Cultured Tobacco Cells Research Articles

Early signaling network in tobacco cells elicited with methyl jasmonate and cyclodextrins

We analyze, for the first time, the early signal transduction pathways triggered by methyl jasmonate (MJ) and cyclodextrins (CDs) in tobacco (Nicotiana tabacum) cell cultures, paying particular attention to changes in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), the production of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO), and late events like the induction of capsidiol. Our data indicate that MJ and CDs trigger a [Ca(2+)](cyt) rise promoted by Ca(2+) influx through Ca(2+)-permeable channels. The joint presence of MJ and CDs provokes a first increase in [Ca(2+)](cyt) similar to that observed in MJ-treated cells, followed by a second peak similar to that found in the presence of CDs alone. Moreover, oxidative burst induced by MJ is more pronounced when tobacco cells are incubated with CDs alone or in combination with MJ. The presence of both elicitors provokes H(2)O(2) production similar to that found in CD-treated cells, and a sustained response similar to that found in MJ-treated cells. In all treatments, H(2)O(2) production is dependent on Ca(2+) influx and protein phosphorylation events. Similarly, the joint action of both elicitors provokes NO accumulation, although to a lesser extent that in MJ-treated cells because CDs alone do not trigger this accumulation. This NO production is dependent on Ca(2+) influx but independent of both H(2)O(2) production and staurosporine-sensitive phosphorylation events. Taken as a whole, these results suggest the existence of different intracellular signaling pathways for both elicitors. Likewise, CDs might act by regulating the signaling pathway triggered by MJ since, in the presence of both compounds, CDs neutralize the strong oxidative and nitrosative bursts triggered by MJ and therefore, they regulate both H(2)O(2) and NO levels.

Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria

With the expansion of the RNA world, antisense strategies have become widespread to manipulate nuclear gene expression but organelle genetic systems have remained aside. The present work opens the field to mitochondria. We demonstrate that customized RNAs expressed from a nuclear transgene and driven by a transfer RNA-like (tRNA-like) moiety are taken up by mitochondria in plant cells. The process appears to follow the natural tRNA import specificity, suggesting that translocation indeed occurs through the regular tRNA uptake pathway. Upon validation of the strategy with a reporter sequence, we developed a chimeric catalytic RNA composed of a specially designed trans-cleaving hammerhead ribozyme and a tRNA mimic. Organelle import of the chimeric ribozyme and specific target cleavage within mitochondria were demonstrated in transgenic tobacco cell cultures and Arabidopsis thaliana plants, providing the first directed knockdown of a mitochondrial RNA in a multicellular eukaryote. Further observations point to mitochondrial messenger RNA control mechanisms related to the plant developmental stage and culture conditions. Transformation of mitochondria is only accessible in yeast and in the unicellular alga Chlamydomonas. Based on the widespread tRNA import pathway, our data thus make a breakthrough for direct investigation and manipulation of mitochondrial genetics.

Molecular Cloning and Partial Characterization of a Peroxidase Gene Expressed in the Roots ofPortulaca oleraceacv., One Potentially Useful in the Remediation of Phenolic Pollutants

Portulaca (Portulaca oleracea cv.) efficiently removes phenolic pollutants from hydroponic solution. In plant roots, peroxidase (PRX) is thought to be involved in the removal of phenolic pollutants by the cross-linking them to cell wall polysaccharides or proteins at the expense of reduction of hydrogen peroxide (H(2)O(2)). In this study, we found that portulaca roots secreted an acidic PRX isozyme that had relatively high H(2)O(2) affinity. We isolated five PRX genes, and the recombinant PRX proteins produced in cultured tobacco cells were partially characterized. Among these genes, PoPRX2 probably encoded the acidic PRX isozyme. PoPRX2 had an extra N-terminal region which has not been reported for other PRX proteins. We found that PoPRX2 oxidized phenolic pollutants, including bisphenol A, octylphenol, nonylphenol, and 17β-estradiol. In addition, we found that the Cys261 residue of PoPRX2 played an important role in the determination of affinity for H(2)O(2) and stability toward H(2)O(2).

Tobacco MYC2 Regulates Jasmonate-Inducible Nicotine Biosynthesis Genes Directly and By Way of the NIC2-Locus ERF Genes

In Arabidopsis, the MYC2-family basic helix-loop-helix transcription factors mediate transcriptional regulation of jasmonate-responsive genes, and their transcriptional activities are suppressed by physical interactions with jasmonate-ZIM domain (JAZ) proteins. Jasmonate-inducible nicotine formation in Nicotiana plants has been shown to be suppressed by tobacco JAZ proteins, and be regulated by both MYC2-related and NIC2-locus ethylene response factor (ERF) transcription factors. We here show that tobacco MYC2 (NtMYC2) recognizes the G-box sequences, 5'-CAC(G/A)T(G/T)-3', found in the proximal promoter regions of several nicotine biosynthesis genes, including Putrescine N-Methyltransferase 2 (PMT2) and Quinolinate Phosphoribosyltransferase 2 (QPT2). Transient transactivation assays using cultured tobacco cells showed that NtMYC2 and NIC2-locus ERF189 additively activated the PMT2 and QPT2 promoters depending on their cognate binding sites. RNA interference (RNAi) silencing of NtMYC2 in tobacco hairy roots strongly decreased transcript levels of jasmonate-responsive structural genes, including those involved in nicotine biosynthesis, as well as the NIC2-locus ERF genes. Conversely, ERF189 was not required for the expression of NtMYC2. NtMYC2, but not ERF189, interacted with tobacoo JAZs in a yeast two-hybrid assay. These results indicate that NtMYC2 controls nicotine biosynthesis genes in two combinatorial ways, by directly binding the G-box in the target promoters and by up-regulating the NIC2-locus ERF genes.

Enhanced accumulation of secreted human growth hormone by transgenic tobacco cells correlates with the introduction of an N-glycosylation site

Extracellular secretion of recombinant proteins from plant cell suspension culture will simplify the protein purification procedure and greatly reduce the production cost. Our early work indicated that presence of hydroxyproline-O-glycosylation at the C- or N-terminus of the target protein boosted the secreted yields in the culture medium. Inspired by early successes, we tested the possibility of introducing an N-glycosylation site to facilitate the secretion of human growth hormone (hGH) from cultured tobacco cells. Three N-glycosylated hGH fusion proteins, designated NAS-EK-hGH, NAS-Kex2-hGH and hGH-NAS, were expressed in tobacco BY-2 cells. Where NAS denotes the “Asn-Ala-Ser” consensus sequence for N-glycosylation; EK denotes an enterokinase cleavage site and Kex2 a sequence to be cleaved by a Golgi-localized Kex2p-like protease. Our results indicated that a single N-glycan attached either at the N-terminus or C-terminus of hGH correlated with enhanced extracellular accumulation of the transgenic proteins; the secreted yield of NAS-EK-hGH and hGH-NAS was 70–90 fold greater than the control targeted, non-glycosylated hGH. NAS-Kex2-hGH was subject to partial cleavage of the N-glycan tag at the Kex2 site in Golgi apparatus, and therefore gave lower yields than the other two constructs.

1,8-Cineole Inhibits Both Proliferation and Elongation of BY-2 Cultured Tobacco Cells

Volatile monoterpenes such as 1,8-cineole inhibit the growth of Brassica campestris seedlings in a dose-dependent manner, and the growth-inhibitory effects are more severe for roots than hypocotyls. The preferential inhibition of root growth may be explained if the compounds inhibit cell proliferation more severely than cell elongation because root growth requires both elongation and proliferation of the constituent cells, whereas hypocotyl growth depends exclusively on elongation of existing cells. In order to examine this possibility, BY-2 suspension-cultured tobacco (Nicotiana tabacum) cells were treated with 1,8-cineole, and the inhibitory effects on cell proliferation and on cell elongation were assessed quantitatively. Treatment with 1,8-cineole lowered both the mitotic index and elongation of the cells in a dose-dependent manner, and the half-maximal inhibitory concentration (IC50) for cell elongation was lower than that for cell proliferation. Moreover, 1,8-cineole also inhibited starch synthesis, with IC50 lower than that for cell proliferation. Thus, the inhibitory effects of 1,8-cineole were not specific to cell proliferation; rather, 1,8-cineole seemed inhibitory to a variety of physiological activities when it was in direct contact with target cells. Based on these results, possible mechanisms for the mode of action of 1,8-cineole and for its preferential inhibition on root growth are discussed.

Vacuole-localized berberine bridge enzyme-like proteins are required for a late step of nicotine biosynthesis in tobacco.

Tobacco (Nicotiana tabacum) plants synthesize nicotine and related pyridine-type alkaloids, such as anatabine, in their roots and accumulate them in their aerial parts as chemical defenses against herbivores. Herbivory-induced jasmonate signaling activates structural genes for nicotine biosynthesis and transport by way of the NICOTINE (NIC) regulatory loci. The biosynthesis of tobacco alkaloids involves the condensation of an unidentified nicotinic acid-derived metabolite with the N-methylpyrrolinium cation or with itself, but the exact enzymatic reactions and enzymes involved remain unclear. Here, we report that jasmonate-inducible tobacco genes encoding flavin-containing oxidases of the berberine bridge enzyme family (BBLs) are expressed in the roots and regulated by the NIC loci. When expression of the BBL genes was suppressed in tobacco hairy roots or in tobacco plants, nicotine production was highly reduced, with a gradual accumulation of a novel nicotine metabolite, dihydromethanicotine. In the jasmonate-elicited cultured tobacco cells, suppression of BBL expression efficiently inhibited the formation of anatabine and other pyridine alkaloids. Subcellular fractionation and localization of green fluorescent protein-tagged BBLs showed that BBLs are localized in the vacuoles. These results indicate that BBLs are involved in a late oxidation step subsequent to the pyridine ring condensation reaction in the biosynthesis of tobacco alkaloids.

Lethal impacts of cigarette smoke in cultured tobacco cells

BackgroundIn order to understand and generalize the toxic mechanism of cigarette smoke in living cells, comparison of the data between animal systems and other biological system such as microbial and plant systems is highly beneficial.ObjectiveBy employing the tobacco cells as model materials for cigarette smoke toxicity assay, the impacts of the combustion by-products such as nitrogen oxides could be highlighted as the toxic impacts of the plant-derived endogenous chemicals could be excluded in the plant cells.MethodsCigarette smoke-induced cell death was assessed in tobacco cell suspension cultures in the presence and absence of pharmacological inhibitors.ResultsCigarette smoke was effective in induction of cell death. The smoke-induced cell death could be partially prevented by addition of nitric oxide (NO) scavenger, suggesting the role for NO as the cell death mediator. Addition of NO donor to tobacco cells also resulted in development of partial cell death further confirming the role of NO as cell death mediator. Members of reactive oxygen species and calcium ion were shown to be protecting the cells from the toxic action of smoke-derived NO.

Production of double repeated B subunit of Shiga toxin2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease.

Pig edema disease is a bacterial disease caused by enterohemorrhagic Escherichia coli. E.coli produces Shiga toxin2e (Stx2e), which is composed of one A subunit (Stx2eA) and five B subunits (Stx2eB). We previously reported production of Stx2eB in lettuce plants as a potential edible vaccine (Matsui etal. in Biosci Biotechnol Biochem 73:1628-1634, 2009). However, the accumulation level was very low, and it was necessary to improve expression of Stx2eB for potential use of this plant-based vaccine. Therefore, in this study, we optimized the Stx2eB expression cassette and found that a double repeated Stx2eB (2× Stx2eB) accumulates to higher levels than a single Stx2eB in cultured tobacco cells. Furthermore, a linker peptide between the two Stx2eB moieties played an important role in maximizing the effects of the double repeat. Finally, we generated transgenic lettuce plants expressing 2× Stx2eB with a suitable linker peptide that accumulate as much as 80mg per 100g fresh weight, a level that will allow us to use these transgenic lettuce plants practically to generate vaccine material.

Microscopic visualization of Pulsed Electric Field induced changes on plant cellular level

Abstract The effects of Pulsed Electric Fields (PEF) on protoplasts from cultured tobacco cells ( Nicotiana tabacum b.y.-2) in comparison to the changes on cultured plant cells with cell walls were visualised in order to study the direct impact of PEF on cell components and to clarify the influence of the cell wall on electroporation. Optical microscopic analyses were carried out and images were recorded during PEF treatment. Results showed higher sensitivity of protoplasts to electric fields related to cells with a cell wall. Protoplasts sizes were measured before and after different treatment intensities and protoplasts shrinkage was used as an indicator for cell rupture. It could be demonstrated that cell volume decrease is influenced by PEF intensity, initial cell size, cell orientation in the electric field and nucleus position. Industrial relevance: Since the beginning of the 20th century the relevance of Pulsed Electric Fields (PEF) technology in food- and biotechnology has increased substantially. However, the mechanism of membrane permeabilization and the PEF induced changes in cell structure remain poorly understood, diminishing the optimal use in food industry. In this study the direct effects of PEF on cultured plant cell material and influencing factors of the degree of membrane disintegration were visualized and identified. The development of new methods to examine cell vitality shall help to convert the basic knowledge into effective processes.

Success stories in molecular farming—a brief overview

Success stories in molecular farming—a brief overview

Acceleration of Cell Growth by Xyloglucan Oligosaccharides in Suspension-Cultured Tobacco Cells

The incorporation of xyloglucan oligosaccharide (XXXG) into the walls of suspension-cultured tobacco cells accelerated cell expansion followed by cell division, changed cell shape from cylindrical to spherical, decreased cell size, and caused cell aggregation. Fluorescent XXXG added to the culture medium was found to be incorporated into the surface of the entire wall, where strong incorporation occurred not only on the surface, but also in the interface walls between cells during cell division. Cell expansion was always greater in the transverse direction than in the longitudinal direction and then, immediately, expansion led to cell division in the presence of XXXG; this process might result in the high level of cell aggregation seen in cultured tobacco cells. We concluded that the integration of this oligosaccharide into the walls could accelerate not only cell expansion, but also cell division in cultured cells.

Aluminium reduces sugar uptake in tobacco cell cultures: a potential cause of inhibited elongation but not of toxicity

Aluminium is well known to inhibit plant elongation, but the role in this inhibition played by water relations remains unclear. To investigate this, tobacco (Nicotiana tabacum L.) suspension-cultured cells (line SL) was used, treating them with aluminium (50 μM) in a medium containing calcium, sucrose, and MES (pH 5.0). Over an 18 h treatment period, aluminium inhibited the increase in fresh weight almost completely and decreased cellular osmolality and internal soluble sugar content substantially; however, aluminium did not affect the concentrations of major inorganic ions. In aluminium-treated cultures, fresh weight, soluble sugar content, and osmolality decreased over the first 6 h and remained constant thereafter, contrasting with their continued increases in the untreated cultures. The rate of sucrose uptake, measured by radio-tracer, was reduced by approximately 60% within 3 h of treatment. Aluminium also inhibited glucose uptake. In an aluminium-tolerant cell line (ALT301) isogenic to SL, all of the above-mentioned changes in water relations occurred and tolerance emerged only after 6 h and appeared to involve the suppression of reactive oxygen species. Further separating the effects of aluminium on elongation and cell survival, sucrose starvation for 18 h inhibited elongation and caused similar changes in cellular osmolality but stimulated the production of neither reactive oxygen species nor callose and did not cause cell death. We propose that the inhibition of sucrose uptake is a mechanism whereby aluminium inhibits elongation, but does not account for the induction of cell death.

Tobacco cell cultures transformed by the hsp 101 gene exhibit an increased resistance to potassium fluoride

Tobacco cell cultures transformed by the hsp 101 gene exhibit an increased resistance to potassium fluoride

Centromere targeting of alien CENH3s in Arabidopsis and tobacco cells

The centromere is a region utilized for spindle attachment on a eukaryotic chromosome and essential for accurate chromatid segregation. In most eukaryotes, centromeres have specific DNA sequences and are capable of assembling specific proteins to form a complex called the kinetochore. Among these proteins, centromeric histone H3 (CENH3) is one of the most fundamental, since CENH3s have been found in all investigated functional centromeres and recruits other centromeric proteins. In this study, the localization of alien CENH3s were analyzed in Arabidopsis and tobacco-cultured cells to determine the interaction between species-specific centromeric DNA and CENH3. Results showed that CENH3 of Arabidopsis and tobacco were localized on centromeres in the tobacco-cultured cells, unlike the case with CENH3 of rice and Luzula. In addition to these CENH3s, CENH3 of Luzula was partially localized in the Arabidopsis cultured cells. These data suggest that only evolutionally close CENH3s are able to target centromeres in alien species. Furthermore, the ability to target alien centromeres of histone fold domains was investigated using amino-terminal deleted CENH3s.

High-level production of bioactive heterodimeric protein human interleukin-12 in rice

Human interleukin-12 (hIL-12), a heterodimeric cytokine comprised of p35 and p40 subunits, was generated previously via tobacco cell suspension culture at a maximum concentration of only 175 μg/L. In order to improve the productivity, the rice α-amylase 3D promoter (Ramy3D) was utilized in order to express recombinant protein at high levels under sucrose starvation conditions. The hIL-12 gene was cloned into the rice expression vector under the control of the Ramy3D promoter. Transgenic rice calli were prepared via particle bombardment-mediated transformation and were screened for rhIL-12p70 expression using ELISA. Western blot and bioassay results confirmed that rhIL-12p70 was expressed and secreted into the culture medium as a functionally active heterodimeric form, and the productivity of rhIL-12p70 was estimated as 12.7 mg/L, which is approximately 73-fold higher than was observed with the tobacco cell culture system [Kwon TH, Seo JE, Kim J, Lee JH, Jang YS, Yang MS. Expression and secretion of the heterodimeric protein interleukin-12 in plant cell suspension culture. Biotechnol Bioeng 2003;81:870–5]. However, the secreted rhIL-12p70 proved unstable in the medium, and degraded rapidly after day 13. In order to stabilize the secreted rhIL-12p70, three stabilizing polymers were tested (polyethylene glycol, polyvinylpyrrolidone, and gelatin). Gelatin was the most effective in stabilizing the secreted rhIL-12p70. After the addition of 2% (w/v) gelatin, the maximum rhIL-12p70 concentration reached 31.0 mg/L, representing a 2.5-fold increase over the control.

Isolation of chloroplast FtsZ and AtpC, and analysis of protein targeting into the complex chloroplast of the haptophyte Pavlova pinguis

Haptophytes are abundant phytoplankton that possess 4 membrane-bound chloroplasts. These chloroplasts originated from a red alga that was taken up by a eukaryotic host cell. No previous work has reported on the protein targeting of haptophytes, except for computational analyses. We isolated the two genes encoding chloroplast proteins AtpC1 and FtsZ from Pavlova pinguis and analyzed their molecular structure. These proteins had a bipartite sequence at the N-terminus, which consisted of an endoplasmic reticulum (ER) signal sequence followed by a chloroplast transit peptide. To demonstrate the functionality of the ER signal sequences and the chloroplast targeting sequences in vivo, we fused the predicted ER signal sequence, chloroplast transit peptide, and bipartite sequence of AtpC1 and FtsZ individually to the N-terminus of green fluorescent protein (sGFP). This was then introduced into cultured tobacco cells. Microscopic observation revealed that the predicted ER signal sequences of AtpC1 and FtsZ had the ability to localize to the ER, and the following amino acids worked as a chloroplast transit peptide in the tobacco cells. We concluded that the AtpC1 and FtsZ of P. pinguis have a bipartite sequence at the N-terminus and a molecular structure in common with other independently established secondarily endosymbiotic algae.

Dicistronic binary vector system—A versatile tool for gene expression studies in cell cultures and plants

Dicistronic binary vector constructs based on pGreenII vectors for Agrobacterium mediated gene transfer alleviate the translational expression monitoring of a target gene in plants. The functionality of the transformation vectors was proven by marker gene constructs containing a mannopine synthase promoter (p-MAS) fused to a beta-glucuronidase ( gus) gene followed by an internal ribosome entry site and a firefly luciferase ( luc) gene. The cap-dependent translation of a physically independent target protein can be monitored by the cap-independently co-translated luciferase, because both mRNAs are located on the same strand. Among three different IRES elements, the tobamo IRES element showed highest activity in transient expression. As a proof of principle for physiological studies the gus gene was replaced by a sodium antiporter gene (At nhx1). Comparative studies with At nhx1 transgenic luc expressing tobacco cell cultures and pea plants ( Pisum sativum L.) showed improved salt tolerance in relation to their wild type counterparts grown under corresponding conditions. A coincidence of the luc gene expression and increased sodium chloride tolerance is demonstrated by measurement of luminescence and cell growth.

The 5′ untranslated region of the VR-ACS1 mRNA acts as a strong translational enhancer in plants

The structure and function of untranslated mRNA leader sequences and their role in controlling gene expression remains poorly understood. Previous research has suggested that the 5' untranslated region (5'UTR) of the Vigna radiata aminocyclopropane-1-carboxylate synthase synthase (VR-ACS1) gene may function as a translational enhancer in plants. To test such hypothesis we compared the translation enhancing properties of three different 5'UTRs; those from the VR-ACS1, the chlorophyll a/b binding gene from petunia (Cab22L; a known translational enhancer) and the Vigna radiata pectinacetylesterase gene (PAE; used as control). Identical constructs in which the coding region of the beta-glucuronidase (GUS) gene was fused to each of the three 5'UTRs and placed under the control of the cauliflower mosaic virus 35S promoter were prepared. Transient expression assays in tobacco cell cultures and mung bean leaves showed that the VR-ACS1 and Cab22L 5'UTRs directed higher levels of GUS activity than the PAE 5'UTR. Analysis of transgenic Arabidopsis thaliana seedlings, as well as different tissues from mature plants, confirmed that while transcript levels were equivalent for all constructs, the 5'UTRs from the VR-ACS1 and Cab22L genes can increase GUS activity twofold to fivefold compared to the PAE 5'UTR, therefore confirming the translational enhancing properties of the VR-ACS1 5'UTR.

Plant cell calcium‐rich environment enhances thermostability of recombinantly produced α‐amylase from the hyperthermophilic bacterium Thermotoga maritime

In the industrial processing of starch for sugar syrup and ethanol production, a liquefaction step is involved where starch is initially solubilized at high temperature and partially hydrolyzed with a thermostable and thermoactive alpha-amylase. Most amylases require calcium as a cofactor for their activity and stability, therefore calcium, along with the thermostable enzyme, are typically added to the starch mixture during enzymatic liquefaction, thereby increasing process costs. An attractive alternative would be to produce the enzyme directly in the tissue to be treated. In a proof of concept study, tobacco cell cultures were used as model system to test in planta production of a hyperthermophilic alpha-amylase from Thermotoga maritima. While comparable biochemical properties to recombinant production in Escherichia coli were observed, thermostability of the plant-produced alpha-amylase benefited significantly from high intrinsic calcium levels in the tobacco cells. The plant-made enzyme retained 85% of its initial activity after 3 h incubation at 100 degrees C, whereas the E. coli-produced enzyme was completely inactivated after 30 min under the same conditions. The addition of Ca(2+) or plant cell extracts from tobacco and sweetpotato to the E. coli-produced enzyme resulted in a similar stabilization, demonstrating the importance of a calcium-rich environment for thermostability, as well as the advantage of producing this enzyme directly in plant cells where calcium is readily available.