Pinoresinol-lariciresinol Reductase Research Articles

Effect of pinoresinol-lariciresinol reductases on biosynthesis of lignans with substrate selectivity in Schisandra chinensis

Schisandra lignans are the main bioactive compounds found in Schisandra chinensis fruits, such as schisandrol lignans and schisandrin lignans, which play important roles in organ protection or other clinical roles. Pinoresinol-lariciresinol reductase (PLR) plays a pivotal role in plant lignan biosynthesis, however, limited research has been conducted on S. chinensis PLR to date. This study identified five genes as ScPLR, successfully cloned their coding sequences, and elucidated their catalytic capabilities. ScPLR3-5 could recognize both pinoresinol and lariciresinol as substrates, and convert them into lariciresinol and secoisolariciresinol, respectively, while ScPLR2 exclusively catalyzed the conversion of (+)-pinoresinol into (+)-lariciresinol. Transcript-metabolite correlation analysis indicated that ScPLR2 exhibited unique properties that differed from the other members. Molecular docking and site-directed mutagenesis revealed that Phe271 and Leu40 in the substrate binding motif were crucial for the catalytic activity of ScPLR2. This study serves as a foundation for understanding the essential enzymes involved in schisandra lignan biosynthesis.

Interactions between second messengers, SA and MAPK6 signaling pathways lead to chitosan-induced lignan production in Linum album cell culture

Lignans along with their special ecological role in plants have received much attention in recent decades due to their anti-cancer properties. However, the mechanisms regulating their biosynthesis are still largely unknown. Here, we investigated the potential role of certain signaling pathways and their relationships in regulating the biosynthesis of 6-methoxypodophyllotoxin (6MPTOX) through pharmacological approaches in chitosan-exposed Linum album cells. The HPLC results revealed that chitosan (100 mg L−1) induces 6MPTOX biosynthesis. Chitosan also elevated the content of second messengers cytosolic free Ca2+, hydrogen peroxide (H2O2) and nitric oxide (NO) by activating plasma-membrane Ca2+ channels, NADPH oxidase and nitrate reductase (NR)/nitric oxide synthase (NOS)-like enzymes, respectively. Co-application of chitosan with EGTA, W-7, imidazole, L-NAME and tungstate inhibitors diminished phenylalanine ammonia-lyase (PAL), pinoresinol-lariciresinol reductase (PLR) and 6MPTOX induction, indicating that Ca2+/CaM, H2O2 and NO are involved in regulating this biosynthetic pathway. Our data suggest that there is a reciprocal relationship between chitosan-induced cytosolic free Ca2+ and H2O2 rising , while NO production is unilaterally regulated by Ca2+ and H2O2. Besides, we found that chitosan also increases salicylic acid (SA) content and correspondingly, exogenous SA stimulates 6MPTOX production by affecting PAL and PLR activity. The level of SA was mutually associated with cytosolic free Ca2+, H2O2 and NO. Real-time PCR results suggest that mitogen-activated protein kinase 6 (MAPK6) acts downstream of second messengers and SA signaling pathways, therefore, it can be a point of integration for signals modulating lignan biosynthesis. Overall, our findings provide new insights into the lignan biosynthesis regulation, wherein Ca2+/CaM, H2O2 and NO signals are mediated, amplified and converged by SA and MAPK6, and the information is passed on to potential downstream targets.

Stress-Responsive cis-Regulatory Elements Underline Podophyllotoxin Biosynthesis and Better Performance of Sinopodophyllum hexandrum Under Water Deficit Conditions

