Nitrogen metabolism in Lignifying Pinus taeda cell cultures.

The primary metabolic fate of phenylalanine, following its deamination in plants, is conscription of its carbon skeleton for lignin, suberin, flavonoid, and related metabolite formation. Since this accounts for 30-40% of all organic carbon, an effective means of recycling the liberated ammonium ion must be operative. In order to establish how this occurs, the uptake and metabolism of various N-labeled precursors (N-Phe, NHCl, N-Gln, and N-Glu) in lignifying Pinus taeda cell cultures was investigated, using a combination of high performance liquid chromatography, N NMR, and gas chromatography-mass spectrometry analyses. It was found that the ammonium ion released during active phenylpropanoid metabolism was not made available for general amino acid/protein synthesis. Rather it was rapidly recycled back to regenerate phenylalanine, thereby providing an effective means of maintaining active phenylpropanoid metabolism with no additional nitrogen requirement. These results strongly suggest that, in lignifying cells, ammonium ion reassimilation is tightly compartmentalized.

The successful colonization of land by vascular plants, from their aquatic forerunners, was in large measure due to elaboration of the phenylpropanoid/phenylpropanoid-acetate pathways. At this critical juncture in evolution, phenylalanine (tyrosine) became the portal entry of phenols into lignins, lignans, flavonoids, suberins, and proanthocyanidins. Vascular plants thus have a very high Phe/Tyr turnover, since ϳ30 -40% of all assimilated carbon in photosynthesis is of phenylpropanoid/ phenylpropanoid-acetate origin (1)(2)(3)(4)(5).
Phenylpropanoid metabolism is not only a feature of normal development, but can also be induced. For example, when loblolly pine (Pinus taeda) cell suspension cultures are exposed to high levels of sucrose (6), there is an induction of lignin synthesis. Curiously, little attention has been paid to the issue of the relationship between phenylpropanoid and nitrogen metabolism (7). This is surprising since there are many indications from physiological studies of significant metabolic relations between nitrogen depletion and the build-up of aromatic compounds, e.g. Lotus pendunculatus produces flavolans under nitrogen-limiting conditions, but not when nitrogen is provided (8).
Scrutiny of the prearomatic pathway, leading to Phe/Tyr, and subsequent phenylpropanoid/phenylpropanoid-acetate metabolism reveals some noteworthy features. First, prephenate accepts an amino group from glutamate via transamination, constituting the point whereby nitrogen is introduced (9 -15). Second, when Phe/Tyr are committed to phenylpropanoid metabolism, rather than to protein or alkaloid synthesis, nitrogen (as the ammonium ion) is immediately removed via the appropriate lyase reaction (16 -18) (Scheme 1). Third, for every mole of cinnamate (p-hydroxycinnamate) formed, an equimolar amount of ammonium ion is generated. Consequently, an efficient means of nitrogen recycling must exist within cells undergoing active phenylpropanoid metabolism, otherwise severe nitrogen deficiency would result. A possible mechanism for recycling is shown in Scheme 2, where the ammonium ion released during lysis is metabolized via glutamine synthetase/ glutamate synthase to generate glutamate (19 -22), thereby permitting arogenate synthesis, and hence completion of the cycle.
The operation of such a phenylpropanoid-nitrogen cycle during lignification was established using actively lignifying P. taeda cell cultures. This was carried out by examining the uptake, metabolism, and product formation of several 15 Nlabeled precursors, in the presence and absence of specific enzyme inhibitors, as described below.

MATERIALS AND METHODS
Plant Materials-Suspension cultures of P. taeda (loblolly pine) were maintained on a modified Brown and Lawrence medium (23) containing 3% sucrose and 2,4-dichlorophenoxyacetic acid (11.3 M) as auxin.
Instrumentation and Chromatography-15 N NMR spectra were recorded at 30.42 MHz on a Bruker AMX 300 spectrometer using a 5-mm diameter broad band frequency probe head employing a Waltz-16 composite pulse sequence. Chemical shifts are quoted relative to the 15 NH 4 ϩ resonance at 0 ppm obtained using 15 NH 4 Cl (100 mM) as an external standard. The resonances in each sample were assigned by comparison of their chemical shifts to authentic standards and published data (24 -32). In order to obtain a good signal/noise ratio, it was necessary to accumulate 3000 -5000 scans.
