©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Nitrogen Metabolism in Lignifying Pinus taeda Cell Cultures (*)

(Received for publication, January 11, 1996)

Pieter S. van Heerden (§) G. H. Neil Towers(¶)(**) Norman G. Lewis (**)

From the Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUDING REMARKS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, NH(4)Cl, 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.


INTRODUCTION

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 30-40% 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 developm ent, 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, 10, 11, 12, 13, 14, 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, 17, 18) (Fig. S1). 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 Fig. S2, where the ammonium ion released during lysis is metabolized via glutamine synthetase/glutamate synthase to generate glutamate(19, 20, 21, 22) , thereby permitting arogenate synthesis, and hence completion of the cycle.


Scheme 1: 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.



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 N-labeled 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 µM) as auxin.

Reagents

All chemicals were either reagent or tissue culture grade unless otherwise stated, whereas HPLC (^1)solvents were of liquid chromatographic grade. L-[N]Phenylalanine, L-Phe (99% N), NH(4)Cl (99.9% N), L-[N]glutamic acid, L-Glu (99% N), and L-[N]glutamine, L-Gln (amide N, 99% N) were purchased from Isotec, Inc. (Miamisburg, OH). Pico-Tag diluent was obtained from Waters (Milford, MA). Phenylisothiocyanate, triethylamine, L-methionine-S-sulfoximine (MSO), azaserine (AZA), and pyridine were purchased from Sigma, while L-2-aminooxy-3-phenylpropanoic acid (L-AOPP) was obtained from Genosys Biotechnologies, Inc. (Woodlands, TX). Anhydrous sodium acetate was purchased from J. T. Baker (Phillipsburg, NJ), while N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) and acetic acid were obtained from Aldrich.

Instrumentation and Chromatography

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 NH(4) resonance at 0 ppm obtained using 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, 25, 26, 27, 28, 29, 30, 31, 32) . In order to obtain a good signal/noise ratio, it was necessary to accumulate 3000-5000 scans.

HPLC was performed on a Waters model 600E system controller, fitted with a Waters Ultra Wisp model 717 sample processor, and a photodiode array detector (Waters model 996) equipped with a NEC Power Mate 2 personal computer. HPLC separations were carried out using a Pico-Tag column (3.9 times 300 mm), with detection at 254 nm, using the procedure of Hagen et al.(33) . The eluent system consisted of two solvent systems: eluent 1, 975 ml of 0.07 N sodium acetate, titrated to pH 6.5 with glacial acetic acid, and mixed with 25 ml of acetonitrile; and eluent 2, 450 ml of acetonitrile, 150 ml of methanol, and 400 ml of Milli-Q water measured separately and then mixed. Elution was carried out in several steps at a flow rate of 1 ml min as follows: 100% eluent 1 to 13.5 min; 13.5 min, step to eluents 1:2 (97:3); 13.5-24 min, concave curve (Waters no. 8) to eluents 1:2 (94:6); 24-30 min, convex curve (Waters no. 5) to eluents 1:2 (91:9); 30-50 min, linear change to eluents 1:2 (66:34); 50-62 min, and held at this condition; 62-62.5 min, linear change to 100% eluent 2; 62.5-66.5 min, held at 100% eluent 2; 66.5-67 min, linear change to 100% eluent 1; 67-87 min, held at 100% eluent 1. The total run time between injections including column reequilibration was 87 min. Quantitation of the Phe, Gln, and Glu pools in the different amino acid samples was made from calibration curves obtained from derivatizing and analyzing known amounts of each amino acid.

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 times 0.25-mm (internal diameter) DB-5MS column (0.25-µm 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 (^14N:N, 336:337); Gln (^14N^14N: ^14NN:NN, 431:432:433); and Glu (^14N:N, 432:433). The following equations for specific isotopic calculations (isotope = I) were used to substract the contribution of natural abundance N, C, ^2H, O, ^18O, Si, and Si as follows (34) . Phe-2TBDMS: (M, m/z 393); [M - C(4)H(9) (m/z 57)] = CH^14NO(2)Si(2) (m/z 336)/CHNO(2)Si(2) (m/z 337); ^14N = I; N = I -{17 times 0.011 (C) + 30 times 0.00015 (^2H) + 0.0037 (N) + 2 times 0.0004 (O) + 2 times 0.051 (S)} I = I - 0.298 I. Gln-3TBDMS: (M, m/z 488); [M - C(4)H(9) (m/z 57)] = CH^14N(2)O(3)Si(3) (m/z 431)/CH^14NNO(3)Si(3) (m/z 432)/CHN(2)O(3)Si(3) (m/z 433; ^14N(2) = I; ^14NN = I - {19 times 0.011 (C) + 43 times 0.00015 (^2H) + 2 times 0.0037 (N) + 3 times 0.0004 (O) + 3 times 0.051 (Si)} I = I - 0.377 I; N(2) = I - 0.373 ^14NN - {0.021(C(2)) + 3 times 0.002 (^18O) + 3 times 0.034 (Si) + 0.0078 (Si(2))} I = I - 0.373 ^14NN - 0.137 I. Glu-3TBDMS: (Mm/z 489; [M - C(4)H(9) (m/z 57)] = CH^14NO(4)Si(3) (m/z 432)/CHNO(4)Si(3) (m/z 433; ^14N = I; N = I - {19 times 0.011 (C) + 42 times 0.00015 (^2H) + 0.0037 (N) + 4 times 0.0004 (O) + 3 times 0.051 (Si)} I = I - 0.374 I.