Sinopodophyllum hexandrum is an endangered medicinal herb known for its bioactive lignan podophyllotoxin (PTOX), which is used for the preparation of anticancer drugs. In its natural habitat, S. hexandrum is exposed to a multitude of adversities, such as fluctuating temperatures, water deficit, and high UV radiations. Transcriptional regulation of genes, which is regulated by the condition-specific binding of transcriptional factors to precise motifs in the promoter region, underlines responses to an environmental cue. Therefore, analysis of promoter sequences could ascertain the spatio-temporal expression of genes and overall stress responses. Unavailability of genomic information does not permit such analysis in S. hexandrum, especially on regulation of PTOX pathway. Accordingly, this study describes isolation and in silico analysis of 5′-upstream regions of ShPLR (PINORESINOL-LARICIRESINOL REDUCTASE) and ShSLD (SECOISOLARICIRESINOL DEHYDROGENASE), the two key genes of the PTOX biosynthetic pathway. Data showed a range of motifs related to basal transcription, stress-responsive elements, such as those for drought, low temperature, and light, suggesting that the expression of these genes and resulting PTOX accumulation would be affected by, at least, these environmental cues. While the impact of temperature and light on PTOX accumulation is well studied, the effect of water deficit on the physiology of S. hexandrum and PTOX accumulation remains obscure. Given the presence of drought-responsive elements in the promoters of the key genes, the impact of water deficit on growth and development and PTOX accumulation was studied. The results showed decline in relative water content and net photosynthetic rate, and increase in relative electrolyte leakage with stress progression. Plants under stress exhibited a reduction in transpiration rate and chlorophyll content, with a gradual increase in osmoprotectant content. Besides, stressed plants showed an increase in the expression of genes involved in the phenylpropanoid pathway and PTOX biosynthesis, and an increase in PTOX accumulation. Upon re-watering, non-irrigated plants showed a significant improvement in biochemical and physiological parameters. Summarily, our results demonstrated the importance of osmoprotectants during water deficit and the revival capacity of the species from water deficit, wherein PTOX synthesis was also modulated. Moreover, isolated promoter sequences could be employed in genetic transformation to mediate the expression of stress-induced genes in other plant systems.

Structure-based engineering of substrate specificity for pinoresinol-lariciresinol reductases

Pinoresinol–lariciresinol reductases (PLRs) are enzymes involved in the lignan biosynthesis after the initial dimerization of two monolignols, and this represents the entry point for the synthesis of 8-8′ lignans and contributes greatly to their structural diversity. Of particular interest has been the determination of how differing substrate specificities are achieved with these enzymes. Here, we present crystal structures of IiPLR1 from Isatis indigotica and pinoresinol reductases (PrRs) AtPrR1 and AtPrR2 from Arabidopsis thaliana, in the apo, substrate-bound and product-bound states. Each structure contains a head-to-tail homodimer, and the catalytic pocket comprises structural elements from both monomers. β4 loop covers the top of the pocket, and residue 98 from the loop governs catalytic specificity. The substrate specificities of IiPLR1 and AtPrR2 can be switched via structure-guided mutagenesis. Our study provides insight into the molecular mechanism underlying the substrate specificity of PLRs/PrRs and suggests an efficient strategy for the large-scale commercial production of the pharmaceutically valuable compound lariciresinol.

Differential expression of pinoresinol-lariciresinol reductase gene in relation to podophyllotoxin accumulation in different plant organs of endangered anticancer species Podophyllum peltatum

Pinoresinol-lariciresinol reductase (PLR) is a key enzyme in podophyllotoxin (PTOX) biosynthesis pathway, which catalyzes the conversion of pinoresinol into secoisolariciresinol, a central precursor for the PTOX biosynthesis. Podophyllum peltatum is an important anticancer species, and PTOX content is obviously different from various plant organs. Therefore, investigation of PLR gene expression facilitates the understanding of the molecular mechanisms of the variation of the PTOX content in different organs from P. peltatum as well as its biosynthesis mechanisms, suggesting that it is an important gene for metabolic engineering of the PTOX. The PTOX was found in all the test organs (root, rhizome, petiole, fruit, leaf and flower), with significant differences among them (p<0.05). The highest PTOX content (2.8018 ± 0.23%) was found in rhizome, which was 30 times higher than the lowest PTOX content in fruit. Furthermore, tissue expression profile showed that P. peltatum PLR (PpPLR) was expressed in all the test organs except for the flower. The highest and lowest expression level was found in rhizome and leaf, respectively. The expression profile of PpPLR was inconsistent with the PTOX content variation in the test organs except for the rhizome, suggesting that the PTOX biosynthetic organ is not necessarily its storage organ. After the initial synthesis, PTOX can be transferred to its storage organ, such as the rhizome.

Assembly of Plant Enzymes in E. coli for the Production of the Valuable (-)-Podophyllotoxin Precursor (-)-Pluviatolide.

