Polyamine metabolism during the cell cycle of synchronized tobacco BY-2 cell line.

The time courses of the contents of free, soluble and insoluble polyamine (PA) conjugates, PA biosynthetic and catabolic enzyme activities and mRNA levels of PA biosynthetic genes were monitored during the cell cycle of synchronized tobacco BY-2 cell line (Nicotiana tabacum L. cv. Bright Yellow 2). Progression through the cell cycle was characterized by specific biphasic changes of PA levels. The first, moderate peak in the amount of free PAs coincided with the S-phase. After a transient decline in G2 phase the contents of free PAs increased rapidly and peaked again during G2/M interface. Then sharply decreased with the minimum at the end of mitosis and during M/G1 transition and started to rise again with the next replication phase. Levels of PA soluble conjugates paralleled those of the free forms. Biosynthetic enzyme activities followed the biphasic manner analogous to the levels of free PAs and seemed to be regulated on both transcriptional and (post)translational level. PA cellular levels were further controlled by both catabolic degradation and conjugation of PAs. PA catabolism played an important role in the PA down-regulation during G2 phase and late mitosis, while the decline in free PAs in G2/M interface and during the whole mitosis resulted mainly from PA conjugation. This study's results demonstrate that during the cell cycle of tobacco BY-2 cells endogenous PA levels are intricately controlled, involving regulation of activities of biosynthetic, catabolic and conjugation enzymes.

Excretion of polyamines in alfalfa and tobacco suspension-cultured cells and its possible role in maintenance of intracellular polyamine contents.

Changes in polyamines (PAs) in cells and cultivation media of alfalfa (Medicago sativa L.) and tobacco bright yellow 2 (BY-2) (Nicotiana tabacum L.) cell suspension cultures were studied over their growth cycles. The total content of PAs (both free and conjugated forms) was nearly 10 times higher in alfalfa, with high level of free putrescine (Put) (in exponential growth phase it represented about 65-73% of the intracellular Put pool). In contrast, the high content of soluble Put conjugates was found in tobacco cells (in exponential phase about 70% of the intracellular Put). Marked differences occurred in the amount of PAs excreted into the cultivation medium: alfalfa cells excreted at the first day after inoculation 2117.0, 230.5, 29.0 and 88.0 nmol g(-1) of cell fresh weight (FW) of Put, spermidine (Spd), spermine (Spm) and cadaverine (Cad), respectively, while at the same time tobacco cells excreted only small amount of Put and Spd (12.7 and 2.4 nmol g(-1) FW, respectively). On day 1 the amounts of Put, Spd, Spm and Cad excreted by alfalfa cells represented 21, 38, 12 and 15% of the total pool (intra- plus extra-cellular contents) of Put, Spd, Spm and Cad, respectively. In the course of lag-phase and the beginning of exponential phase the relative contents of extracellular PAs continually decreased (with the exception of Cad). On day 10, the extracellular Put, Spd, Spm and Cad still represented 11.3, 10.9, 2.1 and 27% of their total pools. The extracellular PAs in tobacco cells represented from day 3 only 0.1% from their total pools. The possible role of PA excretion into the cultivation medium in maintenance of intracellular PA contents in the cells of the two cell culture systems, differing markedly in growth rate and PA metabolism is discussed.

The impact of Cu treatment on phenolic and polyamine levels in plant material regenerated from embryos obtained in anther culture of carrot.

The influence of copper sulphate on the regeneration of carrot (Daucus carota L.) androgenic embryos and changes in the levels of phenolic substances and polyamines that might be indicative of the response to oxidative stress were investigated. The cultivation on the regeneration medium supplemented with Cu(2+) at the concentrations 1 and 10 microM for 15 weeks resulted in significant dose-dependent inhibition of the growth and organogenic ability of carrot embryos. The total content of phenolic acids (represented by the sum of all soluble and insoluble fractions) in the Cu(2+)-treated carrot cultures did not change in comparison with the control (0.1 microM Cu(2+)). However, the levels of phenolic acids in the individual fractions showed significant differences. The cultivation in the presence of increased Cu(2+) evoked first of all the rise of free chlorogenic and caffeic acids, and the increase in soluble ester-bound ferulic acid. Marked dose-dependent decline in the amount of ferulic acid incorporated into the cell walls of the Cu(2+)-treated carrot cultures was partly compensated by the increase in the content of p-hydroxybenzoic acid. Decline in the total polyamine contents in the carrot tissues cultivated in the presence of increased Cu(2+) concentrations was observed. The most abundant polyamine, both in a free and PCA-soluble conjugated forms, was putrescine, the least abundant was spermine, which occurred in free form only. While the levels of free polyamines slightly decreased in a dose-dependent manner in the Cu(2+)-treated cultures, those of PCA-soluble conjugates markedly rose (enhancement to 135 and 170% in 1 and 10 microM Cu(2+), respectively, compared with the control). The decline in the total polyamine contents was caused mainly by the decline in the levels of PCA-insoluble conjugates. The decrease observed in this fraction was approximately to 70 and 50% in 1 and 10 microM Cu(2+)-treated cultures, respectively, when compared with the control. The role of phenolic acids and polyamines in preventing Cu(2+)stress in the carrot tissues is discussed.

Transgenic ipt tobacco overproducing cytokinins overaccumulates phenolic compounds during in vitro growth.

