Lignan Biosynthesis Research Articles

Intrinsic disordered nature and prediction of secondary structures of pinoresinol lariciresinol reductases 2 in Flax (Linum usitatissimum)

Flax (Linum usitatissimum) contains a wide range of essential nutrients, such as proteins, polyunsaturated fatty acids (PUFAs), phenolic compounds, fibers, flavonoids, and lignans. The production of lignans in flax is mainly caused by pinoresinol-lariciresinol reductases (PLRs). Amongst these, PLR2s play a pivotal function by contributing to the further reduction of lariciresinol to secoisolariciresinol in the biosynthesis of lignans. However, the in-silico analysis of PLR2 gene family interlinking the intrinsic disordered nature and secondary structures that could support this hypothesis is still missing in Flax. Herein, we present the first study of Intrinsic disorder proteins (IDPs) in flax to gain an understanding of their biological functions. This study was conducted from June to August 2024 at the Department of Biochemistry, Lovely Professional University, Phagwara, Punjab, India. The flax genome assembly from Phytozome was used to retrieve 30 PLR2 genes which encode for 30 PLR2 proteins. Further, we used PONDR database to identify the intrinsic disordered nature of the proteins while GOR database was used to predict the secondary structures. For identifying the amino acid composition, Protparam database was used. The results of PONDR database revealed that all the proteins are somewhat disordered whereas amino acid composition marked the presence of disorder-promoting residues in almost all the proteins. Secondary structure predictions by GOR revealed the presence of coils, helices and strands. To relate the structural functional relationship, it is of the utmost importance to have an in-depth knowledge of the balance between intrinsic disorder and secondary structure as this will provide key insights into the dynamic functionality and regulatory mechanisms that are the foundation of complex biological processes.

Genome-wide characterization of the DIR gene family in sesame reveals the function of SiDIR21 in lignan biosynthesis

Furofuran-type lignans, mainly sesamin and sesamolin, are the most representative functional active ingredients in sesame (Sesamum indicum L.). Their exceptional antioxidant properties, medicinal benefits, and health-promoting functions have garnered significant attention. Dirigent (DIR) proteins, found in vascular plants, are crucial for the biosynthesis of secondary metabolites, like lignans, and essential for responding to abiotic and biotic stresses. Despite their importance, they have yet to be systematically analyzed, especially those involved in lignan synthesis in sesame. This study unveiled 44 DIR genes in sesame. Phylogenetic analysis categorized these SiDIRs into five subgroups (DIR-a, DIR-b/d, DIR-e, DIR-f, and DIR-g), aligning with conserved motifs and gene structures analyses. Expression analysis unveiled distinct tissue-specific and hormone-responsive expression patterns among the SiDIR gene family members. Particularly, SiDIR21, a member of the DIR-a subgroup, exhibited robust expression in lignan-accumulating tissues and consistently high expression levels in germplasm during seed development with high sesamin content. Furthermore, SiDIR21 overexpression in hairy roots significantly increased sesamin and sesamolin contents, confirming its role in lignan synthesis. Overall, our study offers a valuable resource for exploring SiDIRs’ functions and the lignan biosynthesis pathway in sesame.

Unveiling the Power of Flax Lignans: From Plant Biosynthesis to Human Health Benefits.

Flax (Linum usitatissimum L.) is the richest plant source of lignin secondary metabolites. Lignans from flax have been applied in the fields of food, medicine, and health due to their significant physiological activities. The most abundant lignan is secoisolariciresinol, which exists in a glycosylated form in plants. After ingestion, it is converted by human intestinal flora into enterodiol and enterolactone, which both have physiological roles. Here, the basic structures, contents, synthesis, regulatory, and metabolic pathways, as well as extraction and isolation methods, of flax lignans were reviewed. Additionally, the physiological activity-related mechanisms and their impacts on human health, from the biosynthesis of lignans in plants to the physiological activity effects observed in animal metabolites, were examined. The review elucidates that lignans, as phenolic compounds, not only function as active substances in plants but also offer significant nutritional values and health benefits when flax is consumed.

