Cultured Tobacco Cells Research Articles

A new enzymatic activity from elicitor-treated pear cell cultures converting trans-cinnamic acid to benzaldehyde.

Cell cultures of Asian pear (Pyrus pyrifolia) are known to produce benzoate-derived biphenyl phytoalexins upon elicitor treatment. Although the downstream pathway for biphenyl phytoalexin biosynthesis is almost known, the upstream route of benzoic acid biosynthesis in pear has not been completely elucidated. In the present work, we report benzaldehyde synthase (BS) activity from yeast extract-treated cell suspension cultures of P. pyrifolia. BS catalyzes the in vitro conversion of trans-cinnamic acid to benzaldehyde using a non-oxidative C2 -side chain cleavage mechanism. The enzyme activity was strictly dependent on the presence of a reducing agent, dithiothreitol being preferred. C2 -side chain shortening of the cinnamic acid backbone resembled the mechanisms catalyzed by 4-hydroxybenzaldehyde synthase (HBS) activity in Vanilla planifolia and salicylaldehyde synthase (SAS) activity in tobacco and apple cell cultures. A basal BS activity was also observed in the non-elicited cell cultures. Upon yeast extract-treatment, a 13-fold increase in BS activity was observed when compared to the non-treated control cells. Moreover, feeding of the cell cultures with trans-cinnamic acid, the substrate for BS, resulted in an enhanced level of noraucuparin, a biphenyl phytoalexin. Comparable accumulation of noraucuparin was observed upon feeding of benzaldehyde, the BS product. The preferred substrate for BS was found to be trans-cinnamic acid, for which the apparent Km and Vmax values were 0.5 mM and 50.7 pkat mg-1 protein, respectively. Our observations indicate the contribution of BS to benzoic acid biosynthesis in Asian pear via the CoA-independent and non-β-oxidative route.

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

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

The antibiotic peptaibol alamethicin from Trichoderma permeabilises Arabidopsis root apical meristem and epidermis but is antagonised by cellulase-induced resistance to alamethicin

BackgroundTrichoderma fungi live in the soil rhizosphere and are beneficial for plant growth and pathogen resistance. Several species and strains are currently used worldwide in co-cultivation with crops as a biocontrol alternative to chemical pesticides even though little is known about the exact mechanisms of the beneficial interaction. We earlier found alamethicin, a peptide antibiotic secreted by Trichoderma, to efficiently permeabilise cultured tobacco cells. However, pre-treatment with Trichoderma cellulase made the cells resistant to subsequent alamethicin, suggesting a potential mechanism for plant tolerance to Trichoderma, needed for mutualistic symbiosis.ResultsWe here investigated intact sterile-grown Arabidopsis thaliana seedlings germinated in water or growth medium. These could be permeabilised by alamethicin but not if pretreated with cellulase. By following the fluorescence from the membrane-impermeable DNA-binding probe propidium iodide, we found alamethicin to mainly permeabilise root tips, especially the apical meristem and epidermis cells, but not the root cap and basal meristem cells nor cortex cells. Alamethicin permeabilisation and cellulase-induced resistance were confirmed by developing a quantitative in situ assay based on NADP-isocitrate dehydrogenase accessibility. The combined assays also showed that hyperosmotic treatment after the cellulase pretreatment abolished the induced cellulase resistance.ConclusionWe here conclude the presence of cell-specific alamethicin permeabilisation, and cellulase-induced resistance to it, in root tip apical meristem and epidermis of the model organism A. thaliana. We suggest that contact between the plasma membrane and the cell wall is needed for the resistance to remain. Our results indicate a potential mode for the plant to avoid negative effects of alamethicin on plant growth and localises the point of potential damage and response. The results also open up for identification of plant genetic components essential for beneficial effects from Trichoderma on plants.

