Characterization of Arabidopsis thaliana Pinoresinol Reductase, a New Type of Enzyme Involved in Lignan Biosynthesis

A lignan, lariciresinol, was isolated from Arabidopsis thaliana, the most widely used model plant in plant bioscience sectors, for the first time. In the A. thaliana genome database, there are two genes (At1g32100 and At4g13660) that are annotated as pinoresinol/lariciresinol reductase (PLR). The recombinant AtPLRs showed strict substrate preference toward pinoresinol but only weak or no activity toward lariciresinol, which is in sharp contrast to conventional PLRs of other plants that can reduce both pinoresinol and lariciresinol efficiently to lariciresinol and secoisolariciresinol, respectively. Therefore, we renamed AtPLRs as A. thaliana pinoresinol reductases (AtPrRs). The recombinant AtPrR2 encoded by At4g13660 reduced only (–)-pinoresinol to (–)-lariciresinol and not (+)-pinoresinol in the presence of NADPH. This enantiomeric selectivity accords with that of other PLRs of other plants so far reported, which can reduce one of the enantiomers selectively, whatever the preferential enantiomer. In sharp contrast, AtPrR1 encoded by At1g32100 reduced both (+)- and (–)-pinoresinols to (+)- and (–)-lariciresinols efficiently with comparative kcat/Km values. Analysis of lignans and spatiotemporal expression of AtPrR1 and AtPrR2 in their functionally deficient A. thaliana mutants and wild type indicated that both genes are involved in lariciresinol biosynthesis. In addition, the analysis of the enantiomeric compositions of lariciresinol isolated from the mutants and wild type showed that PrRs together with a dirigent protein(s) are involved in the enantiomeric control in lignan biosynthesis. Furthermore, it was demonstrated conclusively for the first time that differential expression of PrR isoforms that have distinct selectivities of substrate enantiomers can determine enantiomeric compositions of the product, lariciresinol. A lignan, lariciresinol, was isolated from Arabidopsis thaliana, the most widely used model plant in plant bioscience sectors, for the first time. In the A. thaliana genome database, there are two genes (At1g32100 and At4g13660) that are annotated as pinoresinol/lariciresinol reductase (PLR). The recombinant AtPLRs showed strict substrate preference toward pinoresinol but only weak or no activity toward lariciresinol, which is in sharp contrast to conventional PLRs of other plants that can reduce both pinoresinol and lariciresinol efficiently to lariciresinol and secoisolariciresinol, respectively. Therefore, we renamed AtPLRs as A. thaliana pinoresinol reductases (AtPrRs). The recombinant AtPrR2 encoded by At4g13660 reduced only (–)-pinoresinol to (–)-lariciresinol and not (+)-pinoresinol in the presence of NADPH. This enantiomeric selectivity accords with that of other PLRs of other plants so far reported, which can reduce one of the enantiomers selectively, whatever the preferential enantiomer. In sharp contrast, AtPrR1 encoded by At1g32100 reduced both (+)- and (–)-pinoresinols to (+)- and (–)-lariciresinols efficiently with comparative kcat/Km values. Analysis of lignans and spatiotemporal expression of AtPrR1 and AtPrR2 in their functionally deficient A. thaliana mutants and wild type indicated that both genes are involved in lariciresinol biosynthesis. In addition, the analysis of the enantiomeric compositions of lariciresinol isolated from the mutants and wild type showed that PrRs together with a dirigent protein(s) are involved in the enantiomeric control in lignan biosynthesis. Furthermore, it was demonstrated conclusively for the first time that differential expression of PrR isoforms that have distinct selectivities of substrate enantiomers can determine enantiomeric compositions of the product, lariciresinol. Lignans are