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Multi-Site Modulation of Flux during Monolignol Formation in Loblolly Pine (Pinus taeda)
Biochemical and Biophysical Research Communications 261, 652– 657 (1999) Article ID bbrc.1999.1097, available online at http://www.idealibrary.com on
Multi-Site Modulation of Flux during Monolignol Formation in Loblolly Pine (Pinus taeda) Aldwin M. Anterola, Hendrik van Rensburg, Pieter S. van Heerden, Laurence B. Davin, and Norman G. Lewis 1 Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
Received April 29, 1999
Loblolly pine (Pinus taeda L.) cell suspension cultures secrete monolignols when placed in 8% sucrose/20 mM KI solution, and these were used to identify phenylpropanoid pathway flux-modulating steps. When cells were provided with increasing amounts of either phenylalanine (Phe) or cinnamic acid, cellular concentrations of immediate downstream products (cinnamic and p-coumaric acids, respectively) increased, whereas caffeic and ferulic acid pool sizes were essentially unaffected. Increasing Phe concentrations resulted in increased amounts of p-coumaryl alcohol relative to coniferyl alcohol. However, exogenously supplied cinnamic, p-coumaric, caffeic, and ferulic acids resulted only in increases in their intercellular concentrations, but not that of downstream cinnamyl aldehydes and monolignols. Supplying p-coumaryl and coniferyl aldehydes up to 40,000 –320,000-fold above the detection limits resulted in rapid, quantitative conversion into the monolignols. Only at nonphysiological concentrations was transient accumulation of intracellular aldehydes observed. These results indicate that cinnamic and p-coumaric acid hydroxylations assume important regulatory positions in phenylpropanoid metabolism, whereas cinnamyl aldehyde reduction does not serve as a control point. © 1999 Academic Press
genase in tobacco (Nicotiana tabacum) (3), poplar (Populus tremula X Populus alba) (4) and loblolly pine (Pinus taeda) (5). However, the effects of reducing CAD levels (70 –93%) to reduce lignin contents in tobacco, poplar, and loblolly pine through antisense strategies or mutants had little effect on overall lignin deposition. Lignins of gymnosperms, such as loblolly pine, are derived from the monolignols, p-coumaryl and coniferyl alcohols. These monolignols are formed from phenylalanine (Phe) via a series of enzyme catalyzed deamination, hydroxylation, O-methylation, CoA ligation and reductive reactions, which together constitute the phenylpropanoid pathway (Scheme 1). Interestingly, every one of these enzymatic steps has been proposed as having a “key” or regulatory role in monolignol biosynthesis, but without any explicit biochemical data ever provided to demonstrate or support the rate-limiting capacity of any particular step(s). It was therefore the purpose of this study to determine the effects of administering various metabolic precursors to monolignol-forming cells in order to identify fluxmodulating steps in the phenylpropanoid pathway (6). MATERIALS AND METHODS Cell culture. The maintenance of P. taeda cell suspension cultures has been described elsewhere (7).
Lignins are aromatic heterobiopolymers primarily formed in the secondary xylem cell walls of vascular plants. They provide structural support to stems, hydrophobicity to conducting vessels and an effective barrier against pathogen attack (1). Various research efforts around the world have been directed towards modifying lignin (content and monomer composition) in order to obtain woody and/or forage plants that are either more amenable for pulp/paper production or more digestible as animal fodder, respectively (2). Several of these approaches have targeted presumed “key” steps in the pathway, e.g., cinnamyl alcohol dehydro1
Corresponding author. Fax: 509-335-8206. E-mail: [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Chemicals. Phe, cinnamic, p-coumaric, caffeic, ferulic and sinapic acids were purchased from Sigma. 5-Hydroxyferulic acid was synthesized according to (8). CoA esters were gifts from R. E. Kneusel and U. Matern. Cinnamyl, p-coumaryl, 3,4-dihydroxycinnamyl, coniferyl and sinapyl alcohols were obtained via reduction of the methyl esters of their corresponding (hydroxy)cinnamic acids (9). Monolignol glucosides were prepared as described previously (10), whereas the synthesis of p-coumaryl, 3,4-dihydroxycinnamyl, coniferyl and sinapyl aldehydes was carried out as described in (11). Induction of monolignol biosynthesis. P. taeda cell suspension cultures, grown for 7 days in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), were transferred to an autoclaved solution of 8% sucrose and 20 mM KI (1 part cells to 10 parts medium by volume, 25 ml total volume) and incubated at 25°C on a platform shaker set at 115 rpm under continuous light (30-40 mmol s 21 m 22) (7). Cells were filtered after 2, 4, 6, 9, 12, 16, 20 and 24 h of incubation, rinsed with H 2O, freeze-dried and stored at 280°C until
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SCHEME 1. The phenylpropanoid pathway in Pinus taeda affording the monolignols, p-coumaryl and coniferyl alcohols. (a) Phenylalanine ammonia lyase; (b) cinnamate-4-hydroxylase; (c) p-coumarate-3-hydroxylase; (c9) p-coumaroyl CoA 3-hydroxylase; (d) O-methyltransferase; (e) 4-coumarate:CoA ligase; (f) cinnamoyl CoA reductase; (g) cinnamyl alcohol dehydrogenase. (For the purpose of this paper only, steps c– e are currently viewed as being interchangeable.)