We present evidence that overproduction of endogenous cytokinins (CK) caused stress response in non-rooting Pssu-ipt transgenic tobacco (Nicotiana tabacum L.) grown in vitro. It was demonstrated by overaccumulation of phenolic compounds, synthesis of pathogenesis related proteins (PR proteins), and increase in peroxidase (POD) activities. Immunolocalization of zeatin and also PR-1b protein on leaf cryo-sections proved their accumulation in all mesophyll cells of transgenic tobacco contrary to control non-transgenic plants. Intensive blue autofluorescence of phenolic compounds induced by UV in cross-sections of leaf midrib showed enhanced contents of phenolics in transgenic tobacco compared with controls, nevertheless, no significant difference between both plant types was found in leaf total lignin content. Transgenic plantlets exhibited higher peroxidase activities of both soluble and ionically bound fractions compared with controls. HPLC analysis of phenolic acids confirmed the increase in all phenolic acids in transgenic tobacco except for salicylic acid (SA). The effect of high phenolic content on rooting of transgenic tobacco is discussed.

Diurnal changes in polyamine content, arginine and ornithine decarboxylase, and diamine oxidase in tobacco leaves.

Changes in the contents of polyamines (PAs) in tobacco leaves (Nicotiana tabacum L. cv. Wisconsin 38) grown under 16 h photoperiod were correlated with arginine and ornithine decarboxylase (EC 4.1.1.19 and EC 4.1.1.17) and diamine oxidase (EC 1.4.3.6) activities. The maximum of free and soluble conjugated forms of PAs occurred 1-2 h after the middle of the light period and was followed by two distinct peaks at the end of the light and at the beginning of the dark phase. Putrescine was the most abundant and cadaverine the least abundant PA in both free and PCA-soluble forms. However, cadaverine was predominant in PCA-insoluble conjugates, followed by putrescine, spermidine, and spermine. Both arginine and ornithine decarboxylases are involved in putrescine biosynthesis in tobacco leaves. Light dramatically stimulated the activity of ornithine decarboxylase, while no photoinduction of arginine decarboxylase activity was observed. Ornithine decarboxylase was found mainly in the particulate fraction. Only one peak, just after light induction, occurred in the cytosolic fraction, with 35% of the total ornithine decarboxylase activity. By contrast, the total arginine decarboxylase activity was equally divided between the soluble and pellet fractions. A sharp increase in diamine oxidase activity occurred 1 h after exposure to light, concomitant with the light-induced increase in ornithine decarboxylase activity. After a decline, diamine oxidase activity increased again, together with the rise in the amount of free Put. The roles of both conjugation of PAs with hydroxycinnamic acids and oxidative degradation of putrescine in maintaining free PA levels during the 24 h light/dark cycle are discussed. The presented results have shown that the parameters studied here followed rhythmical changes and were not only affected by light.

Polyamine metabolism during the growth cycle of tobacco BY-2 cells.

We studied polyamine (PA) biosynthesis, oxidation and conjugation in asynchronously dividing cells of tobacco BY-2 cell suspension culture (Nicotiana tabacum L.) during 7-day growth cycle. We analyzed the levels of free and conjugated PAs and the activities of biosynthetic and catabolic enzymes during the subculture interval. The contents of free spermidine and spermine started to increase after the inoculation into the fresh medium, positively correlated with the mitotic activity of BY-2 cells and reached their maxima at the beginning of exponential phase on day 3. On the contrary, the endogenous level of free Put showed a transient decline in the lag-phase, and then increased till the end of exponential phase (day 5). The time-course of the content of PCA-soluble conjugates showed a trend similar to that of the free PAs. The inoculation of BY-2 cells into the fresh medium resulted in a sharp increase in the activities of ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50). Arginine decarboxylase (ADC; EC 4.1.1.19) activity remained low during the whole subculture interval. The rise of diamine oxidase (DAO; EC 1.4.3.6) in the first day after subculture coincided with the decrease in free Put level. De novo synthesis of PAs in BY-2 cells after inoculation into the fresh medium and the participation of both PA conjugation with hydroxycinnamic acids and Put oxidative degradation in maintaining of free PA levels during the growth cycle are discussed.

Cytological changes and alterations in polyamine contents induced by cadmium in tobacco BY-2 cells.

Changes in cell viability, proliferation, cell and nuclear morphology including nuclear and DNA fragmentation induced by 0.05 and 1 mM CdSO4 (Cd2+) in tobacco BY-2 cell line (Nicotiana tabacum L.) were studied in the course of 7 days. Simultaneously changes in endogenous contents of both free and conjugated forms of polyamines (PAs) were investigated for 3 days. The application of 0.05 mM Cd2+ evoked decline of cell viability to approximately 60% during the first 24 h of treatment. Later on degradation of cytoplasmic strands, formation of the stress granules and vesicles, modifications in size and shape of the nuclei, including their fragmentation, were observed in the surviving cells. Their proliferation was blocked and cells elongated. Beginning the first day of treatment TUNEL-positive nuclei were detected in cells cultivated in medium containing 0.05 mM Cd2+. Treatment with highly toxic 1 mM Cd2+ induced fast decrease of cell viability (no viable cells remained after 6-h treatment) and cell death occurred before DNA cleavage might be initiated. The exposure of tobacco BY-2 cells to 0.05 mM Cd2+ resulted in a marked accumulation of total PAs (represented by the sum of free PAs and their perchloric acid (PCA)-soluble and PCA-insoluble conjugates) during 3-day treatment. The increase in total PA contents was primarily caused by the increase in putrescine (Put) concentration. The accumulation of free spermidine (Spd) and spermine (Spm) at 12 and 24 h in 0.05 mM Cd2+ treated BY-2 cells and high contents of Spd and especially Spm determined in dead cells after I mM Cd2+ application was observed. The participation of PA conjugation with hydroxycinnamic acids and PA oxidative deamination in maintaining of free PA levels in BY-2 cells under Cd2+-induced oxidative stress is discussed.