Amino Acid Metabolism of Lemna minor L . 1 III

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by aminooxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of a-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate. In a previous paper in this series (26), we have reported on the metabolic changes induced by the irreversible inhibitor of GS,2 MSO, in Lemna minor. It was proposed that glutamate, glutamine, asparagine, aspartate, alanine, and serine may be selectively catabolized to NH4+, either in the photorespiratory nitrogen cycle or by alternative routes (26). These alternative routes include dehydrogenase (e.g. GDH) and amidohydrolase (deamidase) (e.g. asparaginase) pathways (11, 20, 22, 23, 31). We have reasoned that if such alternative pathways of amino acid catabolism to NH4+ are in operation, then these should not be inhibited by the transaminase inhibitor, AOA (9, 15, 16, 36). It follows that if dehydrogenase and amidohydrolase reactions are primarily responsible for the catabolism of amino acids such 'Supported by Purdue University Agricultural Experiment Station funds via a David Ross assistantship awarded to D.G.B. 2 Abbreviations: GS, glutamine synthetase; AOA, aminooxyacetate; ALS, acetolactate synthase; gfw, gram fresh weight; GABA, y-aminobutyrate; GOGAT, glutamate synthase; GDH, glutamate dehydrogenase; MSO, L-methionine-D.L-sulfOximine. as glutamate, alanine, glutamine, and asparagine to NH, in the presence of MSO, then AOA should inhibit neither the MSOinduced depletions of these amino acids nor the MSO-induced accumulation of NH,-. Conversely, if these amino acids are catabolized to NH4+ in the photorespiratory nitrogen cycle, AOA should inhibit both the MSO-induced accumulation of NH, and the MSO-induced depletion of these amino acids (a) as a result of inhibition of glycine decarboxylase by AOA (13, 30, 37) and (b) as a result of inhibition of glyoxylate-dependent aminotransferase reactions (e.g. glutamate:glyoxylate, serine:glyoxylate, and alanine:glyoxylate aminotransferases [2, 9, 10. 24,]), which catalyze the transfer of amino-N to glycine en route to serine and NH4+. Thus, the operation of a photorespiratory pathway of amino acid catabolism to NH4+ might be revealed by AOA inhibition of both NH + accumulation and the depletion of glutamate and alanine normally promoted by MSO. Asparagine can also serve as a nitrogen donor for glycine and, hence, serine and NH4 in photorespiration (12, 16, 33-35), or it may be metabolized to NH4 +independently of photorespiration by transamination reactions yielding oxosuccinamate, which can be further degraded to NH<+ (20, 23, 31). In either case, asparagine catabolism should be inhibited by AOA since the initial step in asparagine catabolism entails transamination. Although inhibition of asparagine catabolism by AOA would rule out an asparaginase pathway, this result would not discriminate between asparagine amino-N transfer to glycine or other amino acids as major pathways of asparagine catabolism (12, 16, 33-35). Similarly, inhibition of alanine catabolism by AOA would not discriminate between alanine amino-N transfer directly to glycine via alanine :glyoxylate aminotransferase or alanine amino-N transfer to glutamate by glutamate:pyruvate aminotransferase (2, 9, 24). Nevertheless, the responses of L. minor to AOA in combination with MSO could shed light on the relative importance of transamination reactions (potentially channelling aminoN to glycine, serine, and NH4 +via the photorespiratory nitrogen cycle), versuis transaminase-independent (i.e. dehydrogenase and amidohydrolase) pathways of amino acid catabolism. With these considerations in mind, we have investigated the effects of AOA. alone or in combination with MSO, on the free amino acid and NH4 +pools of L. minor. In previous reports in this series (26, 27), protein turnover has been defined as a potentially major source of free amino acids accumulated in response to inhibitors of amino acid metabolism. In this paper, we extend these observations on protein turnover in L. minor, demonstrating that the AOA-induced accumulation of the branched-chain amino acids (valine, leucine, and isoleucine) are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron (25, 