Cultured Tobacco Cells Research Articles

Resistance of cellular variants of Nicotiana tabaccum L. plants to water stress

Aim. It has been established that resistance to some heavy metal cations is combined with resistance to osmotic stress. Thus, resistance to barium cations correlates with resistance to water stress. Therefore, the aim of the experiment was to compare the resistance of primary and secondary calli to modelled stresses. Methods. The object of testing was tobacco, a plant sensitive to water deficit. The level of resistance of the variants was assessed by the relative increase in fresh weight under osmotic stresses: (salinity, 25.0 g/l of sea water salts; sodium sulphate, water stress). Both concentrations are lethal for wild-type tobacco cell cultures. Results. Вa-resistant tobacco cell cultures were obtained. Tobacco callus were resistant to lethal modelled stresses. The Ba-resistant culture was developed on medium with the addition of sea water salts and sodium sulphate. Conclusions. The phenomenon of resistance was selected as a result of primary selection on the medium with heavy metal ions. The level of osmotic resistance did not decrease with increasing cultivation time.

The optimised method of HPLC analysis of glutathione allows to determine the degree of oxidative stress in plant cell culture

Redox regulations and antioxidant defence play a central role in the acclimation of plants to their environment. Glutathione represents an essential component of the cellular antioxidant defence system, which keeps levels of reactive oxygen species (ROS) under control. High-performance liquid chromatography (HPLC) separation with fluorescence detection is a sensitive method that enables analysis of reduced and oxidised glutathione levels in small samples of plant tissues or plant cell culture. We aimed to optimise the method to obtain more accurate information about the total level of glutathione and the proportion of the reduced form (GSH) by choosing the most suitable reduction reagent and the conditions under which the reduction occurs. The applicability of the developed method was verified by analysing tobacco cells treated with hydrogen peroxide, which caused a decrease in the GSH/total glutathione ratio. Significant changes in the level of glutathione as well as in the GSH/total glutathione ratio were also observed during tobacco cell culture development.

Senescence metabolomics of <i>Nicotiana tabacum</i> L. VBI-0 heterotrophic suspension cultures

BACKGROUND: Heterotrophic cell cultures are widely used as a model in plant biology. During a culture cycle the composition of the medium changes: the sucrose and other substrates are depleted, metabolism products are accumulated and the density increases. Finally, arrest of a growth is followed by cell death in a short time. These processes are accompanied with physiological alterations, corresponding to senescence. AIM: To resolve metabolic features of tobacco cells in growing and stationary senescent suspension cultures VBI-0. MATERIALS AND METHODS: Nicotiana tabacum VBI-0 cells were cultured in suspension MS medium supplied with 3% sucrose. Cells were sampled at 7th day, during intensive growth, and at 28th day, when the culture was in the stationary phase. The GC-MS method was used to profile the metabolites. RESULTS: Sucrose depletion in media caused starvation of heterotrophic tobacco cell culture and was associated with a decrease in the accumulation of free amino acids. At the same time, the level of pentoses and complex sugars, including sucrose, increased, while the levels of glucose and fructose were not changed significantly and levels of hexose phosphates decreased. During culture senescence cells showed higher levels of accumulation of malate, pyruvate and some other carboxylates. CONCLUSIONS: The metabolomic data indicate that culture senescence was associated with a drop in amino acids metabolism, a decrease in the activity of the upper part of glycolysis, and the accumulation of complex sugars, pentoses and carboxylates.

Elicitor-Mediated Enhancement of α-Tocopherol in Cell Suspension Cultures of Nicotiana tabacum

Plant cell and tissue culture have been used as the alternative and potential renewable source for the production of valuable phytochemicals. Elicitation offers a reliable in vitro approach to produce or enhance potential phytochemicals. α-tocopherol, which is an isoform of vitamin E, is a potent fat-soluble phytochemical known in nature. The present study focused on enhancing the production of α-tocopherol in the cell suspension culture through an elicitation approach. Suspension cultures of Nicotiana tabacum were established from the leaf disk-derived callus. The cell suspension cultures were treated with different elicitors (methyl jasmonate, salicylic acid, and yeast extract) at the lag phase of the cell growth cycle. The effects of elicitors on cell cultures were determined in terms of biomass, and α-tocopherol enhancement was determined using high-performance liquid chromatography (HPLC). Different elicitors depending on the concentration exerted different effects on cell growth and α-tocopherol production. Methyl jasmonate treatment showed the significantly highest increase in α-tocopherol on the 6th day of treatment in tobacco suspension cultures. Methyl jasmonate at the concentration of 150 μM enhanced α-tocopherol content to 24-fold over the control. This study clearly shows that the elicitors had the potential to increase the accumulation of α-tocopherol considerably in tobacco cell cultures. The outcomes of this study could be of considerable importance to the nutraceutical and pharmaceutical industries.

