Assembly and analysis of the complete mitochondrial genome of Forsythia suspensa (Thunb.) Vahl.

BACKGROUND: Forsythia suspensa (Thunb.) Vahl is a valuable ornamental and medicinal plant. Although the nuclear and chloroplast genomes of F. suspensa have been published, its complete mitochondrial genome sequence has yet to be reported. In this study, the genomic DNA of F. suspensa yellowish leaf material was extracted, sequenced by using a mixture of Illumina Novaseq6000 short reads and Oxford Nanopore PromethION long reads, and the sequencing data were assembled and annotated. RESULT: The F. suspensa mitochondrial genome was obtained in the length of 535,692 bp with a circular structure, and the GC content was 44.90%. The genome contains 60 genes, including 36 protein-coding genes, 21 tRNA genes, and three rRNA genes. We further analyzed RNA editing of the protein-coding genes, relative synonymous codon usage, and sequence repeats based on the genomic data. There were 25 homologous sequences between F. suspensa mitochondria and chloroplast genome, which involved the transfer of 8 mitochondrial genes, and 9473 homologous sequences between mitochondrial and nuclear genomes. Analysis of the nucleic acid substitution rate, nucleic acid diversity, and collinearity of protein-coding genes of the F. suspensa mitochondrial genome revealed that the majority of genes may have undergone purifying selection, exhibiting a slower rate of evolution and a relatively conserved structure. Analysis of the phylogenetic relationships among different species revealed that F. suspensa was most closely related to Olea europaea subsp. Europaea. CONCLUSION: In this study, we sequenced, assembled, and annotated a high-quality F. suspensa mitochondrial genome. The results of this study will enrich the mitochondrial genome data of Forsythia, lay a foundation for the phylogenetic development of Forsythia, and promote the evolutionary analysis of Oleaceae species.

Comparative analysis of cold-responsive genes under short-term cold stimulation and cold-adaptive genes under long-term heterogeneous environments reveals a cold adaptation mechanism in weeping forsythia.

Identifying cold-related genes can provide insights into the cold adaptation mechanism of weeping forsythia. In this study, we compared the changes in gene expressions and physiological and biochemical indices under short-term cold stimulation with the changes in gene sequences under a long-term heterogeneous environment to investigate the cold adaptation mechanism in weeping forsythia. The data of adaptive gene sequence changes, e.g., single nucleotide polymorphisms, were obtained from previous landscape genomics studies. The physiological and biochemical indicators and transcriptome results showed that weeping forsythia initiated a series of programs, including increasing cell osmotic pressures, scavenging ROS, activating the defense mechanism that crosses with pathogen infection, and upregulating CBF/DREB1 transcription factor 1, to cope with short-term cold stress. A reanalysis of landscape genomic data suggested that weeping forsythia responded to long-term heterogeneous cold stress by the differentiation of genes related to synthesis of aromatic substances and adenosine triphosphate. Our results supported the hypothesis that the adaptation mechanisms of species to short-term environmental stimulation and long-term stress in heterogeneous environments are different. The differences in cold tolerance among populations are not necessarily obtained by changing cold-responsive gene sequences. This study provides new insights into the cold adaptation mechanisms of plants.

Fibrella aquatilis sp. nov., Fibrella rubiginis sp. nov. and Fibrella forsythiae sp. nov., isolated from freshwater, rusty iron and forsythia flower.