Ectopic and transient expression of VvDIR4 gene in Arabidopsis and grapes enhances resistance to anthracnose via affecting hormone signaling pathways and lignin production.

DIR (Dirigent) proteins play important roles in the biosynthesis of lignin and lignans and are involved in various processes such as plant growth, development, and stress responses. However, there is less information about VvDIR proteins in grapevine (Vitis vinifera L). In this study, we used bioinformatics methods to identify members of the DIR gene family in grapevine and identified 18 VvDIR genes in grapevine. These genes were classified into 5 subfamilies based on phylogenetic analysis. In promoter analysis, various plant hormones, stress, and light-responsive cis-elements were detected. Expression profiling of all genes following Colletotrichum gloeosporioides infection and phytohormones (salicylic acid (SA) and jasmonic acid (JA)) application suggested significant upregulation of 17 and 6 VvDIR genes, respectively. Further, we overexpressed the VvDIR4 gene in Arabidopsis thaliana and grapes for functional analysis. Ectopic expression of VvDIR4 in A. thaliana and transient expression in grapes increased resistance against C. gloeosporioides and C. higginsianum, respectively. Phenotypic observations showed small disease lesions in transgenic plants. Further, the expression patterns of genes having presumed roles in SA and JA signaling pathways were also influenced. Lignin contents were measured before and after C. higginsianum infection; the transgenic A. thaliana lines showed higher lignin content than wild-type, and a significant increase was observed after C. higginsianum infection. Based on the findings, we surmise that VvDIR4 is involved in hormonal and lignin synthesis pathways which regulate resistance against anthracnose. Our study provides novel insights into the function of VvDIR genes and new candidate genes for grapevine disease resistance breeding programs.

Cloning and functional characterization of the pinoresinol-lariciresinol reductase gene IiPLR2 in Isatis indigotica

The pinoresinol-lariciresinol reductase (PLR), a crucial enzyme in the biosynthesis of lignans in plants, catalyzes a two-step reaction to produce lariciresinol and secoisolariciresinol. Lignans such as lariciresinol are the effective components of traditional Chinese medicine Radix Isatidis in exerting antiviral activity. In order to study the function of the key enzyme PLR in the biosynthesis of lariciresinol in Isatis indigotica, the original plant of Radix Isatidis, IiPLR2 was cloned from I. indigotica, with a full length of 954 bp, encoding 317 amino acids. Multiple sequence alignment showed that IiPLR2 contained a conserved nicotinamide adenine dinucleotide phosphate (NADPH)-binding motif. The phylogenetic tree showcased that IiPLR2 shared the same clade with AtPrR1 from Arabidopsis thaliana. The prokaryotic expression vector pET32a-IiPLR2 was constructed and then transformed into Escherichia coli BL21(DE3) competent cells for protein expression. The purified enzyme IiPLR2 could catalyze the conversion of pinoresinol to lariciresinol and the conversion of lariciresinol to secoisolariciresinol. The cloning, sequencing, and catalytic functional analysis of IiPLR2 in this study enrich the understanding of this kind of functional proteins in I. indigotica and supplement the biosynthesis pathways of lignans. Moreover, this study provides a functional module for further research on metabolic regulation and synthetic biology and lays a foundation for comprehensively revealing the relationship between the spatial structures and catalytic functions of such proteins.

In silico prediction of microRNA families involved in the biosynthesis of lignans and cyanogenic glycosides in flax (Linum usitatissimumL.)