needed. The filtrates were directly analyzed by HPLC as described below (HPLC analyses). Administration of monolignol precursors. Phe, cinnamic, p-coumaric, caffeic and ferulic acids, and p-coumaryl and coniferyl aldehydes were individually dissolved in 8% sucrose/20 mM KI solutions to give the following final concentrations: 5, 10, 20, 30, 40, 60 and 80 mM Phe, 0.2, 0.4, 0.8, 1.2, 1.6 and 2.0 mM (hydroxy)cinnamic acids, and 0.4, 0.8, 1.2, 1.6, 2.4, 3.2 and 4.0 mM cinnamyl aldehydes, respectively. Each was then individually used as a bathing medium (25 ml) for the P. taeda suspension cells, under the conditions described above. Cells were harvested at different time points as before, then freeze-dried and stored, with the filtrates subjected to HPLC analysis as described below. For illustrative purposes, only the results of a few representative concentrations are described (see Results and Discussion). Extraction and analysis of intracellular phenylpropanoid metabolites (general procedures). P. taeda cells (50 mg dry wt.) were ground in 0.5 M phosphate buffer (pH 7.0, 1.5 ml), centrifuged at 16,000 g for 10 min with the resulting supernatant (0.75 ml) extracted with ethyl acetate (2 3 0.75 ml). The ethyl acetate solubles were combined, dried in vacuo and reconstituted in 50% aqueous methanol (0.30 ml). The resulting solution was analyzed by HPLC. HPLC and LC-MS analyses. HPLC analyses of plant extracts, and reference phenylpropenoic acids, aldehydes, alcohols, glucosides and cinnamoyl CoA esters were carried out using a Waters Millennium system equipped with a C 18 reversed phase column (Novapak, 3.9 3 300 mm). The eluted compounds were detected at 280 and 254 nm using a Waters 996 photodiode array detector, with confirmatory LC-MS analysis being performed on a Waters Integrity System equipped with a Thermabeam Mass Detector. Using a linear gradient from 3 to 95% acetonitrile in 3% acetic acid in H 2O (reached in 60 min at a flow rate of 1 ml min 21), the various acids, aldehydes, alcohols and glucosides were separated with the following retention volumes (ml): p-coumaryl alcohol glucoside, 18.5; 3,4-dihydroxycinnamyl alcohol, 21.8; coniferyl alcohol glucoside, 24.1; sinapyl alcohol glucoside, 27.2; caffeic acid, 28.8; p-coumaryl alcohol, 30.9; 5-hydroxyferulic acid, 32.1; 3,4-dihydroxycinnamyl aldehyde, 33.3;
coniferyl alcohol, 35.1; sinapyl alcohol, 37.0; p-coumaric acid, 37.8; ferulic acid, 41.6; p-coumaryl aldehyde, 42.2; sinapic acid, 42.8; coniferyl aldehyde, 46.0; sinapyl aldehyde, 47.5; cinnamyl alcohol, 55.5; cinnamic acid, 58.3; and cinnamyl aldehyde, 59.1. The CoA esters, on the other hand, were separated by elution at 1 ml min 21 through the same column using a linear gradient of acetonitrile in 0.1 M KH 2PO 4 (pH 5.3) from 10 to 40% (within 75 min), with the following retention volumes (in ml): caffeoyl CoA, 34.3; 5-hydroxyferuloyl CoA, 36.0; p-coumaroyl CoA, 46.1; feruloyl CoA, 51.5; sinapoyl CoA, 54.8; and cinnamoyl CoA, 68.9, respectively.
RESULTS AND DISCUSSION Induction of monolignol biosynthesis. Cell suspension cultures of loblolly pine (P. taeda), subcultured every 7 days in growth media containing 3% sucrose and 11 mM 2,4-dichlorophenoxyacetic acid, neither produce lignin nor undergo highly active phenylpropanoid metabolism (7). Nor do the cells form secondary walls to any measurable extent, but instead continuously divide with a doubling time of total cell volume of 3 to 4 days. On the other hand, when transferred to a solution of 8% sucrose, the phenylpropanoid pathway is massively induced. Over a 120 h period, the P. taeda cell walls undergo secondary thickening and apparently start to lignify (12). In addition, a cloudy “ligninlike” precipitate accumulates in the bathing solution after ca. 96 h (12). These extracellular metabolites are not lignins proper, and may perhaps more closely resemble formation of the biochemically distinct lignanderived oligomeric substances which accumulate, for example, in pine heartwood tissues (13). In the presence of 20 mM KI (H 2O 2 scavenger), however, both cell wall lignification and formation of the extracellular
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treatment, cellular pool sizes of phenylpropanoid metabolites and the amounts of various substances accumulating in the medium (during the 24 h incubation period) were determined by HPLC and LC/MS analyses. The results are shown in Figs. 1 to 6.
TABLE 1
Representative Pool Sizes of Phenylpropanoid Metabolites in Monolignol-Forming Pinus taeda Cells Metabolite
Amount in 150 mg dry cells (nmol)
Cinnamic Acid p-Coumaric Acid Caffeic Acid Ferulic Acid p-Coumaroyl CoA Caffeoyl CoA Feruloyl CoA p-Coumaryl Aldehyde Coniferyl Aldehyde p-Coumaryl Alcohol Coniferyl Alcohol
100.00 30.00 25.00 7.00 !2.50* !2.50* !2.50* !0.25* !0.25* 30.00 30.00
Note. Pinus taeda cells were incubated in 8% sucrose/20 mM KI for 12 h and then analyzed as described under Materials and Methods. * 5 below detection limits.
precipitate are inhibited and, instead, the monolignols, p-coumaryl and coniferyl alcohols, are secreted into the bathing medium (12). Determination of pool sizes of phenylpropanoid metabolites. All known and potential metabolic intermediates from Phe onward to the monolignols in the phenylpropanoid pathway were obtained as described under Materials and Methods. Next, conditions were identified for the separation of all possible (hydroxy)cinnamic acids, cinnamoyl CoA esters, cinnamyl aldehydes, cinnamyl alcohols, and monolignol glucosides using C 18 reversed phase HPLC. These conditions were then adopted in order to measure the amounts of each possible metabolite accumulating in the cell bathing medium and in the cellular extracts, respectively, which had been obtained from P. taeda cell suspension cultures treated with 8% sucrose and 20 mM KI. Structural confirmation of each metabolite was achieved by LC/MS analysis. Representative metabolic pool sizes of each of the phenylpropanoid compounds observed 12 h following induction of the pathway are shown in Table 1. Only the acids and the monolignols accumulated significantly in the induced cells. The CoA esters and aldehydes, on the other hand, were below the detection limits (2.5 and 0.25 nmol, respectively) of the methods employed. Identification of flux modulating points in the pathway. In order to identify which steps in the pathway might be critical for modulating overall flux and/or monolignol ratios, various precursors (Phe, phenylpropenoic acids [i.e., cinnamic, p-coumaric, caffeic and ferulic acids], as well as feruloyl CoA, p-coumaryl and coniferyl aldehydes) were individually added at different concentrations to P. taeda cells exposed to a solution of 8% sucrose containing 20 mM KI. For each
Effect of exogenously supplied Phe on P. taeda metabolism. P. taeda cells, when placed in 8% sucrose/20 mM KI, excrete monolignols into the medium which only become detectable within ca. 12 h (Fig. 1). From then onwards, p-coumaryl and coniferyl alcohol levels gradually increase from ;0.3 to 0.7 mmol (Fig. 1C, lower trace) and from ;1 to 5 mmol (Fig. 1D, lower trace), respectively, over a 24 h period. However, when Phe was exogenously supplied in amounts ranging from 125 to 1000 mmol (in 25 ml), this resulted in a differential increase in the amounts of monolignols in the bathing solution. Specifically, addition of 125 mmol Phe increased p-coumaryl and coniferyl alcohol levels within 12 and 9 h to 8 and 9 mmol, respectively (Fig. 1C and 1D, middle trace), while addition of 1000 mmol Phe resulted in 18 mmol p-coumaryl and 15 mmol coniferyl alcohols accumulating in the medium (Fig. 1C and 1D, upper trace). Addition of higher concentrations of Phe had no effect on further increasing monolignol levels, indicating that maximum production levels were achieved for these conditions. Significantly, however, increasing the levels (and concentration) of exogenously provided Phe (125 and 1000 mmol), results in differential increases in p-coumaryl and coniferyl alcohol contents of ;26 and ;3 fold within 24 h, respectively. This is a particularly interesting observation given that the lignins in compression wood of gymnosperms have higher p-coumaryl alcohol contents relative to that of “normal” wood (14). This change in monolignol composition might therefore result from an
FIG. 1. Representative effect of exogenously provided phenylalanine on amount of monolignols accumulating in P. taeda cell suspension cultures. Accumulation of intracellular (A) p-coumaryl and (B) coniferyl alcohols when incubated with differing amounts of Phe (Œ: 0 mmol, F: 125 mmol, and ■: 1000 mmol Phe), together with that of extracellular (C) p-coumaryl and (D) coniferyl alcohols. For structures see Scheme 1.
