Lignin and Lignan Biosynthesis - American Chemical Society

Chapter 3

Integrating Nitrogen and Phenylpropanoid Metabolic Pathways in Plants and Fungi Downloaded by STANFORD UNIV GREEN LIBR on September 28, 2012 | http://pubs.acs.org Publication Date: August 13, 1998 | doi: 10.1021/bk-1998-0697.ch003

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G. H. N. Towers , S. Singh , P. S. van Heerden , J. Zuiches , and Norman G. Lewis 1

Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340 2

Phenylpropanoid and phenylpropanoid-acetate pathways are major metabolic sinks for assimilated organic carbon in vascular plants e.g. affording lignins, lignans, flavonoids, suberins, coumarins, and related compounds. Together, they contribute to about 30-40% of all organic plant matter. Their formation was a critical juncture in land plant adaptation, and was achieved by elaboration of complex biochemical pathways initiated by metabolism of the aromatic amino acids, phenylalanine and to a lesser extent tyrosine. Certain fungi, especially wood-decaying basidiomycetes, such as Lentinus lepideus, also biosynthesize phenylpropanoid derivatives in large amounts. The initial step in phenylpropanoid metabolism is catalyzed by phenylalanine and tyrosine ammonia lyases to afford the corresponding cinnamic acid derivatives and an equimolar amount of ammonium ion. In our investigations, N-specifically labeled substrates, i.e. L-[ N]phenylalanine, NH C1, L-[ N]glutamic acid and L-[ N]glutamine were administered to potato and sweet potato tuber disks, loblolly pine cell suspension cultures and L. lepideus mycelia, respectively. Analyses of the resulting amino acid extracts by both N N M R spectroscopy and G C - M S established that the ammonium ion released during active phenylpropanoid metabolism was recycled back for L-phenylalanine regeneration. The significance of this nitrogen cycling process for the evolution and adaptation of plants and fungi to land is discussed. 15

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During evolutionary adaptation from an aquatic to a land-based habitat, plants had to contend with, among other things, different environmental effects resulting from: direct U V - B irradiation, alterations in gravitational load perception and response, metabolism in a desiccating habitat and numerous challenges/encroachments by other organisms leading to a myriad of defense-related responses (7). This successful adaptation to land was achieved primarily by the elaboration of new biochemical pathways leading to the formation of hitherto unknown, so-called secondary, metabolites derived from either phenylalanine, or less frequently, tyrosine (2). The 42

©1998 American Chemical Society

In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

Downloaded by STANFORD UNIV GREEN LIBR on September 28, 2012 | http://pubs.acs.org Publication Date: August 13, 1998 | doi: 10.1021/bk-1998-0697.ch003

