Polyamine Biosynthetic Enzymes in the Cell Cycle of Chlorella

The levels of free amines and the activities of their biosynthetic enzymes were measured in a p-fluorophenylalanine resistant Nicotiana tabacum L. cv Xanthi cell line (TX4) which accumulates high levels of cinnamoylamides, and a wild type cell line (TX1). Putrescine in TXI and spermidine in TXI and TX4 increased 4-fold by day 4 but declined by day 8 of the culture period. Spermine levels were consistently low, while tyramine was not found in TX1 until day 9 when a gradual rise was noted. Ornithine decarboxylase activity in TXI and TX4 increased slightly through day 2 but declined gradually thereafter. S-Adenosylmethionine decarboxylase activity remained low throughout the culture period, and tyrosine and arginine decarboxylases in TX1 were very low in activity. In contrast, the activities of tyrosine and arginine decarboxylases were elevated in TX4, but a 3-fold increase in tyramine after a subculture was not accompanied by a rise in tyrosine decarboxylase. However, tyrosine decarboxylase activity did increase during a second rise in tyramine levels in aging cells, late in the culture period. Although significant differences exist in amine levels, between TX4 and TX1, it is unclear how altered amine metabolism relates to p-fluorophenylalanine resistance.The selection of plant cell cultures resistant to antimetabolites has become a standard procedure for the isolation of biochemically variant cell lines (28). The amino acid analog p-fluorophenylalanine has been used to select a resistant tobacco cell line (TX4) which exhibits enhanced phenylalanine and tyrosine synthesis (21) and accumulates 6 to 10 times more hydroxycinnamoylamide$ (largely caffeoylputrescine) than wild type cells (TX 1) (3-5).Aliphatic amines and tyramine, together with their hydroxycinnamic amides, have also been found to accumulate in the reproductive organs of numerous plant species, including tobacco (10, 22, 27), and increased levels are associated with a wide range of physiological and developmental responses (15,25 tobacco cell lines allow investigation of biochemical patterns in a controlled environment without developmental influences. While the metabolism of phenylalanine in the TX4 line has been examined (6, 7), and the production of the diamine, putrescine, has been studied (1), clarification of subsequent polyamine biosynthesis and hydroxycinnamoylamide accumulation requires more thorough study of amine metabolism in high and low accumulating tissues. This paper reports on the relationship of the levels of putrescine, spermidine, spermine, and tyramine, and of the activities of their biosynthetic enzymes, to the culture growth cycle of PFP-resistant and wild type Nicotiana tabacum cell suspensions. MATERIALS AND METHODSPlant Material. The TX4 culture used in these studies continued to display the traits of PFP-resistance and hydroxycinnamoylamide accumulation (23) initially described by Palmer and Widholm (21). Cell suspensions of TXl and TX4 (Nicotiana tabacum L. cv Xanthi) were generously provided by Dr. J. Berlin, Brauns...

Section: Resultssupporting

confidence: 80%

“…Embryogenesis of carrot cells is associated with elevated polyamine levels and increased ADC activity (15,16). In contrast, ODC activity is more closely related to levels of DNA synthesis (13), as seen in the cell division phase in tomato ovaries following fertilization (1 1) and in meristematic tissues in tomato and potato (12).…”

Section: Resultsmentioning

confidence: 99%

Changes in Amines and Biosynthetic Enzyme Activities in p-Fluorophenylalanine Resistant and Wild Type Tobacco Cell Cultures

Walker¹,

Ellis²,

Dumbroff³

et al. 1986

“…This apparent contradiction may result from subcellular shifts in polyamine location that are reported to accompany various phases of cell division (9,31) and tissue development (1 1). The tobacco cultures that were analyzed had progressed beyond the stage ofrapid cell division and the accompanying demands for high levels of DNA synthesis but such cultures still contain cells at various stages ofgrowth and division.…”

Section: Resultsmentioning

confidence: 95%

Subcellular Localization of Amines and Activities of Their Biosynthetic Enzymes in p-Fluorophenylalanine Resistant and Wild-Type Tobacco Cell Cultures

