Tobacco Cell Suspension Research Articles

Chloroplast‐targeted bacterial RecA proteins confer tolerance to chloroplast DNA damage by methyl viologen or UV‐C radiation in tobacco (Nicotiana tabacum) plants

The nature and importance of the DNA repair system in the chloroplasts of higher plants under oxidative stress or UV radiation-induced genotoxicity was investigated via gain-of-functional approaches exploiting bacterial RecAs. For this purpose, transgenic tobacco (Nicotiana tabacum) plants and cell suspensions overexpressing Escherichia coli or Pseudomonas aeruginosa RecA fused to a chloroplast-targeting transit peptide were first produced. The transgenic tobacco plants maintained higher amounts of chloroplast DNA compared with wild-type (WT) upon treatments with methyl viologen (MV), a herbicide that generates reactive oxygen species (ROS) in chloroplasts. Consistent with these results, the transgenic tobacco leaves showed less bleaching than WT following MV exposure. Similarly, the MV-treated transgenic Arabidopsis plants overexpressing the chloroplast RecA homologue RECA1 showed weak bleaching, while the recA1 mutant showed opposite results upon MV treatment. In addition, when exposed to UV-C radiation, the dark-grown E. coli RecA-overexpressing transgenic tobacco cell suspensions, but not their WT counterparts, resumed growth and greening after the recovery period under light conditions. Measurements of UV radiation-induced chloroplast DNA damage using DraI assays (Harlow et al. 1994) with the chloroplast rbcL DNA probe and quantitative PCR analyses showed that the transgenic cell suspensions better repaired their UV-C radiation-induced chloroplast DNA lesions compared with WT. Taken all together, it was concluded that RecA-overexpressing transgenic plants are endowed with an increased chloroplast DNA maintenance capacity and enhanced repair activities, and consequently have a higher survival tolerance to genotoxic stresses. These observations are made possible by the functional compatibility of the bacterial RecAs in chloroplasts.

GAPDH, NtOSAK and CDC48, a conserved chaperone-like AAA-ATPase, as nitric oxide targets in response to (a)biotic stresses

Increasing evidences support the assumption that nitric oxide (NO) acts as a physiological mediator in plants facing (a)biotic stresses [1], [2]. Understanding its effects requires a deep analysis of the molecular mechanisms underlying its mode of action. In the recent years, efforts have been made in identifying and understanding the function of plant proteins regulated by NO at the post-translational level, notably by S-nitrosylation [3]. We demonstrated that the glycolytic enzyme GAPDH undergoes a fast and transient S-nitrosylation in tobacco cells exposed to a salt stress [4]. S-nitrosylation affects only a small proportion of the GAPDH population and does not affect glycolysis. Interestingly, in vivo GAPDH interacts with the protein kinase NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SnRK2 protein kinase family previously shown to be rapidly activated through NO in response to (a)biotic stresses [5]. Our current hypothesis is that S-nitrosylated GAPDH might acts as a phosphor-relay recruiting protein substrates for NtOSAK. Besides GAPDH, we identified proteins undergoing S-nitrosylation in tobacco cell suspensions exposed to cryptogein, a 10 kDa protein produced by the oomycete Phytophthora cryptogea [6]. These proteins include CDC48, a conserved chaperone-like AAA−ATPases. Using a combination of structural and biochemical analysis, we provided evidence that NO induces a local conformational change within the protein and inhibits its enzymatic activity. The physiological incidence of this process will be discussed.