phenylpropanoid dimers in which the monomers are linked by the central carbon (C8) atoms (1Umezawa T. Phytochem. Rev. 2003; 2: 371-390Crossref Scopus (224) Google Scholar, 2Moss G.P. Pure Appl. Chem. 2000; 72: 1493-1523Crossref Scopus (236) Google Scholar) and are distributed widely in the plant kingdom (1Umezawa T. Phytochem. Rev. 2003; 2: 371-390Crossref Scopus (224) Google Scholar, 3Umezawa T. Wood Res. 2003; 90: 27-110Google Scholar). Biosynthesis of the compounds has been receiving widespread interest, because they have various important features. First, biosynthetic reactions of lignans involve unique stereochemical properties. For example, the initial step of lignan biosynthesis is an enantioselective radical coupling reaction (23Davin L. Lewis N.G. Pytochem. Rev. 2003; 2: 257-288Crossref Scopus (120) Google Scholar), which is of great interest in the field of bioorganic chemistry and may help to provide a model for biomimetic chemistry. Second, lignans have various clinically important biological activities. Some lignans are used in medicines and nutritional supplements, such as the podophyllotoxin-derived semisynthetic lignans used in cancer therapies (4MacRae W.D. Towers G.H.N. Phytochemistry. 1984; 23: 1207-1220Crossref Scopus (574) Google Scholar) and sesamin used in health and nutrition (5Ono E. Nakai M. Fukui Y. Tomimori N. Fukuchi-Mizutani M. Saito M. Satake H. Tanaka T. Katsuta M. Umezawa T. Tanaka Y. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10116-10121Crossref PubMed Scopus (116) Google Scholar). Mechanisms for the control of the gene expression of their biosynthesis are helpful for their biotechnological production. Third, lignans and related compounds, such as norlignans, are often biosynthesized and deposited in significant amounts in the heartwood region of trees as a metabolic event of heartwood formation. Because the metabolic event is specific to trees, biosynthesis of lignans and norlignans can be a clue in helping to elucidate heartwood formation mechanisms. Lignans can be classified into three categories, lignans with 9(9′)-oxygen, lignans without 9(9′)-oxygen, and dicarboxylic acid lignans (1Umezawa T. Phytochem. Rev. 2003; 2: 371-390Crossref Scopus (224) Google Scholar). Of the three categories, the study of the biosynthesis of lignans with 9(9′)-oxygen is the most advanced (1Umezawa T. Phytochem. Rev. 2003; 2: 371-390Crossref Scopus (224) Google Scholar, 6Suzuki S. Umezawa T. J. Wood Sci. 2007; 53: 273-284Crossref Scopus (169) Google Scholar, 23Davin L. Lewis N.G. Pytochem. Rev. 2003; 2: 257-288Crossref Scopus (120) Google Scholar). This type of lignan is formed by the enantioselective dimerization of two coniferyl alcohol units with the aid of dirigent protein (DP) 2The abbreviations used are: DP, dirigent protein; PLR, pinoresinol/lariciresinol reductase; SIRD, secoisolariciresinol dehydrogenase; GC-MS, gas chromatography-mass spectrometry; HPLC, high performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; RT, reverse transcription; MeOH, methyl alcohol; AcOEt, ethyl acetate; qRT, quantitative real time; RT, reverse transcription; PrR, pinoresinol reductase; e.e., enantiomer excess. 2The abbreviations used are: DP, dirigent protein; PLR, pinoresinol/lariciresinol reductase; SIRD, secoisolariciresinol dehydrogenase; GC-MS, gas chromatography-mass spectrometry; HPLC, high performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; RT, reverse transcription; MeOH, methyl alcohol; AcOEt, ethyl acetate; qRT, quantitative real time; RT, reverse transcription; PrR, pinoresinol reductase; e.e., enantiomer excess. to give rise to pinoresinol (furofuran). Pinoresinol is then reduced by pinoresinol/lariciresinol reductase (PLR) via lariciresinol (furan) to secoisolariciresinol (dibenzylbutane), which is in turn oxidized to afford matairesinol (dibenzylbutyrolactone) by secoisolariciresinol dehydrogenase (SIRD) (Fig. 1). The conversion from coniferyl alcohol to matairesinol is believed to be the general biosynthetic pathway for lignans with 9(9′)-oxygen (1Umezawa T. Phytochem. Rev. 2003; 2: 371-390Crossref Scopus (224) Google Scholar, 6Suzuki S. Umezawa T. J. Wood Sci. 2007; 53: 273-284Crossref Scopus (169) Google Scholar, 23Davin L. Lewis N.G. Pytochem. Rev. 2003; 2: 257-288Crossref Scopus (120) Google Scholar). Although DP is an asymmetric inducer, its enantiomeric control is not strong enough to give rise to optically pure pinoresinol. Thus, the enantiomeric composition of pinoresinol from various plant species varies largely (1Umezawa T. Phytochem. Rev. 2003; 2: 371-390Crossref Scopus (224) Google Scholar, 6Suzuki S. Umezawa T. J. Wood Sci. 2007; 53: 273-284Crossref Scopus (169) Google Scholar, 7Umezawa T. Okunishi T. Shimada M. Wood Res. 1997; 84: 62-75Google Scholar). In contrast, dibenzylbutyrolactone lignans, such as matairesinol, are optically pure (1Umezawa T. Phytochem. Rev. 2003; 2: 371-390Crossref Scopus (224) Google Scholar, 6Suzuki S. Umezawa T. J. Wood Sci. 2007; 53: 273-284Crossref Scopus (169) Google Scholar, 7Umezawa T. Okunishi T. Shimada M. Wood Res. 1997; 84: 62-75Google Scholar). These facts unequivocally indicate that not only was the first step mediated by DP, but the subsequent metabolic steps catalyzed by PLR and SIRD were also involved in determining the enantiomeric composition of the lignans (8Umezawa T. Regul. Plant Growth Dev. 2001; 36: 57-67Google Scholar). It was also suggested that a variation in the enantiomeric composition of the upstream lignans (lariciresinol, secoisolariciresinol, and matairesinol) among different plant species may be ascribed, at least in part, to the selectivities of PLR and SIRD isoforms in terms of substrate enantiomers as well as their spatiotemporal expression patterns (8Umezawa T. Regul. Plant Growth Dev. 2001; 36: 57-67Google Scholar). However, this view has not yet been proven to be related to gene expression. Herein, we demonstrate that Arabidopsis thaliana PLRs showed strict substrate preference toward pinoresinol but only weak or no activity toward lariciresinol, unlike conventional PLRs that can reduce both pinoresinol and lariciresinol efficiently. Therefore, we will refer to AtPLRs as A. thaliana pinoresinol reductases (AtPrRs). It was also demonstrated that the enantiomeric composition of lariciresinol in A. thaliana can be controlled by the differential expression of two AtPrRsby using biochemical characterization of their recombinant enzymes and spatiotemporal expression analysis of the genes. Instruments and Chromatography—1H NMR spectra were obtained with a JNM-LA400MK FT-NMR system (JEOL). Chemical shifts and coupling constants (J) were expressed as δ values and in Hz, respectively. Gas chromatography-mass spectrometry (GC-MS) measurement of enzyme assay products was performed on a JMS-DX303HF mass spectrometer (JEOL) equipped with a Hewlett-Packard 5890J gas chromatograph and a JMA-DA5000 mass data system (electron impact mode, 70 eV; gas chromatographic column, Shimadzu HiCap CBP-10 M25-025 (5 m × 0.22 mm); temperature, 40 °C at t = 0–2 min and then to 230 °C at 30 °C min–1; carrier gas, helium; splitless injection). Quantifications of lariciresinol in the roots of A. thaliana wild type and mutants were done with a GC-MS QP5050A mass spectrometer (Shimadzu) equipped with a Shimadzu GC-17A gas chromatograph (electron impact mode, 70 eV; gas chromatographic column, Shimadzu HiCap CBP-10 M25-025 (20 m × 0.22 mm); temperature, 40 °C at t = 0–2 min and then to 230 °C at 30 °C min–1; carrier gas, helium; splitless injection). Reverse-phase high performance liquid chromatography (HPLC) of lariciresinol and pinoresinol and chiral HPLC analyses of