Evidence for the activation of a MAP kinase upon phosphate‐induced cell cycle re‐entry in tobacco cells

Mitogen‐activated‐protein (MAP) kinases are components of signal transduction pathways which respond to a variety of stimuli in different organisms. In quiescent mammalian cells, the reactivation of cell division induced by different mitogenic signals is mediated by the rapid phosphorylation and activation of MAP kinases. We have investigated whether a similar situation occurs in plants, arresting tobacco ( Nicotiana tabacum L.) cells in the G₁ phase of the cell cycle by phosphate starvation, and then inducing them to re‐enter the cell cycle by refeeding with phosphate. The transient activation of a kinase activity with the characteristics of a MAP kinase was observed during the first hour after refeeding, when the cells were still in G₁. Using myelin basic protein (MBP) as substrate, an increase in this phosphorylating activity, with a molecular mass of approximately 45 kDa, was detected in cell extracts between 35 and 55 min after induction, in in‐gel phosphorylation assays and after immunoprecipitation with anti‐MAP kinase antibodies. The specificity of the antibodies against recombinant tobacco MAP kinases suggested that the MAP kinase p45^ntf4 was responsible for the observed activity. These data provide experimental evidence for the activation in vivo of a plant MAP kinase, possibly mediating the reactivation of cell division in G₁‐arrested cells.     

Effect of nalidixic acid and novobiocine on the metabolism of suspension cultured carrot cells

Suspension cells of Daucus carota L. cv. Lunga di Amsterdam are extremely sensitive to both nalidixic acid and novobiocine. The synthesis of DNA, RNA and protein are inhibited within minutes of exposure even at concentrations less than 0.1 mM of both drugs. Moreover, uptake of deoxythymidine by cells is impaired more severely than by sodium azide. Reduction of oxygen consumption caused by the two drugs is slower than in the case of NaN₃ and occurs a few minutes after the inhibition of macromolecular biosynthesis. This excludes the possibility that the block in oxidation is the primary cause of the observed inhibition of cellular functions and suggests that the inhibition of ATP synthesis, which is immediately affected in both cases, may be responsible for these effects. A possible interaction with other cellular targets may be secondary to the uncoupling effect. Cell growth is completely inhibited by 0.3 mM concentration of both drugs, but this inhibition is irreversible only in the case of novobiocine. The irreversibility of cell growth inhibition by novobiocine can be explained by the total disappearance of ATP from the cell.     

Short-term aluminium uptake by tobacco cells: Growth dependence and evidence for internalization in a discrete peripheral region

Short-term uptake and initial localization of aluminium (Al) were investigated in cultured cells of Nicotiana tabacum L. cv. BY-2. Graphite furnace atomic absorption spectrometry and an in vivo Al-sensitive fluorometric assay, employing morin, yielded similar results in all experiments. Aluminium uptake was critically dependent on cell growth. As opposed to negligible uptake in stationary-phase cells, Al uptake (20 μ M AlCl₃, pH 4.5, 23°C) by actively growing cells was detectable within 5 min, with an initial rate of 16 nmol Al (10⁶ cells)⁻¹ h⁻¹. Increased CaCl₂ levels (up to 20 m M ), low temperature (4°C), and pre-chelation of Al to citrate greatly reduced Al uptake (by 75–90%). A pH-associated permeabilization of cells at pH 4.5, as monitored by trypan blue, was observed in some growing cells. Although permeability to trypan blue was not a requirement for Al uptake, enhanced membrane permeability at pH 4.5, relative to pH 5.6, may contribute to Al uptake. Aluminium was observed to localize mainly in a pronounced and discrete fluorescent zone at the cell periphery (2–30 μm wide), presumably in the cortical cytosol and/or the adjoining plasma membrane section, although the possibility cannot be excluded that some Al resided in the cell wall apposing this discrete region. However, as judged by the Al-morin assay, there were no detectable Al levels in the remaining, larger portion of the cell wall. The potential of the Al-morin method in Al toxicity studies is illustrated.     

Changes in the nucleotide pools in leaves and buds of tobacco plants upon treatment with 2,4-dichlorophenoxyacetic acid

Tobacco ( Nicotiana tabacum L. cv. Samsun) plants were treated once with 2,4-dichlorophenoxyacetic acid (2,4-D) at the 8-leaf stage. The effect of the herbicide on leaf metabolism was followed over 7 days by determination of the ribonucleotide pools, including NAD⁺, NADP⁺ and UDP-sugars, by high-preformance liquid chromatography. 2,4-D treatment resulted in large changes in the nucleotide concentrations, the magnitude and sign of which were dependent upon the leafage. The nucleotide pools decreased in the apical tissue, but increased strongly in the mature leaves with the highest relative increase in the oldest leaf tested. The time course of the changes revealed a maximum on day 5 after 2,4-D treatment. The increase in the adenine nucleotide pools, energy charge and the NADVNADP⁺ ratio are interpreted to indicate a stress situation. The different responses of young, mature and senescent tissue to the synthetic auxin could reflect their different inherent sensitivity due to the natural auxin gradient.     

Effects of abscisic acid and proline on adaptation of tobacco callus cultures to salinity and osmotic shock

Tobacco callus ( Nicotina tabacum cv. Badischer Geudertheimer) took up sorbitol rapidly and without a lag period from media with up to 0.7 M of the polyol. Accumulation of proline was greatly enhanced under these conditions and was proportional to the absorbed sorbitol, while the viability of the callus cultures was quite low after a few hours of incubation. Under moderate conditions (0.1 M sorbitol) as well as under severe osmotic shock (0.7 M sorbitol), the cells adapted by adjusting the sorbitol/proline ratio to ca 3. NaCl (0.1 M ) had the same effect as sorbitol (0.7 M ) on the survival rate, but only slightly affected proline synthesis in the first hours of incubation. Addition of 10⁷ or 10 ⁵ M abscisic acid (ABA) did not increase the proline content, but 10 ⁷ M ABA delayed the deleterious effect of NaCl and improved the state of the cells. No influence of abscisic acid during the incubation with sorbitol could be detected. Two different strategies for the adjustment of tobacco callus to salinity and sorbitol are suggested: Non-ionic stress is controlled by the accumulation of proline, whereas ABA could be involved in the adaptation to ionic stress.     