Involvement of phospholipases C and D in the defence responses of riboflavin-treated tobacco cells

Riboflavin is an activator of defence responses in plants that increases resistance against diseases caused by fungal, oomycete, bacterial and viral pathogens. However, the mechanisms driving defence activation by riboflavin are poorly understood. We investigated the signal transduction pathways of phospholipase C (PLC) and phospholipase D (PLD) in tobacco (Nicotiana tabacum) suspension cells using a pharmacological approach to confirm whether riboflavin-mediated activation of the defence response is dependent on both PLC and PLD. The expression patterns analysed by quantitative reverse transcription-polymerase chain reaction demonstrated that the tobacco PLC and PLD gene families were differentially expressed in riboflavin-treated tobacco cells. PLC and PLD expression accompanied defence responses including the expression of defence response genes (PAL, PR-1a and PR-1b), the production of hydrogen peroxide and the accumulation of the phytoalexin scopoletin in tobacco cells treated with riboflavin. These defence responses were significantly inhibited in the presence of the PLC inhibitor U73122 and the PLD inhibitor 1-butanol; however, inhibitor analogues had no effect. Moreover, treating tobacco cells with phosphatidic acid, a signalling molecule produced by phospholipase catalysis, induced the accumulation of the phytoalexin scopoletin and compensated for the suppressive effects of U73122 and 1-butanol on riboflavin-induced accumulation of the phytoalexin. These results offer pharmacological evidence that PLC and PLD play a role in riboflavin-induced defences of tobacco.

DAF-fluorescence without NO: Elicitor treated tobacco cells produce fluorescing DAF-derivatives not related to DAF-2 triazol

Diaminofluorescein-dyes (DAFs) are widely used for visualizing NO production in biological systems. Here it was examined whether DAF-fluorescence could be evoked by other means than nitrosation. Tobacco (Nicotiana tabacum) suspension cells treated with the fungal elicitor cryptogein released compound(s) which gave a fluorescence increase in the cell-free filtrate after addition of DAF-2 or DAF-FM or DAR-4M. DAF-reactive compounds were relatively stable and identified as reaction products of H2O2 plus apoplastic peroxidase (PO). CPTIO prevented formation of these products. Horseradish-peroxidase (HR-PO) plus H2O2 also generated DAF-fluorescence in vitro. Using RP-HPLC with fluorescence detection, DAF derivatives were further analyzed. In filtrates from cryptogein-treated cells, fluorescence originated from two novel DAF-derivatives also obtained in vitro with DAF-2+HR-PO+H2O2. DAF-2T was only detected when an NO donor (DEA-NO) was present. Using high resolution mass spectrometry, the two above-described novel DAF-reaction products were tentatively identified as dimers.In cells preloaded with DAF-2 DA and incubated with or without cryptogein, DAF-fluorescence originated from a complex pattern of multiple products different from those obtained in vitro. One specific peak was responsive to exogenous H2O2, and another, minor peak eluted at or close to DAF-2T.Thus, in contrast to the prevailing opinion, DAF-2 can be enzymatically converted into a variety of highly fluorescing derivatives, both inside and outside cells, of which none (outside) or only a minor part (inside) appeared NO dependent. Accordingly, DAF-fluorescence and its prevention by cPTIO do not necessarily indicate NO production.

PCDD/Fs-induced oxidative damage and antioxidant system responses in tobacco cell suspension cultures

Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) are ubiquitous contaminants and can be considerably accumulated by natural plants. In order to elucidate the biochemical and physiological responses of plant to PCDD/Fs, tobacco Bright Yellow-2 (BY-2) cells were selected as model plant and treated with time- and concentration-dependent PCDD/Fs. The toxic effect and oxidative stress caused by PCDD/Fs were evident, which could be indicted by the reduction in fresh mass, the increase in malondialdehyde (MDA) content, and the damage of tobacco cell ultrastructure. PCDD/Fs tolerance was correlated with changes in antioxidant system and hormones of tobacco cells. Superoxide dismutase (SOD) and peroxidase (POD) exhibited peak enzyme activities at the PCDD/Fs concentration of 1000ng WHO98-TEQg−1fresh weight. Glutathione reductase (GR) enzyme activity increased monotonically at high level PCDD/Fs, but the activity of catalase (CAT) was only slightly affected at all treatment. Meanwhile, the exposure to PCDD/Fs resulted in the changes of hormones content. With the increase of exposure concentration of PCDD/Fs, the levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) increased, whereas the concentration of jasmonates (JAs) decreased. The above results suggest that tobacco cells had the ability to cope with the oxidative stress induced by low concentration of PCDD/Fs through increasing the activities of antioxidant enzymes and alternating plant hormones levels. However, oxidative stress and toxicity would burst out when plant cells were exposed to the high levels of PCDD/Fs.

