Recombinant Pinoresinol-Lariciresinol Reductases from Western Red Cedar (Thuja plicata) Catalyze Opposite Enantiospecific Conversions (1999)

Although the heartwood of woody plants represents the main source of fiber and solid wood products, essentially nothing is known about how the biological processes leading to its formation are initiated and regulated. Accordingly, a reverse transcription-polymerase chain reaction-guided cloning strategy was employed to obtain genes encoding pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) as a means to initiate the study of its heartwood formation. (+)-Pinoresinol-(+)-lariciresinol reductase from Forsythia intermedia was used as a template for primer construction for reverse transcription-polymerase chain reaction amplifications, which, when followed by homologous hybridization cloning, resulted in the isolation of two distinct classes of putative pinoresinol-lariciresinol reductase cDNA clones from western red cedar. A representative of each class was expressed as a fusion protein with β-galactosidase and assayed for enzymatic activity. Using both deuterated and radiolabeled (±)-pinoresinols as substrates, it was established that each class of cDNA encoded a pinoresinol-lariciresinol reductase of different (opposite) enantiospecificity. Significantly, the protein from one class converted (+)-pinoresinol into (–)-secoisolariciresinol, whereas the other utilized the opposite (–)-enantiomer to give the corresponding (+)-form. This differential substrate specificity raises important questions about the role of each of these individual reductases in heartwood formation, such as whether they are expressed in different cells/tissues or at different stages during heartwood development. Although the heartwood of woody plants represents the main source of fiber and solid wood products, essentially nothing is known about how the biological processes leading to its formation are initiated and regulated. Accordingly, a reverse transcription-polymerase chain reaction-guided cloning strategy was employed to obtain genes encoding pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) as a means to initiate the study of its heartwood formation. (+)-Pinoresinol-(+)-lariciresinol reductase from Forsythia intermedia was used as a template for primer construction for reverse transcription-polymerase chain reaction amplifications, which, when followed by homologous hybridization cloning, resulted in the isolation of two distinct classes of putative pinoresinol-lariciresinol reductase cDNA clones from western red cedar. A representative of each class was expressed as a fusion protein with β-galactosidase and assayed for enzymatic activity. Using both deuterated and radiolabeled (±)-pinoresinols as substrates, it was established that each class of cDNA encoded a pinoresinol-lariciresinol reductase of different (opposite) enantiospecificity. Significantly, the protein from one class converted (+)-pinoresinol into (–)-secoisolariciresinol, whereas the other utilized the opposite (–)-enantiomer to give the corresponding (+)-form. This differential substrate specificity raises important questions about the role of each of these individual reductases in heartwood formation, such as whether they are expressed in different cells/tissues or at different stages during heartwood development. Western red cedar (Thuja plicata) lignans, such as plicatic acid 1 (Fig. 1), are deposited in large amount (>20% by dry weight) during its heartwood formation, thereby helping confer protection against invading pathogens (1Gang D.R. Fujita M. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1998; 697: 389-421Crossref Scopus (41) Google Scholar) and contributing significantly to the color, quality, and durability of its highly valued heartwood. Other lignans, such as secoisolariciresinol 2 and matairesinol 3, which are present in certain dietary components (e.g. flax (Linum usitatissimum) seed), are thought to substantially reduce the risk in humans of the onset of breast, prostate, and other hormonally linked cancers, as suggested by epidemiological and tumor-induction suppression studies (2Adlercreutz H. Environ. Health Perspect. 1995; 103 Suppl. 7: 103-112PubMed Google Scholar, 3Adlercreutz H. Honjo H. Higashi A. Fotsis T. Hämäläinen E. Hasegawa T. Okada H. Am. J. Clin. Nutr. 1991; 54: 1093-1100Crossref PubMed Scopus (600) Google Scholar, 4Adlercreutz H. Höckerstedt K. Bannwart C. Bloigu S. Hämäläinen E. Fotsis T. Ollus A. J. Steroid Biochem. 1987; 27: 1135-1144Crossref PubMed Scopus (416) Google Scholar). This so-called chemoprotection occurs through their metabolism by intestinal flora into the compounds enterodiol and enterolactone, respectively, both of which act as phytoestrogens, thereby helping to regulate the hormonal balance in the body. Additionally, the secoisolariciresinol2 and matairesinol 3-derived lignan (5Dewick P.M. Atta-ur-Rahman Studies in Natural Product Chemistry. 