3D fluorescence imaging through a scattering medium using the transport of intensity equation and Fresnel ping-pong algorithm.

A three-dimensional (3D) fluorescent imaging method through a scattering medium has been presented. The proposed method combines digital phase conjugate reconstruction of the scattered light wave by the transport of intensity equation-based 3D fluorescent imaging to the position of scattering medium and followed phase retrieval method by Fresnel ping-pong algorithm. The effectiveness of the proposed method is experimentally verified by measuring the 3D distribution of fluorescent beads and tobacco-cultured cells through a thin diffuser. Compared to other methods, the reconstructed images show enhancement of the detailed structure of the objects blurred by the diffuser in a wide area. In the simulation, the proposed method is capable of recovering images degraded by scattering in the phase modulation range from 0 to 2π. The capability to identify the depth position is also presented.

Monitoring the dynamics of cultured tobacco cells division by TIE-based 3D fluorescence microscopy with two-synchronized image sensors

The dynamic activity of the microtubule cytoskeleton is critical to cellular organization, including structural support, intracellular transport, and engagement in processes such as cell division and cell motility. In this work, we monitor the volumetric cell division process of tobacco BY-2 suspension cells including the dynamics of microtubule in a single-shot by using a 3D epi-fluorescence microscope based on the transport of intensity equation (3DFM-TIE) with two synchronized image sensors. The technique makes it possible to recognize and track cell mobility by examining the fluorescently tagged proteins and studying their behavior and dynamics during the cell division process. In light of experimental findings, the 3DFM-TIE system could be a favored option for live cell imaging and analysis because of its real-time 3D imaging capability and simple and compact optical design, and it has the potential to be employed in both basic and clinical investigations.

3D fluorescence imaging through scattering medium using transport of intensity equation and iterative phase retrieval.

In this paper, we have proposed a method of three-dimensional (3D) fluorescence imaging through a scattering medium. The proposed method combines the numerical digital phase conjugation propagation after measurement of the complex amplitude distribution of scattered light waves by the transport of intensity equation (TIE) with followed iterative phase retrieval to achieve 3D fluorescence imaging through a scattering medium. In the experiment, we present the quantitative evaluation of the depth position of fluorescent beads. In addition, for time-lapse measurement, cell division of tobacco-cultured cells was observed. Numerical results presented the effective range of the phase amount in the scattering medium. From these results, the proposed method is capable of recovering images degraded by a thin scattering phase object beyond a small phase change approximation.

Broad Chain-Length Specificity of the Alkane-Forming Enzymes NoCER1A and NoCER3A/B in Nymphaea odorata.

Many terrestrial plants produce large quantities of alkanes for use in epicuticular wax and the pollen coat. However, their carbon chains must be long to be useful as fuel or as a petrochemical feedstock. Here, we focus on Nymphaea odorata, which produces relatively short alkanes in its anthers. We identified orthologs of the Arabidopsis alkane biosynthesis genes AtCER1 and AtCER3 in N. odorata and designated them NoCER1A, NoCER3A and NoCER3B. Expression analysis of NoCER1A and NoCER3A/B in Arabidopsis cer mutants revealed that the N. odorata enzymes cooperated with the Arabidopsis enzymes and that the NoCER1A produced shorter alkanes than AtCER1, regardless of which CER3 protein it interacted with. These results indicate that AtCER1 frequently uses a C30 substrate, whereas NoCER1A, NoCER3A/B and AtCER3 react with a broad range of substrate chain lengths. The incorporation of shorter alkanes disturbed the formation of wax crystals required for water-repellent activity in stems, suggesting that chain-length specificity is important for surface cleaning. Moreover, cultured tobacco cells expressing NoCER1A and NoCER3A/B effectively produced C19-C23 alkanes, indicating that the introduction of the two enzymes is sufficient to produce alkanes. Taken together, our findings suggest that these N. odorata enzymes may be useful for the biological production of alkanes of specific lengths. 3D modeling revealed that CER1s and CER3s share a similar structure that consists of N- and C-terminal domains, in which their predicted active sites are respectively located. We predicted the complex structure of both enzymes and found a cavity that connects their active sites.