Ultrasound-Microbubble Mediated Cavitation of Plant Cells: Effects on Morphology and Viability

The interaction between ultrasound pulses and microbubbles is known to generate acoustic cavitation that may puncture biological cells. This work presents new experimental findings on the bioeffects of ultrasound-microbubble mediated cavitation in plant cells with emphasis on direct observations of morphological impact and analysis of viability trends in tobacco BY-2 cells that are widely studied in higher plant physiology. The tobacco cell suspensions were exposed to 1 MHz ultrasound pulses in the presence of 1% v/v microbubbles (10% duty cycle; 1 kHz pulse repetition frequency; 70 mm between probe and cells; 1-min exposure time). Few bioeffects were observed at low peak negative pressures (<0.4 MPa) where stable cavitation presumably occurred. In contrast, at 0.9 MPa peak negative pressure (with more inertial cavitation activities according to our passive cavitation detection results), random pores were found on tobacco cell wall (observed via scanning electron microscopy) and enhanced exogenous uptake into the cytoplasm was evident (noted in our fluorescein isothiocyanate dextran uptake analysis). Also, instant lysis was observed in 23.4% of cells (found using trypan blue staining) and programmed cell death was seen in 23.3% of population after 12 h (determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling [TUNEL]). These bioeffects generally correspond in trend with those for mammalian cells. This raises the possibility of developing ultrasound-microbubble mediated cavitation into a targeted gene transfection paradigm for plant cells and, conversely, adopting plant cells as experimental test-beds for sonoporation-based gene therapy in mammalian cells.

Identification of mitochondrial thiamin diphosphate carriers from Arabidopsis and maize

It is currently held that thiamin is made in chloroplasts and converted in the cytosol to the active cofactor thiamin diphosphate (ThDP), and that mitochondria and plastids import ThDP. The organellar transporters that mediate ThDP import in plants have not been identified. Comparative genomic analysis indicated that two members of the mitochondrial carrier family (MCF) in Arabidopsis (At5g48970 and At3g21390) and two in maize (GRMZM2G118515 and GRMZM2G124911) are related to the ThDP carriers of animals and Saccharomyces cerevisiae. Expression of each of these plant proteins in a S. cerevisiae ThDP carrier (TPC1) null mutant complemented the growth defect on fermentable carbon sources and restored the level of mitochondrial ThDP and the activity of the mitochondrial ThDP-dependent enzyme acetolactate synthase. The plant proteins were targeted to mitochondria as judged by dual import assays with purified pea mitochondria and chloroplasts, and by microscopic analysis of the subcellular localization of green fluorescent protein fusions in transiently transformed tobacco suspension cells. Both maize genes were shown to be expressed throughout the plant, which is consistent with the known ubiquity of mitochondrial ThDP-dependent enzymes. Collectively, these data establish that plants have mitochondrially located MCF carriers for ThDP, and indicate that these carriers are highly evolutionarily conserved. Our data provide a firm basis to propagate the functional annotation of mitochondrial ThDP carriers to other angiosperm genomes.

Lysine decarboxylase catalyzes the first step of quinolizidine alkaloid biosynthesis and coevolved with alkaloid production in leguminosae.