43 entry point into these pathways was catalyzed by the action of phenylalanine ammonia lyase (PAL), and to a lesser extent, tyrosine ammonia lyase (TAL), i.e. to give cinnamic and /7-coumaric acids, respectively. In terrestrial plants, of course, these cinnamic acids are precursors of a fantastic array of metabolic products, such as flavonoids, suberins, lignins/lignans and thousands of other phenolics. Indeed, at least 30-40% of the dry weight of all vascular plant material is derived via phenylpropanoid/phenylpropanoid-acetate metabolism. By contrast P A L has not yet been detected in algae with one possible exception (Dunaliella) (3). Some bacteria also employ P A L for the biosynthesis of the photoreceptor, photoactive yellow protein (PYP) (4), and various fungi have the ability to either constitutively or inducibly activate P A L (5). In vascular plants, the flavonoid skeleton is derived from condensation of /7-coumaryl CoA with three malonyl CoA units. Some of the resulting metabolites strongly absorb U V - B (320-400 nm) irradiation and frequently accumulate in epidermal and subepidermal cells of leaves, fruits and stems (6). Consequently, they are considered to be able to protect such organs from U V - B induced damage. Moreover, formation of related metabolites, such as the anthocyanins, can also be induced in response to light, this being a commonly observed phenomenon with roots or developing organs such as young leaves (7). While beyond the scope of this particular chapter, numerous flavonoid-derived substances have crucial roles in plant defense (8), pigmentation (9) and growth/development (10). Additionally, nitrogen fixation during Rhizobium-legumt interactions (77) is also in large part dependent upon the employment of flavonoid signaling molecules, and the transcriptional activation of genes involved in flavonoid biosynthesis is a well-known response of seedlings and many plants (12-14). Vascular plants produce suberin, a complex biopolymeric matrix which is primarily deposited in periderm tissues (2,15-17). For example, a critical function of the barks of trees, as well as in many other types of plants, is to prevent uncontrolled desiccation and water loss by the whole organism and is achieved via formation of suberized layers. Suberized tissue formation is also often implicated in defense, since its structural matrix affords a relatively impenetrable barrier to opportunistic pathogens. The aromatic domain of suberin in potato (Solarium tuberosum) tuber wound-healing suberized tissue was shown to be primarily composed of hydroxycinnamate-derived entities, such as feruloyl tyramine and /7-coumaryl tyramine, although it also apparently contains low levels of monolignol-derived substances. The principal products of phenylpropanoid metabolism, in terms of overall carbon deployment are, however, the polymeric lignins, which account for 20-30% of all vascular plant material (18). They are derived mainly from hydroxycinnamyl alcohols (monolignols), although smaller amounts of hydroxycinnamates are also apparently involved in lignin deposition in grasses as well (18). Closely related metabolites, such as the lignans (2, 79, 20) have various roles such as in plant defense (79, 27), in helping impart color, texture and durability to certain heartwood tissues (22), as antioxidants (23), and so forth (see chapters 22 and 25). Based on these representative examples of phenylpropanoid/phenylpropanoid acetate metabolites and their functions, it should be evident that L-phenylalanine is involved in many metabolic branchpoint pathways, some of which are illustrated in Figure 1. However, both Phe and Tyr are only present in low concentrations in plant cells being actively metabolized via four types of reactions. That is, they are: predominantly converted into £-cinnamate and Zs-/7-coumarate-derived substances through the action of P A L and T A L with concomitant release of ammonium ion (24, 25); incorporated into proteins; decarboxylated and incorporated into aromatic alkaloids (26), as well as undergoing transamination followed by decarboxylation leading to the corresponding phenethyl alcohols, aldehydes and acids which are the precursors of a vast array of phytochemicals (27). As shown in Scheme 1, in the pre-aromatic pathway leading to the formation of Phe and Tyr, prephenate is transaminated via glutamate to yield arogenate (28),


44 Esters Benzoates Proteins Zs-Cinnamate

Lignins, lignans, suberins, flavonoids, coumarins, styrylpyrones, stilbenes, flavanolignans, etc.


L-Phenylalanine . Phenylacetate

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Figure 1. General metabolic fate of phenylalanine in vascular plants.

Lignins Lignans Flavonoids B e n z o i c acids Suberins Coumarins

Scheme 1. Proposed scheme for nitrogen recycling during phenylpropanoid metabolism. Numbers indicate enzymes: 1, shikimatechorismate pathway enzymes; 2, chorismate mutase; 3, prephenate:glutamate aminotransferase; 4, arogenate dehydrogenase; 5, arogenate dehydratase; 6, phenylalanine ammonia lyase (PAL); 7, glutamine synthetase; 8, glutamine 2-oxo-glutarate aminotransferase (GOGAT). (Reproduced with permission from ref. 28.) (Copyright 1986 National Academy of Science.)