HPLC was performed on a Waters GC-MS was performed on a Hewlett Packard 5989A GC-MS system operating in the EI-mode. All separations were performed on a 15 m ϫ 0.25-mm (internal diameter) DB-5MS column (0.25-m filter), with helium as a carrier gas at 5 p.s.i. The source temperature was 250°C, and the injector and interface temperatures were at 275°C, with an electron multiplier voltage of 2200 V for all applications. For analysis of the N-DMTBS amino acid samples, the oven temperature program was raised from 175°C (5 min) to 275°C at 10°C/min and then held for 12.5 min. The mass range scanned was 50 -650 atomic mass units for electron ionization studies. The specific isotopic abundance of Phe, Gln, and Glu was determined by plotting extracted ion current profiles and calculating ratios from the following ion clusters: Phe ( 14 N: 15 Biosynthetic routes leading to the formation of the aromatic amino acids, tyrosine (Tyr) and phenylalanine (Phe), from prephenate. SCHEME 2. Proposed scheme for nitrogen recycling in P. taeda during active phenylpropanoid metabolism. Administration and Metabolism of 15 N-Labeled Substrates-In vivo 15 N-labeling was performed by subculturing 7-day old, 2,4-D treated P. taeda cells (2.5-ml packed cell volume in 25 ml of medium) with a sterile 8% sucrose medium supplemented with the individual 15 N-labeled substrates, in the presence or absence of selected inhibitors (35)(36)(37). 15 N-Phe, 15 NH 4 Cl, 15 N-Gln, and 15 N-Glu were administered at a final concentration of 10 mM at t ϭ 0 h. For inhibitor studies, L-AOPP was administered at a final concentration of 0.1 mM at t ϭ 0 h, whereas MSO and AZA were at 5 mM. In each experiment, the cells were incubated at 25°C, over a time course of 24, 48, 72, and 96 h, on a Lab-Line (Melrose Park, IL) model 3520 orbital shaker (105 rpm) under continuous light provided by two fluorescent lights (40-watt, Philips, Cool White, 25-45 mol s Ϫ1 m Ϫ1 ).
Extraction of Amino Acids from Sucrose-treated Suspension Culture Cells-Following each incubation, suspension culture cells were harvested by filtration of medium on Miracloth, washed with distilled H 2 O (50 ml), weighed, frozen (liquid N 2 ), and stored at Ϫ80°C until needed. Frozen cells were ground in a chilled mortar, extracted with cold EtOH (5 ml), with the resulting slurry transferred, by means of two rinses (3 ml of 95% EtOH each) into a conical tube. The resulting suspension was centrifuged for 10 min (2,200 g, 4°C) in a Beckman model TJ-6 centrifuge, the supernatant was decanted, and the pellet was collected, then resuspended in 95% EtOH (3 ml) and centrifuged for 10 min as before (two times). Supernatants were combined and evaporated to dryness under reduced pressure at 30°C to give the crude amino acid extracts.
Preparation of Amino Acid Extracts for 15 N NMR Spectroscopy-The dried amino acid extracts were individually resuspended in distilled H 2 O (5 ml) and extracted with CHCl 3 (5 ml). The resulting aqueous phase from each experiment was vigorously agitated using a vortex mixer, centrifuged for 10 min (2,200 g, 4°C), with the supernatant frozen (liquid N 2 ) and lyophilized. Each dry amino acid sample was dissolved in 0.1 N HCl (1 ml), containing D 2 O (50 l), and subjected to NMR spectroscopic analysis.
Derivatization with Phenylisothiocyanate and MTBSTFA-Each amino acid extract (initially used for 15 N NMR analyses) was next derivatized, using the MTBSTFA (38) and Pico-Tag (39) methods described previously, with the resulting N-DMTBS and phenylisothiocyanate derivatives analyzed by GC-MS and HPLC, respectively.

RESULTS AND DISCUSSION
The apparent metabolic fate of the ammonium ion released during metabolism of 15 N-Phe was examined, under conditions where cells were undergoing active lignin synthesis. Thus, 10 mM 15 N-Phe (99.9 atom % 15 N) was administered to lignifying P. taeda cell cultures, these then being allowed to metabolize over a 4-day period, with cells removed at 24-h intervals for 15 N NMR spectroscopic analyses. At t ϭ 24 h, three clearly resolved signals (data not shown) were observed at ␦ 91.1, 18.3, and 16.7 ppm. These resonances were assigned to the ␦-amide nitrogen of Gln, ␣-nitrogen atoms of Gln and Glu, and the amino group of Phe, respectively, based on chemical shifts of authentic standards and previously published data (24 -32). By 96 h, additional resonances at ␦ 19.9 and 13.2 ppm were also evident (Fig. 1A); these were attributed to alanine and serine as pre-  viously assigned in nitrogen metabolism studies with white spruce (Picea glauca) buds (24). Importantly, no resonances at any stage corresponding to 15 NH 4 ϩ were observed, in accordance with earlier observations, such as with potato (Solanum tuberosum) slices, where it did not reach detectable levels (7) during active phenylpropanoid-glutamine synthetase/glutamate synthase metabolism.