Three novel strains, designated as HMF5036T, HMF5335T and HMF5405T, were isolated from freshwater, rusty iron and forsythia flower, in Yong-in, Republic of Korea, respectively. They were Gram-stain-negative, facultatively anaerobic, non-motile, reddish-pigmented and rod-shaped bacteria. The predominant fatty acids of three strains were C16 : 1 ω5c and summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c). They were found to contain MK-7 as the predominant menaquinone. The major polar lipids are phosphatidylethanolamine, an unidentified aminophospholipid and an unidentified lipid. Strains HMF5036T, HMF5335T and HMF5405T exhibited the highest 16S rRNA gene sequence similarities of 91.8, 92.6 and 93.6 % to Fibrella aestuarina BUZ 2T and less than 88.7 % to other members of the family Spirosomaceae. Similarity values among the three isolates ranged from 94.9 to 96.6 %. Phylogenetic analysis based on the 16S rRNA gene sequences of the three isolates revealed that they formed a distinct clade within the family Spirosomaceae. The genome sizes of strains HMF5036T, HMF5335T and HMF5405T were 6.8, 6.4 and 7.8 Mbp, and their DNA G+C contents were 54.9, 54.0 and 52.1 mol%, respectively. The average nucleotide identity, digital DNA-DNA hybridization and amino acid identity values between three isolates and F. aestuarina BUZ 2T were 73.8-82.2, 19.6-25.4 and 75.0-87.5 %, respectively. These values were lower than the recommended threshold values for species delimitation. Based on the results of the phenotypic, genotypic, chemotaxonomic and phylogenetic investigations, three novel species, Fibrella aquatilis sp. nov., Fibrella rubiginis sp. nov. and Fibrella forsythiae sp. nov. are proposed. The type strains are HMF5036T (=KCTC 82476T=NBRC 115092T), HMF5335T (=KCTC 82477T=NBRC 115093T) and HMF5405T (=KCTC 82478T=NBRC 115094T), respectively.

Transcriptomic responses to drought stress among natural populations provide insights into local adaptation of weeping forsythia.

BACKGROUND: Understanding the genetic mechanisms of local adaptation is an important emerging topic in molecular ecology and evolutionary biology. RESULTS: Here, we identify the physiological changes and differential expression of genes among different weeping forsythia populations under drought stress in common garden experiments. Physiological results showed that HBWZ might have higher drought tolerance among four populations. RNA-seq results showed that significant differential expression in the genes responding to the synthesis of flavonoids, aromatic substances, aromatic amino acids, oxidation-reduction process, and transmembrane transport occured among four populations. By further reanalysis of results of previous studies, sequence differentiation was found in the genes related to the synthesis of aromatic substances among different weeping forsythia populations. CONCLUSIONS: Overall, our study supports the hypothesis that the dual differentiation in gene efficiency and expression increases among populations in response to heterogeneous environments and is an important evolutionary process of local adaptation. Here, we proposed a new working model of local adaptation of weeping forsythia populations under different intensities of drought stress, which provides new insights for understanding the genetic mechanisms of local adaptation for non-model species.

[Study on molecular of anti-tumor mechanism of Forsythia suspensa based on molecular docking and network pharmacology].

In this paper, the possible molecular mechanism of Forsythia suspensa for the anti-tumor effect was investigated through the network pharmacology and molecular docking. The main components of F. suspensa were obtained by literature mining and TCMSP database. Cancer-related genes were collected with use of GAD and OMIM databases. The interaction network of Compounds-Targets-Pathways was constructed through Cytoscpe software. The targets were analyzed by GO and KEGG means in DAVID database, and the KEGG signal pathways were visualized. Component-Target network analysis results were verified by PyRx molecular docking. The results showed that a total of 26 main components of F. suspensa may act on key targets such as AKT1, IL6, ESR1, EGFR, EGF and CCND1, and were involved in 20 signal pathways. Molecular docking analysis showed that hydrogen bonding, hydrophobic action and Pi-cation bonding maybe the main forms of interaction. In this study, we revealed the anti-tumor effect of F. suspensa through the network of Compounds-Targets-Pathways and molecular docking verification, providing reference and guidance for systematically elucidating the anti-tumor molecular mechanism of the main components of F. suspensa.

The Complete Chloroplast Genome Sequences of the Medicinal Plant Forsythia suspensa (Oleaceae).