45 which in turn can then be either dehydrated to yield L-Phe or decarboxylated/ dehydrated to give L-Tyr (28, 29) in the chloroplast. In the deamination reactions catalyzed by P A L and T A L (Scheme 1), which have no cofactor requirements, the pro-S proton from C-3 of these amino acids is abstracted in an antiperiplanar fashion relative to the (leaving) amino group functionality to generate £ -(fra«5)-cinnamate and £-p-coumarate (24). Although many crude enzyme preparations from plant and fungal sources display tyrosine ammonia lyase (TAL) activity (30), tyrosine is generally speaking not a major source of phenylpropanoid-derived compounds in higher plants and hence is not discussed further. With an equimolar amount of ammonium ion being generated for every phenyl propanoid skeleton formed, clearly there must be a major demand for nitrogen unless some effective nitrogen recycling process is in effect in tissues and cells undergoing active phenylpropanoid metabolism. Recognition that such a mechanism for nitrogen recycling must be in effect has long been recognized (31); however, the actual biochemical processes involved, whereby ammonium ion is assimilated via the glutamine synthase (GS)/glutamine 2-oxoglutarate aminotransferase (GOGAT) system, to generate the amino donor, L-Glu, for regenerating L-Phe has only recently been established (32, 33).


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Results and Discussion Four experimental systems, namely tuber disks of potato (Solarium tuberosum) and sweet potato (Ipomoea batatas), loblolly pine (Pinus taeda) cell suspension cultures and the mycelium of a wood-decaying fungus, Lentinus lepideus, were selected for study. These were chosen since each displays not only highly active phenylpropanoid metabolism under our experimental conditions, but they also engender metabolism into different branches of the pathway. The results obtained for each are described individually below, and are then compared and contrasted. Nitrogen Recycling during Phenylpropanoid Metabolism in Potato Tuber Disks. Wounding and light are known to induce phenylpropanoid metabolism in potato tuber disks. In particular, they engender de novo synthesis of P A L (34), active biosynthesis of chlorogenic acid (35) and other phenolic metabolites, as well as formation of suberized tissues (15-17). It was, therefore, instructive to examine the metabolic fate of the ammonium ion released during induction of the phenylpropanoid pathway in this species. Accordingly, aseptic potato disks were incubated with 25 |LlM L-[ N]phenylalanine for 24 h, following which the resulting amino acid enriched extract was isolated and subjected to N N M R spectroscopic analysis. As can be seen in Figure 2A, only a single enriched N resonance at 90.8 ppm was observed, this presumably corresponding to the amide nitrogen of Gin. Its formation was rationalized as resulting from the assimilation of N H , released during P A L catalysis, and its conversion into N-Glu (amide) by the action of glutamine synthase (GS) (32). This metabolic sequence of events was further confirmed by incubation of potato disks with L-[ N]Phe in the presence of 5 m M methionine-S-sulphoximine (MSO), a known glutamine synthase (GS) inhibitor (36). Under these conditions, the amino acid enriched extract so obtained only displayed resonances corresponding to L-Phe and N H as shown in Figure 2B. These results suggested that GS/GOGAT effectively assimilated any released N H from the P A L reaction. A comparable metabolic fate of N H was observed when potato disks were next incubated with 20 m M NH C1. Following a 24 h period incubation, and isolation of the amino acid enriched extract as before, its N N M R spectrum revealed a resonance at 90.8 ppm again corresponding to L-Gln (amide) (Figure 3A) (32). As before, the addition of MSO inhibited N H assimilation (Figure 3B). Taken together, these results indicated that ammonium ion, either supplied as NH C1 or liberated from L-[ N]Phe during P A L catalysis, was actively assimilated via the GS/GOGAT pathway, thereby providing the amino acid donor for regeneration of arogenate, and 15