pinoresinol were done as previously described (9Okunishi T. Umezawa T. Shimada M. J. Wood Sci. 2000; 46: 234-242Crossref Scopus (25) Google Scholar). Chiral liquid chromatography-mass spectrometry (LC-MS) of lariciresinol was done as follows. The LC-MS system consisted of a Shimadzu LC-10AD HPLC series liquid chromatograph and a Shimadzu LC-MS-2010A single quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization interface and a Q-array-Octapole-Quadrupole mass analyzer. Shimadzu LCMS Solution Version 3.0 was used for data acquisition and processing. LC separation was achieved using a Chiralcel OC column (250 × 2.0 mm, Daicel Chemical Co.) maintained at 40 °C. The mobile phase used for the analysis consisted of EtOH/n-hexane (80:20). The mobile phase was delivered at a flow rate of 0.13 ml/min. The following MS parameters were selected: curved desolvation line temperature, 200 °C; block temperature, 200 °C; probe temperature, 400 °C; and probe voltage, –4.0 kV. Curved desolvation line voltage and Q-array voltage were according to default values by autotuning. Nitrogen served as the nebulizer gas (flow rate, 2.5 liters/min) and curtain gas (pressure, The MS acquisition was in for at a time of The of lariciresinol at and of as an at were column chromatography (20 × T. H. T. T. Shimada M. Scopus Google Scholar), (9Okunishi T. Umezawa T. Shimada M. J. Wood Sci. 2000; 46: 234-242Crossref Scopus (25) Google Scholar), and (9Okunishi T. Umezawa T. Shimada M. J. Wood Sci. 2000; 46: 234-242Crossref Scopus (25) Google Scholar) were and (–)-pinoresinol were obtained by chiral HPLC separation of Plant of and mutants of A. thaliana were with and on a with and acid and on the in were at °C for and then at °C a for The were so that the roots the of the The of the and for At1g32100 and for At4g13660) in the Analysis were obtained from the Arabidopsis of the A. thaliana mutants was done according to M. Phytochemistry. 2006; PubMed Scopus Google Scholar). was from according to the by N. T. Plant J. PubMed Scopus Google Scholar). were by with the and the as for for and for The of the with the and for and and and for the of the The of the were by the of products with the and the the of reverse was using as and for the and and and for the of the of were as and A was obtained by single mutants of and and the by using the described of roots of A. thaliana were with and then with of methyl alcohol The was with units in of at for at °C. The reaction was three with of ethyl and the was The obtained were by a of column chromatography and HPLC to afford pure lariciresinol. × and In roots of the mutants and were with and then with and with as described The obtained were by a of and HPLC to afford pure lariciresinol. in roots of and mutants were by GC-MS as previously N. T. S. Shimada M. Umezawa T. Chem. 2007; PubMed Scopus Google Scholar). roots were with and then with A of of the as was with units in of at for at °C. The reaction was with × and the was The obtained were by The enantiomeric compositions of lariciresinol isolated from wild type and the mutants were by chiral LC-MS analysis using as of pinoresinol from the × was as for lariciresinol. the pinoresinol obtained was to chiral LC-MS analysis to determine its enantiomeric composition as follows. Pinoresinol isolated from the was with and for chiral HPLC The to the (+)- and were and to GC-MS analysis The amounts of (+)-pinoresinol and (–)-pinoresinol were by of the with of the The pinoresinol in the was by GC-MS using as the as in the lariciresinol of by At1g32100 and the At1g32100 was obtained from the Arabidopsis The At4g13660 was by using A. thaliana from their and the and The of the At4g13660 was by The of At1g32100 and At4g13660 were into an expression The At1g32100 and At4g13660 were into E. The and expression of recombinant enzymes were