Lignans are plant secondary metabolites with a wide range of reported health-promoting bioactivities. Traditional routes toward these natural products involve, among others, the extraction from plant sources and chemical synthesis. However, the availability of the sources and the complex chemical structures of lignans often limit the feasibility of these approaches. In this work, we introduce a newly assembled biosynthetic route in E. coli for the efficient conversion of the common higher-lignan precursor (+)-pinoresinol to the noncommercially available (-)-pluviatolide via three intermediates. (-)-Pluviatolide is considered a crossroad compound in lignan biosynthesis, because the methylenedioxy bridge in its structure, resulting from the oxidation of (-)-matairesinol, channels the biosynthetic pathway toward the microtubule depolymerizer (-)-podophyllotoxin. This oxidation reaction is catalyzed with high regio- and enantioselectivity by a cytochrome P450 monooxygenase from Sinopodophyllum hexandrum (CYP719A23), which was expressed and optimized regarding redox partners in E. coli. Pinoresinol-lariciresinol reductase from Forsythia intermedia (FiPLR), secoisolariciresinol dehydrogenase from Podophyllum pleianthum (PpSDH), and CYP719A23 were coexpressed together with a suitable NADPH-dependent reductase to ensure P450 activity, allowing for four sequential biotransformations without intermediate isolation. By using an E. coli strain coexpressing the enzymes originating from four plants, (+)-pinoresinol was efficiently converted, allowing the isolation of enantiopure (-)-pluviatolide at a concentration of 137 mg/L (ee ≥99% with 76% isolated yield).

Pinoresinol‐lariciresinol reductase: Substrate versatility, enantiospecificity, and kinetic properties

Two western red cedar pinoresinol-lariciresinol reductase (PLR) homologues were studied to determine their enantioselective, substrate versatility, and kinetic properties. PLRs are downstream of dirigent protein engendered, coniferyl alcohol derived, stereoselective coupling to afford entry into the 8- and 8'-linked furofuran lignan, pinoresinol. Our investigations showed that each PLR homolog can enantiospecifically metabolize different furofuran lignans with modified aromatic ring substituents, but where phenolic groups at both C4/C4' are essential for catalysis. These results are consistent with quinone methide intermediate formation in the PLR active site. Site-directed mutagenesis and kinetic measurements provided additional insight into factors affecting enantioselectivity and kinetic properties. From these data, PLRs can be envisaged to allow for the biotechnological potential of generation of various lignan skeleta, that could be differentially "decorated" on their aromatic ring substituents, via the action of upstream dirigent proteins.

Differential Gene Profiling of the Heartwood Formation Process in Taiwania cryptomerioides Hayata Xylem Tissues.

Taiwania (Taiwania cryptomerioides) is an important tree species in Taiwan because of the excellent properties of its wood and fascinating color qualities of its heartwood (HW), as well as the bioactive compounds therein. However, limited information is available as to the HW formation of this species. The objective of this research is to analyze the differentially expressed genes (DEGs) during the HW formation process from specific Taiwania xylem tissues, and to obtain genes that might be closely associated with this process. The results indicated that our analyses have captured DEGs representative to the HW formation process of Taiwania. DEGs related to the terpenoid biosynthesis pathway were all up-regulated in the transition zone (TZ) to support the biosynthesis and accumulation of terpenoids. Many DEGs related to lignin biosynthesis, and two DEGs related to pinoresinol reductase (PrR)/pinoresinol lariciresinol reductase (PLR), were up-regulated in TZ. These DEGs together are likely involved in providing the precursors for the subsequent lignan biosynthesis. Several transcription factor-, nuclease-, and protease-encoding DEGs were also highly expressed in TZ, and these DEGs might be involved in the regulation of secondary metabolite biosynthesis and the autolysis of the cellular components of ray parenchyma cells in TZ. These results provide further insights into the process of HW formation in Taiwania.

Insights into Lignan Composition and Biosynthesis in Stinging Nettle (Urtica dioica L.).

Stinging nettle (Urtica dioica L.) has been used as herbal medicine to treat various ailments since ancient times. The biological activity of nettle is chiefly attributed to a large group of phenylpropanoid dimers, namely lignans. Despite the pharmacological importance of nettle lignans, there are no studies addressing lignan biosynthesis in this plant. We herein identified 14 genes encoding dirigent proteins (UdDIRs) and 3 pinoresinol-lariciresinol reductase genes (UdPLRs) in nettle, which are two gene families known to be associated with lignan biosynthesis. Expression profiling of these genes on different organs/tissues revealed a specific expression pattern. Particularly, UdDIR7, 12 and 13 displayed a remarkable high expression in the top internode, fibre tissues of bottom internodes and roots, respectively. The relatively high expression of UdPLR1 and UdPLR2 in the young internodes, core tissue of bottom internode and roots is consistent with the high accumulation of lariciresinol and secoisolariciresinol in these tissues. Lignan quantification showed a high abundance of pinoresinol in roots and pinoresinol diglucosides in young internodes and leaves. This study sheds light on lignan composition and biosynthesis in nettle, providing a good basis for further functional analysis of DIRs and PLRs and, ultimately, engineering lignan metabolism in planta and in cell cultures.