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46 hence completion of the cycle. It is also worth mentioning that in these studies, no significant involvement of glutamate dehydrogenase (GDH) was noted. Nitrogen Recycling during Phenylpropanoid Metabolism in Sweet Potato Tuber Disks. As for potato, both wounding and light exposure of aseptic sweet potato tuber disks enhanced P A L activity and chlorogenic acid accumulation. For example, after 24 h metabolism, the then freshly sliced tuber disks exposed to light now had a 5-fold higher P A L activity than those incubated in the dark (37). Accordingly, a study of nitrogen metabolism in this biological system was next carried out. Incubation of aseptic sweet potato tuber disks with 20 m M L-[ N]phenylalanine for 24 h ultimately gave an amino acid enriched extract, which was subjected to spectroscopic analysis as before. The N N M R spectrum so obtained exhibited resonances corresponding to L-Gln (amide), L-Glu, L - A l a and residual L-phenylalanine, respectively (37). This again suggested that the GS/GOGAT pathway was involved in assimilation of the ammonium ion released during phenylpropanoid metabolism. However, in order to prove unambiguously that this metabolic pathway was in effect, comparable experiments were undertaken but now in the presence of specific inhibitors of GS, GOGAT and P A L , respectively. In this context, addition of MSO (2.5 mM), a glutamine synthase inhibitor (38), to sweet potato tuber disks, which had been administered 2 mM L-phenylalanine as before, now gave an amino acid enriched extract whose N N M R spectrum was devoid of resonances due to N-Gln-(amide-N), N-Glu or N-Ala. By contrast, only signals corresponding to N H (0 ppm) and residual L-[ N]Phe (18.4 ppm) were observed (37). Experiments were also carried out using the specific G O G A T inhibitor, azaserine (AZA) (0.2 mM) (39), and resulted in an amino acid enriched extract whose N N M R spectrum gave N resonances, which only corresponded to L-Gln (90.8 ppm) and residual L-[ N]Phe, i.e. there was no metabolism into either N-Glu or N Ala. Additionally, when N-Phe was administered to aseptic sweet potato tuber disks, but now in the presence of the potent P A L inhibitor, 2-aminoinelan-2phosphonic acid (AIP) (0.05 mM) (40), only a resonance corresponding to Phe was observed, i.e. its deamination was inhibited. Thus, as for potato, these observations clearly suggest the involvement of a GS-GOGAT system for reassimilation of P A L generated N H in sweet potato tuber disks. (As an aside, the incorporation of N into Ala from L-[ N]phenylalanine could be interpreted as suggesting that there may be an active transaminase involving Phe and pyruvate in these disks, or that pyruvate can directly assimilate the N H released during the P A L reaction. However, since there is no transfer of N nitrogen to Ala, when the P A L reaction is blocked, there is no evidence for either process occurring. Consequently, it is apparently formed via further metabolism of L-Glu.)


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In order to ascertain if L-Glu served as nitrogen donor to the aromatic amino acids, i.e. arogenate, Phe and Tyr, it was important to next examine the metabolic fate of N-Glu. Incubation of sweet potato disks with 20 m M L-[ N]Glu for 24 h were next carried out, and only resulted in resonances in the amino acid extract corresponding to Ala and residual Glu. This indicated that transamination reactions were also occurring which afforded Ala during amino acid metabolism in this tissue. To prove that the proposed nitrogen cycle, via GS/GOGAT to arogenate/phenylalanine and tyrosine, was operative, the effect of the P A L inhibitor, AIP, was again examined. In this regard, addition of 0.05 m M AIP (40) to the metabolizing sweet potato tuber disks, ultimately gave an amino acid enriched extract, whose N N M R spectrum revealed a new resonance corresponding to Phe (18.4 ppm). This again showed that Glu acts as an amino donor either directly, or indirectly through arogenate, to permit the continued biosynthesis of Phe in sweet potato tuber disks. Comparable results were obtained when the disks were incubated with 20 m M NH C1. After 24 h, isolation and analysis of the resulting amino acid extract revealed that Gin (amide), Glu and Ala were N-enriched (37). Addition of 2.5 m M M S O as before, however, completely inhibited the assimilation of N H into the above-mentioned amino acids, and only a resonance corresponding to N H was 15

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Figure 2. N N M R spectra of amino acid extracts prepared from potato disks incubated for 24 h with (A) L-[ N]Phe and (B) L-[ N]Phe in the presence of MSO. (Reproduced with permission from ref. 28.) (Copyright 1986 National Academy of Science.)

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Figure 3. N N M R spectra of extracts prepared from potato disks incubated for 24 h with (A) NH C1 and (B) NH C1 in presence of MSO. (Reproduced with permission from ref. 28.) (Copyright 1986 National Academy of Science.)