Forsythia suspensa is an important medicinal plant and traditionally applied for the treatment of inflammation, pyrexia, gonorrhea, diabetes, and so on. However, there is limited sequence and genomic information available for F. suspensa. Here, we produced the complete chloroplast genomes of F. suspensa using Illumina sequencing technology. F. suspensa is the first sequenced member within the genus Forsythia (Oleaceae). The gene order and organization of the chloroplast genome of F. suspensa are similar to other Oleaceae chloroplast genomes. The F. suspensa chloroplast genome is 156,404 bp in length, exhibits a conserved quadripartite structure with a large single-copy (LSC; 87,159 bp) region, and a small single-copy (SSC; 17,811 bp) region interspersed between inverted repeat (IRa/b; 25,717 bp) regions. A total of 114 unique genes were annotated, including 80 protein-coding genes, 30 tRNA, and four rRNA. The low GC content (37.8%) and codon usage bias for A- or T-ending codons may largely affect gene codon usage. Sequence analysis identified a total of 26 forward repeats, 23 palindrome repeats with lengths >30 bp (identity > 90%), and 54 simple sequence repeats (SSRs) with an average rate of 0.35 SSRs/kb. We predicted 52 RNA editing sites in the chloroplast of F. suspensa, all for C-to-U transitions. IR expansion or contraction and the divergent regions were analyzed among several species including the reported F. suspensa in this study. Phylogenetic analysis based on whole-plastome revealed that F. suspensa, as a member of the Oleaceae family, diverged relatively early from Lamiales. This study will contribute to strengthening medicinal resource conservation, molecular phylogenetic, and genetic engineering research investigations of this species.

Molecular data and ecological niche modeling reveal population dynamics of widespread shrub Forsythia suspensa (Oleaceae) in China's warm-temperate zone in response to climate change during the Pleistocene.

BACKGROUND: Despite its high number of endemic deciduous broad-leaved species in China's warm-temperate zone, far less attention has been paid to phylogeographic studies in this region. In this work, the phylogeographic history of Forsythia suspensa endemic to China's warm-temperate zone was investigated to explore the effect of climate change during the Pleistocene on the distribution of this deciduous broad-leaved species in China. RESULTS: The cpDNA data revealed seven phylogeographical groups corresponding to geographical regions. By contrast, the nrDNA data supported the samples clustered into three groups, which was inconsistent with separate geographical regions supported by cpDNA data. Ecological niche modeling showed that the climatically suitable area during the cold period was larger than that during the warm period. CONCLUSIONS: Both molecular data and ecological niche modeling indicated that F. suspensa expanded to nearby low-elevation plains in the glacial periods, and retreated to mountaintops during interglacial warmer stages. This study thus supported that F. suspensa persisted in situ during the glacial of the Pleistocene with enlarged distribution area, contrary to the hypothesis of long distance southward migration or large-scale range contraction.

Seasonal alteration in amounts of lignans and their glucosides and gene expression of the relevant biosynthetic enzymes in the Forsythia suspense leaf.

Lignans of Forsythia spp. are essential components of various Chinese medicines and health diets. However, the seasonal alteration in lignan amounts and the gene expression profile of lignan-biosynthetic enzymes has yet to be investigated. In this study, we have assessed seasonal alteration in amounts of major lignans, such as pinoresinol, matairesinol, and arctigenin, and examined the gene expression profile of pinoresinol/lariciresinol reductase (PLR), pinoresinol-glucosylating enzyme (UGT71A18), and secoisolariciresinol dehydrogenase (SIRD) in the leaf of Forsythia suspense from April to November. All of the lignans in the leaf continuously increased from April to June, reached the maximal level in June, and then decreased. Ninety percent of pinoresinol and matairesinol was converted into glucosides, while approximately 50% of arctigenin was aglycone. PLR was stably expressed from April to August, whereas the PLR expression was not detected from September to November. In contrast, the UGT71A18 expression was found from August to November, but not from April to July. The SIRD expression was prominent from April to May, not detected in June to July, and then increased again from September to November. These expression profiles of the lignan-synthetic enzymes are largely compatible with the alteration in lignan contents. Furthermore, such seasonal lignan profiles are in good agreement with the fact that the Forsythia leaves for Chinese medicinal tea are harvested in June. This is the first report on seasonal alteration in lignans and the relevant biosynthetic enzyme genes in the leaf of Forsythia species.