Subsequent GC-MS and HPLC analyses of the extracts confirmed and extended these observations (see Table I). Thus, both isotopic enrichment and total amounts (micrograms/g fresh weight) of each principal metabolite (Gln and Glu) were determined, following incubation of 15 N-Phe (99.9 atom %, 10 mM) with P. taeda cell cultures, for periods up to 96 h. As can be seen, the phenylalanine present in the soluble pool in the cells was ϳ90 atom % 15 N-enriched at all intervals sampled (24,48,72, and 96 h). But its amount decreased from ϳ677 g/gfw (t ϭ 24 h) to ϳ25 g/gfw (t ϭ 96 h) as a result of the utilization of its carbon skeleton for phenylpropanoid metabolism. However, the Gln/Glu pools were enriched by only ϳ43-53 atom % 15 N, with both nitrogens of Gln labeled in relatively equal amount. In contrast to that of Phe pool sizes, the relative amounts (micrograms/gfw) of Gln/Glu dropped by only about 50% over the duration of the 96-h experiment.
To prove unambiguously that the glutamine synthetase/glutamate synthase pathway was assimilating the ammonium ion released during lignification, incubations of 10 mM 15 N-Phe (99.9 atom % 15 N) were repeated, but now in the presence of specific inhibitors of phenylalanine ammonia lyase, glutamine synthetase, and glutamate synthase, respectively. Thus, when incubations were conducted with lignifying P. taeda cell cultures in the presence of 0.1 mM L-AOPP (35), a known PAL inhibitor, the major resonance now observed was that of unmetabolized 15 N-Phe (Fig. 1B). Small resonances were also noted at ␦ 18.3 ppm, suggesting that phenylpropanoid metabolism was not completely inhibited by L-AOPP, in accordance with previous observations (35,40). This was confirmed by quantitative measurements which revealed that the amounts of Phe (ϳ90% 15 N enriched) remained essentially constant (2926 -2105 g/gfw) throughout the 96-h duration of the experiment. Indeed, the employment of L-AOPP resulted in considerable PAL inhibition as evidenced by the 4-to 100-fold increase in Phe levels over the 24 -96-h time frame examined (cf. Table I  Gln (19 -20%) and Glu (22-25%), respectively (Table I). Nevertheless, these results clearly showed that overall PAL inhibition by L-AOPP adversely affected metabolic flux into Gln/Glu. Interestingly, it had little effect upon the pool sizes of each amino acid relative to that observed previously during active lignification. The effects of treating lignifying P. taeda cell cultures with 5 mM MSO (36), a glutamine synthase inhibitor, was investigated. The results in Fig. 1C show only resonances corresponding to 15 N-Phe and 15 NH 4 ϩ at ␦ 16.7 and 0.0 ppm; signals due to either 15 N-Gln, 15 N-Glu, 15 N-Ala, and 15 N-Ser were absent, indicating that their metabolism from 15 N-Phe was now inhibited. Quantification of both isotopic enrichment and total amounts (micrograms/g fresh weight) supported this conclusion (Table I). The levels of 15 N-Phe (ϳ90 atom % 15 N) rapidly decreased from ϳ2492 g/gfw (at 24 h) down to ϳ34 g/gfw within 96 h, due to the action of PAL. However, as suggested by the NH 4 ϩ resonance at 0.0 ppm, no significant incorporation into 15 N-Gln occurred, as confirmed from its low isotopic enrichment (2-3%). Glutamine synthase inhibition had no apparent effect on the relative pool sizes of Glu which again remained comparable to those previously noted, although, by contrast, the Gln levels dropped to being near undetectable. Interestingly, the Glu isolated was ϳ5-10% enriched, perhaps suggesting that a small amount of Glu formation might occur via transamination of Phe, as suggested earlier (41). In summary, this experiment revealed that inhibition of glutamine synthase prevented an effective assimilation of 15 NH 4 ϩ , released during Phe deamination, into either Gln, Glu, or any other amino acid.