Flaxseed (Linum usitatissimum L.) is renowned for having the highest concentration of lignans among all plant species worldwide. However, it also contains a notable amount of cyanogenic glycosides, prompting efforts to reduce their levels. MicroRNAs, recognized as significant epigenetic factors, hold the potential to serve as genetic markers in achieving this breeding goal. In this study, 44,885 mature microRNAs were utilized alongside one genome and four transcriptomes of flax. Twelve gene sequences were obtained for five lignan enzymes and three enzymes related to cyanogenic glycosides. Based on our in silico approach, we identified 15 microRNA families for the lignan metabolic pathway, 10 for the cyanogenic glycoside pathway, and 6 regulating both pathways. Additionally, we contributed to the annotation of the used transcriptomes and verified the functionality of the “Finding genes by keyword” algorithm available on Phytozome 13. The results obtained led to the design of a unique schema for microRNA mediated regulation in the biosynthetic pathways of lignans and cyanogenic glycosides. This research will enhance our understanding of the regulatory mechanisms of microRNA in these biosynthetic pathways, along with other specialized metabolites. The predicted microRNAs can be employed in marker-assisted selection with the breeding objective of optimizing the ratio of nutritional and antinutritional components in flaxseed while maintaining current physiological parameters.

Integrative analysis of the metabolome and transcriptome provides insights into the mechanisms of lignan biosynthesis in Herpetospermum pedunculosum (Cucurbitaceae).

Herpetospermum pedunculosum (Ser.) C. B. Clarke is a traditional Chinese herbal medicine that heavily relies on the lignans found in its dried ripe seeds (Herpetospermum caudigerum), which have antioxidant and hepatoprotective functions. However, little is known regarding the lignan biosynthesis in H. pedunculosum. In this study, we used metabolomic (non-targeted UHPLC-MS/MS) and transcriptome (RNA-Seq) analyses to identify key metabolites and genes (both structural and regulatory) associated with lignan production during the green mature (GM) and yellow mature (YM) stages of H. pedunculosum. The contents of 26 lignan-related metabolites and the expression of 30 genes involved in the lignan pathway differed considerably between the GM and YM stages; most of them were more highly expressed in YM than in GM. UPLC-Q-TOF/MS confirmed that three Herpetospermum-specific lignans (including herpetrione, herpetotriol, and herpetin) were found in YM, but were not detected in GM. In addition, we proposed a lignan biosynthesis pathway for H. pedunculosum based on the fundamental principles of chemistry and biosynthesis. An integrated study of the transcriptome and metabolome identified several transcription factors, including HpGAF1, HpHSFB3, and HpWOX1, that were highly correlated with the metabolism of lignan compounds during seed ripening. Furthermore, functional validation assays revealed that the enzyme 4-Coumarate: CoA ligase (4CL) catalyzes the synthesis of hydroxycinnamate CoA esters. These results will deepen our understanding of seed lignan biosynthesis and establish a theoretical basis for molecular breeding of H. pedunculosum.

Phytochemical and Gene Network Analysis Elucidating the Key Genes Involved in the Biosynthesis of Gomisin J in Schisandra sphenanthera

The biosynthesis and distribution of lignans in medicinal plants, particularly in Schisandra sphenanthera, hold significant pharmacological importance. This study bridges the knowledge gap in understanding the tissue-specific biosynthesis and distribution of these compounds, with a focus on Gomisin J. Our phytochemical analysis revealed a distinct accumulation pattern of Gomisin J, predominantly in the roots, contrasting with the distribution of Pregomisin and Dihydroguaiaretic acid. This finding highlights the roots’ unique role in lignan storage and biosynthesis. Further, differential gene expression analysis across various tissues illuminated the biosynthetic pathways and regulatory mechanisms of these lignans. Utilizing Weighted Gene Co-expression Network Analysis (WGCNA), we identified the MEtan module as a key player, strongly correlated with Gomisin J levels. This module’s in-depth examination revealed the crucial involvement of four cytochrome P450 (CYP) enzymes and eight transcription factors. Notably, the CYP genes DN6828 and DN2874-i3 exhibited up-regulation in roots across both male and female plants, while DN51746 was specifically up-regulated in male roots, indicating a potential gender-specific aspect in Gomisin J biosynthesis. Comparative analysis with functionally characterized CYP71A homologs suggests these CYP genes might be involved in distinct biosynthetic pathways, including terpenoids, alkaloids, and phenylpropanoids, and potentially in lignan biosynthesis. This hypothesis, supported by their more than 55% identity with CYP71As and strong correlation with Gomisin J concentration, opens avenues for novel discoveries in lignan biosynthesis, pending further functional characterization. Our research provides a comprehensive understanding of the genetic and metabolic mechanisms underlying the tissue-specific distribution of lignans in Schisandra sphenanthera, offering valuable insights for their pharmacological exploitation.