[Molecular identification of raw materials from lian qiao bai du wan].

Lian Qiao Bai Du Wan was used to study the identification of Chinese patent medicine by molecular marker technique. DNA was extracted through modified CTAB method. The psbA-trnH and rbcL sequences were gradient amplified, and PCR products were ligated with the pEASY-T5 vector and then transformed into Trans1-T1 cells, respectively. Clones were selected randomly and sequenced. All sequences were analyzed by BlastN and the neighbor-joining (NJ) phylogenetic tree was constructed by MEGA 4.0. The results showed that nine kinds of medicinal materials can be identified by psbA-trnH sequences, and six kinds of medicinal materials by rbcL sequences from Lian Qiao Bai Du Wan. Molecular marker technique can stably and accurately distinguish multi-origin medicinal materials in Chinese patent medicine.

Expression dosage effects of a small number of genes after the artificial doubling of weeping forsythia.

Whole genome doubling (WGD) plays a critical role in plant evolution, yet the mechanisms underlying the maintenance of overall equilibrium following an artificial doubling event, as well as its impact on phenotype and adaptability, remain unclear. By comparing the gene expression of naturally occurring weeping forsythia diploids and colchicine-induced autotetraploids under normal growth conditions and cold stress, we identified gene expression dosage responses resulting from ploidy change. Only a small proportion of effectively expressed genes showed dosage effect, and most genes did not exhibit significant expression differences. However, the genes that showed expression dosage effect were largely random. The autotetraploids had slower overall growth rates, possibly resulting from negative gene dosage effects on zeatin synthesis and multiple metabolic delays caused by other negative dosage genes. Our comparative analysis of cold response genes in diploids and autotetraploids revealed that genes related to "response to abscisic acid" and "cold acclimation" were key factors contributing to greater cold tolerance in the autotetraploids. In particular, gene expression related to "cold acclimation" might mitigate the effects of cold stress. Taken together, our findings suggested that overall gene expression equilibrium following WGD of weeping forsythia autotetraploids was achieved through the inactivation of the majority of duplicated genes. Our research provides new insights into the mechanisms regulating expression dosage balance following polyploidization events.

Identification of yield-related genes through genome-wide association: case study of weeping forsythia, an emerging medicinal crop.

KEY MESSAGE: This study identified candidate genes related to fruit yield for an emerging medicinal crop, weeping forsythia. BACKGROUND: The genetic basis of crop yield is an agricultural research hotspot. Identifying the genes related to yield traits is the key to increase the yield. Weeping forsythia is an emerging medicinal crop that currently lacks excellent varieties. The genes related to fruit yield in weeping forsythia have not been identified. OBJECTIVE: Thus, we aimed to screen the candidate genes related to fruit yield of weeping forsythia by using genome-wide association analysis. METHODS: Here, 60 samples from the same field and source of weeping forsythia were collected to identify its yield-related candidate genes. Association analysis was performed on the variant loci and the traits related to yield, i.e., fruit length, width, thickness, and weight. RESULTS: Results from admixture, neighbor-joining, and kinship matrix analyses supported the non-significant genetic differentiation of these samples. Significant association was found between 2 variant loci and fruit length, 8 loci and fruit width, 24 loci and fruit thickness, and 13 loci and fruit weight. Further search on the 20 kb up/downstream of these variant loci revealed 1 gene related to fruit length, 16 genes related to fruit width, 12 genes related to fruit thickness, and 13 genes related to fruit weight. Among which, 4 genes, namely, WRKY transcription factor 35, salicylic acid-binding protein, auxin response factor 6, and alpha-mannosidase were highly related to the fruit development of weeping forsythia. CONCLUSION: This study identify four candidate genes related to fruit development, which will provide useful information for the subsequent molecular-assisted and genetic breeding of weeping forsythia.

Transgenic Forsythia plants expressing sesame cytochrome P450 produce beneficial lignans.