TcMYB1, TcMYB4, and TcMYB8 participate in the regulation of lignan biosynthesis in Taiwania cryptomerioides Hayata

Taiwania (Taiwania cryptomerioides Hayata) is a conifer species that is rich in bioactive secondary metabolites. Lignans, which are the major extracts, are especially important, but the biosynthetic gene regulation and downstream biosynthesis pathways remain unclear. In a previous study, we identified three pinoresinol-lariciresinol reductase (PLR) genes in Taiwanina, and in the present study, the regulation of these PLR genes was investigated. Through next-generation transcriptome sequencing, gene co-expression network analysis was performed to predict the relationship between putative transcription factor genes and upstream biosynthetic genes. Three MYB genes were identified as putative lignan biosynthetic regulators and named TcMYB1, TcMYB4, and TcMYB8. The gene expression of TcMYBs and TcPLRs was further confirmed with real-time PCR. Finally, an Agrobacteria-mediated transient overexpression experiment was conducted, and the activation of proTcPLR3::YFP by TcMYBs suggested that TcMYB1, TcMYB4, and TcMYB8 are biosynthetic regulators of lignan. These lignan regulators can be used in further research and applied to achieve a higher lignan content.

Characterization of LuWRKY36, a flax transcription factor promoting secoisolariciresinol biosynthesis in response to Fusarium oxysporum elicitors in Linum usitatissimum L. hairy roots.

The involvement of a WRKY transcription factor in the regulation of lignan biosynthesis in flax using a hairy root system is described. Secoisolariciresinol is the main flax lignan synthesized by action of LuPLR1 (pinoresinol-lariciresinol reductase 1). LuPLR1 gene promoter deletion experiments have revealed a promoter region containing W boxes potentially responsible for the response to Fusarium oxysporum. W boxes are bound by WRKY transcription factors that play a role in the response to stress. A candidate WRKY transcription factor, LuWRKY36, was isolated from both abscisic acid and Fusarium elicitor-treated flax cell cDNA libraries. This transcription factors contains two WRKY DNA-binding domains and is a homolog of AtWRKY33. Different approaches confirmed LuWRKY36 binding to a W box located in the LuPLR1 promoter occurring through a unique direct interaction mediated by its N-terminal WRKY domain. Our results propose that the positive regulator action of LuWRKY36 on the LuPLR1 gene regulation and lignan biosynthesis in response to biotic stress is positively mediated by abscisic acid and inhibited by ethylene. Additionally, we demonstrate a differential Fusarium elicitor response in susceptible and resistant flax cultivars, seen as a faster and stronger LuPLR1 gene expression response accompanied with higher secoisolariciresinol accumulation in HR of the resistant cultivar.

The control exerted by ABA on lignan biosynthesis in flax (Linum usitatissimum L.) is modulated by a Ca2+ signal transduction involving the calmodulin-like LuCML15b

The LuPLR1 gene encodes a pinoresinol lariciresinol reductase responsible for the biosynthesis of (+)-secoisolariciresinol, a cancer chemopreventive lignan, highly accumulated in the seedcoat of flax (Linum usitatissimum L.). Abscisic acid (ABA) plays a key role in the regulation of LuPLR1 gene expression and lignan accumulation in both seeds and cell suspensions, which require two cis-acting elements (ABRE and MYB2) for this regulation. Ca2+ is a universal secondary messenger involved in a wide range of physiological processes including ABA signaling. Therefore, Ca2+ may be involved as a mediator of LuPLR1 gene expression and lignan biosynthesis regulation exerted by ABA. To test the potential implication of Ca2+ signaling, a pharmacological approach was conducted using both flax cell suspensions and maturing seed systems coupled with a ß-glucuronidase reporter gene experiment, RT-qPCR analysis, lignan quantification as well as Ca2+ fluorescence imaging. Exogenous ABA application results in an increase in the intracellular Ca2+ cytosolic concentration, originating mainly from the extracellular medium. Promoter-reporter deletion experiments suggest that the ABRE and MYB2 cis-acting elements of the LuPLR1 gene promoter functioned as Ca2+-sensitive sequences involved in the ABA-mediated regulation. The use of specific inhibitors pointed the crucial roles of the Ca2+ sensors calmodulin-like proteins and Ca2+-dependent protein kinases in this regulation. This regulation appeared conserved in the two different studied systems, i.e. cell suspensions and maturing seeds. A calmodulin-like, LuCML15b, identified from gene network analysis is proposed as a key player involved in this signal transduction since RNAi experiments provided direct evidences of this role. Taken together, these results provide new information on the regulation of plant defense and human health-promoting compounds, which could be used to optimize their production.