Alternative carbon sources for the production of plant cellular agriculture: a case study on acetate.

Plant cellular agriculture aims to disrupt the way plant derived products are produced. Plant cell cultures are typically grown with sucrose as the primary carbon and energy source, but alternative carbon sources may have advantages over sucrose including less strain on food systems, lower costs, and more sustainable sourcing. Here we review carbon and energy sources that may serve as alternatives to sucrose in the cultivation of plant cell cultures. We identified acetate as a promising candidate and took the first steps to evaluate its potential for use in growing tobacco plant cell cultures. When added to media containing sucrose, acetate concentrations above 8 mM completely inhibit growth. Lower concentrations of acetate (2-4 mM) can support an increase in dry weight without sucrose but do not provide enough energy for substantial growth. 13C labeling indicates that tobacco plant cell cultures can incorporate carbon from exogenous acetate into proteins and carbohydrates. Analysis of transcriptome data showed that genes encoding glyoxylate cycle enzymes are expressed at very low levels compared to genes from the TCA cycle and glycolysis. Adaptive laboratory evolution experiments were able to increase tobacco cell cultures tolerance to acetate, demonstrating the potential for this type of approach going forward. Overall, our results indicate that acetate can be metabolized by plant cell cultures and suggest that further adaptive laboratory evolution or strain engineering efforts may enable acetate to serve as a sole carbon and energy source for tobacco plant cell cultures. This assessment of acetate provides a framework for evaluating other carbon and energy sources for plant cell cultures, efforts that will help reduce the costs and environmental impact, and increase the commercial potential of plant cellular agriculture.

Deep learning based image quality improvement of a light-field microscope integrated with an epi-fluorescence microscope

Light-field three-dimensional (3D) fluorescence microscopes can acquire 3D fluorescence images in a single shot, and followed numerical reconstruction can realize cross-sectional imaging at an arbitrary depth. The typical configuration that uses a lens array and a single image sensor has the trade-off between depth information acquisition and spatial resolution of each cross-sectional image. The spatial resolution of the reconstructed image degrades when depth information increases. In this paper, we use U-net as a deep learning model to improve the quality of reconstructed images. We constructed an optical system that integrates a light-field microscope and an epifluorescence microscope, which acquire the light-field data and high-resolution two-dimensional images, respectively. The high-resolution images from the epifluorescence microscope are used as ground-truth images for the training dataset for deep learning. The experimental results using fluorescent beads with a size of 10 µm and cultured tobacco cells showed significant improvement in the reconstructed images. Furthermore, time-lapse measurements were demonstrated in tobacco cells to observe the cell division process.

Denovo induction of a DNA-histone H3K9 methylation loop on synthetic human repetitive DNA in cultured tobacco cells.

Genetic modifications in plants are crucial tools for fundamental and applied research. Transgene expression usually varies among independent lines or their progeny and is associated with the chromatin structure of the insertion site. Strategies based on understanding how to manipulate the epigenetic state of the inserted gene cassette would help to ensure transgene expression. Here, we report a strategy for chromatin manipulation by the artificial tethering of epigenetic effectors to a synthetic human centromeric repetitive DNA (alphoid DNA) platform in plant Bright-Yellow-2 (BY-2) culture cells. By tethering DNA-methyltransferase (Nicotiana tabacum DRM1), we effectively induced DNA methylation and histone methylation (H3K9me2) on the alphoid DNA platform. Tethering of the Arabidopsis SUVH9, which has been reported to lack histone methyltransferase activity, also induced a similar epigenetic state on the alphoid DNA in BY-2 cells, presumably by activating the RNA-dependent DNA methylation (RdDM) pathway. Our results emphasize that the interplay between DNA and histone methylation mechanisms is intrinsic to plant cells. We also found that once epigenetic modification states were induced by the tethering of either DRM1 or SUVH9, the modification was maintained even when the direct tethering of the effector was inhibited. Our system enables the analysis of more diverse epigenetic effectors and will help to elucidate the chromatin assembly mechanisms of plant cells.

Introduction of a long synthetic repetitive DNA sequence into cultured tobacco cells.

