Polyamine Metabolism in Embryogenic Cells of Daucus carota

Abstract

Changes in the metabolsm of polyamines, which seem to be involved in transcription and translation in animal systems, have been studied in cultured cells of Daucus carota (carrot) undergoing embryogenesis. Putrescine levels were elevated by as much as 2-fold over the control within 24 hours after transfer of the celis to embryogenic medium. Spermidine levels were elevated also but spermine levels appeared to be lower in embryogenic celis. Embryogenic celis incorporated ["4Clarginine into putrescine at two times the rate of control celis. These changes suggest that polyamines may be involved in celiular differentiation during embryogenesis.The metabolism of the polyamines, putrescine, spermidine, and spermine, has been the subject of considerable investigation, especially in animal systems (2). Elevated levels of at least one of these compounds have been noted in developing rat fetuses (15), in sea urchin embryos (11), following partial hepatectomy (14), and in transformed cells (3). In addition, there is evidence that polyamines are involved in the regulation of transcription (9,13) and translation (8,10 (5, 16). After addition of an equal volume of 10%1o (w/v) trichloroacetic acid, the homogenates were heated at 80 C for 10 min. The cooled mixture was then centrifuged at 20,000g for 20 min and the supernatant retained for further extraction. After noting the volume of supernatant, a l-ml aliquot was withdrawn for the dansylation procedures (3, 6) used to quantitate the polyamines.A 0.2-ml volume of the supernatant obtained after deproteination corresponding to approximately 7 mg dry cell weight was sufficient for densylation. After making this volume ofsupernatant basic (pH greater than 9) with 18.5 mg of anhydrous Na2CO3, 0.4 ml of dansyl chloride in acetone (30 mg/ml) was added. The stoppered tubes were incubated overnight at 27 C. The excess dansyl chloride was destroyed by the addition of 0.1 ml of a proline solution (100 mg/ml in H20) and the dansylated products were extracted into 0.5 ml of benzene. Aliquots of 10 to 15 [LI of the benzene extract were supplied to activated (45 min at 100 C) silica gel TLC plates. The plates were developed for 5 hr in ethyl acetate-cyclohexane (2:3, v/v) and immediately sprayed with 10% (v/v) triethanolamine in isopropyl alcohol. The polyamines were quantified with a Turner model III fluorometer equipped with a Camag T Scanner Mark III (activation at 365 mm) and peak areas (>512 nm) from the extracts were compared to those obtained from dansylation of standards. It was necessary to include at least two standard spots on each plate along with the extract samples because there was a variation of fluorescence both between plates and upon a single plate.In order to determine radioactivity present in the polyamines after exposure to ["Clarginine, 2.5 ml of the supernatant obtained after deproteination were extracted four times with 2 volumes of ether to remove the trichloroacetic acid. Then, the extract was made basic by the addition of 0.05 ml of 10 N NaOH per ml an...

Translational Modification of an 18 Kilodalton Polypeptide by Spermidine in Rice Cell Suspension Cultures

“…Recent evidence suggests a possible role for polyamines, especially Spd, in reproductive differentiation in various organisms (2,7,10,11,15 eluted with a protein fraction having a molecular mass in excess of 45,000 in oat and petunia protoplasts (14). Because there was a large difference in the relative molecular mass in the polypeptides from tobacco thin-layer culture and oat and petunia protoplasts, a need exists to establish unambiguously the size of proteins that are labeled by Spd, as was also suggested by Mizrahi et al (14).…”

“…Recent evidence suggests a possible role for polyamines, especially Spd, in reproductive differentiation in various organisms (2,7,10,11,15). Application of exogenous Spd causes floral initiation in cultures which would otherwise form vegetative buds.…”

Participation of Ornithine Decarboxylase in Early Stages of Tomato Fruit Development

“…Such information is lacking for plant systems. Several reports (4,8,14,15,(20)(21)(22) do, however, describe the presence of various polyamines in plants and the occurrence of enzymes involved in polyamine biosynthesis, e.g. ODC3 and ADC.…”

“…ODC3 and ADC. It is commonly accepted that ADC is the enzyme responsible for the production of putrescine in plants and that ODC is of lesser importance (14,15,(20)(21)(22)). …”

Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture

“…3). The pattern of changes in ADC activity was also reported to be similar to that in ODC activity during the cell cycle in H. tuberosus explants (30 (25), the main route for production of PUT seems to be via ADC in carrot (13). In a detailed analysis of ODC and ADC in carrot tissues, it was observed that the undifferentiated cells grown in culture only contained active ADC, while ODC activity increased sharply during differentiation to somatic embryos (4).…”