nanoparticles on tobacco cell suspension cultures. Both types of CuO nanoparticles significantly dropped the viability of the cells in a dose and time dependent manner. Accordingly, tobacco cells were found to increase the activity of antioxidant enzymes after 48 h of exposure to nanoparticles. The production of reactive oxygen species (ROS) and malondialdehyde (MDA) in a dose dependent manner was also observed. Assessment of the toxicity of CuO NPs revealed that chemically synthesized NPs were more toxic than biologically synthesized ones. It can be concluded that the organic components of the plant extract as capping agents that remain on the surface of green synthesized CuO NPs may reduce their toxicity effects.

Mechanism for temperature-shift-responsive acute Ca^ uptake in suspension-cultured tobacco and rice cells

Mechanism for temperature-shift-responsive acute Ca^ uptake in suspension-cultured tobacco and rice cells

Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco.

Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of α-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in α-tubulin can be used to prepare particular isotypes and specifically engineered versions of α-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures.