Suspension-cultured Tobacco Cells Research Articles

Zinc-Dependent Protection of Tobacco and Rice Cells From Aluminum-Induced Superoxide-Mediated Cytotoxicity.

Al3+ toxicity in growing plants is considered as one of the major factors limiting the production of crops on acidic soils worldwide. In the last 15 years, it has been proposed that Al3+ toxicity are mediated with distortion of the cellular signaling mechanisms such as calcium signaling pathways, and production of cytotoxic reactive oxygen species (ROS) causing oxidative damages. On the other hand, zinc is normally present in plants at high concentrations and its deficiency is one of the most widespread micronutrient deficiencies in plants. Earlier studies suggested that lack of zinc often results in ROS-mediated oxidative damage to plant cells. Previously, inhibitory action of Zn2+ against lanthanide-induced superoxide generation in tobacco cells have been reported, suggesting that Zn2+ interferes with the cation-induced ROS production via stimulation of NADPH oxidase. In the present study, the effect of Zn2+ on Al3+-induced superoxide generation in the cell suspension cultures of tobacco (Nicotiana tabacum L., cell-line, BY-2) and rice (Oryza sativa L., cv. Nipponbare), was examined. The Zn2+-dependent inhibition of the Al3+-induced oxidative burst was observed in both model cells selected from the monocots and dicots (rice and tobacco), suggesting that this phenomenon (Al3+/Zn2+ interaction) can be preserved in higher plants. Subsequently induced cell death in tobacco cells was analyzed by lethal cell staining with Evans blue. Obtained results indicated that presence of Zn2+ at physiological concentrations can protect the cells by preventing the Al3+-induced superoxide generation and cell death. Furthermore, the regulation of the Ca2+ signaling, i.e., change in the cytosolic Ca2+ ion concentration, and the cross-talks among the elements which participate in the pathway were further explored.

Mitigation of copper toxicity by DNA oligomers in green paramecia

Impact of transition metals which catalyze the generation of reactive oxygen species (ROS), on activation of cell death signaling in plant cells have been documented to date. Similarly in green paramecia (Paramecium bursaria), an aquatic protozoan species harboring symbiotic green algae in the cytoplasm, toxicities of various metallic ions have been documented. We have recently examined the effects of double-stranded GC-rich DNA fragments with copper-binding nature and ROS removal catalytic activity as novel plant cell-protecting agents, using the suspension-cultured tobacco cells. Here, we show that above DNA oligomers protect the cells of green paramecia from copper-induced cell death, suggesting that the phenomenon firstly observed in tobacco cells is not limited only within higher plants but it could be universally observable in wider range of organisms.

Plant-based vaccines for animals and humans: recent advances in technology and clinical trials.

It has been about 30 years since the first plant engineering technology was established. Although the concept of plant-based pharmaceuticals or vaccines motivates us to develop practicable commercial products using plant engineering, there are some difficulties in reaching the final goal: to manufacture an approved product. At present, the only plant-made vaccine approved by the United States Department of Agriculture is a Newcastle disease vaccine for poultry that is produced in suspension-cultured tobacco cells. The progress toward commercialization of plant-based vaccines takes much effort and time, but several candidate vaccines for use in humans and animals are in clinical trials. This review discusses plant engineering technologies and regulations relevant to the development of plant-based vaccines and provides an overview of human and animal vaccines currently under clinical trials.

Involvement of sulfhydryl compounds and antioxidant enzymes in H2S-induced heat tolerance in tobacco (Nicotiana tabacum L.) suspension-cultured cells

Hydrogen sulfide (H2S) is a multifunctional second messenger involved in plant growth, development, and acquisition of stress tolerance, including heat tolerance, but the mechanism of H2S-induced heat tolerance in tobacco suspension-cultured cells is not completely clear. This study investigated the effects of pretreatment with the H2S donor sodium hydrosulfide (NaHS) and its precursors cysteine and potassium bisulfite (KHSO3) on the heat tolerance of tobacco suspension-cultured cells and the involvement of sulfhydryl compounds and antioxidant enzymes in conferring heat tolerance. Pretreatment with NaHS, cysteine, and KHSO3 significantly increased the survival percentage of tobacco suspension-cultured cells under heat stress, while treatment with the H2S scavenger hypotaurine in combination with NaHS eliminated heat tolerance induced by treatment with NaHS alone. In addition, NaHS treatment increased the levels of water-soluble sulfhydryl compounds such as H2S, total sulfhydryl compounds, total sulfhydryl proteins, cysteine, and glutathione (GSH) as well as the activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and glutathione reductase (GR) under normal culture conditions (26°C). Under heat stress at 43°C, the levels of water-soluble sulfhydryl compounds and the activities of antioxidant enzymes all dropped, but the cells treated with NaHS sustained significantly higher levels of water-soluble sulfhydryl compounds and activities of antioxidant enzymes than the control. These results suggest that the pretreatment with NaHS could improve the heat tolerance of tobacco suspension-cultured cells and that the acquisition of this heat tolerance is caused by the elevated levels of water-soluble sulfhydryl compounds and elevated activities of antioxidant enzymes induced by NaHS.

Production of the dammarene sapogenin (protopanaxadiol) in transgenic tobacco plants and cultured cells by heterologous expression of PgDDS and CYP716A47.

Protopanaxadiol (PPD) is an aglycone of dammarene-type ginsenoside and has high medicinal values. In this work, we reported the PPD production in transgenic tobacco co-overexpressing PgDDS and CYP716A47. PPD is an aglycone of ginsenosides produced by Panax species and has a wide range of pharmacological activities. PPD is synthesized via the hydroxylation of dammarenediol-II (DD) by CYP716A47 enzyme. Here, we established a PPD production system via cell suspension culture of transgenic tobacco co-overexpressing the genes for PgDDS and CYP716A47. The concentration of PPD in transgenic tobacco leaves was 2.3-5.7 µg/g dry weight (DW), depending on the transgenic line. Leaf segments were cultured on medium with various types of hormones to induce callus. Auxin treatment, particularly 2,4-D, strongly enhanced the production of DD (783.8 µg g(-1) DW) and PPD (125.9 µg g(-1) DW). Treatment with 2,4-D enhanced the transcription of the HMG-Co reductase (HMGR) and squalene epoxidase genes. PPD production reached 166.9 and 980.9 µg g(-1) DW in a 250-ml shake flask culture and in 5-l airlift bioreactor culture, respectively.

Extracellular ATP is Involved in the Salicylic Acid-Induced Cell Death in Suspension-Cultured Tobacco Cells

Extracellular ATP (eATP) can function as a signaling molecule to regulate a wide range of cellular processes. We investigated the regulatory role of eATP in the cell death induced by salicylic acid (SA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. Treatment of tobacco suspension cells with SA induced cell death. The same treatment lowered the levels of eATP, accompanied by a decrease of both the respiratory O2 uptake and intracellular ATP levels in tobacco suspension cells. Treatment with β,γ-methyleneadenosine 5’-triphosphate (AMP-PCP), which is the non-hydrolysable analogue of ATP and can exclude eATP from binding sites of eATP receptors, also induced cell death in tobacco cell cultures. Treatment with exogenous ATP partially alleviated the cell death induced by SA. These observations suggest that eATP is involved in the SA-induced cell death in tobacco cell cultures.

Changes in cell wall properties coincide with overexpression of extensin fusion proteins in suspension cultured tobacco cells.

Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.

Molecular Farming – Production of pharmaceuticals in plants and plant cells

Many different plant-based systems have been used to produce valuable small molecules or recombinant proteins but only a small number have made the leap from an experimental platform to a viable commercial process. This reflects a combination of factors, principally the technical issues that must be addressed to achieve competitive performance, the economic principles that need to be satisfied to ensure manufacturing processes are financially viable and sustainable, and the regulatory demands that must be met to ensure that products manufactured in plants are safe, efficacious and meet the quality standards demanded by the regulators [1,2]. In general, plants and plant cells are ideal production platforms, which can be easily scaled up to provide valuable products for pharmaceutical, cosmetic and industrial applications. Typically plant systems are grown in containment under defined conditions, either in the greenhouse or in bioreactors, allowing process controls to regulate growth and product formation, thus ensuring regulatory compliance. In this presentation, the advantages and disadvantages of commonly used plant-based systems are presented as well as strategies for recovering the final product. In many cases production and downstream processes have to be optimized to establish a commercially viable platform and therefore novel strategies for strain and process optimization, helping to increase yields and scalability are presented. These approaches include the optimization of expression vectors and recombinant protein targeting [3], the testing of different plant platforms [4], the use of statistical experimental techniques to optimise medium and cultivation conditions [5], establishment of elite lines by flow cytometry [6], as well as strategies for scale up and GMP-compliant production [7].

Assessment of cultivation factors that affect biomass and geraniol production in transgenic tobacco cell suspension cultures.

A large-scale statistical experimental design was used to determine essential cultivation parameters that affect biomass accumulation and geraniol production in transgenic tobacco (Nicotiana tabacum cv. Samsun NN) cell suspension cultures. The carbohydrate source played a major role in determining the geraniol yield and factors such as filling volume, inoculum size and light were less important. Sucrose, filling volume and inoculum size had a positive effect on geraniol yield by boosting growth of plant cell cultures whereas illumination of the cultures stimulated the geraniol biosynthesis. We also found that the carbohydrates sucrose and mannitol showed polarizing effects on biomass and geraniol accumulation. Factors such as shaking frequency, the presence of conditioned medium and solubilizers had minor influence on both plant cell growth and geraniol content. When cells were cultivated under the screened conditions for all the investigated factors, the cultures produced ∼5.2 mg/l geraniol after 12 days of cultivation in shaking flasks which is comparable to the yield obtained in microbial expression systems. Our data suggest that industrial experimental designs based on orthogonal arrays are suitable for the selection of initial cultivation parameters prior to the essential medium optimization steps. Such designs are particularly beneficial in the early optimization steps when many factors must be screened, increasing the statistical power of the experiments without increasing the demand on time and resources.

Fungal cellulase is an elicitor but its enzymatic activity is not required for its elicitor activity

Plant-pathogenic fungi produce cellulases. However, little information is available on cellulase as an elicitor in plant-pathogen interactions. Here, an endocellulase (EG1) was isolated from Rhizoctonia solani. It contains a putative protein of 227 amino acids with a signal peptide and a family-45 glycosyl hydrolase domain. Its aspartic acid (Asp) residue at position 32 was changed to alanine (Ala), resulting in full loss of its catalytic activity. Wild-type and mutated forms of the endoglucanase were expressed in yeast and purified to homogeneity. The purified wild-type and mutant forms induced cell death in maize, tobacco and Arabidopsis leaves, and the transcription of three defence marker genes in maize and tobacco and 10 genes related to defence responses in maize. Moreover, they also induced the accumulation of reactive oxygen species (ROS), medium alkalinization, Ca(2+) accumulation and ethylene biosynthesis of suspension-cultured tobacco cells. Similarly, production of the EG1 wild-type and mutated forms in tobacco induced cell death using the Potato virus X (PVX) expression system. In vivo, expression of EG1 was also related to cell death during infection of maize by R. solani. These results provide direct evidence that the endoglucanase is an elicitor, but its enzymatic activity is not required for its elicitor activity.

The S-Domain Receptor Kinase Arabidopsis Receptor Kinase2 and the U Box/Armadillo Repeat-Containing E3 Ubiquitin Ligase9 Module Mediates Lateral Root Development under Phosphate Starvation in Arabidopsis.

When plants encounter nutrient-limiting conditions in the soil, the root architecture is redesigned to generate numerous lateral roots (LRs) that increase the surface area of roots, promoting efficient uptake of these deficient nutrients. Of the many essential nutrients, reduced availability of inorganic phosphate has a major impact on plant growth because of the requirement of inorganic phosphate for synthesis of organic molecules, such as nucleic acids, ATP, and phospholipids, that function in various crucial metabolic activities. In our screens to identify a potential role for the S-domain receptor kinase1-6 and its interacting downstream signaling partner, the Arabidopsis (Arabidopsis thaliana) plant U box/armadillo repeat-containing E3 ligase9 (AtPUB9), we identified a role for this module in regulating LR development under phosphate-starved conditions. Our results show that Arabidopsis double mutant plants lacking AtPUB9 and Arabidopsis Receptor Kinase2 (AtARK2; ark2-1/pub9-1) display severely reduced LRs when grown under phosphate-starved conditions. Under these starvation conditions, these plants accumulated very low to no auxin in their primary root and LR tips as observed through expression of the auxin reporter DR5::uidA transgene. Exogenous auxin was sufficient to rescue the LR developmental defects in the ark2-1/pub9-1 lines, indicating a requirement of auxin accumulation for this process. Our subcellular localization studies with tobacco (Nicotiana tabacum) suspension-cultured cells indicate that interaction between ARK2 and AtPUB9 results in accumulation of AtPUB9 in the autophagosomes. Inhibition of autophagy in wild-type plants resulted in reduction of LR development and auxin accumulation under phosphate-starved conditions, suggesting a role for autophagy in regulating LR development. Thus, our study has uncovered a previously unknown signaling module (ARK2-PUB9) that is required for auxin-mediated LR development under phosphate-starved conditions.

Isolation, culture, and transient transformation of plant protoplasts.

Transient gene expression in protoplasts, which has been used in several plant species, is an important and versatile tool for rapid functional gene analysis, protein subcellular localization, and biochemical manipulations. This unit describes transient gene expression by electroporation of DNA into protoplasts of Arabidopsis or tobacco suspension-cultured cells and by polyethylene glycol (PEG)-mediated DNA transformation into protoplasts derived from rice leaf sheaths. PEG-mediated DNA transformation for transient gene expression in rice protoplasts in suspension culture is also described as an alternative technique. Methods for collecting intracellular and secreted proteins are also provided.

ARTICLE RETRACTED BY AUTHORS]Optimization of Parameters for Isolation of Viable Protoplasts by Surgical Paper Tape Tobacco Leaf Sandwich Method and Cell Suspension Culture of Nicotiana tabacum: A Rapid and Simple Method of Protoplasts Isolation

ARTICLE RETRACTED BY AUTHORS]Optimization of Parameters for Isolation of Viable Protoplasts by Surgical Paper Tape Tobacco Leaf Sandwich Method and Cell Suspension Culture of Nicotiana tabacum: A Rapid and Simple Method of Protoplasts Isolation

Molecular pharming in plants and plant cell cultures: a great future ahead?

Plant biotechnology may not be a familiar concept to the general public, but it is a rapidly developing field of research that involves the use of plants, plant tissues and plant cell cultures to make or modify products and processes. The versatility of plants and plant cells can be harnessed to produce diverse products, including valuable proteins. This is often described as ‘molecular farming’ and it requires the introduction of foreign DNA into plants or plant cells, turning them into factories for the production of specific recombinant protein products. The term ‘molecular pharming’ is often used instead to highlight the production of protein-based biopharmaceuticals, which contributes to the sustainable production of drugs that promote human and animal wellbeing. Both terms also apply to the production of valuable secondary metabolites such as the anticancer drugs paclitaxel, vincristine and vinblastine, but we will focus on recombinant proteins and their use as biopharmaceuticals in this article. The biopharmaceutical markets have expanded rapidly over the last 20 years, and are projected to more than double in volume over the next decade from US$200 billion in 2013 to at least US$500 billion in 2020. The two major biopharmaceutical production systems are microbes (mainly Escherichia coli and yeast) and mammalian cells such as the Chinese hamster ovary platform. In both cases, productivity has increased substantially over the last decade due to process optimization, platform standardization and genetic improvements. Both the US FDA and European Medicines Agency are familiar with these systems, and standard protocols can be followed to ensure the approval of new products. However, equivalent protocols are only just emerging for plant-based production systems, and only one plant-derived biopharmaceutical protein is currently on the market. With their established production infrastructure and regulatory framework, microbial and mammalian production systems have raced far ahead of their plant-based counterparts. No company will change their production host without a clear economic benefit, nor will they consider plants and plant cells for new products if there is no advantage over their incumbent technology. Furthermore, new companies will not base their manufacturing on a second-best option. Therefore, plant-based systems must begin to compete head-to-head with the established systems and, on a technological basis, we can already identify the areas where plantbased systems have the advantage, namely in terms of speed, improved product quality and scalability. The international success story of molecular pharming began in 2006 with the US Department of Agriculture approval of a poultry vaccine against Newcastle disease developed by Dow AgroSciences (IN, USA) [1,2]. The vaccine was manufactured in transgenic tobacco cell suspension cultures and was a benchmark for the regulatory acceptance of plants as a manufacturing platform, Molecular pharming in plants and plant cell cultures: a great future ahead?

Protection of tobacco cells from oxidative copper toxicity by catalytically active metal-binding DNA oligomers

The impact of copper ions on the oxidative and calcium signal transductions, leading to cell death in plant cells, have been documented. Copper induces a series of biological and chemical reactions in plant cells including the oxidative burst reflecting the production of reactive oxygen species and the stimulation of calcium channel opening allowing a transient increase in cytosolic calcium concentrations. These early events, completed within a few minutes after the contact with copper, are known to trigger the development of cell death. The effects of DNA fragments with copper-binding motifs as novel plant cell-protecting agents were assessed using cell suspension cultures of transgenic tobacco (Nicotiana tabacum L., cell line BY-2) expressing the aequorin gene. The addition of GC-rich double-stranded DNA fragments, prior to the addition of copper ions, effectively blocked both the copper-induced calcium influx and cell death. In addition, the DNA-Cu complex examined was shown to possess superoxide-scavenging catalytic activity, suggesting that DNA-mediated protection of the cells from copper toxicity is due to the removal of superoxide. Lastly, a possible mechanism of DNA-Cu interaction and future applications of these DNA fragments in the protection of plant roots from metal toxicity or in aid of phyto-remediation processes are discussed.

A Novel Wild-Land Fire-Fighting Foam for Minimizing the Phytotoxicity of Wood Burning-Derived Smoke Tested in Living Plant Cells

Impact of wild-land fires to the ecosystem is highly complex. Damages to the ecosystem can be attributed not only to the direct impact of fire and release of toxic post-combustion gasses but also to the spraying of fire-fighting chemicals. Fire-fighting foam (FFF) agents are frequently applied for controls in wild-land fires including forest fire. However, effects of FFFs on the composition of the post-combustion gasses and the phytotoxicity of smoke derived from burning woods have not been determined to date. In the present study, with Fourier transform infrared spectroscopy (FT-IR), we have analyzed the chemical composition of the gasses derived from wood slices exposed to two distinct manners of combustion, namely, smoldering (gradual combustion without flame) and rapid burning (combustion with flame). Tested samples include slices of Japanese cedar, Japanese cypress, and Western hemlock. The amount of hydrocarbons, detected in the post-combustion gas such as methane, ethane, ethylene, propane, hexane, formaldehyde, acrolein and phenol, were higher in the gasses from smoldered samples. The major hydrocarbon found in the post-combustion gases processed in the presence of pilot flame was methane. Other hydrocarbons were hardly detectable. Addition of FFFs, namely, a soap-based FFF (designated as MK-08) and a detergent co cocktail-based FFF (Phos-chek) onto wooden slices resulted in slight increase in other hydrocarbons in the gasses derived from flame-driven combustion of wood slices. Interestingly, addition of Phos-chek drastically elevated the phytotoxicity of post-combustion gas derived from Western hemlock slices heated in the presence of pilot flame when assessed using the suspension cultured tobacco cells. In contrast, the soap-based FFF tested here did not alter the phytotoxicity of the post-combustion gasses, suggesting that soap-based FFF might minimize the impact of the fire-fighting activity to the living plants consisting the ecosystem in the forests and wild-land.

Grape marc extract causes early perception events, defence reactions and hypersensitive response in cultured tobacco cells

Grape marc extract (GME) showed elicitor activity on suspension-cultured cells of tobacco. The BY-2 cells reacted to GME (0.25% and 0.125%) with a long-sustained pH rise in their growth medium. Using EGTA or LaCl3, we showed that extracellular alkalinization depended on Ca2+ mobilization. The tobacco BY-2 cells challenged with GME promoted cell death and the upregulation of defence-related genes such as PR3, PAL and CCoAOMT. Cell death rate was quantified using an experimental calibrated Evans Blue assay. The GME-induced cell death was dose-dependent and occurred in 24 h. Longer exposure increased the extent of tobacco cell death. To investigate a potential hypersensitive reaction, we tested the effect of various inhibitors of protein synthesis (cycloheximide) and proteases (aprotinin, pepstatin and E-64) on GME-induced cell death. All these chemicals reduced GME-induced cell death rate in 30 min. Overall, our findings indicate that GME elicits early perception events, defence reactions and cell death requiring protein synthesis and proteases.