Administration and Metabolism of N-Labeled Substrates

In vivoN-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 N-labeled substrates, in the presence or absence of selected inhibitors(35, 36, 37) . N-Phe, NH(4)Cl, N-Gln, and N-Glu were administered at a final concentration of 10 mM at t = 0 h. For inhibitor studies, L-AOPP was administered at a final concentration of 0.1 mM at t = 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 µmol sm).

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 -80 °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 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 µl), and subjected to NMR spectroscopic analysis.

Derivatization with Phenylisothiocyanate and MTBSTFA

Each amino acid extract (initially used for 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 N-Phe was examined, under conditions where cells were undergoing active lignin synthesis. Thus, 10 mMN-Phe (99.9 atom % 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 N NMR spectroscopic analyses. At t = 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, alpha-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, 25, 26, 27, 28, 29, 30, 31, 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 previously assigned in nitrogen metabolism studies with white spruce (Picea glauca) buds(24) . Importantly, no resonances at any stage corresponding to NH(4) 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.


Figure 1: Representative N-NMR spectra of P. taeda cellular amino acid extracts obtained after administration of A, 10 mMN-Phe (96 h); B, 10 mMN-Phe and 0.1 mML-AOPP (96 h); C, 10 mMN-Phe and 5 mM MSO (72 h); and D, 10 mMN-Phe and 5 mM AZA (72 h).



Subsequent GC-MS and HPLC analyses of the extracts confirmed and extended these observations (see Table 1). Thus, both isotopic enrichment and total amounts (micrograms/g fresh weight) of each principal metabolite (Gln and Glu) were determined, following incubation of 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 90 atom % N-enriched at all intervals sampled (24, 48, 72, and 96 h). But its amount decreased from 677 µg/gfw (t = 24 h) to 25 µg/gfw (t = 96 h) as a result of the utilization of its carbon skeleton for phenylpropanoid metabolism. However, the Gln/Glu pools were enriched by only 43-53 atom % 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 mMN-Phe (99.9 atom % 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 mML-AOPP(35) , a known PAL inhibitor, the major resonance now observed was that of unmetabolized 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 (90% N enriched) remained essentially constant (2926-2105 µg/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 1, Phe with and without L-AOPP). But PAL was not inhibited fully, as revealed by the small isotopic enhancement of both Gln (19-20%) and Glu (22-25%), respectively (Table 1). 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 N-Phe and NH(4) at 16.7 and 0.0 ppm; signals due to either N-Gln, N-Glu, N-Ala, and N-Ser were absent, indicating that their metabolism from N-Phe was now inhibited. Quantification of both isotopic enrichment and total amounts (micrograms/g fresh weight) supported this conclusion (Table 1). The levels of N-Phe (90 atom % N) rapidly decreased from 2492 µg/gfw (at 24 h) down to 34 µg/gfw within 96 h, due to the action of PAL. However, as suggested by the NH(4) resonance at 0.0 ppm, no significant incorporation into 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 5-10% 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 NH(4), released during Phe deamination, into either Gln, Glu, or any other amino acid.

The effects of treating lignifying P. taeda cells with 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 N-Phe and N-Gln (-amide), with only a very small signal at 18.3 ppm, due to either H(2)N-N-Gln, or N-Glu if incomplete inhibition occurred. This interpretation was confirmed by quantification, as shown in Table 1; azaserine treatment had little effect upon carbon metabolism into cinnamate, as evidenced by rapid depletion of Phe from 1162 to 70 µg/g fresh weight over the 96-h duration. Glutamine levels were now higher (315-120 µg/gfw) than previously noted, due to glutamate synthase inhibition, with the 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 (N,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 NH(4) into Glu occurred.

While the above experiments provided convincing evidence for the metabolic sequence N-Phe NH(4) N(-amide)-Gln 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 NH(4)Cl, N(-amide)-Gln, and N-Glu to lignifying P. taeda cell cultures was next investigated, to ascertain whether Phe would accumulate in a 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 mMNH(4)Cl for 96 h, with samples removed at 24-h intervals and analyzed. As can be seen in Fig. 2A, N NMR spectroscopic analyses of the extracts were devoid of any signal corresponding to N-Phe. Instead, a range of resonances attributed to N-Gln, N-Glu, NH(4)Cl, the and ,` nitrogens of arginine ( 66.8 and 53.4 ppm), N-Ser, the alpha-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 N-Phe-derived ammonium ion which resulted in the enhancement of 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.