Pinoresinol-lariciresinol reductases, key to the lignan synthesis in plants.

This paper provides an overview on activity, stereospecificity, expression and regulation of pinoresinol-lariciresinol reductases in plants. These enzymes are shared by the pathways to all 8-8' lignans derived from pinoresinol. Pinoresinol-lariciresinol reductases (PLR) are enzymes involved in the lignan biosynthesis after the initial dimerization of two monolignols. They catalyze two successive reduction steps leading to the production of lariciresinol or secoisolariciresinol from pinoresinol. Two secoisolariciresinol enantiomers can be synthetized with different fates. Depending on the plant species, these enantiomers are either final products (e.g., in the flaxseed where it is stored after glycosylation) or are the starting point for the synthesis of a wide range of lignans, among which the aryltetralin type lignans are used to semisynthesize anticancer drugs such as Etoposide®. Thus, the regulation of the gene expression of PLRs as well as the possible specificities of these reductases for one reduction step or one enantiomer are key factors to fine-tune the lignan synthesis. Results published in the last decade have shed light on the presence of more than one PLR in each plant and revealed various modes of action. Nevertheless, there are not many results published on the PLRs and most of them were obtained in a limited range of species. Indeed, a number of them deal with wild and cultivated flax belonging to the genus Linum. Despite the occurrence of lignans in bryophytes, pteridophytes and monocots, data on PLRs in these taxa are still missing and indeed the whole diversity of PLRs is still unknown. This review summarizes the data, published mainly in the last decade, on the PLR gene expression, enzymatic activity and biological function.

A variable loop involved in the substrate selectivity of pinoresinol/lariciresinol reductase from Camellia sinensis

Pinoresinol/lariciresinol reductase (PLR), an NADPH-dependent reductase that catalyzes the sequential reduction of pinoresinol into secoisolariciresinol via Lariciresinol, can lead to the structural and stereochemical diversity of lignans. The relationship between substrate-selective reaction of PLR and sequence homology still remains unclear. In this study, we focused on the contribution of the variable region between PLRs in determining substrate selectivity. Here, two CsPLRs (CsPLR1 and CsPLR2) were identified in the tea plant (Camellia sinensis var. sinensis cv. Shuchazao). In vitro enzymatic assays showed that CsPLR1 could convert (+)- and (−)-pinoresinol into lariciresinol or secoisolariciresinol, whereas CsPLR2 catalyzed (+)-pinoresinol enantioselectively into (−)-secoisolariciresinol. Homology modeling and site-directed mutagenesis were used to examine the role of a variable loop in catalysis and substrate selectivity. The L174I mutant in CsPLR1 lost the capacity to reduce either (+)- or (−)-pinoresinol but retained the ability to catalyze the reduction of (−)-lariciresinol. These findings provide a basis for better understanding of the substrate-selective reaction of PLR.

RNAi-mediated silencing of pinoresinol lariciresinol reductase in Linum album hairy roots alters the phenolic accumulation in response to fungal elicitor