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

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

Colour bio-factories: Towards scale-up production of anthocyanins in plant cell cultures

Anthocyanins are widely distributed, glycosylated, water-soluble plant pigments, which give many fruits and flowers their red, purple or blue colouration. Their beneficial effects in a dietary context have encouraged increasing use of anthocyanins as natural colourants in the food and cosmetic industries. However, the limited availability and diversity of anthocyanins commercially have initiated searches for alternative sources of these natural colourants. In plants, high-level production of secondary metabolites, such as anthocyanins, can be achieved by engineering of regulatory genes as well as genes encoding biosynthetic enzymes. We have used tobacco lines which constitutively produce high levels of cyanidin 3-O-rutinoside, delphinidin 3-O-rutinoside or a novel anthocyanin, acylated cyanidin 3-O-(coumaroyl) rutinoside to generate cell suspension cultures. The cell lines are stable in their production rates and superior to conventional plant cell cultures. Scale-up of anthocyanin production in small scale fermenters has been demonstrated. The cell cultures have also proven to be a suitable system for production of 13C-labelled anthocyanins. Our method for anthocyanin production is transferable to other plant species, such as Arabidopsis thaliana, demonstrating the potential of this approach for making a wide range of highly-decorated anthocyanins. The tobacco cell cultures represent a customisable and sustainable alternative to conventional anthocyanin production platforms and have considerable potential for use in industrial and medical applications of anthocyanins.

Stereo-Specific Transcript Regulation of the Polyamine Biosynthesis Genes by Enantiomers of Ornithine in Tobacco Cell Culture.

BackgroundOrnithine (Orn) plays an essential role in the metabolism of plant cells through incorporation in polyamines biosynthesis, the urea cycle and nitrogen metabolism. Physiological response of the plant cells to its two enantiomers have not been widely investigated yet.ObjectivesThis study aimed to evaluate effect of ornithine enantiomers on expression of certain polyamine (PAs) biosynthetic genes in tobacco cells.Materials and MethodsSuspension-cultured tobacco cells were treated with different concentrations of L- and D- Orn for 24 h. Cell viability was assayed by Evans Blue and hydrogen peroxide content. The changes of gene expression were analyzed by semi-quantitative RT-PCR.ResultsExogenous D-Orn resulted in enhancement of expression of genes involved in Orn, arginine and S-adenosyl methionine metabolism. Additionally, exogenous D-Orn treatment resulted in sustained viability of cultured tobacco cells and normal levels of hydrogen peroxide were maintained. Supplied L-Orn increased the hydrogen peroxide level and lowered viability of cells. Treatment with L-Orn had a negative effect on the transcript levels for most analyzed PA-related genes. It was also illustrated that transcription of putrescine methyl transferase, key enzyme for nicotine production, was highly upregulated by L-Orn.ConclusionsBased on the results, D-Orn was shown to have a stereo-selective function in regulation of the PAs-related genes.

Metabolomics-guided investigations of unintended effects of the expression of the hydroxycinnamoyl quinate hydroxycinnamoyltransferase (hqt1) gene from Cynara cardunculus var. scolymus in Nicotiana tabacum cell cultures

Chlorogenic acids (CGAs) are phenolic compounds biosynthesized in the phenylpropanoid pathway, with hydroxycinnamoyl quinate hydroxycinnamoyltransferase (HQT) as the key enzyme. Variation of CGAs has been noted in different plants, with globe artichoke (Cynara cardunculus var. scolymus L.) producing high amounts and a diverse spectrum of CGAs in its leaves. In the current study, the effect of overexpression of the hqt1 transgene from globe artichoke in tobacco was evaluated at the metabolome level. Here, metabolomic approaches based on ultra-high performance liquid chromatography coupled to mass spectrometry, together with chemometric models such as principal component analysis and orthogonal partial least square discriminant analysis, were employed to evaluate altered metabolic changes due to hqt1 overexpression. CGA profiles (caffeoylquinic acids: 3-CQA, 4-CQA and 5-CQA; p-coumaroylquinic acids: 4-pCoQA and 5-pCoQA; and 4,5-di-caffeoylquinic acid) of transgenic tobacco cell cultures were detected at lower concentrations than in the wild type. Interestingly, the cells were found to rather accumulate, as an unintended effect, abscisic acid - and benzoic acid derivatives. The results suggest that insertion of hqt1 in tobacco, and overexpression in undifferentiated cells, led to rechannelling of the phenylpropanoid pathway to accumulate benzoic acids. These findings proved to be contrary to the results shown elsewhere in leaf tissues, thus indicating differential metabolic control and regulation in the undifferentiated cell culture system.