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51 evident. These observations are again in agreement with the hypothesis that the GS/GOGAT enzyme system is involved in reassimilation of N H in sweet potato disks, and thereby enables active phenylpropanoid metabolism to continue. +

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Nitrogen Recycling during Phenylpropanoid Metabolism in Loblolly Pine Cells. Phenylpropanoid metabolism in loblolly pine (Pinus taeda) cell suspension cultures can be actively induced by subculturing in solutions containing high levels of sucrose (33, 41, 42), i.e. to give both lignified cell walls and an 'extracellular lignin precipitate' (see chapter 25). It was, therefore, next of interest to establish the metabolic fate of 10 m M L-[ N]Phe. Thus, following a 24 h period, the resulting amino acid enriched extract so obtained was subjected to N N M R spectroscopic analysis, and revealed N resonances corresponding to the amide group of Gin, the a amino groups of Gin and Glu and the amino group of Phe (33), respectively. On the other hand, when metabolism was extended to 96 h, resonances corresponding to Ala and Ser were also evident. Quantitative measurements of both pool size and isotopic enrichment of these amino acids were also carried out at different metabolic intervals and again clearly implicated the involvement of the GS/GOGAT pathway in the assimilation of P A L generated N H . Moreover, when experiments were next conducted in the presence of the known P A L inhibitor, 0.1 mM L-aminooxy phenylpropionic acid (AOPP) (43), the dominant resonance observed was that of unmetabolized L-[ N]Phe together with a very small signal presumably due to Gin and Glu amino groups, i.e. P A L was substantially inhibited by addition of the known inhibitor, L - A O P P . When experiments were next carried out using the GS inhibitor, MSO (5 mM), this resulted in only L-[ N]Phe and N H resonances being observed, i.e. no incorporation of N into Gin, Glu, Ala and Ser was noted (33). Additionally, the employment of A Z A (5 mM), an inhibitor of G O G A T (39), substantially inhibited incorporation of the ammonium ion from N-Phe into the a amino group of Glu. Taken together, these results, using specific inhibitors, indicate that the primary metabolic fate of the nitrogen of N-Phe is via its assimilation into Gin and Glu by the action of GS/GOGAT. Again, no evidence for N H assimilation by G D H was observed. In order to establish the further channeling of nitrogen from Glu back to Phe to complete the cycle, it was necessary to establish the metabolic fate of N H , L-[ N]Gln and L-[ N]Glu. Initial experiments were conducted using NH C1, whose metabolism over a 96 h time period gave a N N M R spectrum of its amino acid extract revealing resonances attributable to Gin, Glu, Ala, Arg, Pro, Lys, Orn, y-aminobutyric acid (GABA) and N H . This suggested that the N H derived from NH C1 was available for utilization in the various general pools used for amino acid and protein biosynthesis. On the other hand, the N H ion released from Phe during phenylpropanoid metabolism was only incorporated into Gin, Glu, Ala and Ser, i.e. it was not apparently made available for general amino acid/protein synthesis. Additionally, incubation of loblolly pine cell cultures with 10 m M NH C1 as before, but now in the presence of 0.1 m M of L-AOPP (43), gave only a resonance corresponding to N-Phe. Similarly, when L-[ N]Gln or L - p N ] G l u were incubated with the induced P. taeda cell cultures, in the presence of L-AOPP (0.1 mM), only accumulation of N-Phe was observed. (This was further established by quantitation of the amino acid pool sizes by HPLC and GC-MS isotopic enrichment.) Taken together, these results again prove that the ammonium ion released is transferred to L-Glu, which then functions as the amino donor to form Phe. In summary, employing N-substrates with and without specific inhibitors of P A L , GS and G O G A T , and with subsequent analysis of the resulting metabolic products by HPLC, N N M R spectroscopy and GC-MS, resulted in the discovery of a novel mechanism for the recycling of nitrogen during active phenylpropanoid metabolism in loblolly pine cell cultures. The ammonium ion released is first assimilated by the GS/GOGAT pathway resulting in the synthesis of L-Glu, which then serves as the amino donor to Phe. The efficient recycling of N H , at low nitrogen levels, would allow for lignin formation, as well as that of other 15


