Interference of L-a-Aminooxy-/?-Phenylpropionic Acid with Phenylalanine Metabolism in Buckwheat Heike Holländer, Hans-Hermann Kiltz *, and Nikolaus Amrhein Arbeitsgruppe Hormonphysiologie der Pflanzen, Lehrstuhl für Pflanzenphysiologie, and * Lehr­ stuhl für Biochemie, Ruhr-Universität Bochum, Postfach 102148, D-4630 Bochum Z. Naturforsch. 34 c, 1162— 1173 (1979); received August 29, 1979 Phenylalanine Ammonia-Lyase, L-ar-Aminooxy-/?-phenylpropionic Acid, Aromatic Amino Acids, Shikimic Acid, Fagopyrum esculentum L-a-Aminooxy-/?-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 concentra­ tion, 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 mM 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 phenylala­ nine 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 metab­ olized by control tissue, but accumulated in AOPP treated tissue. In the presence of AOPP radio­ activity from [14C] shikimate accumulated predominantly in phenylalanine, and the flow of shi­ kimate 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. Introduction phenylalanine, L-a-aminooxy-/?-phenylpropionic acid (AOPP) as a highly potent competitive inhibitor of Structural analogues of amino acids have been PAL [8], which appears to have the desired specif­ widely used in biochemical and biological studies of icity to be applicable in in vivo studies [7, 9 —11]. metabolism and development [1-3]. In partic­ Thus, it was shown that seeds germinate and devel­ ular, analogues of phenylalanine, such as e. g. p- op normally in the presence of concentrations of fluoro-phenylalanine, have been of interest with re­ AOPP, which suppress phenylpropanoid synthesis spect to their activation and incorporation into pro­ [9, 12], but apparently did not interfere with protein teins [4] as well as tools in investigations of aromatic synthesis. In fact, Norris et al. [13] have shown that amino acid biosynthesis [3]. In higher plants phenyl­ AOPP is neither activated by nor does it inhibit alanine is the immediate precursor of cinnamic acid, phenylalanyl-tRNA-synthetase from Phaseolus au­ which is the parent compound of a multitude of reus seeds. It is known, however, that AOPP interferes phenolic substances, including lignins and flavonoids. with the transamination of phenylalanine in extracts Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) ca­ from Phaseolus aureus seedlings [11], and also inhi­ talyzes the first committed step in the biosynthesis of bits pig heart glutamate-oxalacetate transaminase as these substances, and the biochemistry and physiolo­ well as glutamate-pyruvate transaminase (Amrhein gy of this enzyme have been studied in considerable and Striiber, unpublished). The potency of inhibi­ detail [5, 6]. As outlined elsewhere [7], specific inhi­ tion was, however, far less pronounced than in the bitors of this enzyme would be of value in the case of phenylalanine deamination. Nevertheless, the elucidation of various aspects of the biochemistry interference of AOPP in vivo with the formation and and physiology of phenylpropanoid metabolism. We metabolism of amino acids other than phenylalanine have recently introduced the aminooxy analogue of has to be taken into account. We, therefore, decided to investigate the effect of AOPP on the levels of soluble amino acids in buckwheat hypocotyls. Fur­ Abbreviations: AOA, a-aminooxyacetic acid; AOPP, L-ar- aminooxy-/?-phenylpropionic acid; PAL, phenylalanine thermore, we hoped to gain insight into the regula­ ammonia-lyase (EC 4.3.1.5). tion of the shikimate pathway under conditions, Reprint requests to Prof. Dr. N. Amrhein. which prevent the flow of phenylalanine into the 0341-0382/79/1200-1162 $01.00/0 phenylpropanoid pathway. H. Holländer et al. ■ L-ar-Aminooxy-/?-Phenylpropionic Acid and Phenylalanine Metabolism 1163 Materials and Methods 25 °C and under continuous illumination from fluo­ rescent tubes (5000 lux). Leucine and phenylalanine Plant material were fed at 1 pCi/vial (leucine concentration: 1.4 pM; Buckwheat (Fagopyrum esculentum Moench) seeds phenylalanine concentration: 0.98 pM ), shikimic acid were germinated in moist Vermiculite and grown in at0.2-0.5 pCi/vial (concentration: 1.2 — 3 pM). the dark for 6 days at 23.5 ± 1 °C. For the experi­ ments described in Table III seeds of the various Extraction and measurement of amino acids species were allowed to germinate for 5 days on filter Buckwheat hypocotyls, entire seedlings, or leaf paper soaked with either H20 or 0.3 mM l-AOPP in disks (fresh weights 0.1 -1.2 g) were extracted plastic dishes in a growth chamber under a 16 h twice for 20 min with 20 ml of boiling 80% ethanol photoperiod at 22 ± 1 °C. Mung bean seeds had under reflux. The extracts were dried in an air previously been soaked overnight in running tap wa­ stream and residues taken up in 8 ml HzO. After ter. The leaf material was from plants grown in the acidification with 1 n HC1 and three extractions with greenhouse (wheat, barley, tobacco) or in the Botan­ 4 ml ethylacetate the aqueous phases were trans­ ical Garden (Catalpa ). ferred to 1 x 5 cm columns of Dowex 50 W x 8 equilibrated with 0.01 N HC1. The columns were Chemicals washed with 30 ml 0.01 n HC1 followed by 5 ml The enantiomers of a-aminooxy-/?