27). Thus, valine, leucine, and isoleucine accumulate in response to AOA independently of de novo synthesis, and hence most probably from protein turnover. These investigations of the responses of L. miinor to 447 www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 1988 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 87, 1988 AOA, alone or in combination with chlorsulfuron (and vice versa), confirm that the chlorsulfuron-induced accumulation of a-aminon-butyrate (27) is inhibited by AOA and therefore most likely entails transamination of 2-oxobutyrate, which is accumulated in the presence of sulfonyl urea herbicides (19). MATERIALS AND METHODS Organism and Growth Conditions. Lemna minor L. was grown essentially as described previously (26, 27) on the basal medium described by Stewart (32), using 5 mm KNO3 as sole nitrogen source in all investigations reported here. Chemicals. AOA and MSO were obtained from Sigma Chemical Co. (St. Louis, MO). Chlorsulfuron was a gift from Dr. Philip Haworth, Sandoz Crop Protection Corp., Zoecon Research Institute, Palo Alto, CA 94304. All other chemicals were obtained from Sigma unless otherwise stated. Free Amino Acid Extraction and Quantification. Lemna fronds were harvested for amino acid analysis essentially as described previously (27, 28), by filtering, washing with distilled H20, blotting dry on tissue paper, weighing, and extracting in methanol (0.2-0.4 gfw/10 ml methanol). After storage for at least 48 h at 40C, the methanol extracts were phase-separated by addition of 5 ml chloroform and 6 ml H20; the upper aqueous phase was rotary evaporated to dryness and redissolved in 2 ml H20. At this point, known amounts of internal standards were added. For the acidic amino acids, 250 nmol of L-a-aminoadipic acid was employed. For the neutral and basic amino acids, 250 nmol of either L-pipecolic acid or L-a-amino-n-butyrate was employed. L-a-Amino-n-butyrate was used as internal standard only in experiments in which chlorsulfuron was not supplied (27). Pipecolic acid was used in experiments in which chlorsulfuron was supplied and where endogenous accumulation of a-amino-n-butyrate was expected (27). The choice of a-amino-n-butyrate as an internal standard for routine studies of the effects of AOA and MSO on amino acid metabolism in L. minor was based on preliminary observations which indicated that this compound was a negligible component of the free amino acid pool of L. minor following 24 h treatment with 10-4 M AOA and/or 10-4 M MSO (cf. small but significant accumulation of pipecolic acid in response to MSO [26]). After addition of internal standards and removal of 50 ,A aliquots for NH4+ determination as described previously (26), the samples were applied to 1 x 2 cm columns of Dowex-50H+, and amino acids were eluted with NH40H as described previously (27). Following concentration of the NH40H eluates to dryness and redissolving in 1 ml H20, samples were applied to 1 x 2 cm columns of Dowex-1-acetate, and neutral plus basic and acidic amino acid fractions were recovered as described previously (27). Amino acids were derivatized for gas chromatography analysis exactly as described before (27). Glutamine Synthetase Determination. Glutamine synthetase (transferase) activity was determined essentially as described by Rhodes et al. (29). Fronds (0.4 gfw) were harvested in a strainer, washed with distilled H20 (50 ml), blotted dry on tissue paper, weighed and placed in a prechilled (4°C) mortar and pestle, and ground with ice-cold extraction buffer (50 mm Imidazole-acetate pH 7.2 containing 0.5 mm EDTA and 1 mm DTT). The slurry was filtered through Whatman No. 1 filter paper, and the filtrate was collected in prechilled vials. The GS reaction mixture consisted of 1.0 ml of a solution containing 1.64 g L-glutamine, 196 mg hydroxylamine, 80 mg MnCl2, 20 mg ADP in 80 ml 0.1 M Tris/acetate (pH 6.4) (pH adjusted to 6.4 with NaOH), to which was added 0.25 ml sodium arsenate (264 mm in 0.1 M Tris/acetate, pH 6.4) plus 0.65 ml 0.1 M Tris/acetate (pH 6.4), plus 0.1 ml enzyme extract. Reactions were initiated by addition of enzyme extract and incubated at 30°C for 0 or 10 min. Reactions were stopped at either zero time or after 10 min incubation by addition