Genome information has been accumulated for many species, and these genes and regulatory sequences are expected to be applied in plants by enhancing or creating new metabolic pathways. We hypothesized that manipulating a long array of repetitive sequences using tethered chromatin modulators would be effective for robust regulation of gene expression in close proximity to the arrays. This approach is based on a human artificial chromosome made of long synthetic repetitive DNA sequences in which we manipulated the chromatin by tethering the modifiers. However, a method for introducing long repetitive DNA sequences into plants has not yet been established. Therefore, we constructed a bacterial artificial chromosome-based binary vector in Escherichia coli cells to generate a construct in which a cassette of marker genes was inserted into 60-kb synthetic human centromeric repetitive DNA. The binary vector was then transferred to Agrobacterium cells and its stable maintenance confirmed. Next, using Agrobacterium-mediated genetic transformation, this construct was successfully introduced into the genome of cultured tobacco BY-2 cells to obtain a large number of stable one-copy strains. ChIP analysis of obtained BY-2 cell lines revealed that the introduced synthetic repetitive DNA has moderate chromatin modification levels with lower heterochromatin (H3K9me2) or euchromatin (H3K4me3) modifications compared to the host centromeric repetitive DNA or an active Tub6 gene, respectively. Such a synthetic DNA sequence with moderate chromatin modification levels is expected to facilitate manipulation of the chromatin structure to either open or closed.

'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.

Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei.

The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

Genetic and Pharmacological Inhibition of Autophagy increases the Monoubiquitination of Non-Photosynthetic Phosphoenolpyruvate Carboxylase.

Phosphoenolpyruvate carboxylase (PEPC) is an enzyme with key roles in carbon and nitrogen metabolisms. The mechanisms that control enzyme stability and turnover are not well known. This paper investigates the degradation of PEPC via selective autophagy, including the role of the monoubiquitination of the enzyme in this process. In Arabidopsis, the genetic inhibition of autophagy increases the amount of monoubiquitinated PEPC in the atg2, atg5, and atg18a lines. The same is observed in nbr1, which is deficient in a protein that recruits monoubiquitinated substrates for selective autophagy. In cultured tobacco cells, the chemical inhibition of the degradation of autophagic substrates increases the quantity of PEPC proteins. When the formation of the autophagosome is blocked with 3-methyladenine (3-MA), monoubiquitinated PEPC accumulates as a result. Finally, pull-down experiments with a truncated version of NBR1 demonstrate the recovery of intact and/or fragmented PEPC in Arabidopsis leaves and roots, as well as cultured tobacco cells. Taken together, the results show that a fraction of PEPC is cleaved via selective autophagy and that the monoubiquitination of the enzyme has a role in its recruitment towards this pathway. Although autophagy seems to be a minor pathway, the results presented here increase the knowledge about the role of monoubiquitination and the regulation of PEPC degradation.

Synthesis and biological effects of small molecule enhancers for improved recombinant protein production in plant cell cultures.

Histone deacetylases are involved in chromatin remodelling and thus play a vital role in the epigenetic regulation of gene expression. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various cellular events such as transcription. Novel HDAC inhibitors were designed and synthesised to promote higher levels of recombinant protein production in tobacco cell cultures. The effect of these chemical enhancers on the epigenetic profiles in plant cells has been evaluated by molecular docking, in vitro and in vivo studies. The addition of these novel enhancers led to an increase in histone H3 acetylation levels that promoted an increase in the accumulation levels of the recombinant protein in cell culture. These results can pave the way for the application of these enhancers to improve the production of high value products in plant cell based systems.

Chemical Screening Pipeline for Identification of Specific Plant Autophagy Modulators.

Autophagy is a major catabolic process in eukaryotes with a key role in homeostasis, programmed cell death, and aging. In plants, autophagy is also known to regulate agronomically important traits such as stress resistance, longevity, vegetative biomass, and seed yield. Despite its significance, there is still a shortage of reliable tools modulating plant autophagy. Here, we describe the first robust pipeline for identification of specific plant autophagy-modulating compounds. Our screening protocol comprises four phases: (1) high-throughput screening of chemical compounds in cell cultures of tobacco (Nicotiana tabacum); (2) confirmation of the identified hits in planta using Arabidopsis (Arabidopsis thaliana); (3) further characterization of the effect using conventional molecular biology methods; and (4) verification of chemical specificity on autophagy in planta. The methods detailed here streamline the identification of specific plant autophagy modulators and aid in unraveling the molecular mechanisms of plant autophagy.