Isolation and characterization of mutants resistant to amino acid analogues obtained from Saccharomyces cerevisiae strains

Mutants resistant to the amino acid analogues dl-thiaisoleucine, dl-4-azaleucine, 5,5,5-trifluoro-dl-leucine and l-O-methylthreonine, were isolated from Saccharomyces cerevisiae wine yeast strains. The fermentative production of secondary metabolites by the mutants was tested in grape must. Higher alcohols, acetaldehyde and acetic acid concentration varied depending on strain and analogue. Most of the mutants produced increased amounts of amyl alcohol. A remarkable variability in the level of n-propanol, isobutanol, acetaldehyde and acetic acid was observed. In practical application, the use of mutants resistant to amino acid analogues can improve the quality of wines by reducing or increasing the presence of some secondary compounds.     

The expression of the 14D9 catalytic antibody in suspended cells of Nicotiana tabacum cultures increased by the addition of protein stabilizers and by transference from erlenmeyer flasks to a 2‐L bioreactor

The effect of two protein stabilizers (polyvinylpyrrolidone [PVP] and gelatine) on growth and 14D9 yield of Nicotiana tabacum cell suspension cultures (Ab‐KDEL and sec‐Ab) was analyzed. The addition of PVP at a concentration of 1.0 g L^?1 produced the highest total 14D9 yield (biomass + culture medium) in the Ab‐KDEL line (4.82% total soluble protein [TSP]). With the addition of gelatine, the highest total 14D9 yield (2.48% TSP) was attained in the Ab‐KDEL line at 5.0 g L^?1 gelatine. When the Ab‐KDEL suspended cells were cultured in a 2‐L bioreactor, the highest 14D9 yield was 8.1% TSP at a 5% w/v inoculum size, which was the best 14D9 yield so far obtained in the platforms tested (E. coli, N. tabacum leaves and seeds, N. tabacum hairy roots, and cell suspension cultures). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1185–1189, 2014     

Generation and properties of ascorbic acid-deficient transgenic tobacco cells expressing antisense RNA for L-galactono-1,4-lactone dehydrogenase

In higher plants, the terminal step of L-ascorbic acid (AsA) biosynthesis is catalyzed by the enzyme L-galactono-1,4-lactone dehydrogenase (EC 1.3.2.3, GalLDH). We generated AsA-deficient transgenic tobacco BY-2 cell lines by antisense expression of the GalLDH cDNA that was amplified from BY-2 cells using PCR. Two transgenic cell-lines, AS1-1 and AS2-2, having a marked expression of antisense RNA were analyzed. Antisense suppression of GalLDH mRNA led to a significant decline in the GalLDH activity. The AsA levels in the transgenic cell lines were found to be 30% lower than the wild-type BY-2 cells. In synchronous cultures, division of AS1-1 and AS2-2 cells was restrained with a concomitant decrease in mitotic index that was probably due to a decline in AsA levels. The rate of cell growth was also found to be less than that of the wild-type cells. Interestingly, there was a significant phenotypic difference between the transgenic and wild-type cells. The calli of AS1-1 and AS2-2 appeared to be sticky and soft. Back extrusion method also showed that AsA-deficient BY-2 callus was rheologically soft. Furthermore, microscopic analysis revealed that AS1-1 and AS2-2 cells were abnormally slender, suggesting a potential for a significant and a uni-axial elongation. Thus, we observed that decline in the AsA levels has an adverse effect on the division, growth and structure of a plant cell.     

Boron is required for the stimulation of the ferricyanide-induced proton release by auxins in suspension-cultured cells of Daucus carota and Lycopersicon esculentum

Boron deficiency reduces the ferricyanide-induced net proton release of suspension-cultured carrot ( Daucus carota L.) and tomato ( Lycopersicon esculentum Mill.) cells by more than 50%. This effect is reversed within 60 to 90 min by the addition of B. Vanadate (400 μ M ) completely suppresses the proton release, indicating an ATPasedriven process. The differences between B treatments do not appear when auxins are omitted from the experimental solution, but can be observed within less than 30 min after the addition of auxin to auxin-deficient cell cultures. This suggests, that an adequate supply of B is required for the auxin action to take place. The results are discussed with respect to the primary functions of B in membranes and transport processes, and its possible influence on auxin-induced metabolic events.     

Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for <Emphasis Type="SmallCaps">l</Emphasis>-galactono-1,4-lactone dehydrogenase

l-Galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) is the last enzyme in the putative l-ascorbic acid (AsA) biosynthetic pathway of plants. Here, we show for the first time that the overexpression of GalLDH can increase AsA content in tobacco (Nicotiana tabacum L.) BY-2 cells. To see the effect, we analyzed the properties of these AsA-overproducing transgenic cell lines, especially in relation to AsA content of cells, cell division, senescence and resistance to oxidative stress. The mitotic index in AsA-overproducing cells was higher than in wild-type cells. Moreover, the browning of these cells was markedly restrained, and the proportion of dead cells was reduced, especially in the later period of culture. These AsA-overproducing cells also acquired resistance to paraquat (methyl viologen), which produces active oxygen species. These results contribute to the previous insights about AsA and raise the possibility of the generation of plants that have resistance to environmental stresses by increasing their AsA content.