Lysine decarboxylase (LDC) catalyzes the first-step in the biosynthetic pathway of quinolizidine alkaloids (QAs), which form a distinct, large family of plant alkaloids. A cDNA of lysine/ornithine decarboxylase (L/ODC) was isolated by differential transcript screening in QA-producing and nonproducing cultivars of Lupinus angustifolius. We also obtained L/ODC cDNAs from four other QA-producing plants, Sophora flavescens, Echinosophora koreensis, Thermopsis chinensis, and Baptisia australis. These L/ODCs form a phylogenetically distinct subclade in the family of plant ornithine decarboxylases. Recombinant L/ODCs from QA-producing plants preferentially or equally catalyzed the decarboxylation of L-lysine and L-ornithine. L. angustifolius L/ODC (La-L/ODC) was found to be localized in chloroplasts, as suggested by the transient expression of a fusion protein of La-L/ODC fused to the N terminus of green fluorescent protein in Arabidopsis thaliana. Transgenic tobacco (Nicotiana tabacum) suspension cells and hairy roots produced enhanced levels of cadaverine-derived alkaloids, and transgenic Arabidopsis plants expressing (La-L/ODC) produced enhanced levels of cadaverine, indicating the involvement of this enzyme in lysine decarboxylation to form cadaverine. Site-directed mutagenesis and protein modeling studies revealed a structural basis for preferential LDC activity, suggesting an evolutionary implication of L/ODC in the QA-producing plants.

Calcium- and ROS-mediated defence responses in BY2 tobacco cells by nonpathogenic Streptomyces sp

The early molecular events underlying the elicitation of plant defence reactions by Gram-positive bacteria are relatively unknown. In plants, calcium and reactive oxygen species are commonly involved as cellular messengers of a wide range of biotic stimuli from pathogenic to symbiotic bacteria. In the present work, we checked whether nonpathogenic Streptomyces sp. strains could induce early signalling events leading to defence responses in BY2 tobacco cell suspensions. We have demonstrated that nonpathogenic Streptomyces sp. OE7 strain induced a cytosolic Ca(2+) increase and a biphasic oxidative burst in the upstream signalling events, leading to defence responses in BY2 tobacco cell suspensions. Streptomyces sp. OE7 also elicited delayed intracellular free scopoletin production and programmed cell death. In agreement with scopoletin production, OE7 induced accumulation of PAL transcripts and increased accumulation of transcripts of EREBP1 and AOX genes that are known to be regulated by the jasmonate/ethylene pathway. Transcript levels of PR1b and NIMIN2α, both salicylic acid pathway-linked genes, were not modified. Moreover, Streptomyces sp. OE7 culture filtrates could reduce Pectobacterium carotovorum- and Pectobacterium atrosepticum-induced death of BY2 cells and soft rot on potato slices. New insights are thus provided into the interaction mechanisms between Streptomyces sp. and plants; Streptomyces sp. could be sensed by plant cells, and through cytosolic Ca(2+) changes and the generation of reactive oxygen species, defence responses were induced. These induced defence responses appeared to participate in attenuating Pectobacterium-induced diseases in plants. Thus, Streptomyces sp. OE7 could be a biocontrol agent against Pectobacterium sp.

Oligochitosan induces programmed cell death in tobacco suspension cells

Oligochitosan has been proved to trigger plant cell death. to gain some insights into the mechanisms of oligochitosan-induced cell death, the nature of oligochitosan-induced cell death and the role of calcium (ca2+), nitric oxide (no) and hydrogen peroxide (h2o2) were studied in tobacco suspension cells. oligochitosan-induced cell death occurred in cytoplasmic shrinkage, phosphatidylserine externalization, chromatin condensation, tunel-positive nuclei. cytochrome c release and induction of programmed cell death (pcd)-related gene hsr203j, suggesting the activation of pcd pathway. pretreatment cells with cyclosporin a, resulted in reducing oligochitosan-induced cytochrome c release and cell death, indicating oligochitosan-induced pcd was mediated by cytochrome c. in the early stage, cells undergoing pcd showed an immediate burst in free cytosolic ca2+ ([ca2+](cyt)) elevation, no and h2o2 production. further study showed that these three signals were involved in oligochitosan-induced pcd, while ca2+ and no played a negative role in this process by modulating cytochrome c release. (c) 2011 elsevier ltd. all rights reserved.