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FIG. 2. Representative effect of exogenously provided phenylalanine on intracellular levels of (hydroxy)cinnamic acids in P. taeda cell cultures. (A) cinnamic; (B) p-coumaric; (C) caffeic; and (D) ferulic acids. Legend: Œ, 0 mmol; F, 125 mmol; and ■, 1000 mmol Phe. For structures see Scheme 1.
increase in Phe availability, as demonstrated in this study. Analysis of the cellular extracts also revealed that increasing amounts of exogenously supplied Phe had no measurable effect on intracellular coniferyl alcohol levels, but, on the other hand, p-coumaryl alcohol content increased 4 fold (Fig. 1A and 1B). Additionally, increasing the availability of Phe resulted in significant increases in the cellular pool sizes of both cinnamic and p-coumaric acids. That is, under conditions whereby maximum monolignol formation occurred (16 h), the levels of cinnamic acid rose by as much as 16-fold from 0.05 mmol to 0.8 mmol within this time period and then plateaued (Fig. 2A), whereas p-coumaric acid levels increased ;10 fold from 0.02 mmol to 0.2 mmol (Fig. 2B). The accumulation of these metabolites is contrary, however, to what is expected if Phe ammonia-lyase (PAL) is truly controlling flux into the pathway, as has been frequently suggested (15, 16). Indeed, the very low metabolic pool sizes of Phe in actively lignifying cells (17) might suggest that the deamination of Phe to form cinnamic acid is not ratelimiting. Additionally, extracellular caffeic and ferulic acid levels were not significantly increased, staying at ca. 0.03 and 0.007 mmol, respectively (Fig. 2C and 2D), regardless of the amount of Phe available. Moreover, under none of the conditions examined were either cinnamoyl CoA esters or cinnamyl aldehydes detected in the cells (the analytical protocol employed has detection limits of 2.5 and 0.25 nmol, respectively). Accordingly, these data suggest that the regulatory point(s) in the phenylpropanoid pathway, while under various conditions of Phe availability, are situated prior to O-methylation, CoA ester and aldehyde reduction steps.
Effect of exogenously provided (hydroxy)cinnamic acids on P. taeda metabolism. When exogenously supplied (hydroxy)cinnamic acids were next provided at different concentrations to P. taeda cells, no measurable changes in the amounts of monolignols in the bathing medium were noted, under any of the conditions employed. Examination of the intracellular constituents, on the other hand, revealed that each exogenously supplied metabolite accumulated to high levels inside the cells. For example, cinnamic acid contents increased to 0.6 and 5 mmol (from 0.03 mmol basal level) within 2 h, when 5 and 40 mmol cinnamic acid were administered to P. taeda cells (Fig. 3A), representing ;20 and ;150 fold increases, respectively. As can be seen, however, cinnamic acid underwent further metabolism dropping to near basal levels within 12 h. Concomitant with this decline in cinnamic acid content is the formation of cinnamic acid glucose esters [as indicated from mass spectral analysis under atmospheric pressure chemical ionization (APCI) conditions, data not shown] that accumulate both in the cells and in the bathing medium (data not shown). These data also indicate that cinnamic acid derivatives, if not correctly compartmentalized, are shunted, perhaps via a detoxification mechanism to afford glucose esters, as has been previously noted in French bean (Phaseolus vulgaris) (18). Nevertheless, together with the observed results from Phe metabolism, these data reveal that rate of formation of p-coumaric acid, by the action of cinnamate 4-hydroxylase, limits flux during monolignol biosynthesis. Addition of 40 mmol cinnamic acid also increased intracellular p-coumaric acid levels ;25 fold up to ;0.5 mmol within 6 h, which gradually decreased to ;0.1 mmol at 12 h (Fig. 3B). This, in turn, indicated that the 3-hydroxylation step might also be limiting, particularly since levels of other downstream metabolites
FIG. 3. Representative effect of exogenously provided cinnamic acid on intracellular levels of (hydroxy)cinnamic acids in P. taeda cell cultures. (A) cinnamic; (B) p-coumaric; (C) caffeic; and (D) ferulic acids. Legend: Œ, 0 mmol; F, 5 mmol; and ■, 40 mmol cinnamic acid. For structures see Scheme 1.
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FIG. 4. Representative effect of exogenously provided p-coumaric and ferulic acids on their intracellular levels in P. taeda cell cultures. (A) p-coumaric and (B) ferulic acids. Legend: Œ, 0 mmol; F, 5 mmol; and ■, 40 mmol p-coumaric and ferulic acids, respectively. For structures see Scheme 1.
[e.g., caffeic and ferulic acids (Fig. 3C and 3D)] were not significantly affected. When 40 mmol p-coumaric and ferulic acids were next provided, their intracellular levels increased ;150 and ;450 fold from 0.02 and 0.007 mmol basal levels, respectively, to 3 mmol within 2 h (Fig. 4). However, this had no observable effect on the pool sizes of the rest of the downstream intercellular phenylpropanoid metabolites, except again for both intra- and extracellular accumulation of glucose esters (identified using APCI mass fragmentation patterns, data not shown) of the exogenously supplied hydroxycinnamic acids. Interestingly, exogenously supplied caffeic acid did not accumulate intracellularly to the same extent as the other phenylpropenoic acids, rising only to 0.5 mmol from 0.025 mmol basal level (20 fold increase) in the 24 h incubation period (Fig. 5A), nor did ferulic acid levels increase beyond basal levels (0.007 mmol) (Fig. 5B). Instead, glucose esters of caffeic acid were also rapidly formed and excreted into the bathing medium (data not shown). Given that caffeic acid did not accumulate in any of the other precursor administration experiments, there was no evidence obtained suggesting that the O-methylation of caffeic acid to form ferulic acid is rate-limiting.
FIG. 5. Representative effect of exogenously provided caffeic acid on intracellular levels of (A) caffeic and (B) ferulic acids, respectively, in P. taeda cell cultures. Legend: Œ, 0 mmol; F, 5 mmol; and ■, 40 mmol caffeic acid. For structures see Scheme 1.
decline of the aldehydic precursors. As can be seen, neither p-coumaryl nor coniferyl aldehydes were detectable in the cell bathing medium after 2 h incubation (Fig. 6A and 6B). However, when the supply of exogenously provided p-coumaryl and coniferyl aldehydes was further increased to 30 mmol (representing 140,000 times above the detection limit), a transient accumulation of the intracellular levels of aldehydes to ;0.1 mmol from 2 to 9 h was detected (data not shown), this representing at least 40 times above the detection limit. Within 2 h, however, the amounts of monolignols in the medium increased up to ;30 to 35 mmol (Fig. 6C and 6D, middle traces), while the aldehydes in the medium were again no longer detectable (Fig. 6A and 6B, middle traces). The maximal formation of monolignols accumulating in the cell bathing medium was ;80 to 95 mmol, this being achieved when 80 mmol of each aldehyde was supplied to P. taeda cells (Fig. 6C and 6D, upper traces), and which corresponds to ;100 and ;20 fold increases in p-coumaryl and coniferyl alcohol contents, respectively, relative to their levels in the induced cells
Feruloyl CoA ester administration to P. taeda cells. Feruloyl CoA was also administered to the cells, but it was not effectively taken up by the cells, as revealed by HPLC analysis (data not shown). Effect of exogenously supplied cinnamyl aldehydes to P. taeda metabolism. For each of the sets of experiments described above, the intracellular levels of p-coumaryl and coniferyl aldehydes were below the detection limit (0.25 nmol) of the methods employed. Moreover, they remained undetectable in the cells even when 10 mmol cinnamyl aldehydes were exogenously supplied to P. taeda cells, this representing a 40,000 fold increase above the detection limit (data not shown). On the other hand, the amounts of the corresponding monolignols in the medium rapidly rose to 10-12 mmol within 2 h (Fig. 6C and 6D, lower traces) in an essentially equimolar manner relative to that of the
FIG. 6. Representative effect of exogenously provided cinnamyl aldehydes on extracellular levels of monolignols in P. taeda cell cultures. Time course of depletion of (A) p-coumaryl and (B) coniferyl aldehydes in the extracellular medium. Time course of formation of (C) p-coumaryl and (D) coniferyl alcohols in the extracellular medium. Legend: Œ, 0 mmol; F, 10 mmol; ■, 30 mmol; and }, 80 mmol cinnamyl aldehydes. For structures see Scheme 1.
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(i.e., in 8% sucrose/20 mM KI). Addition of more than 80 mmol of either aldehyde did not increase further the monolignol levels in either the cells or bathing medium. At 80 mmol exogenously provided aldehyde, intracellular p-coumaryl and coniferyl aldehyde levels accumulated up to ;2 and 3 mmol reaching maximum values at ;12 h (data not shown), while 5 and 10 mmol remained in the medium after 24 h, respectively (Fig. 6A and 6B). Thus, an ;320,000 fold increase in the amount of available aldehydes (above detection limit) was required in order to saturate the rate-limiting capacity of P. taeda cinnamyl alcohol dehydrogenase. By comparison, increasing the amount of available cinnamic acid 50 – 400 fold (5– 40 mmol) did not increase the monolignol levels significantly, and this situation also held true for the other phenylpropenoic acids. Thus, compared with cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase, the ability of cinnamyl alcohol dehydrogenase to control the metabolic flux to monolignols is insignificant. Based on these results, the control of flux during monolignol biosynthesis does not rely solely on one enzyme (which is often described as being PAL) but is rather distributed among the other enzymes including cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase. Moreover, the increased availability of Phe significantly influences the ratio of monolignols formed by P. taeda cells, suggesting possible regulatory roles for the enzymes in the preceding pathways (e.g., shikimate pathway) and nitrogen recycling (17). Cinnamyl alcohol dehydrogenase, on the other hand, is not ratelimiting even with P. taeda. Indeed, these data provide a straightforward explanation as to why downregulating cinnamyl alcohol dehydrogenase, by as much as 70 to 93%, had no significant effects on the amount of lignins formed (3–5). ACKNOWLEDGMENTS The authors thank the United States National Aeronautics and Space Administration (NAG100164 and NAG21198), the Arthur M. and Kate Eisig Tode Foundation, as well as the Lewis B. and Dorothy Cullman and G. Thomas Hargrove Center for Land Plant Adaptation
Studies. Thanks are also extended to Dr. Ulrich Matern and R. E. Kneusel for authentic standards of the various CoA esters.