-phenylpropi- HzO. Amino acids were eluted with 50 ml 2 n onic acid and its N-benzyloxycarbonyl derivatives as NH4OH, and the eluates were dried in an air stream. well as the enantiomers of a-hydrazino-/?-phenylpro- The residues were taken up in 1 ml HzO, and ali­ pionic acid were synthesized according to proce­ quots of 20 to 40 pi were subjected to amino acid dures kindly made available to us by Dr. J. S. analysis in a BC 200 amino acid analyzer (LKB, Morley, ICI Pharmaceuticals Division, Macclesfield, München). An amino acid standard chromatogram Cheshire, U. K. Aminooxy-acetic acid, semihydro­ is found in [14]. Incorporation of radioactivity into chloride, was obtained from Sigma, St. Louis, MO., the aromatic amino acids was determined either and Rhodotorula glutinis PAL, Cat. No. 0810, from after separation of the amino acids by paper chro­ P-L Biochemicals Inc., Milwaukee, WI. l-[U -14C]- matography in «-propanol: NH3 (7 : 3, v/v) or in the Leucine (sp. A. 354 mCi/mmol), l-[U -14C] phenyl­ eluates of the amino acid analyzer as described in alanine (sp. A. 513 mCi/mmol), and d-[2,3,4,5(«)]- ref. [14]. [14C]shikimic acid (sp. A. 84 mCi/mmol) were pro­ vided by the Radiochemical Centre, Amersham, U. Other measurements K. The radiopurity of the compounds was checked Radioactivity in ethanol-extracted dried hypoco­ by paper and thin layer chromatography. Chemicals tyls was determined after combustion in a Mikromat- and biochemicals in reagent grade were purchased Analyzer, Berthold-Frieseke, Wildbad. Incorporation from Merck, Darmstadt, Dowex 50 W x 8 from of radioactivity into protein was determined as the Serva, Heidelberg. activity released by hydrolysis of the ethanol-ex­ tracted hypocotyls in 6 N HC1 at 110 °C for 48 h. Incubation of buckwheat hypocotyls Radioactivity was determined in a Berthold Frie- Excised hypocotyls from 6-day-old buckwheat seed­ seke BF 5000 scintillation counter. Anthocyanin ex­ lings were incubated in petri dishes as described tractions and measurements were made according to previously (for references see [8]). Hypocotyls for in­ ref. [15]. Apparent inhibitor constants for buckwheat cubations in the dark were prepared under a dim PAL were determined as described in ref. [8]. green safety light. For feeding experiments 20 de­ rooted 6-day-old seedlings were placed with the cut Results lower hypocotyl end in a glass scintillation vial con­ Effect of AOPP on amino acid levels taining 2 ml of either 0.1 m potassium phosphate in excised buckwheat hypocotyls buffer, pH 5.5, or, in addition, 1 mM l-AOPP. After a 4 h preincubation the radioactive precursor was When hypocotyls of 6-day-old etiolated buck­ added and the incubation continued for 24 h at wheat seedlings were excised and incubated for 24 h 1164 H. Holländer et al. • L-ar-Aminooxy-/?-Phenylpropionic Acid and Phenylalanine Metabolism dure of Uchiyama et al. [16] using Rhodotorula PAL, and the fluorescence assay of Wong et al. [17] — produced similar values within a range of ± 30%, but difficulties arose also with low phenylalanine concentrations. Moreover, AOPP from AOPP-treated plants severely interfered with the enzymatic proce­ dure, because it is a potent inhibitor of Rhodotorula PAL [8]. AOPP gives, however, only a slight reaction with ninhydrin and did not interfere with the deter­ mination of phenylalanine in the amino acid ana­ lyzer. Amino acid measurements in general were the same within the variations given in Table I, indepen­ dent on whether the hypocotyls were floated in the Time (h ) incubation solutions in petri dishes or placed with the cut lower ends in the solutions in small vials. The latter mode of incubation, which deviates from our standard procedure [18], became necessary, when it was found that labelled shikimic acid was taken up effectively only with the transpiration stream. When 1 mM l-AOPP was included in the incubation medi­ um, phenylalanine levels rose dramatically, while all other amino acids were hardly affected (Table I).