Lignans are widely distributed plant secondary metabolites that have received attention for their benefits to human health. Sesamin is a furofran lignan that is conventionally extracted from Sesamum seeds and shows anti-oxidant and anti-inflammatory activities in the human liver. Sesamin is biosynthesized by the Sesamum-specific enzyme CYP81Q1, and the natural sources of sesamin are annual plants that are at risk from climate change. In contrast, Forsythia species are widely distributed perennial woody plants that highly accumulate the precursor lignan pinoresinol. To sustainably supply sesamin, we developed a transformation method for Forsythia leaf explants and generated transgenic Forsythia plants that heterologously expressed the CYP81Q1 gene. High-performance liquid chromatography (HPLC) and LC-mass spectrometry analyses detected sesamin and its intermediate piperitol in the leaves of two independent transgenic lines of F. intermedia and F. koreana. We also detected the accumulation of sesamin and piperitol in their vegetatively propagated descendants, demonstrating the stable and efficient production of these lignans. These results indicate that CYP81Q1-transgenic Forsythia plants are promising prototypes to produce diverse lignans and provide an important strategy for the cost-effective and scalable production of lignans.

Molecular phylogeny of tribe Forsythieae (Oleaceae) based on nuclear ribosomal DNA internal transcribed spacers and plastid DNA trnL-F and matK gene sequences.

The tribe Forsythieae comprises 2 genera (Forsythia and Abeliophyllum) and 14 species distributed mostly in the Far East. Although Forsythieae is considered monophyletic, with many symplesiomorphic characters, the phylogenetic status of Abeliophyllum remains controversial. We assessed the phylogenetic relationships of Forsythieae, based on a 3.3-kb plastid fragment (trnL-F region and matK gene) and nuclear internal transcribed spacer (ITS) region DNA sequences. We obtained a highly resolved and strongly supported topology with possible outgroups. The topology of the combined tree was congruent with those of the ITS region and matK gene. Maximum parsimony, maximum likelihood, and Bayesian inference tree analyses for the combined data also yielded identical relationships. Combined sequence data strongly supported the monophyly of Forsythieae and the close relationship between Fontanesia and Jasminum. Oleaceae, not Fontanesia, was found to be a sister group to Forsythieae. Moreover, the genus Abeliophyllum was distinctly independent of Forsythia. Three Forsythia lineages were suggested: (a) ONJ (ovata-nakaii-japonica clade), (b) VGE (viridissima-giraldiana-europaea), and (c) KISS (koreana-intermedia-saxatilis-suspensa). Our results indicated that F. × intermedia is not a hybrid between F. suspensa and F. viridissima, but further studies are needed to determine its taxonomic identity. Furthermore, the diverse fruit shapes in Oleaceae are assumed to be the result of parallelism or convergence.

Application of genetic marker and real-time polymerase chain reaction for discrimination between Forsythia viridissima and Forsythia suspensa.

Forsythiae Fructus has been used as a herbal medicine for a fruit of Forsythia viridissima LINDLEY or Forsythia suspensa VAHL (Oleaceae). In Korea, the fruit of Forsythia viridissima is used and in China, the fruit of Forsythia suspensa is used generally. There are differences in the amount and distribution of constituents between Forsythia viridissima (FV) and Forsythia suspensa (FS). Accordingly, a discrimination of these two herbal drugs is needed. In this study, we designed FV genetic marker based on the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA that can discriminate Forsythia viridissima and Forsythia suspensa and species-specific amplification product 252 bp was confirmed. Using the real-time polymerase chain reaction (PCR) (allelic discrimination) analysis, an accurate discrimination between Forsythia viridissima and Forsythia suspensa was accomplished. Accordingly, with the use of PCR analysis based on ITS region sequence of ribosomal DNA and the real-time PCR analysis which can efficiently discriminate between Forsythia viridissima and Forsythia suspensa was developed.

Metabolic engineering of lignan biosynthesis in Forsythia cell culture.