of 2 ml FeCl3 reagent (8 g TCA, 5.2 g FeCl3, 16 ml concentrated HCl in 200 ml H,O). After centrifugation for 5 min at 1500 rpm in a Hamilton Bell model 1550 centrifuge, the absorbance was determined at 500 nm in a Perkin Elmer Lamda 4B spectrophotometer. Transferase activity is expressed as ,umol -y-glutamyl hydroxamate/h-gfw. Total Soluble Protein Extraction and Determination. Total soluble protein was extracted from L. minor essentially as described for GS extraction except the extraction buffer employed was 100 mm potassium phosphate (pH 7.5) containing 0.5 mm EDTA, 0.2 mm DTT, and 20 mm (NH4)2S04. Aliquots of 0.1 ml filtrate were precipitated with 0.1 ml 20% w/v TCA in 1 ml Beckman polyethylene microfuge tubes. After storage for at least 1 h on ice, the samples were centrifuged in a Beckman Microfuge II for 5 min at 10,000 rpm. The protein pellets were washed three times with 0.2 ml absolute ethanol, centrifuging between washes. The protein pellets were redissolved in 0.2 ml 0.36 N NaOH, transferred to 1 ml microreaction vessels (Supelco, Bellefonte, PA) washing the microfuge tubes with a further 0.1 ml 0.36 N NaOH. To the 0.3 ml 0.36 N NaOH protein samples was added 0.3 ml concentrated (= 12 M) HCl; the microreaction vessels were capped and heated at 120°C for 16 h in a heating block. The hydrolyzed protein samples were concentrated to dryness under a stream of compressed, dry air and redissolved in 0.5 ml H2O, 250 nmol pipecolic acid was added as internal standard, and the samples were applied to 1 x 2 cm columns of Dowex-50 H +. After washing the columns with 6 ml H2O, amino acids were eluted with 6 ml 6 M NH40H, concentrated to dryness, and derivatized for gas chromatography analysis exactly as described for free amino acids (27). Protein contents were determined from the sum of the amino acids recovered in the protein hydrolysates. RESULTS AND DISCUSSION Initial growth studies indicated that AOA was inhibitory to the growth of L. minor at concentrations of 10-5 M and above, when 5 mm KNO3 was used as sole nitrogen source (Table I). At 10-4 M, AOA produced >99% growth inhibition (Table I), and this concentration was chosen for subsequent studies of the effects of AOA on amino acid metabolism. Changes in Free Amino Acid Pools Induced by AOA. Several striking changes in amino acid metabolism of nitrate-grown L. minor were induced by 10 -4 M AOA (Table II). These responses elicited by AOA included rapid depletion of aspartate and serine and pronounced accumulations of amino acids such as,B-alanine. valine, threonine, alanine, glycine, leucine, isoleucine, proline, phenylalanine, lysine, and tyrosine (Table II). The ratios of glycine:serine and glutamate:aspartate increased dramatically (>10fold) in response to AOA (Table II). These responses appear consistent with AOA inhibition of glycine decarboxylase (30, 37) and glutamate:oxaloacetate aminotransferase (9). Presumably, serine and aspartate utilization can continue in the presence of AOA. This would simply require the occurrence of active pathways of serine and aspartate utilization which are not transaminase dependent (e.g. serine decarboxylation to ethanolamine (4, 18) and aspartate conversion to asparagine via asparaginesynthetase [20, 23]). The accumulations of alanine, valine, leucine, and isoleucine induced by AOA (Table II) would not be intuitively expected from AOA inhibition of transaminases (e.g. alanine-, valine-, and leucine-transaminases [9, 23]). The most logical explanation for these amino acid accumulations is that they do not involve de novo synthesis, but rather originate from protein turnover (6-8, 26). Presumably, AOA would inhibit the reverse transamination of amino acids such as alanine, valine, leucine, and isoleucine (derived from protein hydrolysis) to glutamate (or other amino acids). Because serine and aspartate are the only amino acids to deplete in response to AOA (Table II), it seems likely that these are the only amino acids released from protein 448 BRUNK AND RHODES www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 1988 American Society of Plant Biologists. All rights reserved. AMINO ACID METABOLISM OF LEMNA MINOR Table I. Growth of L. minor in the Presence of Different Concentrations ofAOA