REFERENCES 1. Lewis, N. G., and Yamamoto, E. (1990) Ann. Rev. Plant Phys. Plant Mol. Biol. 41, 455– 496. 2. Boudet, A. M., and Grima-Pettenati, J. (1996) Mol. Breed. 2, 25–39. 3. Halpin, C., Knight, M. E., Foxon, G. A., Campbell, M. M., Boudet, A. M., Boon, J. J., Chabbert, B., Tollier, M.-T., and Schuch, W. (1994) Plant J. 6, 339 –350. 4. Baucher, M., Chabbert, B., Pilate, G., Van Doorsselaere, J., Tollier, M.-T., Petit-Conil, M., Cornu, D., Monties, B., Van Montagu, M., Inze´, D., Jouanin, L., and Boerjan, W. (1996) Plant Physiol. 112, 1479 –1490. 5. Ralph, J., MacKay, J. J., Hatfield, R. D., O’Malley, D. M., Whetten, R. W., and Sederoff, R. R. (1997) Science 277, 235–239. 6. Lewis, N. G. (1998) Presented at the 215th American Chemical Society National Meeting, Dallas, Texas, March 29 –April 2, 1998. 7. Eberhardt, T. L., Bernards, M. A., He, L., Davin, L. B., Wooten, J. B., and Lewis, N. G. (1993) J. Biol. Chem. 268, 21088 –21096. 8. Ohashi, H., Yamamoto, E., Lewis, N. G., and Towers, G. H. N. (1987) Phytochemistry 26, 1915–1916. 9. Morelli, E., Rej, R. N., Just, G., and Lewis, N. G. (1986) Phytochemistry 25, 1701–1705. 10. Lewis, N. G., Inciong, M. A. J., Dhara, K. P., and Yamamoto, E. (1989) J. Chromatogr. 479, 345–352. 11. Freudenberg, K., and Hubner, H. H. (1952) Chem. Ber. 85, 1181– 1191. 12. Nose, M., Bernards, M. A., Furlan, M., Zajicek, J., Eberhardt, T. L., and Lewis, N. G. (1995) Phytochemistry 39, 71–79. 13. Gang, D. R., Fujita, M., Davin, L. B., and Lewis, N. G. (1998) in Lignin and Lignan Biosynthesis (Lewis, N. G., and Sarkanen, S., Eds.), Vol. 697, pp. 389 – 421, ACS Symposium Series, Washington, DC. 14. Timell, T. E. (1986) Compression Wood in Gymnosperms, 3 Vols., Springer-Verlag, Berlin. 15. Jones, D. H. (1984) Phytochemistry 23, 1349 –1359. 16. Bate, N. J., Orr, J., Ni, W., Meromi, A., Nadler-Hassar, T., Doerner, P. W., Dixon, R. A., Lamb, C. J., and Elkind, Y. (1994) Proc. Natl. Acad. Sci. USA 91, 7608 –7612. 17. van Heerden, P. S., Towers, G. H. N., and Lewis, N. G. (1996) J. Biol. Chem. 271, 12350 –12355. 18. Edwards, R., Mavandad, M., and Dixon, R. A. (1990) Phytochemistry 29, 1867–1873.
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Multi-Site Modulation of Flux during Monolignol Formation in Loblolly Pine (Pinus taeda) Aldwin M. Anterola, Hendrik van Rensburg, Pieter S. van Heerden, Laurence B. Davin, and Norman G. Lewis 1 Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
Received April 29, 1999
Loblolly pine (Pinus taeda L.) cell suspension cultures secrete monolignols when placed in 8% sucrose/20 mM KI solution, and these were used to identify phenylpropanoid pathway flux-modulating steps. When cells were provided with increasing amounts of either phenylalanine (Phe) or cinnamic acid, cellular concentrations of immediate downstream products (cinnamic and p-coumaric acids, respectively) increased, whereas caffeic and ferulic acid pool sizes were essentially unaffected. Increasing Phe concentrations resulted in increased amounts of p-coumaryl alcohol relative to coniferyl alcohol. However, exogenously supplied cinnamic, p-coumaric, caffeic, and ferulic acids resulted only in increases in their intercellular concentrations, but not that of downstream cinnamyl aldehydes and monolignols. Supplying p-coumaryl and coniferyl aldehydes up to 40,000 –320,000-fold above the detection limits resulted in rapid, quantitative conversion into the monolignols. Only at nonphysiological concentrations was transient accumulation of intracellular aldehydes observed. These results indicate that cinnamic and p-coumaric acid hydroxylations assume important regulatory positions in phenylpropanoid metabolism, whereas cinnamyl aldehyde reduction does not serve as a control point. © 1999 Academic Press
genase in tobacco (Nicotiana tabacum) (3), poplar (Populus tremula X Populus alba) (4) and loblolly pine (Pinus taeda) (5). However, the effects of reducing CAD levels (70 –93%) to reduce lignin contents in tobacco, poplar, and loblolly pine through antisense strategies or mutants had little effect on overall lignin deposition. Lignins of gymnosperms, such as loblolly pine, are derived from the monolignols, p-coumaryl and coniferyl alcohols. These monolignols are formed from phenylalanine (Phe) via a series of enzyme catalyzed deamination, hydroxylation, O-methylation, CoA ligation and reductive reactions, which together constitute the phenylpropanoid pathway (Scheme 1). Interestingly, every one of these enzymatic steps has been proposed as having a “key” or regulatory role in monolignol biosynthesis, but without any explicit biochemical data ever provided to demonstrate or support the rate-limiting capacity of any particular step(s). It was therefore the purpose of this study to determine the effects of administering various metabolic precursors to monolignol-forming cells in order to identify fluxmodulating steps in the phenylpropanoid pathway (6). MATERIALS AND METHODS Cell culture. The maintenance of P. taeda cell suspension cultures has been described elsewhere (7).
Lignins are aromatic heterobiopolymers primarily formed in the secondary xylem cell walls of vascular plants. They provide structural support to stems, hydrophobicity to conducting vessels and an effective barrier against pathogen attack (1). Various research efforts around the world have been directed towards modifying lignin (content and monomer composition) in order to obtain woody and/or forage plants that are either more amenable for pulp/paper production or more digestible as animal fodder, respectively (2). Several of these approaches have targeted presumed “key” steps in the pathway, e.g., cinnamyl alcohol dehydro1
Corresponding author. Fax: 509-335-8206. E-mail: [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Chemicals. Phe, cinnamic, p-coumaric, caffeic, ferulic and sinapic acids were purchased from Sigma. 5-Hydroxyferulic acid was synthesized according to (8). CoA esters were gifts from R. E. Kneusel and U. Matern. Cinnamyl, p-coumaryl, 3,4-dihydroxycinnamyl, coniferyl and sinapyl alcohols were obtained via reduction of the methyl esters of their corresponding (hydroxy)cinnamic acids (9). Monolignol glucosides were prepared as described previously (10), whereas the synthesis of p-coumaryl, 3,4-dihydroxycinnamyl, coniferyl and sinapyl aldehydes was carried out as described in (11). Induction of monolignol biosynthesis. P. taeda cell suspension cultures, grown for 7 days in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), were transferred to an autoclaved solution of 8% sucrose and 20 mM KI (1 part cells to 10 parts medium by volume, 25 ml total volume) and incubated at 25°C on a platform shaker set at 115 rpm under continuous light (30-40 mmol s 21 m 22) (7). Cells were filtered after 2, 4, 6, 9, 12, 16, 20 and 24 h of incubation, rinsed with H 2O, freeze-dried and stored at 280°C until
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SCHEME 1. The phenylpropanoid pathway in Pinus taeda affording the monolignols, p-coumaryl and coniferyl alcohols. (a) Phenylalanine ammonia lyase; (b) cinnamate-4-hydroxylase; (c) p-coumarate-3-hydroxylase; (c9) p-coumaroyl CoA 3-hydroxylase; (d) O-methyltransferase; (e) 4-coumarate:CoA ligase; (f) cinnamoyl CoA reductase; (g) cinnamyl alcohol dehydrogenase. (For the purpose of this paper only, steps c– e are currently viewed as being interchangeable.)