Lignans are a large class of secondary metabolites in plants, with numerous biological effects in mammals, including antitumor and antioxidant activities. Sesamin, the most abundant furofuran-class lignan in sesame seeds (Sesamum plants), is produced by the cytochrome P450 enzyme CYP81Q1 from the precursor lignan, pinoresinol. In contrast, Forsythia plants produce dibenzylbutyrolactone-class lignans, such as matairesinol, from pinoresinol via the catalysis of pinoresinol/lariciresinol reductase (PLR) and secoisolariciresinol dehydrogenase. Here we present the engineering of lignan biosynthesis in Forsythia cell suspension cultures for the development of an efficient production method of beneficial lignans. A suspension cell culture prepared from leaves of Forsythia koreana produced lignans, mainly pinoresinol and matairesinol glucosides, at levels comparable with that obtained from the leaves. In an attempt to increase the pinoresinol content in Forsythia, we generated a transgenic cell line overexpressing an RNA interference (RNAi) construct of PLR (PLR-RNAi). Down-regulation of PLR expression led to a complete loss of matairesinol and an accumulation of approximately 20-fold pinoresinol in its glucoside form in comparison with the non-transformant. Moreover, the Forsythia transgenic cells co-expressing CYP81Q1 and PLR-RNAi exhibited production of sesamin as well as accumulation of pinoresinol glucoside. These data suggest Forsythia cell suspension to be a promising tool for the engineering of lignan production. To the best of our knowledge, this is the first report on transgenic production of an exogenous lignan in a plant species.

[Identify the original plant of Forsythia suspensa by nucleotide sequence].

OBJECTIVE: To investigate the means of distinguishing the original plant of Forsythia suspensa from confusion. METHODS: To amplify the chloroplast matK gene by PCR using a consensus primer set and determine their nucleotide sequence by PCR direct sequencing. The ITS sequences were gained from NCBI. The characteristic analysis of matK and ITS sequences were generated using Clustal aligned. RESULTS: There were 30 bp and 8 bp unique nucleotide in ITS and matK sequence in Forsythia suspensa. The matK gene and ITS sequences might be good molecular marker to be used to identify the original plant of Forsythia suspensa. CONCLUSION: The sequence analysis of matK gene ITS sequences might become the mean to identify the original plant of Forsythia suspensa.

Enhancing lignan biosynthesis by over-expressing pinoresinol lariciresinol reductase in transgenic wheat.

Lignans are phenylpropane dimers that are biosynthesized via the phenylpropanoid pathway, in which pinoresinol lariciresinol reductase (PLR) catalyzes the last steps of lignan production. Our previous studies demonstrated that the contents of lignans in various wheat cultivars were significantly associated with anti-tumor activities in APC(Min) mice. To enhance lignan biosynthesis, this study was conducted to transform wheat cultivars ('Bobwhite', 'Madison', and 'Fielder', respectively) with the Forsythia intermedia PLR gene under the regulatory control of maize ubiquitin promoter. Of 24 putative transgenic wheat lines, we successfully obtained 3 transformants with the inserted ubiquitin-PLR gene as screened by PCR. Southern blot analysis further demonstrated that different copies of the PLR gene up to 5 were carried out in their genomes. Furthermore, a real-time PCR indicated approximately 17% increase of PLR gene expression over the control in 2 of the 3 positive transformants at T(0) generation. The levels of secoisolariciresinol diglucoside, a prominent lignan in wheat as determined by HPLC-MS, were found to be 2.2-times higher in one of the three positive transgenic sub-lines at T(2 )than that in the wild-type (117.9 +/- 4.5 vs. 52.9 +/- 19.8 mug/g, p <0.005). To the best of our knowledge, this is the first study that elevated lignan levels in a transgenic wheat line has been successfully achieved through genetic engineering of over-expressed PLR gene. Although future studies are needed for a stably expression and more efficient transformants, the new wheat line with significantly higher SDG contents obtained from this study may have potential application in providing additive health benefits for cancer prevention.

De Novo Transcriptomes of Forsythia koreana Using a Novel Assembly Method: Insight into Tissue- and Species-Specific Expression of Lignan Biosynthesis-Related Gene.