The effects of treating lignifying P. taeda cells with 15 N-Phe, in the presence of AZA (37), an inhibitor of glutamate synthase, was also examined. The results, illustrated in Fig. 1D, show that the predominant resonances were due to 15 N-Phe and 15 N-Gln (␦-amide), with only a very small signal at ␦ 18.3 ppm, due to either H 2 N-15 N-Gln, or 15 N-Glu if incomplete inhibition occurred. This interpretation was confirmed by quantification, as shown in Table I; azaserine treatment had little effect upon carbon metabolism into cinnamate, as evidenced by rapid depletion of Phe from ϳ1162 to 70 g/g fresh weight over the 96-h duration. Glutamine levels were now higher (315-120 g/gfw) than previously noted, due to glutamate synthase inhibition, with the 15 N-Gln essentially being only singly labeled (at the ␦-amide-nitrogen); this established that further cycling of the nitrogen through glutamate synthase to ultimately afford the corresponding ( 15 N, 15 N) double-labeled species was greatly reduced. Significantly, the effect of AZA on Glu levels resulted in a 4 -9-fold depletion from previous levels.
Taken together, these results indicated that the primary metabolic fate of the nitrogen of Phe, following its release as ammonium ion during active phenylpropanoid metabolism, was sequential assimilation into glutamine and then glutamate. There was no evidence for ammonium ion assimilation by glutamine dehydrogenase, since in the presence of either MSO or AZA, neither of which inhibits glutamine dehydrogenase, essentially no detectable incorporation of 15 NH 4 ϩ into Glu occurred.
While the above experiments provided convincing evidence for the metabolic sequence 15 N-Phe 3 15 NH 4 ϩ 3 15 N(␦-amide)-Gln 3 15 N-Glu, they did not establish that the ammonium ion, liberated during deamination, was recycled back to Phe during active phenylpropanoid metabolism. Consequently, the fate of exogenously provided 15 NH 4 Cl, 15 N(␦-amide)-Gln, and 15 N-Glu to lignifying P. taeda cell cultures was next investigated, to ascertain whether Phe would accumulate in a 15 N-enriched form. Experiments were carried out in the presence and absence of the PAL inhibitor, L-AOPP, as before.
P. taeda cell cultures were first administered 10 mM 15 NH 4 Cl for 96 h, with samples removed at 24-h intervals and analyzed. As can be seen in Fig. 2A, 15 N NMR spectroscopic analyses of the extracts were devoid of any signal corresponding to 15 N-Phe. Instead, a range of resonances attributed to 15 N-Gln, 15 N-Glu, 15 NH 4 Cl, the ␦ and ,Ј nitrogens of arginine (␦ 66.8 and 53.4 ppm), 15 N-Ser, the ␣-nitrogen of proline (␦ 32.4 ppm), and the side chain amino groups of Lys (⑀), Orn (␦), and ␥-aminobutyric acid at ␦ 11.7 ppm were evident. Thus, when an ammonium source, such as NH 4 Cl, was administered to lignifying P. taeda cell cultures, it was made available to general pools for amino acid/protein synthesis, i.e. its fate differed from that of 15 N-Phe-derived ammonium ion which resulted in the enhancement of 15 N signals only in Glu, Gln, Ala, and Ser. This strongly suggests that the ammonium ion generated during lignin synthesis is tightly compartmentalized, and not made available for general amino acid metabolism/protein synthesis.
Interestingly, as can be seen from Table II, administration of   15 NH 4 Cl (10 mM) to the lignifying P. taeda cell cultures resulted in a rapid 6-fold increase in Gln levels (495 g/gfw) followed by its gradual decline to ϳ98 g/gfw over 96 h, with both nitrogens being enriched (79 -52%). This was expected since an increased availability of NH 4 ϩ stimulates glutamine synthetase, with a concomitant increase in Gln accumulation (42,43). By contrast, both the amount and isotopic enrichment of Glu were only slightly elevated over previous levels (see Table I). As before, the 15 N-Phe pool size was very small, although significantly it was now partially enriched (19 -37% 15 N), thus providing the first hint of evidence in support of the proposed phenylpropanoid-nitrogen cycle.