5. University of Karachi, Karachi, Pakistan1990: 459-503Google Scholar), podophyllotoxin 4 (as its etoposide or teniposide derivatives) from Podophyllum species, is one of only five or so plant-derived substances used in cancer treatment today (6Stähelin H. Eur. J. Cancer. 1973; 9: 215-221Abstract Full Text PDF PubMed Scopus (199) Google Scholar, 7Ross W. Rowe T. Glisson B. Yalowich J. Liu L. Cancer Res. 1984; 44: 5857-5860PubMed Google Scholar, 8Richter A. Strausfeld U. Knippers R. Nucleic Acids Res. 1987; 15: 3455-3468Crossref PubMed Scopus (44) Google Scholar). As shown in Fig. 1, it is proposed that these distinct plant metabolites are biochemically interrelated. The entry point into each of the above 8,8′-linked lignans, a branch of the general phenylpropanoid pathway, occurs via stereoselective coupling of two molecules of E-coniferyl alcohol5 to give pinoresinol 6 (9Davin L.B. Wang H.-B. Crowell A.L. Bedgar D.L. Martin D.M. Sarkanen S. Lewis N.G. Science. 1997; 275: 362-366Crossref PubMed Scopus (580) Google Scholar,10Davin L.B. Lewis N.G. An. Acad. Bras. Cienc. 1995; 67, Suppl. 3: 363-378Google Scholar). 1D. R. Gang, M. A. Costa, M. Fujita, A. T. Dinkova-Kostova, H.-B. Wang, W. Martin, V. Burlat, S. Sarkanen, L. B. Davin, and N. G. Lewis, submitted for publication. In Forsythiasp., an ∼78-kDa protein confers the regio- and stereoselectivity of coupling to give only the (+)-enantiomeric form. This firstcase of control of bimolecular phenoxy radical coupling is considered to involve a unique biochemical mechanism of radical capture (9Davin L.B. Wang H.-B. Crowell A.L. Bedgar D.L. Martin D.M. Sarkanen S. Lewis N.G. Science. 1997; 275: 362-366Crossref PubMed Scopus (580) Google Scholar), and the term “dirigent protein” was introduced to describe this phenomenon. The corresponding gene encoding the protein has been cloned, and the functional recombinant protein was obtained using aSpodoptera/baculovirus expression system.1 Additionally, several cDNAs in western red cedar that are homologous to the dirigent protein (11.Lewis, N. G., Davin, L. B., Dinkova-Kostova, A. T., Fujita, M., Gang, D. R., and Sarkanen, S. (1997) U. S. Patent Application PCT/US97/20391.Google Scholar) have been identified. The regio- and enantioselective control of coupling conferred by these homologous proteins, as well as their temporal and tissue-specific expression, are under current investigation. In Forsythia intermedia, (+)-pinoresinol 6a can undergo enantiospecific reduction to first give (+)-lariciresinol7a and then (–)-secoisolariciresinol 2a (see Fig. 2 A). This reductive sequence is catalyzed by the enantiospecific, bifunctional, NADPH-dependent (+)-pinoresinol-(+)-lariciresinol reductase (PLR-Fi1) 2The abbreviations used are: PLR, pinoresinol-lariciresinol reductase; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; pfu, plaque-forming unit; IFR, isoflavone reductase. (12Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), which has considerable homology (∼ 50% identity, ∼65% similarity) to isoflavone reductases (Ref. 13Gang D.R. A.T. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1997; Scopus Google and under and in formation. then occurs via enantiospecific of (–)-secoisolariciresinol 2a T. Davin L.B. E. Lewis N.G. J. Chem. Soc. Chem. Google Scholar). other can the opposite of these (e.g. in flax seed), that their formation is by genes encoding dirigent and As an other species, such have been proposed to pinoresinol-lariciresinol which using T. T. M. Am. Chem. Soc. Symp. Ser. 1998; 697: Scholar). is that both and reductases are present in such with each expressed in and The of the plicatic acid 1 and its occurs during western red cedar heartwood this formation is proposed to from the metabolism of secoisolariciresinol 2 and matairesinol as shown (Fig. (1Gang D.R. Fujita M. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1998; 697: 389-421Crossref Scopus (41) Google Scholar). has been that these metabolites are in the the and the deposited into the from the via an (see D.R. Fujita M. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1998; 697: 389-421Crossref Scopus (41) Google and This of to is known as which of woody the of this of to heartwood formation and its to that heartwood as their main the wood and or genes have been that are This can by the in with in from their that are high in The of this was to whether the genes encoding pinoresinol-lariciresinol present in T. as a first into the biochemical processes heartwood formation. the of two classes of pinoresinol-lariciresinol reductases from T. the first (+)-pinoresinol 6a to give (–)-secoisolariciresinol 2a (Fig. 2 whereas the to (Fig. 2 As the of these two classes of from its the biochemical in control of coupling and metabolism in Western red cedar plicata) in University and used or polymerase and and obtained from and from and radiolabeled was from for polymerase chain reaction and by used for of with the by to a in and acid sequence using the for the Scholar, The Scholar) and the University and University of and at a 6 A was used for of for employed a followed by