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.

Transcriptomic Insights and Cytochrome P450 Gene Analysis in Kadsura coccinea for Lignan Biosynthesis.

Kadsura coccinea is a medicinal plant from the Schisandraceae family that is native to China and has great pharmacological potential due to its lignans. However, there are significant knowledge gaps regarding the genetic and molecular mechanisms of lignans. We used transcriptome sequencing technology to analyze root, stem, and leaf samples, focusing on the identification and phylogenetic analysis of Cytochrome P450 (CYP) genes. High-quality data containing 158,385 transcripts and 68,978 unigenes were obtained. In addition, 36,293 unigenes in at least one database, and 23,335 across five databases (Nr, KEGG, KOG, TrEMBL, and SwissProt) were successfully annotated. The KEGG pathway classification and annotation of these unigenes identified 10,825 categorized into major metabolic pathways, notably phenylpropanoid biosynthesis, which is essential for lignan synthesis. A key focus was the identification and phylogenetic analysis of 233 Cytochrome P450 (CYP) genes, revealing their distribution across 38 families in eight clans, with roots showing specific CYP gene expression patterns indicative of their role in lignan biosynthesis. Sequence alignment identified 22 homologous single genes of these CYPs, with 6 homologous genes of CYP719As and 1 of CYP81Qs highly expressed in roots. Our study significantly advances the understanding of the biosynthesis of dibenzocyclooctadiene lignans, offering valuable insights for future pharmacological research and development.

A Comprehensive Analysis of Transcriptomics and Metabolomics Revealed Key Pathways Involved in Saccharum spontaneum Defense against Sporisorium scitamineum.

Sugarcane smut, caused by Sporisorium scitamineum, poses a severe threat to sugarcane production. The genetic basis of sugarcane resistance to S. scitamineum remains elusive. A comparative transcriptomic and metabolomic study was conducted on two wild Saccharum species of S. spontaneum with contrast smut resistance. Following infection, the resistant line exhibited greater down-regulation of genes and metabolites compared to the susceptible line, indicating distinct biological processes. Lignan and lignin biosynthesis and SA signal transduction were activated in the resistant line, while flavonoid biosynthesis and auxin signal transduction were enhanced in the susceptible line. TGA2.2 and ARF14 were identified as playing positive and negative roles, respectively, in plant defense. Exogenous auxin application significantly increased the susceptibility of S. spontaneum to S. scitaminum. This study established the significant switching of defense signaling pathways in contrast-resistant S. spontaneum following S. scitamineum infection, offering a hypothetical model and candidate genes for further research into sugarcane smut disease.

Transcriptome-wide analysis of PIP reductase gene family identified a phenylpropene synthase crucial for the biosynthesis of dibenzocyclooctadiene lignans in Schisandra chinensis

Phenylpropenes, such as isoeugenol and eugenol, are produced as defend compounds, floral attractants, and flavor constituents by phenylpropene synthases belonging to the PIP reductase family. Moreover, isoeugenol is proposed to be involved in the biosynthesis of dibenzocyclooctadiene lignans, the main active compounds of Schisandra chinensis (Turcz.) Baill. fruits (SCF). S. chinensis, a woody vine plant, is widely used for its medicinal, horticultural, edible, and economic values. In this study, nine ScPIP genes were identified and characterized from the transcriptome datasets of SCF. The expression profiles revealed that ScPIP genes were differentially expressed during different developmental stages of SCF. Three ScPIPs were selected and cloned as candidate genes encoding phenylpropene synthases according to phylogenetic analysis. ScPIP1 was proved to function as isoeugenol synthase (IGS) and designated as ScIGS1 through in vivo functional characterization in Escherichia coli. Subcellular localization analysis demonstrated that ScIGS1 was localized in both the cytoplasm and nucleus. The three-dimensional (3D) model of ScIGS1 was obtained using homology modeling. Site-directed mutagenesis experiments revealed that the substitution of residues at positions 110 and 113 impacted the product specificity of ScIGS1 and the mutation of Lys157 to Ala abolishing catalytic function. Moreover, the kcat values of mutants were lower than that of ScIGS1 using a deep learning approach. In conclusion, this study provides a basis for further research on PIP reductases and the biosynthetic pathway of dibenzocyclooctadiene lignans.