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phenylpropanoid products, to continue under conditions where there is limited nitrogen availability in either the whole plant or in cell cultures. Nitrogen Recycling during Phenylpropanoid Metabolism in the Basidiomycete, Lentinus lepideus. Lentinus lepideus is a wood-decaying basidiomycete which produces a 'brown' rot of wood. It is often described as a 'woody' bracket fungus, but does not produce the lignins present in higher plants. Nevertheless, it displays high P A L activity and it biosynthesizes significant amounts of methylated phenolic acids in culture medium, e.g. methyl /7-methoxy cinnamate (44). Since it grows on wood which is a poor nitrogen substrate, it was anticipated that some mechanism for the conservation of nitrogen should also be in place in the rapidly growing mycelium. To investigate this possibility, four-day old mycelium was incubated with N labeled substrates, in an analogous manner to that described for the plant specimens. Accordingly, in a time course study of L-[ N]Phe metabolism, it was noted that after 2 h, the first amino acid to be detected, labeled with N , was Gin (amide-N). After 12 h, additional N resonances corresponding to Glu, Ala, residual Phe as well as an unidentified resonance at 91.19 ppm were also evident. Interestingly, following 24 and 48 h of incubation with L-[ N]Phe, an additional resonance attributed to y-aminobutyric acid (GABA) was also observed. Specific inhibitors of P A L and GS were next used to investigate whether nitrogen recycling was continuing as before. Thus, addition of MSO (GS inhibitor) to the L-[ N]Phe-treated mycelia completely inhibited incorporation into Gin and Glu, although resonances attributed to G A B A , Ala and Phe were evident. Similar results were observed when AIP (PAL inhibitor) was used. Therefore, it appears that in Lentinus, apart from the GS/GOGAT pathway operating, additional mechanisms for ammonium assimilation may also be in effect. 1 5

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Concluding Remarks In summary, our results with potato and sweet potato root tuber disks, loblolly pine cells and Lentinus lepideus mycelia demonstrate that PAL-generated ammonium ion is actively assimilated via G S / G O G A T metabolism. The L-Glu formed as a consequence of GS/GOGAT activity appears to serve as a nitrogen donor for aromatic amino acid biosynthesis, leading to arogenate, Phe and Tyr. However, in the basidiomycete, L. lepideus, ammonium ion produced from Phe is apparently assimilated by GS/GOGAT pathway, as well as by additional mechanism(s) which need to be further clarified. No evidence supporting a role of G D H in the reassimilation of PAL-liberated ammonium ion in any of these species was found. A general mechanism for recycling the liberated ammonium ion back to Phe for further phenylpropanoid metabolism in plant and fungal species is illustrated in Scheme 1. This efficient nitrogen recycling mechanism explains why plants and fungi do not experience any obvious symptoms of nitrogen limitation under conditions of active phenylpropanoid metabolism. Experimental L. lepideus was subcultured on solid potato dextrose agar and then maintained on liquid culture medium containing (per L): dextrose (20 g), yeast extract (5 g), peptone (2 g), M g S 0 . 7 H 0 (0.5 g), K H P 0 (0.45 g) and K H P 0 (0.5 g). Maintenance of suspension cultures of loblolly pine (33, 41, 42), potato (32, 45) and sweet potato tuber (37) slices were as previously reported. The general procedure for studying the fate of the N H ion released from the P A L reaction was as follows: N-labeled substrates, e.g. L-phenylalanine, L-glutamic acid, L-glutamine (amide N) or NH C1 were administered to cells or tissues under aseptic conditions. In certain experiments synthetic inhibitors of P A L , (AIP, 2-aminoindan-2-phosphonic acid and AOPP, L-aminooxy phenylpropionic acid), GS 4