according to L. J. Umezawa T. Plant 2001; PubMed Google Scholar). by × for the was for using the system according to the were using the 72: PubMed Scopus Google Scholar) with as a of activity was according to M. Lewis N.G. J. Chem. PubMed Scopus Google Scholar) with The assay consisted of mm, recombinant enzyme 400 and NADPH. The reaction was performed at 30 °C for and The reaction was by the of of The were and to GC-MS For of the enantiomeric composition of enzyme assay enzyme were as described but with of of enzyme assay products were by HPLC, and by chiral For of and (+)-pinoresinol and (–)-pinoresinol were used as or and substrate were for min at 30 °C to assay by NADPH. The reaction was performed at 30 °C for or and values were from and was by by the enzyme Analysis of At1g32100 and were from the and of A. thaliana using the Plant of the was reverse using reverse and according to the The was performed on a system in a reaction of of 200 and of with a step of min at by 40 of °C for and °C for The used were as reverse reverse the of the of the the was performed by the at the of the For were using recombinant the The was as an to the in For the analysis of the reaction was of and of with a step of min at by 40 at °C for and °C for Analysis of At1g32100 and acid and the were using the Res. 1997; Scopus Google Scholar). The were using the Appl. Google Scholar). Analysis of Lignans in A. analysis of the from the A. thaliana indicated the presence of lariciresinol, the other lignans, such as secoisolariciresinol, and matairesinol, were not the other of lignans were in the was isolated from the and its was by of the NMR with of the Chiral LC-MS analysis showed that the enantiomeric composition of isolated lariciresinol was in of the (Fig. Analysis of At1g32100 and genes (At1g32100 and At4g13660) annotated as PLR were by the A. thaliana for At1g32100 and for analysis (Fig. indicated that both genes in which were in This was in with data M. M. M. PubMed Scopus Google Scholar). of AtPrR1 and the biochemical characterization of the enzymes encoded by At1g32100 and the recombinant enzymes using E. were in for their activity toward pinoresinol and lariciresinol 1). First, using they were to reduce pinoresinol efficiently to lariciresinol. both unlike conventional not reduce to secoisolariciresinol of time. of to only amounts of secoisolariciresinol not were with both recombinant and it was that the activity of the enzyme encoded by At1g32100 toward lariciresinol was that of pinoresinol 1). the other the enzyme encoded by At4g13660 not reduce lariciresinol to secoisolariciresinol of 1). These demonstrated that enzymes showed strict substrate toward pinoresinol and only weak or no activity toward lariciresinol. This is in sharp contrast to conventional PLRs that can reduce both pinoresinol and lariciresinol efficiently rise to Thus, the enzymes encoded by At1g32100 and which were annotated as a type of PLR in terms of substrate Therefore, we will refer to this PLR as pinoresinol reductase AtPrR1 for At1g32100 and AtPrR2 for of recombinant AtPrR1 and AtPrR2 toward and and recombinant enzyme encoded by At1g32100 and AtPrR1 and recombinant enzyme encoded by At1g32100 and in a we the enantiomeric selectivity of the in the Chiral LC-MS analysis showed that the enantiomeric composition of lariciresinol formed following of with AtPrR1 was in of the (Fig. and the other AtPrR2 (–)-lariciresinol the assay (Fig. and an of (+)- and (–)-pinoresinols with AtPrR1 formed efficiently (+)- and on the other AtPrR2 reduced only (–)-pinoresinol and not compositions of pinoresinol and lariciresinol isolated from A. thaliana and formed thaliana in a of AtPrR1 and analysis of AtPrR1 and AtPrR2 using both enantiomers of pinoresinol as was values of AtPrR1 were for (+)-pinoresinol and for the other the of AtPrR2 