Anthriscus sylvestris Deoxypodophyllotoxin Synthase Involved inthe Podophyllotoxin Biosynthesis.

Tetrahydrofuran ring formation from dibenzylbutyrolactone lignans is a key step in the biosynthesis of aryltetralin lignans including deoxypodophyllotoxin and podophyllotoxin. Previously, Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2-ODD) from Podophyllum hexandrum (Himalayan mayapple, Berberidaceae) was found to catalyze the cyclization of a dibenzylbutyrolactone lignan, yatein, to give deoxypodophyllotoxin and designated as deoxypodophyllotoxin synthase (DPS). Recently, we reported that the biosynthesis of deoxypodophyllotoxin and podophyllotoxin evolved in a lineage-specific manner in phylogenetically unrelated plant species such as P. hexandrum and Anthriscus sylvestris (cow parsley, Apiaceae). Therefore, a comprehensive understanding of the characteristics of DPSs that catalyze the cyclization of yatein to deoxypodophyllotoxin in various plant species is important. However, for plant species other than P. hexandrum, the isolation of the DPS enzyme gene and the type of the enzyme, e.g.whether it is 2-ODD or another type of enzyme such as cytochrome P-450, have not been reported. In this study, we report the identification and characterization of A. sylvestris DPS (AsDPS). Phylogenetic analysis showed that AsDPS belonged to the 2-ODD superfamily and shared moderate amino acid sequence identity (40.8%) with P. hexandrum deoxypodophyllotoxin synthase (PhDPS). Recombinant protein assay indicated that AsDPS and PhDPS differ in terms of the selectivity of substrate enantiomers. Protein modeling using AlphaFold2 and site-directed mutagenesis indicated that the Tyr305 residue of AsDPS probably contributes to substrate recognition. This study advances our understanding of the podophyllotoxin biosynthetic pathway in A. sylvestris and provides new insight into 2-ODD involved in plant secondary (specialized) metabolism.

Current understanding of lignan biosynthesis

Current understanding of lignan biosynthesis

Combined metabolomic and transcriptomic analysis reveals the characteristics of the lignan in Isatis indigotica Fortune

Isatis indigotica Fortune is a plant species containing lignan compounds of significant economic value. Its root plays a crucial role in treating viruses and exhibits antitumor, anti-inflammatory, antibacterial, and other biological activities. Now, I. indigotica has been included in Isatis tinctoria Linnaeus. In this study, the roots of diploid I. indigotica, tetraploid I. indigotica, and Isatis tinctoria Linnaeus were analyzed using metabolome and transcriptome analysis. The metabolomic analysis detected 48 lignan metabolites, including Lirioresinol A, Vladinol A, Syringaresinol, Arctigenin, Acanthoside B, and Sesamin as characteristic compounds, without significant variations among the remaining metabolites. The transcriptomic analysis identified 41 differentially expressed phenylpropanoid synthase genes, which were further analyzed for variations in lignan transcriptome profiles across different samples. RT-qPCR analysis also revealed differential genes expression related to lignan biosynthesis pathway among the three sample groups. The analysis of transcription factors showed that the AP2-EREBP family (Iin24319), MYB family (Iin24843), and WRKY family (Iin08158) displayed expression patterns similar to Iin14549. Phylogenetic analyses also indicate that Iin14549 may play a role in lignan synthesis. These transcription factor families exhibited high expression in tetraploid I. indigotica, moderate expression in diploid I. indigotica, and low expression in I. tinctoria. The findings of this study can serve as a reference for improving the quality of I. indigotica and developing germplasms with high lignan content. Additionally, these results lay a foundation for the functional characterization of UGTs in lignan biosynthesis pathway.