Detection of bacterial aggregation in tobacco cell suspensions treated with pathogenic bacteria

Previous studies of this model system involving plant cell suspensions inoculated with bacteria, have documented that interactions with incompatible pathogens, which cause a hypersensitive response on whole plants, will cause a transient increase in oxygen uptake 2–4 h after inoculation. The initial objective of this study was to determine whether this oxygen uptake burst was a result of increased bacterial multiplication, possibly due to nutrient leakage from plant cells. The adaptation of flow cytometry and the use of fluorescent nucleic acid stains provided the precision needed to monitor bacterial concentrations in tobacco suspension cells inoculated with pathogenic and non-pathogenic Pseudomonas species. Surprisingly, there was a transient decrease in the planktonic, or free-living, bacteria in cell suspensions inoculated with isolate Pseudomonas syringae pv. syringae WT (HR+), an incompatible pathogen of tobacco. This decrease in planktonic numbers was followed by an apparent increase in bacterial multiplication. Examination of the samples with fluorescent microscopy revealed the formation of bacterial aggregates in the extracellular fluid of the Pss WT (HR+) inoculated plant cells. The size of the aggregates increased at the onset of the oxygen uptake response, and contained increasing numbers of bacterial cells. These aggregated bacterial cells appear to be removed along with plant cells, as a result of filtration during sample preparation, causing the apparent decrease in planktonic bacteria detected by flow cytometry. This bacterial aggregation was also observed with the compatible Pseudomonas tabaci pathogen, which does not induce a noticeable oxygen uptake burst. No aggregation was observed with suspension inoculated with Pseudomonas fluorescens, a saprophyte, or Pss B7 (HR−), a Tn5 mutant of P. s. syringae. This aggregation response was rapid, once initiated, and appeared similar to reports of adhesion involving Hrp pili.

Regulatory dephosphorylation of CDK at G₂/M in plants: yeast mitotic phosphatase cdc25 induces cytokinin-like effects in transgenic tobacco morphogenesis.

During the last three decades, the cell cycle and its control by cyclin-dependent kinases (CDKs) have been extensively studied in eukaryotes. This endeavour has produced an overall picture that basic mechanisms seem to be largely conserved among all eukaryotes. The intricate regulation of CDK activities includes, among others, CDK activation by CDC25 phosphatase at G₂/M. In plants, however, studies of this regulation have lagged behind as a plant Cdc25 homologue or other unrelated phosphatase active at G₂/M have not yet been identified. Failure to identify a plant mitotic CDK activatory phosphatase led to characterization of the effects of alien cdc25 gene expression in plants. Tobacco, expressing the Schizosaccharomyces pombe mitotic activator gene, Spcdc25, exhibited morphological, developmental and biochemical changes when compared with wild type (WT) and, importantly, increased CDK dephosphorylation at G₂/M. Besides changes in leaf shape, internode length and root development, in day-neutral tobacco there was dramatically earlier onset of flowering with a disturbed acropetal floral capacity gradient typical of WT. In vitro, de novo organ formation revealed substantially earlier and more abundant formation of shoot primordia on Spcdc25 tobacco stem segments grown on shoot-inducing media when compared with WT. Moreover, in contrast to WT, stem segments from transgenic plants formed shoots even without application of exogenous growth regulator. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size due to a shortening of the G₂ phase together with high activity of cyclin-dependent kinase, NtCDKB1, in early S-phase, S/G₂ and early M-phase. Spcdc25-expressing tobacco ('Samsun') cell suspension cultures showed a clustered, more circular, cell phenotype compared with chains of elongated WT cells, and increased content of starch and soluble sugars. Taken together, Spcdc25 expression had cytokinin-like effects on the characteristics studied, although determination of endogenous cytokinin levels revealed a dramatic decrease in Spcdc25 transgenics. The data gained using the plants expressing yeast mitotic activator, Spcdc25, clearly argue for the existence and importance of activatory dephosphorylation at G₂/M transition and its interaction with cytokinin signalling in plants. The observed cytokinin-like effects of Spcdc25 expression are consistent with the concept of interaction between cell cycle regulators and phytohormones during plant development. The G₂/M control of the plant cell cycle, however, remains an elusive issue as doubts persist about the mode of activatory dephosphorylation, which in other eukaryotes is provided by Cdc25 phosphatase serving as a final all-or-nothing mitosis regulator.