needed. The filtrates were directly analyzed by HPLC as described below (HPLC analyses). Administration of monolignol precursors. Phe, cinnamic, p-coumaric, caffeic and ferulic acids, and p-coumaryl and coniferyl aldehydes were individually dissolved in 8% sucrose/20 mM KI solutions to give the following final concentrations: 5, 10, 20, 30, 40, 60 and 80 mM Phe, 0.2, 0.4, 0.8, 1.2, 1.6 and 2.0 mM (hydroxy)cinnamic acids, and 0.4, 0.8, 1.2, 1.6, 2.4, 3.2 and 4.0 mM cinnamyl aldehydes, respectively. Each was then individually used as a bathing medium (25 ml) for the P. taeda suspension cells, under the conditions described above. Cells were harvested at different time points as before, then freeze-dried and stored, with the filtrates subjected to HPLC analysis as described below. For illustrative purposes, only the results of a few representative concentrations are described (see Results and Discussion). Extraction and analysis of intracellular phenylpropanoid metabolites (general procedures). P. taeda cells (50 mg dry wt.) were ground in 0.5 M phosphate buffer (pH 7.0, 1.5 ml), centrifuged at 16,000 g for 10 min with the resulting supernatant (0.75 ml) extracted with ethyl acetate (2 3 0.75 ml). The ethyl acetate solubles were combined, dried in vacuo and reconstituted in 50% aqueous methanol (0.30 ml). The resulting solution was analyzed by HPLC. HPLC and LC-MS analyses. HPLC analyses of plant extracts, and reference phenylpropenoic acids, aldehydes, alcohols, glucosides and cinnamoyl CoA esters were carried out using a Waters Millennium system equipped with a C 18 reversed phase column (Novapak, 3.9 3 300 mm). The eluted compounds were detected at 280 and 254 nm using a Waters 996 photodiode array detector, with confirmatory LC-MS analysis being performed on a Waters Integrity System equipped with a Thermabeam Mass Detector. Using a linear gradient from 3 to 95% acetonitrile in 3% acetic acid in H 2O (reached in 60 min at a flow rate of 1 ml min 21), the various acids, aldehydes, alcohols and glucosides were separated with the following retention volumes (ml): p-coumaryl alcohol glucoside, 18.5; 3,4-dihydroxycinnamyl alcohol, 21.8; coniferyl alcohol glucoside, 24.1; sinapyl alcohol glucoside, 27.2; caffeic acid, 28.8; p-coumaryl alcohol, 30.9; 5-hydroxyferulic acid, 32.1; 3,4-dihydroxycinnamyl aldehyde, 33.3;
coniferyl alcohol, 35.1; sinapyl alcohol, 37.0; p-coumaric acid, 37.8; ferulic acid, 41.6; p-coumaryl aldehyde, 42.2; sinapic acid, 42.8; coniferyl aldehyde, 46.0; sinapyl aldehyde, 47.5; cinnamyl alcohol, 55.5; cinnamic acid, 58.3; and cinnamyl aldehyde, 59.1. The CoA esters, on the other hand, were separated by elution at 1 ml min 21 through the same column using a linear gradient of acetonitrile in 0.1 M KH 2PO 4 (pH 5.3) from 10 to 40% (within 75 min), with the following retention volumes (in ml): caffeoyl CoA, 34.3; 5-hydroxyferuloyl CoA, 36.0; p-coumaroyl CoA, 46.1; feruloyl CoA, 51.5; sinapoyl CoA, 54.8; and cinnamoyl CoA, 68.9, respectively.
RESULTS AND DISCUSSION Induction of monolignol biosynthesis. Cell suspension cultures of loblolly pine (P. taeda), subcultured every 7 days in growth media containing 3% sucrose and 11 mM 2,4-dichlorophenoxyacetic acid, neither produce lignin nor undergo highly active phenylpropanoid metabolism (7). Nor do the cells form secondary walls to any measurable extent, but instead continuously divide with a doubling time of total cell volume of 3 to 4 days. On the other hand, when transferred to a solution of 8% sucrose, the phenylpropanoid pathway is massively induced. Over a 120 h period, the P. taeda cell walls undergo secondary thickening and apparently start to lignify (12). In addition, a cloudy “ligninlike” precipitate accumulates in the bathing solution after ca. 96 h (12). These extracellular metabolites are not lignins proper, and may perhaps more closely resemble formation of the biochemically distinct lignanderived oligomeric substances which accumulate, for example, in pine heartwood tissues (13). In the presence of 20 mM KI (H 2O 2 scavenger), however, both cell wall lignification and formation of the extracellular
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treatment, cellular pool sizes of phenylpropanoid metabolites and the amounts of various substances accumulating in the medium (during the 24 h incubation period) were determined by HPLC and LC/MS analyses. The results are shown in Figs. 1 to 6.
TABLE 1
Representative Pool Sizes of Phenylpropanoid Metabolites in Monolignol-Forming Pinus taeda Cells Metabolite
Amount in 150 mg dry cells (nmol)
Cinnamic Acid p-Coumaric Acid Caffeic Acid Ferulic Acid p-Coumaroyl CoA Caffeoyl CoA Feruloyl CoA p-Coumaryl Aldehyde Coniferyl Aldehyde p-Coumaryl Alcohol Coniferyl Alcohol
100.00 30.00 25.00 7.00 !2.50* !2.50* !2.50* !0.25* !0.25* 30.00 30.00
Note. Pinus taeda cells were incubated in 8% sucrose/20 mM KI for 12 h and then analyzed as described under Materials and Methods. * 5 below detection limits.