Savinin Triggers Programmed Cell Death of Ray Parenchyma Cells in Heartwood Formation of Taiwania cryptomerioides Hayata.

The purpose of this study was to investigate the relationship between lignan biosynthesis and programmed cell death (PCD) of ray parenchyma cells during the heartwood formation of Taiwania (Taiwania cryptomerioides Hayata). Since the PCD of ray parenchyma cells and the synthesis of lignans are the two main processes involved in the formation of heartwood, both of which need to be completed through gene regulation. Based on the results of genomics and bioinformatics analysis, that the PCD of tracheids are induced by genotoxic, and the PCD of ray parenchyma cells is induced by biological factors, such as fungi, bacteria, and viruses, which could induce oxidative stress. According to the results of time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis, lignans are produced in ray parenchyma cells, and the accumulation of savinin and its downstream lignans might be the cause of PCD in ray parenchyma cells. An in vitro experiment further confirmed that the accumulation of savinin could cause protoplasts of Taiwania's xylem to produce taiwanin A, which is the marker of heartwood formation in Taiwania. Resulting in an increase in reactive oxygen species (ROS) content, which could induce oxidative stress in ray parenchyma cells and potentially lead to PCD. Based on these findings, we conclude that accumulation of savinin could be induced PCD of ray parenchyma cells in heartwood formation in Taiwania.

Establishment of Agrobacterium rhizogenes-mediated RNAi of Phryma leptostachya accelerates the functional identification of key genes of the furofuran lignan biosynthetic pathway

Phryma leptostachya is a well-known medicinal plant that produces furofuran lignans with various biological activities. However, the biosynthetic pathways of these natural products in P. leptostachya remain unclear. Hairy root cultures provide a novel method for elucidating the biosynthetic pathways of secondary metabolites. Here, we established a hairy root system for P. leptostachya by optimizing five parameters: Agrobacterium strains, explant types, infection time, A. rhizogenes cell density, and acetosyringone (AS) concentration. The highest transformation frequency (74.51 %) was achieved when the stem explants were infected with ATCC15834 (OD600 = 0.8) strain of A. rhizogenes for 30 min on 1/2 MS medium supplemented with 200 µM AS. Based on the optimal protocol, RNAi hairy roots were induced by silencing the cytochrome P450 gene, PlCYP81Q38, which converts (+)-pinoresinol to (+)-sesamin by means of (+)-piperitol in P. leptostachya, to determine its in vivo role in the lignan biosynthetic pathway and verify the availability of this system. PlCYP81Q38 silencing resulted in significant down-regulation of the PlCYP81Q38 gene at the transcriptional level, consistent with a remarkable decrease in the content of leptostachyol acetate and 6-desmethoxy-leptostachyol acetate, demonstrating its significance in the production of lignans. In conclusion, the approaches and results presented here provide a solid foundation for elucidating the role of key genes involved in the lignan biosynthesis pathway in P. leptostachya and for the industrial production of lignan through plant biotechnology.

Identification and functional characterization of the dirigent gene family in Phryma leptostachya and the contribution of PlDIR1 in lignan biosynthesis