R. K. in and Scholar) or with using an performance liquid was as (12Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). employed a with an of acid in at a of of and 2a and and and 6a and using a with and of and using a with was a weight) was obtained from of a western red cedar plicata) to the of E. R. Biol. Scopus Google Scholar). T. cDNA was using of with the the and the with a of for the The was used for (12Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). A cDNA with a for the for the was and in the as A to obtain the as intermedia (+)-pinoresinol-(+)-lariciresinol reductase cDNA (12Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) was used to a for T. cDNA A. cDNA of intermedia (+)-pinoresinol-(+)-lariciresinol reductase was used as the template in each and of with the primer and the primer as (12Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). in a as of 1 at 2 at and at with at and an at 4 the in a a corresponding to was The was used with the and to to a radiolabeled which was 6 and to of T. A for as (12Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). which then to The two of cDNA was to the and in one by for 4 at with The for with at in and and for with at in and in a (see was for for and to a hybridization at in a The to this which was then with was for at with The in and for at to and and at for with the from the and with to they to for at through two of with hybridization as cDNA clones from the in The cDNAs in using T. reductase cDNA was obtained from T. by a reverse transcription-polymerase chain reaction was from the used for the cDNA was with from to for and The of template and was then with and of in a of The reaction was at for and then by 1 was to the and at for The reaction was used as the template in each and of with primer and The sequence of primer was the of the cDNAs obtained in the first of (see was in a as (12Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) for the at in at two the The from the The then into the and into to The and of the cDNA using the and the primer and to The primer of the was the of primer of the The the cDNAs with and the cDNAs using five individual cDNA that for different of The was used to as cDNA B. cDNAs and under and for a of these cDNA The of one of the clones encoding was to in with the β-galactosidase gene in for expression of the pinoresinol-lariciresinol reductase as a fusion protein (12Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). In for the of to in with the β-galactosidase gene was from the with and and then into with these two and each into to The for each at in with with to The by for and to an of in with and The to at and then by and in of 2 and the by of at 4 for to the for each was and assayed for pinoresinol-lariciresinol reductase as and in protein from from E. the of (±)-pinoresinols was with for as as or the the cDNA of reductase was in with the β-galactosidase gene in reductase was assayed by the formation of and and and (12Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google A. A. Davin L.B. Bedgar D.L. Lewis N.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). each of of Chu A. Davin L.B. Lewis N.G. 1998; Scopus Google Scholar) in and the protein from E. The enzymatic reaction was initiated by of in and at with for several different and which the was with and as for the and the was of each the with an for of its using liquid The of the was to in in and to and from the and for by as above using as and as at for the was with The to in and to corresponding to secoisolariciresinol2 and pinoresinol 6 in and to with at and in the of western red cedar plicata) heartwood occurs through the of the plicatic and its into is known about and both heartwood and its in woody This is a only its general biological its to and which essentially only heartwood. a heartwood formation is initiated in the and initiated to in the as the plant the its formation of metabolites and other into and these can for of the dry of the H. J. Scholar, H. K. J. Chem. 44: Google Scholar, H. K. J. Chem. 44: Google Scholar, H. J. Chem. Google Scholar, H. K. J. Chem. Google Scholar, H. J. Chem. Google Scholar, H. J. Chem. Google Scholar, H. J. Chem. 44: Google Scholar, Scopus Google Scholar). The and of these metabolites can from plant to and these to a large the of the different in the plant The general of leading to heartwood to well in the plant and was first by J. Res. Ser. Biol. Scopus Google Scholar, Scopus Google Scholar) and then by H. J. Chem. Scopus Google Scholar, Sarkanen and Scholar, Am. Chem. Soc. Symp. Ser. Scholar). As heartwood formation it is by the of and a or is the heartwood from the This is to in the of heartwood such as the in western red cedar. are in this that to heartwood (see D.R. Fujita M. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1998; 697: 389-421Crossref Scopus (41) Google and Although the is in heartwood is about the that at the gene or (1Gang D.R. Fujita M. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1998; 697: 389-421Crossref Scopus (41) Google Scholar). of to the and biochemical and processes in heartwood formation, using lignan in western red cedar plicata) as a means to this As several genes from western red cedar woody (11.Lewis, N. G., Davin, L. B., Dinkova-Kostova, A. T., Fujita, M., Gang, D. R., and Sarkanen, S. (1997) U. S. Patent Application PCT/US97/20391.Google Scholar) that have homology and to the dirigent protein from for (+)-pinoresinol 6a formation in that western red cedar dirigent protein genes are of one or with the of its heartwood lignan formation. the of its heartwood (e.g. plicatic acid Fig. 1), a proposed of the pinoresinol-lariciresinol was as in its formation. this was first from western red cedar and used to a cDNA A). Using intermedia (+)-pinoresinol-(+)-lariciresinol reductase cDNA as a cDNAs of the encoded only the first of the putative pinoresinol-lariciresinol reductase the as by to the intermedia (+)-pinoresinol-(+)-lariciresinol reductase the of these cDNA by by in cDNA The other clones encoded cDNAs homologous to other this strategy was in a it the sequence of a putative T. pinoresinol-lariciresinol reductase. This sequence was used to a primer under which then used in an to obtain a A T. pinoresinol-lariciresinol reductase cDNA was obtained from by an strategy R. K. in and Scholar, J. T. A Scholar). first cDNA utilized reverse and the from the (see under was for (see under using two for hybridization to the of the and the of the cDNA clones obtained (as to other from the first T. (see under The then into a and both cDNAs from these products, they only in of with corresponding to acid and homology (see to the pinoresinol-lariciresinol reductase cDNA from intermedia acid in The of was then used to a T. cDNA (see under this cDNAs that homology to the reductase. that the cDNAs into two the first class five of which to each other and one of which was unique the class of which to and 2 of which to each other two classes reductases are by the high of homology the of is and to whereas is and to (see The acid of the putative encoded by these different cDNAs are shown in Fig. 3, as with the intermedia pinoresinol-lariciresinol reductase (PLR-Fi1) and a from which a pinoresinol-lariciresinol reductase (see 13Gang D.R. A.T. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1997; Scopus Google and As can in both classes of T. reductases have high of homology to and similarity) and of homology when with each other and cDNAs have homology to the isoflavone reductase and and a and that they to the class of known as pinoresinol-lariciresinol of PLR, IFR, and protein acid of and with of which the two classes of T. pinoresinol-lariciresinol are and using the of the using the of and in a with of which the two classes of T. pinoresinol-lariciresinol are and using the of the using the of and was to the functional expression of a representative of each of these two classes of T. putative pinoresinol-lariciresinol the of was in with the β-galactosidase present in the cloning and under the control of the corresponding recombinant protein as a fusion protein in E. (12Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). In an in for the of to in with the β-galactosidase gene was from its cloning and into as under both cDNAs under control of the or and with to the fusion control or the to to in and assayed for pinoresinol-lariciresinol reductase activity. protein for each of the fusion and the with used as (see under by each of and as with the in and by of and was for both fusion proteins, and as well as for the control of into or was for the control (see under A was for both the and fusion proteins, with formation and substrate and of in in as in was for fusion protein this the two classes reductases present in these protein shown to pinoresinol-lariciresinol reductases as distinct their the of the catalyzed by these two first as A the of both with reductase using with as at for with the and fusion proteins, by with with the and in The pinoresinol 6 substrate and secoisolariciresinol 2 by and then to with both at and by in the Fig. 4 the of the pinoresinol with the fusion protein in which essentially only the was to the of In Fig. 4 the substrate with the fusion protein in which the of 6a been 2 have of and Fig. 4 the of both and with at Additionally, Fig. 4 its with obtained at of of 2 and a under these the using for Accordingly, Fig. 4 the of the secoisolariciresinol 2 obtained at a with the fusion which that only been The of the was by of its with at and with the at of the Fig. 4 in the of the secoisolariciresinol 2 a with the fusion that only been As its in Fig. 4 at and with the at of the and catalyzed formation of the opposite of secoisolariciresinol2 from pinoresinol 6 (see Fig. The of each class of T. reductase was using with as of deuterated by each of and as with the in and by as The products, corresponding to in and to with both and of the obtained of fusion protein with is shown in in which only into the of secoisolariciresinol is the other Fig. the of the