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.

Cryptogein, a fungal elicitor, remodels the phenylpropanoid metabolism of tobacco cell suspension cultures in a calcium‐dependent manner

Plant cells use calcium-based signalling pathways to transduce biotic and/or abiotic stimuli into adaptive responses. However, little is known about the coupling between calcium signalling, transcriptional regulation and the downstream biochemical processes. To understand these relationships better, we challenged tobacco BY-2 cells with cryptogein and evaluated how calcium transients (monitored through the calcium sensor aequorin) impact (1) transcript levels of phenylpropanoid genes (assessed by RT-qPCR); and (2) derived-phenolic compounds (analysed by mass spectrometry). Most genes of the phenylpropanoid pathway were up-regulated by cryptogein and cell wall-bound phenolic compounds accumulated (mainly 5-hydroxyferulic acid). The accumulation of both transcripts and phenolics was calcium-dependent. The transcriptional regulation of phenylpropanoid genes was correlated in a non-linear manner with stimulus intensity and with components of the cryptogein-induced calcium signature. In addition, calmodulin inhibitors increased the sensitivity of cells to low concentrations of cryptogein. These results led us to propose a model of coupling between the cryptogein signal, calcium signalling and the transcriptional response, exerting control of transcription through the coordinated action of two decoding modules exerting opposite effects.

An extracellular hydrophilic carboxy-terminal domain regulates the activity of TaALMT1, the aluminum-activated malate transport protein of wheat

Al³+ -resistant cultivars of wheat (Triticum aestivum L.) release malate through the Al³+ -activated anion transport protein Triticum aestivum aluminum-activated malate transporter 1 (TaALMT1). Expression of TaALMT1 in Xenopus oocytes and tobacco suspension cells enhances the basal transport activity (inward and outward currents present in the absence of external Al³+, and generates the same Al³+ -activated currents (reflecting the Al³+-dependent transport function) as observed in wheat cells. We investigated the amino acid residues involved in this Al³+-dependent transport activity by generating a series of mutations to the TaALMT1 protein. We targeted the acidic residues on the hydrophilic C-terminal domain of TaALMT1 and changed them to uncharged residues by site-directed mutagenesis. These mutant proteins were expressed in Xenopus oocytes and their transport activity was measured before and after Al³+ addition. Three mutations (E274Q, D275N and E284Q) abolished the Al³+-activated transport activity without affecting the basal transport activity. Truncation of the hydrophilic C-terminal domain abolished both basal and Al³+-activated transport activities. Al³+-dependent transport activity was recovered by fusing the N-terminal region of TaALMT1 with the C-terminal region of AtALMT1, a homolog from Arabidopsis. These findings demonstrate that the extracellular C-terminal domain is required for both basal and Al³+-dependent TaALMT1 activity. Furthermore, we identified three acidic amino acids within this domain that are specifically required for the activation of transport function by external Al³+.

Plant-derived vaccines and other therapeutics produced in contained systems

The use of contained plant systems for the production of biopharmaceuticals represents a powerful alternative to current methods, combining the benefits of whole-plant systems and cell cultures. In vitro contained production systems include plant cell suspensions, hairy root cultures, novel plants grown in contained conditions and microalgae. These systems show intrinsic advantages, such as control over growth conditions, production in compliance with good manufacturing practice and avoidance of political resistance to the release of genetically modified field crops. At present, one of the two plant-produced vaccine-related products that have gone all the way through production and regulatory hurdles derives from tobacco cell suspensions, and the second is a human therapeutic enzyme, which is expected to reach commercial development soon and derives from carrot suspension cells. In the future, several other products from contained systems are expected to reach the clinical trial stage.

