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

Abstract

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 [...

Polyamine oxidase activity and polyamine content in maize during seed germination

“…On the other hand, the changes in polyamine content, which are almost the same in shoots and roots, cannot be correlated to the pattern of PAo activity. This fact and the cell wall localization of PAO once again favour the hypothesis of a compartmentation of the polyamine metabolic machinery, as already demonstrated in plants for their biosynthetic enzymes (Torrigiani et al 1986, Walker et al 1987.…”

Arginine Decarboxylase Is Localized in Chloroplasts

“…Previous work attempting the subcellular localization of ADC involved the measurement of enzyme activity (Torrigiani et al, 1986; Walker et al, 1987). However, these studies have been questioned because of artifacts resulting from the use of 14 C trapping (Birecka and Birecki, 1993;Smith, 1993).…”