precipitate are inhibited and, instead, the monolignols, p-coumaryl and coniferyl alcohols, are secreted into the bathing medium (12). Determination of pool sizes of phenylpropanoid metabolites. All known and potential metabolic intermediates from Phe onward to the monolignols in the phenylpropanoid pathway were obtained as described under Materials and Methods. Next, conditions were identified for the separation of all possible (hydroxy)cinnamic acids, cinnamoyl CoA esters, cinnamyl aldehydes, cinnamyl alcohols, and monolignol glucosides using C 18 reversed phase HPLC. These conditions were then adopted in order to measure the amounts of each possible metabolite accumulating in the cell bathing medium and in the cellular extracts, respectively, which had been obtained from P. taeda cell suspension cultures treated with 8% sucrose and 20 mM KI. Structural confirmation of each metabolite was achieved by LC/MS analysis. Representative metabolic pool sizes of each of the phenylpropanoid compounds observed 12 h following induction of the pathway are shown in Table 1. Only the acids and the monolignols accumulated significantly in the induced cells. The CoA esters and aldehydes, on the other hand, were below the detection limits (2.5 and 0.25 nmol, respectively) of the methods employed. Identification of flux modulating points in the pathway. In order to identify which steps in the pathway might be critical for modulating overall flux and/or monolignol ratios, various precursors (Phe, phenylpropenoic acids [i.e., cinnamic, p-coumaric, caffeic and ferulic acids], as well as feruloyl CoA, p-coumaryl and coniferyl aldehydes) were individually added at different concentrations to P. taeda cells exposed to a solution of 8% sucrose containing 20 mM KI. For each
Effect of exogenously supplied Phe on P. taeda metabolism. P. taeda cells, when placed in 8% sucrose/20 mM KI, excrete monolignols into the medium which only become detectable within ca. 12 h (Fig. 1). From then onwards, p-coumaryl and coniferyl alcohol levels gradually increase from ;0.3 to 0.7 mmol (Fig. 1C, lower trace) and from ;1 to 5 mmol (Fig. 1D, lower trace), respectively, over a 24 h period. However, when Phe was exogenously supplied in amounts ranging from 125 to 1000 mmol (in 25 ml), this resulted in a differential increase in the amounts of monolignols in the bathing solution. Specifically, addition of 125 mmol Phe increased p-coumaryl and coniferyl alcohol levels within 12 and 9 h to 8 and 9 mmol, respectively (Fig. 1C and 1D, middle trace), while addition of 1000 mmol Phe resulted in 18 mmol p-coumaryl and 15 mmol coniferyl alcohols accumulating in the medium (Fig. 1C and 1D, upper trace). Addition of higher concentrations of Phe had no effect on further increasing monolignol levels, indicating that maximum production levels were achieved for these conditions. Significantly, however, increasing the levels (and concentration) of exogenously provided Phe (125 and 1000 mmol), results in differential increases in p-coumaryl and coniferyl alcohol contents of ;26 and ;3 fold within 24 h, respectively. This is a particularly interesting observation given that the lignins in compression wood of gymnosperms have higher p-coumaryl alcohol contents relative to that of “normal” wood (14). This change in monolignol composition might therefore result from an
FIG. 1. Representative effect of exogenously provided phenylalanine on amount of monolignols accumulating in P. taeda cell suspension cultures. Accumulation of intracellular (A) p-coumaryl and (B) coniferyl alcohols when incubated with differing amounts of Phe (Œ: 0 mmol, F: 125 mmol, and ■: 1000 mmol Phe), together with that of extracellular (C) p-coumaryl and (D) coniferyl alcohols. For structures see Scheme 1.
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FIG. 2. Representative effect of exogenously provided phenylalanine on intracellular levels of (hydroxy)cinnamic acids in P. taeda cell cultures. (A) cinnamic; (B) p-coumaric; (C) caffeic; and (D) ferulic acids. Legend: Œ, 0 mmol; F, 125 mmol; and ■, 1000 mmol Phe. For structures see Scheme 1.
increase in Phe availability, as demonstrated in this study. Analysis of the cellular extracts also revealed that increasing amounts of exogenously supplied Phe had no measurable effect on intracellular coniferyl alcohol levels, but, on the other hand, p-coumaryl alcohol content increased 4 fold (Fig. 1A and 1B). Additionally, increasing the availability of Phe resulted in significant increases in the cellular pool sizes of both cinnamic and p-coumaric acids. That is, under conditions whereby maximum monolignol formation occurred (16 h), the levels of cinnamic acid rose by as much as 16-fold from 0.05 mmol to 0.8 mmol within this time period and then plateaued (Fig. 2A), whereas p-coumaric acid levels increased ;10 fold from 0.02 mmol to 0.2 mmol (Fig. 2B). The accumulation of these metabolites is contrary, however, to what is expected if Phe ammonia-lyase (PAL) is truly controlling flux into the pathway, as has been frequently suggested (15, 16). Indeed, the very low metabolic pool sizes of Phe in actively lignifying cells (17) might suggest that the deamination of Phe to form cinnamic acid is not ratelimiting. Additionally, extracellular caffeic and ferulic acid levels were not significantly increased, staying at ca. 0.03 and 0.007 mmol, respectively (Fig. 2C and 2D), regardless of the amount of Phe available. Moreover, under none of the conditions examined were either cinnamoyl CoA esters or cinnamyl aldehydes detected in the cells (the analytical protocol employed has detection limits of 2.5 and 0.25 nmol, respectively). Accordingly, these data suggest that the regulatory point(s) in the phenylpropanoid pathway, while under various conditions of Phe availability, are situated prior to O-methylation, CoA ester and aldehyde reduction steps.
Effect of exogenously provided (hydroxy)cinnamic acids on P. taeda metabolism. When exogenously supplied (hydroxy)cinnamic acids were next provided at different concentrations to P. taeda cells, no measurable changes in the amounts of monolignols in the bathing medium were noted, under any of the conditions employed. Examination of the intracellular constituents, on the other hand, revealed that each exogenously supplied metabolite accumulated to high levels inside the cells. For example, cinnamic acid contents increased to 0.6 and 5 mmol (from 0.03 mmol basal level) within 2 h, when 5 and 40 mmol cinnamic acid were administered to P. taeda cells (Fig. 3A), representing ;20 and ;150 fold increases, respectively. As can be seen, however, cinnamic acid underwent further metabolism dropping to near basal levels within 12 h. Concomitant with this decline in cinnamic acid content is the formation of cinnamic acid glucose esters [as indicated from mass spectral analysis under atmospheric pressure chemical ionization (APCI) conditions, data not shown] that accumulate both in the cells and in the bathing medium (data not shown). These data also indicate that cinnamic acid derivatives, if not correctly compartmentalized, are shunted, perhaps via a detoxification mechanism to afford glucose esters, as has been previously noted in French bean (Phaseolus vulgaris) (18). Nevertheless, together with the observed results from Phe metabolism, these data reveal that rate of formation of p-coumaric acid, by the action of cinnamate 4-hydroxylase, limits flux during monolignol biosynthesis. Addition of 40 mmol cinnamic acid also increased intracellular p-coumaric acid levels ;25 fold up to ;0.5 mmol within 6 h, which gradually decreased to ;0.1 mmol at 12 h (Fig. 3B). This, in turn, indicated that the 3-hydroxylation step might also be limiting, particularly since levels of other downstream metabolites
FIG. 3. Representative effect of exogenously provided cinnamic acid on intracellular levels of (hydroxy)cinnamic acids in P. taeda cell cultures. (A) cinnamic; (B) p-coumaric; (C) caffeic; and (D) ferulic acids. Legend: Œ, 0 mmol; F, 5 mmol; and ■, 40 mmol cinnamic acid. For structures see Scheme 1.