for (–)-pinoresinol was In addition, kcat/Km values of AtPrR1 were for (+)-pinoresinol and for the of AtPrR2 for (–)-pinoresinol was for there was no significant in (+)- and (–)-pinoresinols with to kcat/Km values. In contrast, the kcat/Km of AtPrR2 for (–)-pinoresinol was that of AtPrR1 for of recombinant AtPrR1 and in a of the of AtPrR1 and AtPrR2 in the A. thaliana we isolated the functionally deficient mutants of AtPrR1 and and for AtPrR1 and one line for AtPrR2 were obtained from the Arabidopsis of the the of the was by of the products with the and in and were to be in the of the AtPrR1 and AtPrR2 genes (Fig. that in was to be in an of the AtPrR1 gene (Fig. The of AtPrR1 and AtPrR2 in the was by in three mutants (Fig. In addition, an was obtained by the single mutants of and and the by using the described The of AtPrR1 and AtPrR2 in the was by (Fig. Analysis of Lignans in of the lariciresinol in the roots of the and single mutants as well as the were GC-MS analysis of the indicated that there was no significant the single mutants and the wild type the other GC-MS analysis of of the showed that lariciresinol biosynthesis in the was This was for by the of pinoresinol of which was not in the wild type or the single Therefore, it was that both AtPrR1 and AtPrR2 were involved in lariciresinol of wild type and in the of thaliana in a of Lignans in Arabidopsis compositions of lariciresinol isolated from and mutants were and in of (–)-lariciresinol (Fig. and and lariciresinol isolated from the in of the (Fig. and The enantiomeric composition of the pinoresinol isolated from the roots was in of the The pathway from coniferyl alcohol to matairesinol via lariciresinol, and secoisolariciresinol (Fig. has been as the general pathway for lignans with 9(9′)-oxygen (1Umezawa T. Phytochem. Rev. 2003; 2: 371-390Crossref Scopus (224) Google Scholar, 6Suzuki S. Umezawa T. J. Wood Sci. 2007; 53: 273-284Crossref Scopus (169) Google Scholar). It was that PLR and SIRD are enzymes that control the enantiomeric compositions of the lignans in the pathway (8Umezawa T. Regul. Plant Growth Dev. 2001; 36: 57-67Google Scholar). However, the of gene expression to the have been because gene and for plants that are as lignan were gene for the of the have been S. E. 2007; PubMed Scopus Google Scholar). A. thaliana is the most widely used model plant in plant bioscience Thus, the genome is and its and are GC-MS analysis of from plants of A. thaliana showed the presence of amounts of lariciresinol not However, M. Phytochemistry. 2006; PubMed Scopus Google Scholar) that which to of phenylpropanoid compounds, were in the plant These to lignans in the and GC-MS analysis of the A. thaliana showed the presence of significant amounts of lariciresinol we isolated lariciresinol from the roots and conclusively it by its NMR with that of an the were by GC-MS without lariciresinol was not that lariciresinol in A. thaliana as a This is the first on the of lariciresinol in A. GC-MS analysis indicated that the lignan was in roots M. Phytochemistry. 2006; PubMed Scopus Google Scholar), other lignans, such as secoisolariciresinol, or matairesinol, were in the roots in the the data the presence of lariciresinol in A. thaliana in we the genes A. thaliana genes (At1g32100 and which were annotated as PLR, were by the A. thaliana The expression analysis of the two genes using indicated that both genes in which were in (Fig. Furthermore, the gene expression well with the that lariciresinol was in the roots of A. thaliana, that the genes were involved in the biosynthesis of lariciresinol. we the biochemical characterization of recombinant encoded by the genes. The recombinant pinoresinol to lariciresinol efficiently 1). the other the specific activity toward lariciresinol to give rise to secoisolariciresinol was weak or not unlike