Lignans are diphenolic compounds produced in plants via coupling of two coniferyl alcohol molecules with the aid of a dirigent protein to form pinoresinol (PINO). The latter is reduced via lariciresinol (LARI) to secoisolariciresinol by the bifunctional pinoresinol–lariciresinol reductase (PLR). In this study, we clarified the consequences of altered lignan biosynthesis on amino acids, phenolics compounds and lignin in the hairy roots of Linum album with an ihpRNAi construct to silence PLR gene expression. Down-regulation of PLR-La1 resulted in up to an 8.3 and 3.3-time increased PINO and LARI content respectively, and reduced levels of podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (6-MPTOX). By Suppression of PLR expression, the metabolites belonging to shikimate and phenylpropanoid pathways are conducted to phenolic compounds and lignin accumulations. Although PINO and LARI were induced in response to fungal elicitor, the accumulation of PTOX and 6-MPTOX did not occur in PLR down-regulated roots. Our result also demonstrated variation in amino acids, phenolic compounds and lignin levels in presence of the fungal elicitation in PLR down regulated-roots. This data assert the accumulation of aryltetralin lignans in interactions with plant pathogens by PLR activity and the importance this enzyme for defense against pathogens in L. album.

Mapping podophyllotoxin biosynthesis and growth-related transcripts with high elevation in Sinopodophyllum hexandrum

Sinopodophyllum hexandrum, a perennial rhizomatous herb that produces the anti-cancer metabolite podophyllotoxin (PPT), is distributed in the Himalayan region. While greater plant growth and PPT accumulation is observed at higher compared with lower elevations, the mechanism responsible for elevation-dependent growth promotion and PPT biosynthesis has not been reported. Here, the de novo sequenced transcriptome of S. hexandrum is described. When plants were grown at 3300 versus 2300 m above sea level (asl), a total of 53,691 unigenes were generated and 736 differentially expressed genes (DEGs) were observed. DEGs (357) were identified with 234 characterized genes showing greater up than down regulation, 171 versus 63, respectively. Functional annotation classified 23 DEGs involved in PPT biosynthesis, including phenylpropanoid enzymes: phenylalanine ammonia layse (PAL), cinnamyl alcohol-dehydrogenase (CAD), dirigent protein (DIR), pinoresinol-lariciresinol reductase (PLR), CYP719A23, CYP71CU1, and 2-oxoglutarate/Fe(II)-dependent dioxygenase (2-ODD) which directly participate in PPT biosynthesis. Over 200 DEGs that participate in plant growth and development are categorized into: cell morphogenesis, bio-signaling, primary metabolism, photosynthesis/energy, transcription/polynucleotide metabolism, translation/protein synthesis, transport, and stress tolerance. This transcriptomic analysis provides insight into the mechanism that enhances plant growth and PPT accumulation in S. hexandrum under higher elevation conditions.

The gene expression and enzymatic activity of pinoresinol-lariciresinol reductase during wood formation in Taiwania cryptomerioides Hayata

Abstract Taiwania (Taiwania cryptomerioides Hayata) is an indigenous conifer species of Taiwan. Various secondary metabolites of Taiwania with diverse bioactivities have been identified, and lignans are especially abundant in the heartwood (hW). In the present study, the wood of this species was separated to cambium (Cam), sapwood (sW), transition zone (TZ) and hW and their transcriptomes were sequenced. Three pinoresinol-lariciresinol reductases (PLRs; designated TcPLR1, TcPLR2.2 and TcPLR3), which are responsible for lignan biosynthesis, were cloned and their expressions in wood tissues were detected. TcPLRs had higher expression levels in Cam and sW in RNA-seq and reverse transcriptase-polymerase chain reaction (RT-PCR) results. Liquid chromatography-mass spectrometry (LC-MS) analysis of the reaction products of TcPLRs revealed that TcPLR1 can reduce (+)-pinoresinol to lariciresinol, and both TcPLR2.2 and TcPLR3 could reduce (+)-pinoresinol to lariciresinol and secoisolariciresinol.

Piriformospora indica cell wall modulates gene expression and metabolite profile in Linum album hairy roots.

