The nonselective herbicide glyphosate (N-lphosphonomethyljglycine) 3 To whom reprint requests should be addressed.were added to the growth medium, but no effect of glyphosate was found on chorismate mutase, prephenate dehydrogenase, and prephenate dehydratase. High concentrations of the herbicide inhibited the two initial enzymes of the shikimate pathway, but this inhibition was apparently too slight to account for growth inhibition caused by low glyphosate concentrations. Inhibition by glyphosate of the growth of carrot and tobacco cells was alleviated by the addition of the three aromatic amino acids, but Haderlie et al. (15) hesitated to conclude that the synthesis of the aromatic amino acids was inhibited by glyphosate because glyphosate did not appreciably reduce the cellular concentrations of these amino acids. Just recently, Greshoff (14) extended the earlier studies of Jaworski (20) and Haderlie et al. (15) with an investigation on glyphosate's growth inhibition of E. coli, Chlamydomonas reinhardtii, carrot and soybean cell cultures, and Arabidopsis thaliana seedlings. In all cases, phenylalanine and tyrosine acted synergistically in the alleviation of the inhibitory effect of glyphosate. It was suggested that some essential process or compound derived from phenylalanine and/or tyrosine was inhibited by glyphosate or one of its metabolites (14). Nilsson (23) reported an increase in the amount of free amino acids and in NH3 in wheat plants sprayed with glyphosate, but the percentage of phenylalanine and tyrosine of the total amino acids was strongly reduced. In a series of recent papers, 16,17) hypothesized that glyphosate might exert its effect at least in part through induction of PAL4 activity, resulting in one or more of the following growth-limiting conditions: (a) depletion of free phenylalanine and, possibly, tyrosine levels and coincident inhibition of protein synthesis; (b) toxic levels of ammonia provided that the rate of deamination exceeds the rate of amination; and (c) accumulation of growth-limiting phenolics derived from trans-cinnamic acid, the product of the PAL reaction. In spite of the attractiveness of this proposal, there are alternative explanations for the induction of PAL by glyphosate (see under "Discussion"), and this hypothesis would not explain the growth-inhibitory action of glyphosate in organisms, such as bacteria, which do not have PAL. Furthermore, if conditions b or c were pertinent, one would expect that phenylalanine would increase the inhibitory effect of glyphosate rather than alleviate it.PAL, however, may contribute only partially to aromatic amino acid depletion, as was recently pointed out by Duke et al. (11). It was also suggested (11) that glyphosate's effect on aromatic compounds may not be its principal mode of action. In the present HOLLANDER AND AMRHEIN study, we used an approach basically similar to that of Duke and Hoagland (9,16,17). We reasoned that, due to the possible functioning of a homeostatic control system and/or the possible involvemen...
ʟ-α-Aminooxy-β-phenylpropionic acid (AOPP), a potent competitive inhibitor of phenylalanine ammonia-lyase (PAL), blocked light-induced phenylpropanoid synthesis in excised buckwheat hypocotyls and produced an up to 40-fold increase in the endogenous phenylalanine concentration, while the level of all other amino acids was hardly affected. After a 24 h incubation in the light in the presence of 0.3 or 1 mᴍ AOPP phenylalanine alone constituted about 25% of the total soluble amino acids, compared to appr. 1% in the controls. In the presence of AOPP illuminated hypocotyls accumulated nearly 3 times more phenylalanine than hypocotyls kept in the dark, indicating an enhancing effect of light on the flow of carbon through the shikimate pathway. Exogenously added [14C] phenylalanine was extensively metabolized by control tissue, but accumulated in AOPP treated tissue. In the presence of AOPP radioactivity from [14C] shikimate accumulated predominantly in phenylalanine, and the flow of shikimate into tyrosine and phenylalanine was not affected by the inhibitor. Therefore, under these conditions no feedback control of phenylalanine and tyrosine synthesis from shikimate is apparent in buckwheat hypocotyls.
Both enantiomers of α-aminooxy-β-phenylpropionic acid (AOPP), potent inhibitors of L-phenylalanine ammonia-lyase, and their N-benzyloxycarbonyl (N-BOC) derivatives inhibit anthocyanin formation in developing flowers of Ipomoea tricolor Cav. and Catharanthus roseus Don. as well as in seedlings of Brassica oleracea var. caulo-rapa DC (kohlrabi) and B. oleracea var. capitata L. (red cabbage) with little interference with their normal development. Kohlrabi seedlings tolerate up to 0.3 mM L-AOPP and N-BOC-L-AOPP without a reduction of fresh weight or chlorophyll content, while anthocyanin is reduced to less than 20%.
A previously described procedure for the estimation of relative activities of phenylalanine ammonia-lyase (EC 4.3.1.5) in intact plant cells (Amrhein et al. (1976) Planta 131, 33-40) was reexamined for its specificity and its applicability to various tissues. In buckwheat hypocotyl segments (3)H is stereospecifically released from the pro-3S-position of L-[2,3-(3)H]phenylalanine and is thus due to phenylalanine ammonia-lyase activity. In buck wheat and sunflower leaf disks, however, (3)H release occurs from both the 2- and 3-positions of the labeled substrate and can only partially be attributed to phenylalanine ammonia-lyase activity.
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