PLRs of other plants that can reduce both pinoresinol and lariciresinol efficiently M. Lewis N.G. J. Chem. PubMed Scopus Google Scholar, S. E. 2007; PubMed Scopus Google Scholar, A. Lewis N.G. J. Chem. PubMed Scopus Google Scholar, E. Phytochemistry. PubMed Scopus Google Scholar). Therefore, we refer to A. thaliana pinoresinol/lariciresinol reductase as A. thaliana pinoresinol reductase The substrate of can for or matairesinol was not in A. In addition, AtPrR1 encoded by At1g32100 is different from AtPrR2 encoded by At4g13660 with to selectivity of substrate AtPrR2 showed strong preference for Thus, (–)-lariciresinol with was formed following of with the enzyme In addition, (+)- and (–)-pinoresinols were with the enzyme (–)-pinoresinol was reduced to (–)-lariciresinol with a kcat/Km of (+)-pinoresinol was This is in line with the selectivity of substrate enantiomers of PLRs from other PLR A. Lewis N.G. J. Chem. PubMed Scopus Google Scholar), M. Lewis N.G. J. Chem. PubMed Scopus Google Scholar), and E. Phytochemistry. PubMed Scopus Google Scholar) catalyzed the of (+)-pinoresinol to and T. M. Lewis N.G. J. Chem. PubMed Scopus Google Scholar), and E. Phytochemistry. PubMed Scopus Google Scholar) reduced (–)-pinoresinol to (–)-lariciresinol and (Fig. that a PLR from L. (+)-pinoresinol in the first step but (–)-lariciresinol in the step S. E. 2007; PubMed Scopus Google Scholar) (Fig. It is that (+)-pinoresinol and (–)-lariciresinol have at and the preferential substrate PLRs reduced one of the enantiomers In sharp contrast, AtPrR1 reduced to give rise to lariciresinol In addition, (+)- and (–)-pinoresinols were with the enzyme both were reduced efficiently with comparative kcat/Km values min–1; Although the acid of AtPrR1 high with that of the selectivities of substrate enantiomers of are different from AtPrR1 and AtPrR2 of but they have different acid Therefore, acid are involved in determining the in the enantiomeric In addition, analysis that PLR and AtPrR2 into a and into (Fig. the enantiomeric selectivity and of of PLR and PrR are to determine the acid for their enantiomeric In to the substrate enantiomer selectivity in the in with enantiomeric compositions of lariciresinol in A. thaliana plants and to the of AtPrR1 and AtPrR2 in the we isolated the and and an GC-MS analysis indicated that the lariciresinol in the single and were not but with the the other lariciresinol was not in the significant amounts of of pinoresinol were in the These indicate the of the two genes. isolated lariciresinol from the as well as the wild type and their enantiomeric The enantiomeric composition of lariciresinol in the wild type was in of the the other lariciresinol isolated from and mutants showed and in of the which are that isolated from the wild type This well with the that AtPrR1 both (+)- and (–)-pinoresinols The of the was for by the AtPrR2 that rise to which in the of the in of the the other lariciresinol from an in of the which is that of the wild type this can be for by the that AtPrR2 (–)-pinoresinol The of the was for by AtPrR1 that both (+)- and (–)-pinoresinols most in the of the in of with the wild type Although AtPrR1 reduced to afford lariciresinol in of the the of the formed lariciresinol is This can be to the enantioselective pinoresinol formation mediated by the In pinoresinol isolated from the in of the (–)-pinoresinol as (–)-pinoresinol with a high is enantioselective AtPrR1 can (–)-lariciresinol with a high as in the demonstrate that PrRs together with DP are involved in enantiomeric control in lignan biosynthesis. In addition, differential expression of PrR isoforms that have different selectivity in terms of substrate enantiomers can determine the enantiomeric compositions of the lignan are to for Chemical for the for the of A. thaliana also for Chemical for the of from A.