Germination of Phaseolus vulgaris II. Stimulation of Axis Growth by dl-Fluorophenylalanines.

The initiation and continued elongation of excised embryonic axes from Phascolus vulgaris L. (var. WVhite Marrowfat) apparently depend on protein synthesis since puromycin, cycloheximide and actinomycin D strongly inhibit growth as well as amino acid incorporation into protein (9). In an attempt to extend these findings, I have tested the effects on axis growth of various amino acid analogtues reported to inhibit growth in higher plants and/or bacteria (1, 2, 6, 7, 8). The analogues tested were fluorophenylalanine, /3-phenylserine, f8-2-thienylalanine, which are analogues of phenylalanine, and hydroxyproline, canavanine, ethionine, norletucine and 5-methyltryptophan. The embryonic axes were excised from dry seeds and incubated for 11 hourrs (9). The initiation of axis elongation began after approximately 4.5 hoturs of incubation and the fresh weight increase was linear over the next 6.5 hours with a resultant increase of approximately 50 % in fresh weight. After incubation, axes were embedded in paraffin ancl sectioned, and the I)NA was stained by the Feuilgeln method (4). p-Fluoro-DL-phenylalanine was reer :-stallized twice from water. Thinlayer chromatography in several solvent systems indicated the presence of only 1 compound, whether the spot was madle visible by ninhydrin or by fluorescence quienching. Of the compotunds tested only the fluorophenylalanines and /3-2-thienylalanine, analogues of phenylalanine, significantly affected the increase in axis freslh weight. DL-Fluoropheaylalanine stimilated the fresh weight increase while /3-2-thienylalanine had an inhibitory effect (table I). At 5 X 10-4 a, the concentration at which the majority of experiments were condtucted, p-fltioro-DL-phenylalanine always increasedl the fresh weight gain in comparison with the controls. The stimtulation ATariedl from 20 to 40 %. The ortho ancl meta isomers also appeared to be stimtlatory, bhtt at 5 X 10-4 -.I slhowe(I onlv abouit 50 % of the activity of the para isomer. The stimultationi by p-flntoro DL-phenvlalanine wVas essentially reversed by L-phenylalanine, although the slightly inhibitory effect caused by 10-3 M L-phenylalanine alone makes these results less clear-cuit (table II). Tyrosine had no effect oni either the endogenous fresh weight increase or the p-fltlorophenylalanine-stimLulated increase. A kinetic study of the effects of 5 X 10-4 al p-fluoro-DL-phenylalanine on growth showed that stimulation took place over the entire period of elongation, although there was no indication that initiation of elongation began earlier than uslial. Measurements of the intact axes; indicated that the increased weight was due to an increased length. No mitotic figures were seen in either control axes or those treated with p-fluoro-DL-phenylalanine and presumably the effect of p-fluorophenylalanine was on cell elongation. Since it has been reported that phenylalanine hydroxylase converts p-fluorophenylalanine to tyrosine and fluoride ion in rats (3), I tested the effects of varying concentrations of NaF on axis growth. No effects were noted between 5 " 10M and 5 X 10-4 M, while at 5 X 10-3 At growth was inhibited by 90 %. It seems highly unlikely, therefore, that the stimulation of axis growth by fluorophenylalanine is due to the formation of fluoride ion. The results of this study were unexpected since it was found that p-fluorophenylalanine stimulates axis growth by 20 to 40 % at concentrations which have been found to inhibit the growth of other tissuies by 50 to 70 % (6). In addition, the other amino acid analogues tested, with the exception of ,8-2-thienylalanine, failed to inhibit growth. The inhibitory effect of fluol ophenylalanine on growth is generally attributed to reduced enzymatic activities resulting from the replacement of phenylalanine by fluorophenylalanine in proteins (7). In higher plants, however, phenylalanine is also a metabolic intermediate for other types of compotunds, including phenolics, coumnarins and flavonoids (5). Therefore, the effects of fluorophenylalanine onl plant growth may bJe complicated by its effects on these metabolic conversions of phenylalanine. Watkins and Magrill have recently reportecl that chromatographs of extracts from plants fe(d '4C-p-fluiorophenylalanine showed new spots as compared with chromatographs of those fe(d 14C_ phenylalanine (10). They also suiggested that biosynthesis of flavonoids from p-ftiorophenylalanine is blocked when a hydroxylation is required at the 4' position. These results suggest that the stimulation of axis growth by fluorophenylalanine may involve other metabolic effects in addition to replacement of phenylalanine in proteins.