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

Abstract

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...

Effect of polyamine biosynthetic inhibitors on alkaloids and organogenesis in tobacco callus cultures

“…In this investigation, we show that pyrrolidine alkaloids (nornicotine + nicotine) and Put (especially its bound fraction) titers are also more effectively reduced by D F M A and D-Arg than by DFMO treatments in the NTW-38 callus strain. Others have also reported that DFMO does not inhibit the levels of Put conjugated to cinnamic acids [1] and have suggested a major role for ADC in the supply of Put for biosynthesis of conjugates in tobacco cell cultures [34]. Since, as discussed above, the pyrrolidine ring of tobacco alkaloids may arise from Put in conjugates, the present results further support the view [31] that ADC, not ODC, has a major role in the generation of Put going into the tobacco alkaloids.…”

Hydroxycinnamoyl-CoA:Putrescine Hydroxycinnamoyltransferase in Tobacco Cell Cultures with High and Low Levels of Caffeoylputrescine

“…ODC may have a similar function, as it was also increased in TX4 cells, but this pattern may be complicated by the involvement of ODC in other pathways. Recently, Walker et al (22) detected distinctly higher arginine decarboxylase (ADC) activity in TX4 cells and concluded that putrescine may be formed mainly by ADC rather than by ODC. We therefore also measured ADC activity on several days during the kinetic study.…”

Secondary Products from Plant Cell Cultures

“…Though the biochemical comparison of parent and variant lines provided some clues as to the biochemical requirements of overproducing lines, we did not analyze the system at the molecular level. One reason for this was the finding that not only the hydroxycinnamoyl putrescine pathway but also other enzyme activities such as tyrosine decarboxylase (WALKER et al, 1986) were altered in the pfluorophenylalanine resistant cell line. Another reason was the fact that not only one but several enzyme activities should be "coenhanced" in order to improve hydroxycinnamoyl putrescine production by genetic techniques.…”