Incubation of the lignifying P. taeda cell cultures with 15 NH 4 Cl (10 mM), in the presence of L-AOPP (0.1 mM), was next carried out. As can be seen in Fig. 2B and Table II, the most notable feature was the steady growth of 15 N-Phe (201-1301 g/gfw), this ultimately resulting in the dominant signal (at ␦ 16.7 ppm) in the NMR spectrum. Additionally, all three metabolites, Glu (48 -70% 15 N), Phe (51-64% 15 N), and Gln (68 -89% total 15 N) were isotopically enriched. Thus, proof that NH 4 Cl was normally destined for the phenylpropanoid pathway was in hand.
Existence of this phenylpropanoid-nitrogen cycle was proven further by incubating the lignifying cells with 15 N(␦)-Gln (10 mM) and 15 N-Glu (10 mM), respectively, in the presence and absence of L-AOPP (0.1 mM). As noted previously for 15 NH 4 Cl metabolism, administration of 15 N(␦)-Gln (10 mM) did not result in any resonance corresponding to 15 N-Phe at any time points examined. Only signals due to 15 N-Gln and 15 N-Glu were evident by 24 h (data not shown), with prolonged incubation (72-96 h) resulting in additional resonances for 15 N-Ala, 15 N-Ser, and 15 N-Asn (Fig.  3A), respectively. (The 15 N-Asn resonance was not unexpected, since the Gln pool can serve as amino donor to aspartate, which in turn undergoes transamination to give Asn-amino N; hence it is also assumed that the resonance at ␦ 18.3 ppm contains a minor contribution due to Asp(␣) and Asn(␣) (43).) Determination of pool sizes and isotopic enrichment as before verified that Phe concentrations were low (7-13 g/gfw) but enriched (23-28% 15 N), whereas 15 N-Gln and 15 N-Glu levels remained fairly constant (cf. 15 NH 4 Cl metabolism); note also that the isotopic enrichments of both were lower than before, with the 15 N-Gln being predominantly singly labeled.
In a somewhat analogous manner, administration of 15 N-Glu (10 mM) to P. taeda cell cultures, gave amino acid extracts devoid of 15 N-Phe resonances at any of the sampling points, whereas signals due to 15 N-Glu, 15 N-Ala, 15 N-Ser, and 15 N-Pro were readily detected (Fig. 4A). This was further verified by quantification which again revealed a small Phe pool size (6 -10 g/gfw of 42-47% 15 N enrichment) (Table II).
On the other hand, when P. taeda cells were incubated with 15 N(␦)-Gln and 15 N-Glu in the presence of 0.1 mM L-AOPP, resonances due to L-Phe (␦ 16.7 ppm) were now readily evident (Figs. 3B and 4B). This was further proven by quantification which revealed a ϳ100-fold increase in its amount (ϳ1012-1074 g/gfw from 72-96-h duration) with an isotopic enrichment of 29 -75% 15 N, i.e. again confirming the proposed phenylpropanoid-nitrogen cycle.
Resonances due to 15 N-Gln, 15 N-Glu, 15 N-Ala, and 15 N-Ser were also observed, as well as minor signals due to 15 N Pro, Lys, Orn, and ␥-aminobutyric acid. Moreover, although Gln pool sizes were close to those previously noted (Table II), there was a significant increase in the extent of formation of doublelabeled 15 N-Gln. This is presumed to result via limited catabolism of Glu to ammonium ion and 2-oxoglutarate, with subsequent reassimilation by glutamine synthetase to yield 15 N(␣), 15 N(␦)-Gln.

CONCLUDING REMARKS
With the combined use of HPLC, 15 N NMR, and GC-MS, the mechanism for recycling of ammonia during phenylpropanoid metabolism in lignifying P. taeda (loblolly pine) cell cultures was unambiguously established. The use of enzyme inhibitors was necessary to elucidate this mechanism, with metabolite accumulations being consistent with the known mode of action of L-AOPP, MSO, and AZA; namely inhibition of PAL, glutamine synthetase, and glutamate synthase, respectively. The ammonium ion generated during active phenylpropanoid biosynthesis is first incorporated into the ␦-amide of glutamine, followed in turn by the ␣-amino position of glutamate, which then acts as an amino donor for a range of transamination products, including the aromatic amino acids, arogenate, and phenylalanine. This nitrogen cycle explains how optimum use is made of the plant's available nitrogen, so that active phenylpropanoid metabolism and lignification can continue, even at low nitrogen levels.