BackgroundFurofuran lignans, the main insecticidal ingredient in Phryma leptostachya, exhibit excellent controlling efficacy against a variety of pests. During the biosynthesis of furofuran lignans, Dirigent proteins (DIRs) are thought to be dominant in the stereoselective coupling of coniferyl alcohol to form ( ±)-pinoresinol. There are DIR family members in almost every vascular plant, but members of DIRs in P. leptostachya are unknown. To identify the PlDIR genes and elucidate their functions in lignan biosynthesis, this study performed transcriptome-wide analysis and characterized the catalytic activity of the PlDIR1 protein.ResultsFifteen full-length unique PlDIR genes were identified in P. leptostachya. A phylogenetic analysis of the PlDIRs classified them into four subfamilies (DIR-a, DIR-b/d, DIR-e, and DIR-g), and 12 conserved motifs were found among them. In tissue-specific expression analysis, except for PlDIR7, which displayed the highest transcript abundance in seeds, the other PlDIRs showed preferential expression in roots, leaves, and stems. Furthermore, the treatments with signaling molecules demonstrated that PlDIRs could be significantly induced by methyl jasmonate (MeJA), salicylic acid (SA), and ethylene (ETH), both in the roots and leaves of P. leptostachya. In examining the tertiary structure of the protein and the critical amino acids, it was found that PlDIR1, one of the DIR-a subfamily members, might be involved in the region- and stereo-selectivity of the phenoxy radical. Accordingly, LC–MS/MS analysis demonstrated the catalytic activity of recombinant PlDIR1 protein from Escherichia coli to direct coniferyl alcohol coupling into ( +)-pinoresinol. The active sites and hydrogen bonds of the interaction between PlDIR1 and bis-quinone methide (bisQM), the intermediate in ( +)-pinoresinol formation, were analyzed by molecular docking. As a result, 18 active sites and 4 hydrogen bonds (Asp-42, Ala-113, Leu-138, Arg-143) were discovered in the PlDIR1-bisQM complex. Moreover, correlation analysis indicated that the expression profile of PlDIR1 was closely connected with lignan accumulations after SA treatment.ConclusionsThe results of this study will provide useful clues for uncovering P. leptostachya's lignan biosynthesis pathway as well as facilitate further studies on the DIR family.

Changes in growth characteristics and secondary metabolites in Sinopodophyllum hexandrum with increasing age

Sinopodophyllum hexandrum is a perennial rhizomatous herb that produces the highly valued lignin podophyllotoxin (PPT). Although the effects of environmental factors on growth and secondary metabolites accumulation have been revealed, the changes with increasing plant age have not yet been conducted. Here, growth characteristics, contents of lignans and flavonoids, and expression levels of key genes in 2- to 9-year-old plants were systematically examined. The growth parameters significantly increased, and there were three distinctly morphological variants with single, two, and three leaves with increasing plant age. The accumulation of two lignans PPT and 4'-demethylepipodophyllotoxin (4'-DMEP), three target flavonoids, total flavonoids and phenolic, as well as in vitro antioxidant capacity maximized at 5–6 years. The expression levels of 8 key genes participating in lignan and flavonoid biosynthesis were up-regulated with increasing plant age. The findings provide the compelling evidence to guide sustainable cultivation and collection of S. hexandrum.

Identification of regulatory pathways of miRna and phasiRna related to flax lignan synthesis

ABSTRACT Lignan is an important functional health care substance. At present, research on the molecular mechanism of flax lignan biosynthesis is primarily focused at the transcriptional level, with no research on the regulatory mechanism of small RNAs, particularly phased small interfering RNAs (phasiRNAs) related to lignan content. In this study, we used flax seeds at different developmental stages as materials to construct small RNA libraries and degradation libraries. Eighteen known and 4 new miRNA families containing 40 and 4 miRNA members, respectively, were identified. A total of 104 target genes were identified. Using small RNA data for a genome-wide search, a total of twenty-two 21-nt PHAS loci and twenty-eight 24-nt PHAS loci were obtained. Five miRNAs capable of triggering target genes to produce phasiRNAs were identified. These 5 miRNAs triggered their target genes to produce 39 phasiRNAs. Three complete miRNA-PHAS loci-phasiRNA-target gene functional regulatory pathways were identified. Furthermore, qRT‒PCR confirmed an obvious regulatory relationship among the regulatory pathways. In this study, the molecular regulatory network among flax miRNAs, phasiRNAs and target genes was described from the perspective of lignan synthesis, providing an important scientific basis for the study of flax molecular breeding for high lignan content.