Walker

Ellis²,

Chapple³

et al. 1987

Three levels of free amines and the activities of their biosynthetic enzymes were measured in subcellular fractions of two cell lines of Nicotiana tabacum L. cv Xanthi. The TX4 cell line, a p-fluorophenylalanine resistant culture which accumulates high levels of cinnamoylamides, was compared to the wild-type culture TX1. In cells harvested on day 6 of the growth cycle, nearly all free putrescine, spermidine, and tyramine was found in the supernatant fraction of both cell lines. Although a consistent portion of ornithine decarboxylase activity was detected in the nuclear-enriched fractions of TX1 and TX4, the larest levels of activity were in the supernatants of both lines. In TX1, arginine decarboxylase activity was low relative to that of ornithine decarboxylase, but, in the TX4 line arginine decarboxylase levels in the cytosol were substantially elevated. Tyrosine decarboxylase was not detected in 6-day-old TX1 cells, but significant amounts of activity were measured in the lOOOg and supernatant fractions of TX4. S-Adenosylmethionine decarboxylase activity was low in both cell lines and was located predominantly in the supernatant.Recently, we reported on changes in levels of polyamines and tyramine, and on the activities of their biosynthetic enzymes (ADC,3 ODC, SDC, and TDC), during the growth cycle of PFPresistant (TX4) and wild-type (TX 1) cell suspension cultures of Nicotiana tabacum (34). The biosynthetic activity of ADC was significantly higher in TX4 than TX 1, suggesting that it provided the putrescine moiety required to maintain high levels of cinnamoylputrescine found in the TX4 line. These basic differences in biosynthetic activity make the TX cell lines useful model systems in which to study regulatory mechanisms in amine metabolism. In the present study, we have investigated the compartmentation of specific enzymes involved in amine biosynthesis and the endogenous levels of mono-, di-, and polyamines.ODC activity has been reported to occur in plant nuclei (14,21,29). Along with ADC, ODC has also been detected in the cytoplasm, chloroplasts, and mitochondria (29,33 MATERIALS AND METHODS Plant Material. Cell suspensions of TX 1 and TX4 (Nicotiana tabacum L. cv Xanthi) were generously provided by Dr. J. Berlin, Braunschweig, Federal Republic of Germany. The cultures were maintained at 24°C in an MX medium supplemented with 2 FM 2,4-D under continuous illumination. TX 1 and TX4 were subcultured every 7 and 10 d by transferring 1.5 and 2.5 g fresh weight, respectively, to 50 ml of fresh media. Data represent the average of at least three replicate determinations. The TX4 culture used in these studies continued to display the traits of PFP-resistance and hydroxycinnamoylamide accumulation (23) initially described by Palmer and Widholm (20). Subcellular Fractionation. For TDC preparations, 80 g fresh weight of 6-d-old cells were collected by vacuum filtration and disrupted by grinding twice for 3 min in a prechilled mortar together with 40 ml of extraction buffer (100 mm pH 7.5; 20% glycerol [...

“…This situation might explain why there have been so few reports in which significant growth inhibition has been shown to result from markedly decreased PA titers accomplished through the use of specific inhibitors of PA biosynthesis in vivo. Indeed, it has been reported that in log phase cultures of Chlorella grown in the presence of difluoromethylornithine, a significant increase in generation time is not observed until the second cell cycle, suggesting that endogenous PA titers are sufficient to support cell division and related biosynthetic activities for a considerable time (8).…”

Section: Discussionmentioning

confidence: 99%

Changes in Polyamine Biosynthesis Associated with Postfertilization Growth and Development in Tobacco Ovary Tissues

Slocum¹,

Galston²

1985

122

Polyamine (PA) titers and the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxyhse (ODC, EC 4.1.1.17), enzymes which catalyze rate-limiting steps in PA biosynthesis, were monitored during tobacco ovary maturation. In the period between anthesis and fertilization, the protein content of ovary tissues rapidly increased by about 40% and was accompanied by approximately a 3-fold increase in ODC activity, while ADC activity remained nearly constant. PA titers also remained relatively unchanged until fertilization, at which time they increased dramatically and the DNA content of ovary tissues doubled. This increase in PA biosynthesis was correhted with a further 3-fold increase in ODC activity, reaching a mximum 3 to 4 days after fertilization. During this time, ADC