CONTROL OF FIRE BLIGHT INFECTIONS WITH SYNTHETIC PEPTIDES THAT ELICIT PLANT DEFENSE RESPONSES

Antimicrobial peptides (AMPs) are reliable compounds to develop strategies for integrated plant disease management. These peptides can act directly against the pathogens and/or induce plant defense responses. In this work, 33 sequences including linear and cyclic peptides, linear and cyclic peptidotriazoles, and cyclic lipopeptides were tested for induction of plant defense. A first screening was carried out with BY2 tobacco cell cultures by determining the alkalinization and production of hydrogen peroxide. Peptides BP13, BP100, BP143 and BPC200W were slightly positive in these assays and were tested by RT-qPCR for expression of 10 defense-related genes in tomato, which are representative of the jasmonic acid, salicylic acid and ethylene pathways. BP13 and BPC200W induced overexpression of several genes that are also overexpressed by reference plant defense peptide elicitors. Plant assays with potted pear plants in the greenhouse confirmed the effect of peptides BP13 and BPC200W in controlling Erwinia amylovora infections, putatively through plant defense induction.

Melatonin Protects Cultured Tobacco Cells against Lead-Induced Cell Death via Inhibition of Cytochrome c Translocation.

Melatonin was discovered in plants more than two decades ago and, especially in the last decade, it has captured the interests of plant biologists. Beyond its possible participation in photoperiod processes and its role as a direct free radical scavenger as well as an indirect antioxidant, melatonin is also involved in plant defense strategies/reactions. However, the mechanisms that this indoleamine activates to improve plant stress tolerance still require identification and clarification. In the present report, the ability of exogenous melatonin to protect Nicotiana tabacum L. line Bright Yellow 2 (BY-2) suspension cells against the toxic exposure to lead was examined. Studies related to cell proliferation and viability, DNA fragmentation, possible translocation of cytochrome c from mitochondria to cytosol, cell morphology after fluorescence staining and also the in situ accumulation of superoxide radicals measured via the nitro blue tetrazolium reducing test, were conducted. This work establishes a novel finding by correcting the inhibition of release of mitochondrial ctytocrome c in to the cytoplasm with the high accumulation of superoxide radicals. The results show that pretreatment with 200 nm of melatonin protected tobacco cells from DNA damage caused by lead. Melatonin, as an efficacious antioxidant, limited superoxide radical accumulation as well as cytochrome c release thereby, it likely prevents the activation of the cascade of processes leading to cell death. Fluorescence staining with acridine orange and ethidium bromide documented that lead-stressed cells additionally treated with melatonin displayed intact nuclei. The results revealed that melatonin at proper dosage could significantly increase BY-2 cell proliferation and protected them against death. It was proved that melatonin could function as an effective priming agent to promote survival of tobacco cells under harmful lead-induced stress conditions.

Aluminium-induced cell death requires upregulation of NtVPE1 gene coding vacuolar processing enzyme in tobacco (Nicotiana tabacum L.)

Cell death mechanism triggered by aluminium (Al) ion was investigated at root apex of tobacco (cultivar Bright Yellow) and in cultured tobacco cell line BY-2 derived from Bright Yellow, focusing on VPE genes (NtVPE1a, NtVPE1b, NtVPE2, NtVPE3). Cell death was detected as a loss of integrity of the plasma membrane by vital staining with fluorescein diacetate (in root apex) and Evans blue (in BY-2), respectively. At root apex, the upregulation of gene expression of VPE1a and VPE1b was observed significantly after 9h of Al exposure in parallel with an enhancement of cell death, while the upregulation of VPE2 and VPE3 were observed later. Similarly, in BY-2 cells, the upregulation of VPE1a and VPE1b and the enhancement of cell death were synchronously observed after 3-h exposure to Al, while the upregulation of VPE2 and VPE3 occurred later. RNA interference (RNAi) lines of each of the VPEs were constructed in BY-2 cells. Comparative studies between wild-type and the RNAi lines indicated that both Al-enhanced VPE activity and Al-induced cell death were significantly suppressed in the RNAi lines of VPE1 (dual suppressor of VPE1a and VPE1b), but not in the RNAi lines of VPE2 and that of VPE3. Taken together, we conclude that the upregulation of VPE1 gene expression and following enhancement of VPE activity under Al stress cause cell death in actively growing or elongating cells of tobacco.