Novel fluorescent conjugate containing glucose and NBD and its carrier-mediated uptake by tobacco cells

Some compounds that contain glucose groups can be transported across the plasma membrane into the cells through hexose transporters. To test the hypothesis that glucose-conjugated insecticides also have similar uptake and translocation properties, a novel fluorescent conjugate ( 12) was prepared by conjugating glucose and 7-nitrobenz-2-oxa-1,3-diazole with 4-iodo-1-phenylpyrazoles. Its fluorescence spectra and uptake by suspension-cultured tobacco ( Nicotiana tabacum L.cv.) cells were studied. The fluorescence spectra showed long wavelengths with maximum emission at 530 nm. After incubating tobacco cell suspensions in 10 μM conjugate for 0.5 h, green fluorescence of 12 was clearly visible in the cells under fluorescence microscopy. After 2 h of incubation, more than 70% of 12 was absorbed. Carbonyl cyanide m-chlorophenylhydrazone, phloridzin and glucose drastically inhibited uptake. In concentration-dependent uptakes, the uptake rate of 12 showed a saturable component and was in accordance with Michaelis–Menten kinetics. The results proved that the glucose moiety can guide 12 into tobacco cells and that hexose transporters mediated the uptake.

Determination of Capsidiol in Tobacco Cells Culture by HPLC

Capsidiol is a bicyclic sesquiterpene, which accumulates extracellularly in plants, and has been isolated from many types of Solanaceae. It acts as a phytoalexin produced by Nicotiana tabacum in response to pathogens. Capsidiol has antifungal activity and is formed first in tobacco and pepper plants after infestation. The amount of capsidiol in tobacco cell suspension culture has been previously determined by solid-phase extraction and organic solvent extraction with thin-layer chromatography or gas chromatography analysis. A high-performance liquid chromatography method with UV detection at 210 nm on a C(8) column utilizing both extraction methods was developed to analyze capsidiol in suspension cell culture. The HPLC method was linear in the concentration range of 0.1-2.0 mg/L. The lower limit of quantitation was 0.1 mg/L. Organic solvent extraction and solid-phase extraction methods were compared. Both methods are generally similar in their overall efficiency (82% and 75%, respectively), but eliminations of interfering compounds are different. The relative standard deviation across five extractions of known amounts of capsidiol from plant sample was less than 5.1%. The relative standard deviation across five elicitations of cell cultures was less than 5.9%. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analysis of capsidiol was performed, and corresponding mass spectra are presented.

The biosynthesis of acetovanillone in tobacco cell-suspension cultures

A soluble enzyme, extracted from tobacco cell-suspension cultures 24 h after treatment with 100 μM methyl jasmonate, has been shown to synthesize acetovanillone (apocynin) from feruloyl-CoA in the presence of NAD. The enzyme displayed Michaelis–Menten kinetics with apparent K m values of 5.6 μM for feruloyl-CoA and 260 μM for NAD and exhibited very high specificity for its substrates. The increase in acetovanillone synthase activity was followed by an increase in the concentration of both acetovanillone and acetosyringone in the culture medium. No intermediate could be detected when analysing the reaction medium by HPLC during the formation of acetovanillone in cell-free extracts. The apparent molecular mass estimated by gel permeation on an FPLC column was ca. 79 kDa. To our knowledge, this is the first report of an enzymic system catalysing the synthesis of an acetophenone. This work demonstrates that the biosynthesis of acetophenones in tobacco proceeds from hydroxycinnamic acids through a CoA-dependent β-oxidation pathway. Interestingly in methyl jasmonate-treated cells, which synthesize very large amounts of hydroxycinnamoylputrescines, inhibition of the synthesis of these conjugates increased the concentration of acetovanillone and acetosyringone in the culture medium, suggesting that the two metabolic pathways can compete for their common precursors, i.e. hydroxycinnamoyl-CoA thioesters.

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.