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FIG. 4. Representative effect of exogenously provided p-coumaric and ferulic acids on their intracellular levels in P. taeda cell cultures. (A) p-coumaric and (B) ferulic acids. Legend: Œ, 0 mmol; F, 5 mmol; and ■, 40 mmol p-coumaric and ferulic acids, respectively. For structures see Scheme 1.
[e.g., caffeic and ferulic acids (Fig. 3C and 3D)] were not significantly affected. When 40 mmol p-coumaric and ferulic acids were next provided, their intracellular levels increased ;150 and ;450 fold from 0.02 and 0.007 mmol basal levels, respectively, to 3 mmol within 2 h (Fig. 4). However, this had no observable effect on the pool sizes of the rest of the downstream intercellular phenylpropanoid metabolites, except again for both intra- and extracellular accumulation of glucose esters (identified using APCI mass fragmentation patterns, data not shown) of the exogenously supplied hydroxycinnamic acids. Interestingly, exogenously supplied caffeic acid did not accumulate intracellularly to the same extent as the other phenylpropenoic acids, rising only to 0.5 mmol from 0.025 mmol basal level (20 fold increase) in the 24 h incubation period (Fig. 5A), nor did ferulic acid levels increase beyond basal levels (0.007 mmol) (Fig. 5B). Instead, glucose esters of caffeic acid were also rapidly formed and excreted into the bathing medium (data not shown). Given that caffeic acid did not accumulate in any of the other precursor administration experiments, there was no evidence obtained suggesting that the O-methylation of caffeic acid to form ferulic acid is rate-limiting.
FIG. 5. Representative effect of exogenously provided caffeic acid on intracellular levels of (A) caffeic and (B) ferulic acids, respectively, in P. taeda cell cultures. Legend: Œ, 0 mmol; F, 5 mmol; and ■, 40 mmol caffeic acid. For structures see Scheme 1.
decline of the aldehydic precursors. As can be seen, neither p-coumaryl nor coniferyl aldehydes were detectable in the cell bathing medium after 2 h incubation (Fig. 6A and 6B). However, when the supply of exogenously provided p-coumaryl and coniferyl aldehydes was further increased to 30 mmol (representing 140,000 times above the detection limit), a transient accumulation of the intracellular levels of aldehydes to ;0.1 mmol from 2 to 9 h was detected (data not shown), this representing at least 40 times above the detection limit. Within 2 h, however, the amounts of monolignols in the medium increased up to ;30 to 35 mmol (Fig. 6C and 6D, middle traces), while the aldehydes in the medium were again no longer detectable (Fig. 6A and 6B, middle traces). The maximal formation of monolignols accumulating in the cell bathing medium was ;80 to 95 mmol, this being achieved when 80 mmol of each aldehyde was supplied to P. taeda cells (Fig. 6C and 6D, upper traces), and which corresponds to ;100 and ;20 fold increases in p-coumaryl and coniferyl alcohol contents, respectively, relative to their levels in the induced cells
Feruloyl CoA ester administration to P. taeda cells. Feruloyl CoA was also administered to the cells, but it was not effectively taken up by the cells, as revealed by HPLC analysis (data not shown). Effect of exogenously supplied cinnamyl aldehydes to P. taeda metabolism. For each of the sets of experiments described above, the intracellular levels of p-coumaryl and coniferyl aldehydes were below the detection limit (0.25 nmol) of the methods employed. Moreover, they remained undetectable in the cells even when 10 mmol cinnamyl aldehydes were exogenously supplied to P. taeda cells, this representing a 40,000 fold increase above the detection limit (data not shown). On the other hand, the amounts of the corresponding monolignols in the medium rapidly rose to 10-12 mmol within 2 h (Fig. 6C and 6D, lower traces) in an essentially equimolar manner relative to that of the
FIG. 6. Representative effect of exogenously provided cinnamyl aldehydes on extracellular levels of monolignols in P. taeda cell cultures. Time course of depletion of (A) p-coumaryl and (B) coniferyl aldehydes in the extracellular medium. Time course of formation of (C) p-coumaryl and (D) coniferyl alcohols in the extracellular medium. Legend: Œ, 0 mmol; F, 10 mmol; ■, 30 mmol; and }, 80 mmol cinnamyl aldehydes. For structures see Scheme 1.
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(i.e., in 8% sucrose/20 mM KI). Addition of more than 80 mmol of either aldehyde did not increase further the monolignol levels in either the cells or bathing medium. At 80 mmol exogenously provided aldehyde, intracellular p-coumaryl and coniferyl aldehyde levels accumulated up to ;2 and 3 mmol reaching maximum values at ;12 h (data not shown), while 5 and 10 mmol remained in the medium after 24 h, respectively (Fig. 6A and 6B). Thus, an ;320,000 fold increase in the amount of available aldehydes (above detection limit) was required in order to saturate the rate-limiting capacity of P. taeda cinnamyl alcohol dehydrogenase. By comparison, increasing the amount of available cinnamic acid 50 – 400 fold (5– 40 mmol) did not increase the monolignol levels significantly, and this situation also held true for the other phenylpropenoic acids. Thus, compared with cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase, the ability of cinnamyl alcohol dehydrogenase to control the metabolic flux to monolignols is insignificant. Based on these results, the control of flux during monolignol biosynthesis does not rely solely on one enzyme (which is often described as being PAL) but is rather distributed among the other enzymes including cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase. Moreover, the increased availability of Phe significantly influences the ratio of monolignols formed by P. taeda cells, suggesting possible regulatory roles for the enzymes in the preceding pathways (e.g., shikimate pathway) and nitrogen recycling (17). Cinnamyl alcohol dehydrogenase, on the other hand, is not ratelimiting even with P. taeda. Indeed, these data provide a straightforward explanation as to why downregulating cinnamyl alcohol dehydrogenase, by as much as 70 to 93%, had no significant effects on the amount of lignins formed (3–5). ACKNOWLEDGMENTS The authors thank the United States National Aeronautics and Space Administration (NAG100164 and NAG21198), the Arthur M. and Kate Eisig Tode Foundation, as well as the Lewis B. and Dorothy Cullman and G. Thomas Hargrove Center for Land Plant Adaptation
Studies. Thanks are also extended to Dr. Ulrich Matern and R. E. Kneusel for authentic standards of the various CoA esters.
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