The use of FM dyes to analyze plant endocytosis.

FM (Fei-Mao) styryl dyes are compounds of amphiphilic character that are used for the fluorescence tracking of endocytosis and related processes, i.e., the internalization of membrane vesicles from the plasma membrane (PM) and dynamics of endomembranes. Staining with FM dyes and subsequent microscopical observations could be performed both on the tissue and cellular level. Here, we describe simple procedures for the effective FM dye staining and de-staining in root epidermal cells of Arabidopsis thaliana seedlings and suspension-cultured tobacco cells. The progression of FM dye uptake, reflected by an increased amount of the dye in the endosomal compartments, is monitored under the fluorescence microscope in a time-lapse manner. The data obtained can be used for the characterization of the rate of endocytosis and the function of components of endosomal recycling machinery.

Production of dammarenediol-II triterpene in a cell suspension culture of transgenic tobacco

Dammarenediol-II is biologically active tetracyclic triterpenoid, which is basic compound of ginsenoside saponin. Here, we established the dammarenediol-II production via a cell suspension culture of transgenic tobacco overexpressing PgDDS. Dammarenediol-II synthase catalyzes the cyclization of 2,3-oxidosqualene to dammarenediol-II, which is the basic triterpene skeleton in dammarene-type saponin (ginsenosides) in Panax ginseng. Dammarenediol-II is a useful candidate both for pharmacologically active triterpenes and as a defense compound in plants. Dammarenediol-II is present in the roots of P. ginseng in trace amounts because it is an intermediate product in triterpene biosynthesis. In this work, we established the production of dammarenediol-II via cell suspension culture of transgenic tobacco. The dammarenediol-II synthase gene (PgDDS) isolated from P. ginseng was introduced into the Nicotiana tobacum genome under the control of 35S promoter by Agrobacterium-mediated transformation. Accumulation of dammarenediol-II in transgenic tobacco plants occurred in an organ-specific manner (roots > stems > leaves > flower buds), and transgenic line 14 (T14) exhibited a high amount (157.8 μg g⁻¹ DW) of dammarenediol-II in the roots. Dammarenediol-II production in transgenic tobacco plants resulted in reduced phytosterol (β-sitosterol, campesterol, and stigmasterol) contents. A cell suspension culture was established as a shake flask culture of a callus derived from root segments of transgenic (T14) plants. The amount of dammarenediol-II production in the cell suspension reached 573 μg g⁻¹ dry weight after 3 weeks of culture, which is equivalent to a culture volume of 5.2 mg dammarenediol-II per liter. Conclusively, the production of dammarenediol-II in a cell suspension culture of transgenic tobacco can be applied to the large-scale production of this compound and utilized as a source of pharmacologically active medicinal materials.

Metabolomic study reveals a selective accumulation of l-arginine in the d-ornithine treated tobacco cell suspension culture

The non-protein amino acid ornithine (Orn) plays essential roles in regulation of the urea cycle and polyamines biosynthesis in tobacco. Herein, we show that d-enantiomer of Orn, can actively participate in metabolites production in tobacco cells, functioning a positive role in plant cells metabolism, as opposed to the common l-enantiomer. Using a comprehensive amino acids and amines profiling method by liquid chromatography–electrospray ionization triple quadruple mass spectrometry (LC–ESI-QqQ-MS) in combination with chiral LC–ESI-MS, it was shown that d-Orn has a potential advantage in promoting selective and large accumulation of l-arginine (l-Arg) in tobacco cells. Exogenous d-Orn resulted in a selective up-regulation of l-Arg by 80-fold, while l-Orn slightly increased the levels of all amino acids. Changes of all the urea cycle related intermediates, e.g. citrulline, Arg and Orn were also shown to be critical following change of Orn's stereochemistry. GC/MS profiling of the metabolites revealed that high nicotine production was the dominant change driven by l-Orn treatments. From these observations, d-Orn was shown to be a selective regulator of l-Arg biosynthesis and the urea cycle. We propose that d-Orn has a potential function in the tobacco cells which through some previously unidentified mechanism result in l-Arg accumulation.