Figure 2: Representative N-NMR spectra of P. taeda cellular amino acid extracts obtained after administration of A, 10 mMNH(4)Cl (72 h); and B, 10 mMNH(4)Cl in the presence of 0.1 mML-AOPP (96 h).



Interestingly, as can be seen from Table 2, administration of NH(4)Cl (10 mM) to the lignifying P. taeda cell cultures resulted in a rapid 6-fold increase in Gln levels (495 µg/gfw) followed by its gradual decline to 98 µg/gfw over 96 h, with both nitrogens being enriched (79-52%). This was expected since an increased availability of NH(4) 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 1). As before, the N-Phe pool size was very small, although significantly it was now partially enriched (19-37% 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 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 2, the most notable feature was the steady growth of N-Phe (201-1301 µg/gfw), this ultimately resulting in the dominant signal (at 16.7 ppm) in the NMR spectrum. Additionally, all three metabolites, Glu (48-70% N), Phe (51-64% N), and Gln (68-89% total 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 N()-Gln (10 mM) and N-Glu (10 mM), respectively, in the presence and absence of L-AOPP (0.1 mM). As noted previously for NH(4)Cl metabolism, administration of N()-Gln (10 mM) did not result in any resonance corresponding to N-Phe at any time points examined. Only signals due to N-Gln and N-Glu were evident by 24 h (data not shown), with prolonged incubation (72-96 h) resulting in additional resonances for N-Ala, N-Ser, and N-Asn (Fig. 3A), respectively. (The 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(alpha) and Asn(alpha) (43) .)


Figure 3: Representative N-NMR spectra of P. taeda cellular amino acid extracts obtained after administration of A, 10 mMN-Gln (96 h); and B, 10 mMN-Gln in the presence of 0.1 mML-AOPP (96 h).



Determination of pool sizes and isotopic enrichment as before verified that Phe concentrations were low (7-13 µg/gfw) but enriched (23-28% N), whereas N-Gln and N-Glu levels remained fairly constant (cf.NH(4)Cl metabolism); note also that the isotopic enrichments of both were lower than before, with the N-Gln being predominantly singly labeled.

In a somewhat analogous manner, administration of N-Glu (10 mM) to P. taeda cell cultures, gave amino acid extracts devoid of N-Phe resonances at any of the sampling points, whereas signals due to N-Glu, N-Ala, N-Ser, and N-Pro were readily detected (Fig. 4A). This was further verified by quantification which again revealed a small Phe pool size (6-10 µg/gfw of 42-47% N enrichment) (Table 2).


Figure 4: Representative N-NMR spectra of P. taeda cellular amino acid extracts obtained after administration of A, 10 mM N-Glu (72 h); and B, 10 mMN-Glu in the presence of 0.1 mML-AOPP (96 h).



On the other hand, when P. taeda cells were incubated with N()-Gln and N-Glu in the presence of 0.1 mML-AOPP, resonances due to L-Phe ( 16.7 ppm) were now readily evident (Fig. 3B and 4B). This was further proven by quantification which revealed a 100-fold increase in its amount (1012-1074 µg/gfw from 72-96-h duration) with an isotopic enrichment of 29-75% N, i.e. again confirming the proposed phenylpropanoid-nitrogen cycle.

Resonances due to N-Gln, N-Glu, N-Ala, and N-Ser were also observed, as well as minor signals due to N Pro, Lys, Orn, and -aminobutyric acid. Moreover, although Gln pool sizes were close to those previously noted (Table 2), there was a significant increase in the extent of formation of double-labeled 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 N(alpha),N()-Gln.


CONCLUDING REMARKS

With the combined use of HPLC, 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 alpha-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.


FOOTNOTES

*
This work was supported in part by United States Department of Energy Grant DE-FG06-91ER20022, National Aeronautic and Space Administration Grant NAG10-0164), and the Arthur M. and Katie E. Tode Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Chemistry, University of the Orange Free State, P. O. Box 339, Bloemfontein, 9300, South Africa.

Present address: Dept. of Botany, University of British Columbia, BC, V6T 1Z4, Canada.

**
Authors to whom correspondence should be addressed. Tel.: 509-335-2682; Fax: 509-335-7643.

(^1)
The abbreviations used are: HPLC, high performance liquid chromatography; MSO, L-methionine-S-sulfoximine; AZA, azaserine; L-AOPP, L-2-aminooxy-3-phenylpropanoic acid; MTBSTFA, N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide; GC-MS, gas chromatography-mass spectroscopy; PAL, phenylalanine ammonia lyase; gfw, gram fresh weight; N-DMTBS, N(O)-dimethyl-tert-butylsilyl; TBDMS, tert-butyldimethylsilyl.


ACKNOWLEDGEMENTS

Thanks are extended to Dr. W. F. Siems for the GC-MS determinations, and to K. Sikes for maintenance of the P. taeda cell cultures.


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