Engineered gray mold resistance, antioxidant capacity, and pigmentation in betalain-producing crops and ornamentals

Betalains are tyrosine-derived red-violet and yellow plant pigments known for their antioxidant activity, health-promoting properties, and wide use as food colorants and dietary supplements. By coexpressing three genes of the recently elucidated betalain biosynthetic pathway, we demonstrate the heterologous production of these pigments in a variety of plants, including three major food crops: tomato, potato, and eggplant, and the economically important ornamental petunia. Combinatorial expression of betalain-related genes also allowed the engineering of tobacco plants and cell cultures to produce a palette of unique colors. Furthermore, betalain-producing tobacco plants exhibited significantly increased resistance toward gray mold (Botrytis cinerea), a pathogen responsible for major losses in agricultural produce. Heterologous production of betalains is thus anticipated to enable biofortification of essential foods, development of new ornamental varieties, and innovative sources for commercial betalain production, as well as utilization of these pigments in crop protection.

Arabinogalactan Proteins Are Involved in Salt-Adaptation and Vesicle Trafficking in Tobacco by-2 Cell Cultures.

Arabinogalactan proteins (AGPs) are a highly diverse family of glycoproteins that are commonly found in most plant species. However, little is known about the physiological and molecular mechanisms of their function. AGPs are involved in different biological processes such as cell differentiation, cell expansion, tissue development and somatic embryogenesis. AGPs are also involved in abiotic stress response such as salinity modulating cell wall expansion. In this study, we describe how salt-adaptation in tobacco BY-2 cell cultures induces important changes in arabinogalactan proteins distribution and contents. Using the immuno-dot blot technique with different anti-AGP antibodies (JIM13, JIM15, and others), we observed that AGPs were highly accumulated in the culture medium of salt-adapted tobacco cells, probably due to the action of phospholipases. We located these AGP epitopes using immunogold labeling in the cytoplasm associated to the endoplasmic reticulum, the golgi apparatus, and vesicles, plasma membrane and tonoplast. Our results show that salt-adaptation induced a significant reduction of the cytoplasm, plasma membrane and tonoplast content of these epitopes. Yariv reagent was added to the control and salt-adapted tobacco cell cultures, leading to cell death induction in control cells but not in salt-adapted cells. Ultrastructural and immunogold labeling revealed that cell death induced by Yariv reagent in control cells was due to the interaction of Yariv reagent with the AGPs linked to the plasma membranes. Finally, we propose a new function of AGPs as a possible sodium carrier through the mechanism of vesicle trafficking from the apoplast to the vacuoles in salt-adapted tobacco BY-2 cells. This mechanism may contribute to sodium homeostasis during salt-adaptation to high saline concentrations.

Biotechnological production of recombinant tissue plasminogen activator protein (reteplase) from transplastomic tobacco cell cultures

Transplastomic plants are a system of choice for the mass production of biopharmaceuticals due to the polyploidy of the plastid genome and the low risk of pollen-mediated outcrossing because of maternal inheritance. However, as field-grown plants, they can suffer contamination by agrochemicals and fertilizers, as well as fluctuations in yield due to climatic changes and infections. Tissue-type plasminogen activator (tPA), a protein used to treat heart attacks, converts plasminogen into plasmine, which digests fibrin and induces the dissolution of fibrin clots. Recently, we obtained transplastomic tobacco plants carrying the K2S gene encoding truncated human tPA (reteplase) with improved biological activity, and confirmed the presence of the target protein in the transgenic plant leaves. Considering the advantages of plant cell cultures for biopharmaceutical production, we established a cell line derived from the K2S tobacco plants. The active form of reteplase was quantified in cultures grown in light or darkness, with production 3-fold higher in light.

Immunocytochemical detection of rhamnogalacturonan II on forming cell plates in cultured tobacco cells.