Elicitation of Linum album hairy roots by Piriformospora indica cell wall induced the target genes and specific metabolites in phenylpropanoid pathway and shifted the amino acid metabolism toward the phenolic compound production. Plants have evolved complex mechanisms to defend themselves against various biotic stresses. One of these responses is the production of metabolites that act as defense compounds. Manipulation of plant cell cultures by biotic elicitors is a useful strategy for improving the production of valuable secondary metabolites. This study focused on hairy root culture of Linum album, an important source for lignans. The effects of cell wall elicitor extracted from Piriformospora indica on phenylpropanoid derivatives were evaluated to identify metabolic traits related to biotic stress tolerance. Significant increases in lignin, lignans; lariciresinol, podophyllotoxin, and 6-methoxy podophyllotoxin; phenolic acids: cinnamic acid, ferulic acid, and salicylic acid; flavonoids: myricetin, kaempferol, and diosmin were observed in response to the fungal elicitor. In addition, the gene expression levels of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, cinnamoyl-CoA reductase, and pinoresinol-lariciresinol reductase significantly increased after elicitation. The composition of free amino acids was altered under the elicitation. Phenylalanine and tyrosine, as precursors of phenylpropanoid metabolites, were increased, but alanine, serine, and glutamic acid significantly decreased in response to the fungal elicitor, suggesting that the amino acid pathway may be shifted toward biosynthesis of aromatic amino acids and precursors of the phenylpropanoid pathway. These results provided evidence that up-regulation of genes involved in the phenylpropanoid pathway in response to the fungal elicitor resulted in enhanced metabolic responses associated with the protection in L. album. This approach can also be applied to improve lignan production.

Functional characterization of the pinoresinol-lariciresinol reductase-2 gene reveals its roles in yatein biosynthesis and flax defense response.

This study provides new insights into the biosynthesis regulation and in planta function of the lignan yatein in flax leaves. Pinoresinol-lariciresinol reductases (PLR) catalyze the conversion of pinoresinol into secoisolariciresinol (SECO) in lignan biosynthesis. Several lignans are accumulated in high concentrations, such as SECO accumulated as secoisolariciresinol diglucoside (SDG) in seeds and yatein in aerial parts, in the flax plant (Linum usitatissimum L.) from which two PLR enzymes of opposite enantioselectivity have been isolated. While LuPLR1 catalyzes the biosynthesis of (+)-SECO leading to (+)-SDG in seeds, the role(s) of the second PLR (LuPLR2) is not completely elucidated. This study provides new insights into the in planta regulation and function of the lignan yatein in flax leaves: its biosynthesis relies on a different PLR with opposite stereospecificity but also on a distinct expression regulation. RNAi technology provided evidence for the in vivo involvement of the LuPLR2 gene in the biosynthesis of (-)-yatein accumulated in flax leaves. LuPLR2 expression in different tissues and in response to stress was studied by RT-qPCR and promoter-reporter transgenesis showing that the spatio-temporal expression of the LuPLR2 gene in leaves perfectly matches the (-)-yatein accumulation and that LuPLR2 expression and yatein production are increased by methyl jasmonate and wounding. A promoter deletion approach yielded putative regulatory elements. This expression pattern in relation to a possible role for this lignan in flax defense is discussed.

In vitro polyploidy induction: changes in morphology, podophyllotoxin biosynthesis, and expression of the related genes in Linum album (Linaceae).

Induction of tetraploidy was performed and podophyllotoxin production increased by upregulating the expression level and enzyme activity of genes related to its biosynthesis in tetraploid compared to diploid Linum album. Linum album is a valuable medicinal plant that produces antiviral and anticancer compounds including podophyllotoxin (PTOX). To achieve homogeneous materials, in vitro diploid clones were established, and their nodal segments were exposed to different concentrations and durations of colchicine. This resulted in successful tetraploidy induction, confirmed by flow cytometry, and is being reported for the first time. The highest efficiency of tetraploid induction (22%) was achieved after 72h exposure to 2.5-mM colchicine treatment. The stable tetraploids were produced after being subcultured three times, and their ploidy stability was confirmed after each subculture. The effects of autopolyploidy were measured on the morphological and phytochemical characteristics, as well as enzyme activity and the expression levels of some key genes involved in the PTOX biosynthetic pathway, including phenylalanine ammonia-lyase (PAL), cinnamoyl-Coa reductase (CCR), cinnamyl-alcohol dehydrogenase (CAD), and pinoresinol-lariciresinol reductase (PLR). The tetraploid plants had larger leaves and stomata (length and width) and lower density stomata. Increasing the ploidy level from diploid to tetraploid resulted in 1.39- and 1.23-fold enhancement of PTOX production, respectively, in the leaves and stem. The increase in PTOX content was associated with upregulated activities of some enzymes studied related to its biosynthetic pathway and the expression of the corresponding genes. The expression of the PAL gene and PLR enzymatic activity had the most positive correlation with the ploidy level in both leaf and stem tissues. Our results verified that autotetraploid induction is a useful breeding method, remarkably increasing the PTOX content in the leaves and stem of L. album.