Effect of Ag-doping on cytotoxicity of SnO2 nanoparticles in tobacco cell cultures

SnO2 nanoparticles (NPs) are promising materials for electrochemical, catalytic, and biomedical applications due to their high photosensitivity, suitable stability characteristics, wide band gap energy potential, and low cost. Doping SnO2 NPs with metallic elements such as Ag has been used to improve their efficiency. Despite their commercial importance, the current literature lacks investigations to determine their toxic effects on plant systems. In this study, SnO2 and Ag/SnO2 NPs were synthesized using polymer pyrolysis method and characterized by means of XRD, TEM, SEM, EDX, and DLS techniques. Subsequently, the toxicity of the synthesized NPs on cell viability, cell proliferation, and a number of oxidative stress markers were measured in tobacco cell cultures. SnO2 and Ag/SnO2 NPs were found to be polygonal in shape with the size range of 10–30 nm. Both NPs induced cytotoxicity by reducing the cell viability and cell proliferation in a dose-dependent manner. Furthermore, the generation of H2O2, phenolics, flavonoids, and increased activities of superoxide dismutase (SOD) and peroxidase (POD) were observed. According to the results, Ag-doping played a key role in the induction of toxicity in tobacco cell cultures. The obtained results confirmed that SnO2 and Ag/SnO2 NPs induced cytotoxicity in tobacco cells through oxidative stress.

Spatial and Temporal Profile of Glycine Betaine Accumulation in Plants Under Abiotic Stresses.

Several halophytes and a few crop plants, including Poaceae, synthesize and accumulate glycine betaine (GB) in response to environmental constraints. GB plays an important role in osmoregulation, in fact, it is one of the main nitrogen-containing compatible osmolytes found in Poaceae. It can interplay with molecules and structures, preserving the activity of macromolecules, maintaining the integrity of membranes against stresses and scavenging ROS. Exogenous GB applications have been proven to induce the expression of genes involved in oxidative stress responses, with a restriction of ROS accumulation and lipid peroxidation in cultured tobacco cells under drought and salinity, and even stabilizing photosynthetic structures under stress. In the plant kingdom, GB is synthesized from choline by a two-step oxidation reaction. The first oxidation is catalyzed by choline monooxygenase (CMO) and the second oxidation is catalyzed by NAD+-dependent betaine aldehyde dehydrogenase. Moreover, in plants, the cytosolic enzyme, named N-methyltransferase, catalyzes the conversion of phosphoethanolamine to phosphocholine. However, changes in CMO expression genes under abiotic stresses have been observed. GB accumulation is ontogenetically controlled since it happens in young tissues during prolonged stress, while its degradation is generally not significant in plants. This ability of plants to accumulate high levels of GB in young tissues under abiotic stress, is independent of nitrogen (N) availability and supports the view that plant N allocation is dictated primarily to supply and protect the growing tissues, even under N limitation. Indeed, the contribution of GB to osmotic adjustment and ionic and oxidative stress defense in young tissues, is much higher than that in older ones. In this review, the biosynthesis and accumulation of GB in plants, under several abiotic stresses, were analyzed focusing on all possible roles this metabolite can play, particularly in young tissues.

Energy saving and improvement of metabolism of cultured tobacco cells upon exposure to 2-D clinorotation

Studies have confirmed that on the ground, the plant cells must expend energy to maintain positional homeostasis against gravity. Under microgravity conditions, such energy may be saved for other process such as biosynthesis of beneficial metabolites for growth. This hypothesis was examined on a cell line of tobacco (Nicotiana tabacum cv. Burley 21). The cells were continuously treated with 2-D clinostat for 1 week. Exposure to clinorotation conditions increased biomass and total protein. Total content of soluble sugar also increased which may provide more precursors for Krebs cycle and adenosine triphosphate (ATP) production. In the case of 2-D clinorotation, the expression and activity of glutamate producing enzyme, glutamate dehydrogenase (GDH) increased, whereas the activity of glutamate decarboxylase (GAD) decreased. Regarding the role of GAD in initiation of gamma amino butyric acid (GABA) shunt, it is plausible that under clinorotation condition, the tobacco cells directed their metabolism toward saving energy for Krebs cycling and more production of ATP rather than shifting to side paths such as GABA shunt.Improvement of radical scavenging enzymes activity and increase of the contents of phenolic compounds and certain peroxide neutralizing amino acids, e.g., His, Pro, Ser, and Asp under clinorotation conditions decreased membrane lipid peroxidation and maintained the growth potential of tobacco cells.