obtained from of fusion protein with only into the (–)-enantiomer established the of two distinct classes of pinoresinol-lariciresinol reductases in western red as in Fig. the first class of reductase Fig. 2 reduction of into whereas the other Fig. 2 formation of (–)-secoisolariciresinol in a reaction to that of the intermedia reductase the corresponding to from the above with and to as of each As shown in Fig. of the obtained from of fusion protein with that only the was the opposite from that of the by this of the by of during reduction of was as an to In Fig. the of the obtained from of fusion protein with which into both and of with the obtained from with both and respectively, (as and for formation to that in with to of the the is converted into whereas the 6a is only into In these of the of was the other with of the resulted in of both and in which the then converted into (–)-secoisolariciresinol the was into that and enantiospecific with and of pinoresinol that distinct from that for from intermedia, the only the (+)-enantiomeric of pinoresinol (Fig. 2 (12Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, A. A. Davin L.B. Bedgar D.L. Lewis N.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). western red cedar pinoresinol-lariciresinol reductases can reduce both and of they are highly enantiospecific As this differential a role in heartwood formation. As in (1Gang D.R. Fujita M. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1998; 697: 389-421Crossref Scopus (41) Google Scholar), formation of heartwood is a highly with metabolites deposited into at and and D.R. A.T. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1997; Scopus Google Scholar). two different lignans, matairesinol and are deposited into the of a western Scopus Google Scholar). deposited into in the heartwood and of or classes of The different pinoresinol-lariciresinol reductases as well as the several different dirigent protein that such lignan expressed in the of T. The differential these two classes of reductases raises important questions about their to the two reductase from a cDNA from from of a at distinct pinoresinol-lariciresinol reductase genes expressed in its at the that the was that of these reductases are in form. that both reductase classes are in lignan in western red cedar leading to the of different heartwood in different Although this the of it the control of heartwood and that is in the two classes of pinoresinol-lariciresinol reductase from western red cedar in with present in intermedia, it was to the sequence homology of these with other reductases D.R. A.T. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1997; Scopus Google Scholar). This resulted in a the acid sequence of pinoresinol-lariciresinol isoflavone reductases and a of of As can the pinoresinol-lariciresinol reductases a 1), with of a from L. which a pinoresinol-lariciresinol reductase. their different both classes of western red cedar pinoresinol-lariciresinol reductases (e.g. and are to each other to intermedia pinoresinol-lariciresinol reductase (PLR-Fi1) or The of the western red cedar reductases to the that these are from the and that of the in the the pinoresinol-lariciresinol reductases in Fig. 6 important for substrate enantiospecificity. is that the the western red cedar reductases have their to of pinoresinol that they are enantiospecific the The isoflavone reductases a the from (see Fig. the other with the of the from the so-called and distinct Fig. that these In this of using a from that this protein converted into R. Gang, H. K. G. W. M. L. B. Davin, and N. G. Lewis, submitted for publication. Fig. the catalyzed by pinoresinol-lariciresinol reductases isoflavone reductases is shown in and the (Fig. As can in Fig. reductive processes are in of both substrate and the (1Gang D.R. Fujita M. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1998; 697: 389-421Crossref Scopus (41) Google Scholar, A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 13Gang D.R. A.T. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1997; Scopus Google Scholar). is that of these distinct metabolites as of in the of (e.g. in P.M. The in Google Scholar), metabolites in (1Gang D.R. Fujita M. Davin L.B. Lewis N.G. Am. Chem. Soc. Symp. Ser. 1998; 697: 389-421Crossref Scopus (41) Google Scholar, M. M. J. Lewis N.G. 1995; PubMed Scopus Google Scholar), and plicatic acid in western red cedar J. Chem. Scopus Google Scholar, Sarkanen and Scholar, Am. Chem. Soc. Symp. Ser. Accordingly, a of the role of these reductases in of their and tissue-specific expression, as well as their substrate is an of the processes heartwood formation can formation is one of the important biological processes the of woody this has been to study from a biochemical to the and of the heartwood Accordingly, an cloning strategy genes in the formation of metabolites deposited into western red cedar heartwood. The recombinant encoded by these genes and in of their their are to to how heartwood formation is and using western red cedar as a