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. The RG-II region serves as the site of borate cross-linking within pectin, via which pectin macromolecules link together to form a gel. In this study, we examined whether RG-II is present in the cell plate, the precursor of primary cell walls that forms during cytokinesis. A structure inside dividing cells was labeled with a rabbit polyclonal anti-RG-II antibody and detected by immunofluorescence microscopy. An antibody against callose, a marker polysaccharide for the cell plate, also labeled the structure. In immunoelectron microscopy analyses using the anti-RG-II antibody, gold particles were distributed in electron-lucent vesicular structures that appeared to correspond to the forming cell plates in late anaphase cells. Together, these results suggest that RG-II is present in cell plates from the early phase of their assembly.

Effector-activated immune responses in potato and tobacco cell cultures caused by phytopathogen Clavibacter michiganensis ssp. sepedonicus

Generation of reactive oxygen species (ROS) in tobacco (Nicotiana tabacum L.) cell cultures and potato (Solanum tuberosum L.) of two varieties experiencing the action of bacterial pathogen Clavibacter michiganensis ssp. sepedonicus (Cms) was investigated. The intensity and dynamics of the changes in hydrogen peroxide concentration observed in these cultures provided evidence for the development in tobacco of the effector-activated immune responses and the induction of the same type of responses but with low intensity for resistant potato variety and the inhibition of the defense mechanisms for its susceptible variety. This is in accordance with the data concerning the dynamics of plant cell culture death as well as the results obtained earlier on the whole plants. The experiments performed had also the purpose to elucidate whether the development of the above responses on the ability of bacteria Cms to form biofilms during plant infection. It was shown that this ability of Cms is significantly inhibited upon the combined cultivation of it with the plant cells exerting the responses of the effector-activated immunity and represented by the cells of tobacco and resistant potato variety. In the case of susceptible potato variety, the process of the biofilm formation was suppressed by the plant only to a slight extent. In addition, the fact concerning the participation of heat shock proteins (HSPs) in the development of the effector-activated immune responses was revealed.

Coordinated protein co-expression in plants by harnessing the synergy between an intein and a viral 2A peptide.

SummaryA novel approach is developed for coordinated expression of multiple proteins from a single transgene in plants. An Ssp DnaE mini‐intein variant engineered for hyper‐N‐terminal autocleavage is covalently linked to the foot‐and‐mouth disease virus 2A (F2A) peptide with unique ribosome skipping property, via a peptide linker, to create an ‘IntF2A’ self‐excising fusion protein domain. This IntF2A domain acts, in cis, to direct highly effective release of its flanking proteins of interest (POIs) from a ‘polyprotein’ precursor in plants. This is successfully demonstrated in stably transformed cultured tobacco cells as well as in different organs of transgenic tobacco plants. Highly efficient polyprotein processing mediated by the IntF2A domain was also demonstrated in lettuce and Nicotiana benthamiana based on transient expression. Protein constituents released from the polyprotein precursor displayed proper function and accumulated at similar levels inside the cells. Importantly, no C‐terminal F2A extension remains on the released POIs. We demonstrated co‐expression of as many as three proteins in plants without compromising expression levels when compared with those using single‐protein vectors. Accurate differential cellular targeting of released POIs is also achieved. In addition, we succeeded in expressing a fully assembled and functional chimeric anti‐His Tag antibody in N. benthamiana leaves. The IntF2A‐based polyprotein transgene system overcomes key impediments of existing strategies for multiprotein co‐expression in plants, which is particularly important for gene/trait stacking.

Bioconversion of stilbenes in genetically engineered root and cell cultures of tobacco

It is currently possible to transfer a biosynthetic pathway from a plant to another organism. This system has been exploited to transfer the metabolic richness of certain plant species to other plants or even to more simple metabolic organisms such as yeast or bacteria for the production of high added value plant compounds. Another application is to bioconvert substrates into scarcer or biologically more interesting compounds, such as piceatannol and pterostilbene. These two resveratrol-derived stilbenes, which have very promising pharmacological activities, are found in plants only in small amounts. By transferring the human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) gene to tobacco hairy roots and cell cultures, we developed a system able to bioconvert exogenous t-resveratrol into piceatannol in quantities near to mg L−1. Similarly, after heterologous expression of resveratrol O-methyltransferase from Vitis vinifera (VvROMT) in tobacco hairy roots, the exogenous t-resveratrol was bioconverted into pterostilbene. We also observed that both bioconversions can take place in tobacco wild type hairy roots (pRiA4, without any transgene), showing that unspecific tobacco P450 hydroxylases and methyltransferases can perform the bioconversion of t-resveratrol to give the target compounds, albeit at a lower rate than transgenic roots.

Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture

Thioredoxins (Trxs), key components of cellular redox regulation, act by controlling the redox status of many target proteins, and have been shown to play an essential role in cell survival and growth. The presence of a Trx system in the nucleus has received little attention in plants, and the nuclear targets of plant Trxs have not been conclusively identified. Thus, very little is known about the function of Trxs in this cellular compartment. Previously, we studied the intracellular localization of PsTrxo1 and confirmed its presence in mitochondria and, interestingly, in the nucleus under standard growth conditions. In investigating the nuclear function of PsTrxo1 we identified proliferating cellular nuclear antigen (PCNA) as a PsTrxo1 target by means of affinity chromatography techniques using purified nuclei from pea leaves. Such protein–protein interaction was corroborated by dot-blot and bimolecular fluorescence complementation (BiFC) assays, which showed that both proteins interact in the nucleus. Moreover, PsTrxo1 showed disulfide reductase activity on previously oxidized recombinant PCNA protein. In parallel, we studied the effects of PsTrxo1 overexpression on Tobacco Bright Yellow-2 (TBY-2) cell cultures. Microscopy and flow-cytometry analysis showed that PsTrxo1 overexpression increases the rate of cell proliferation in the transformed lines, with a higher percentage of the S phase of the cell cycle at the beginning of the cell culture (days 1 and 3) and at the G2/M phase after longer times of culture (day 9), coinciding with an upregulation of PCNA protein. Furthermore, in PsTrxo1 overexpressed cells there is a decrease in the total cellular glutathione content but maintained nuclear GSH accumulation, especially at the end of the culture, which is accompanied by a higher mitotic index, unlike non-overexpressing cells. These results suggest that Trxo1 is involved in the cell cycle progression of TBY-2 cultures, possibly through its link with cellular PCNA and glutathione.

INSULINA ESTIMULA LA PROLIFERACIÓN CELULAR DEPENDIENTE DE AUXINAS EN LOS CULTIVOS DE CÉLULAS EN SUSPENSIÓN DE TABACO NT-1

La insulina regula el crecimiento en plantas como maíz y frijol, a través de la proliferación celular. En este estudio observamos que en cultivos de tabaco NT-1, la hormona regula la proliferación celular de manera dependiente de la presencia de auxinas. Los cultivos donde se limitó la concentración de auxinas, presentaron una baja actividad mitótica, la cual fue re establecida por la adición de 1.23 nM de insulina a través de la expresión de los genes ciclina B, CDKB, E2F, subunidad β- de la proteína G ARC A y glutation-S-transferasa (GST). Los cultivos también presentaron un incremento en peso seco y células pequeñas que corresponden a un fenotipo con alta división celular. Por otra parte, se ha reportado que la ausencia de auxinas en este tipo de cultivos de células de tabaco en suspensión conduce a una sincronización parcial, y en este sistema la insulina también promovió la proliferación, incrementando la expresión del gen E2F.

The effect of Bacillus subtilis producing Surfactin in ROS production and transformation efficiency of tobacco cells

Surfactin secreted by bacilli has biological functions in plant. Surfactin C14 and C15 have the highest effect on inducing hydrogen peroxide species release in the plant. Surfactin production in the two Bacillus strains ACCT21332 and FKR3 were analysed by HPLC and the phytotoxicity of the Bacilli-derived surfactins was determined in Tobacco cell culture. Surfactin C14 and C15 were detected in ACCT21332 but not in FKR3 strain. Extracellular hydrogen peroxide produced by tobacco cell culture cells exposed to ATCC21332 and FKR3 strains increased compared to untreated ones. The Agrobacterium mediated transformation rate of tobacco cells drops from 4% transformed cells to 0.8 and 1.2% when pretreated with ATCC21332 or FKR3 strain, respectively. The strong drop in transformation rate of plant cell culture after FKR3 strain pre-treatment indicates that Surfactin C14 and C15 are not the major or the only cause in protecting plant cells from Agrobacterial infection and transformation.