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53 (MSO, methionine-S-sulphoximine) and G O G A T ( A Z A , azaserine) were administered along with N-labeled substrates (32, 33, 37). Immediately after each incubation metabolism period, the corresponding plant or fungal material was rinsed with distilled water and frozen (liq. N ). The amino acid enriched extracts from the soluble pool were extracted with ethanol and evaporated under reduced pressure at 30°C. The corresponding dried amino acid extracts were then resuspended in 5 ml of distilled water and extracted with an equal volume of CHC1 . The aqueous phase so obtained was next centrifuged for 10 min (9,000 rpm, 4°C), with the resulting supernatants frozen (liq. N ) and lyophilized. The lyophilized amino acid samples were dissolved in 0.1 N HC1 (1 ml), containing D 0 (50 jil), and subjected to analysis by N-nuclear magnetic resonance ( N NMR) spectroscopy (28, 29). N N M R spectroscopic measurements were performed on a Bruker A M X 500 spectrometer operating at 50.68 M H z at 298K, with broad band coupling, employing a Waltz-16 composite pulse sequence. Chemical shifts relative to the N H resonance at 0 ppm were obtained using NH C1 (250 mM) as external standard. Assignment of resonances in each sample was made by comparison with authentic N-labeled amino acids. The amino acid extracts for GC-MS analysis were prepared by the method similar to that described above for the N M R spectroscopic analyses. Amino acid extracts were derivatized, using the M T B S T F A reagent (46) with the resulting N-DMTBS derivatives of amino acids in the extracts analyzed by GC-MS (32, 33). GC-MS was performed on a Hewlett Packard 5989A GC-MS system operating in the El-mode. The specific isotopic abundances of various amino acids (e.g. Phe, Gin and Glu) were determined by plotting ion current profiles and calculating ratios of the N : N from the selected ion clusters as described elsewhere (33). The total pools of amino acids in loblolly pine cell extracts were determined after derivatization with phenylisothiocyanate using the pico-tag method and analyzed by HPLC (45). 15

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Acknowledgments We thank the U.S. Department of Energy (DE-FG06-91ER20022), the National Aeronautic and Space Administration (NAG 100164) and the Arthur M . and Katie Eisig-Tode Foundation (to P. S. van Heerden) for generous support of this research. Literature Cited 1. Lewis, N . G.; Davin, L . B . In Isoterpenoids and Other Natural Products: Evolution and Function; Nes, W. D., Ed.; ACS Symposium Series; Washington, DC, 1994, Vol. 562, pp 202-246. 2. Davin, L . B.; Lewis, N . G. In Recent Advances in Phytochemistry; Stafford, H . A., Ibrahim, R. K., Eds.; Plenum Press: New York, N Y , 1992; pp 325-375. 3. Löffelhardt, W.; Lugwig. B.; Kindl, H.; Hoppe-Seylers Z. Physiol. Chem. 1973, 354, 1006-1012. 4. Hoff, W. D.; Dux, P.; Hard, K.; Devreese, Nugeren-Roodzant, I. M.; Crielaard, W.; Boelens, R.; Kaptein, R.; Van Beeuman, J.; Hellingwerf, K . J. Biochemistry 1994, 33, 13959-13962. 5. Sikora, L. A.; Marzluf, G.A. J. Bacteriol. 1982, 150, 1287-1291. 6. Strack, D.; Wray, V . In The Flavonoids. Advances in Research since 1986; Harborne, J. B., Ed.; Chapman and Hall: London, 1993; pp 1-22. 7. Toguri, T.; Umemoto, N . ; Kabayashi, O.; Ohtani, T. Plant Mol. Biol. 1993, 23, 933-946. 8. Lamb, C. J.; Lawton, M . A.; Dron, M . ; Dixon, R. A. Cell, 1989, 56, 215-224. 9. Brouillard, R.; Dangles, O. In The Flavonoids. Advances in Research since 1986; Harborne, J. B., Ed.; Chapman and Hall: London, 1993; pp 565-588. 10. Vogt, T.; Pollack, P.; Taglyn, N.; Taylor, L. P. Plant Cell, 1994, 6, 11-23. In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


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