Forsythia spp. are perennial woody plants which are one of the most extensively used medicinal sources of Chinese medicines and functional diets owing to their lignan contents. Lignans have received widespread attention as leading compounds in the development of antitumor drugs and healthy diets for reducing the risks of lifestyle-related diseases. However, the molecular basis of Forsythia has yet to be established. In this study, we have verified de novo deep transcriptome of Forsythia koreana leaf and callus using the Illumina HiSeq 1500 platform. A total of 89 million reads were assembled into 116,824 contigs using Trinity, and 1,576 of the contigs displayed the sequence similarity to the enzymes responsible for plant specialized metabolism including lignan biosynthesis. Notably, gene ontology (GO) analysis indicated the remarkable enrichment of lignan-biosynthetic enzyme genes in the callus transcriptome. Nevertheless, precise annotation and molecular phylogenetic analyses were hindered by partial sequences of open reading frames (ORFs) of the Trinity-based contigs. To obtain more numerous contigs harboring a full-length ORF, we developed a novel overlapping layout consensus-based procedure, virtual primer-based sequence reassembly (VP-seq). VP-seq elucidated 709 full-length ORFs, whereas only 146 full-length ORFs were assembled by Trinity. The comparison of expression profiles of leaf and callus using VP-seq-based full-length ORFs revealed 50-fold upregulation of secoisolariciresinol dehydrogenase (SIRD) in callus. Expression and phylogenetic cluster analyses predicted candidates for matairesinol-glucosylating enzymes. We also performed VP-seq analysis of lignan-biosynthetic enzyme genes in the transcriptome data of other lignan-rich plants, Linum flavum, Linum usitatissimum and Podophyllum hexandrum. The comparative analysis indicated both common gene clusters involved in biosynthesis upstream of matairesinol such as SIRD and plant lineage-specific gene clusters, in particular, genes responsible for biosynthetic pathways for production of podophyllotoxin; CYP71BE54, a key enzyme gene for podophyllotoxin biosynthesis in P. hexandrum, was not found in L. flavum, although both P. hexandrum. and L. flavum yield podophyllotoxin. Altogether, these data have established the fruitful molecular basis of Forsythia and provided insight into the molecular evolution and diversity of lignan biosynthetic pathways.

Generation of Triple-Transgenic Forsythia Cell Cultures as a Platform for the Efficient, Stable, and Sustainable Production of Lignans.

Sesamin is a furofuran lignan biosynthesized from the precursor lignan pinoresinol specifically in sesame seeds. This lignan is shown to exhibit anti-hypertensive activity, protect the liver from damages by ethanol and lipid oxidation, and reduce lung tumor growth. Despite rapidly elevating demand, plant sources of lignans are frequently limited because of the high cost of locating and collecting plants. Indeed, the acquisition of sesamin exclusively depends on the conventional extraction of particular Sesamum seeds. In this study, we have created the efficient, stable and sustainable sesamin production system using triple-transgenic Forsythia koreana cell suspension cultures, U18i-CPi-Fk. These transgenic cell cultures were generated by stably introducing an RNAi sequence against the pinoresinol-glucosylating enzyme, UGT71A18, into existing CPi-Fk cells, which had been created by introducing Sesamum indicum sesamin synthase (CYP81Q1) and an RNA interference (RNAi) sequence against pinoresinol/lariciresinol reductase (PLR) into F. koreanna cells. Compared to its transgenic prototype, U18i-CPi-Fk displayed 5-fold higher production of pinoresinol aglycone and 1.4-fold higher production of sesamin, respectively, while the wildtype cannot produce sesamin due to a lack of any intrinsic sesamin synthase. Moreover, red LED irradiation of U18i-CPi-Fk specifically resulted in 3.0-fold greater production in both pinoresinol aglycone and sesamin than production of these lignans under the dark condition, whereas pinoresinol production was decreased in the wildtype under red LED. Moreover, we developed a procedure for sodium alginate-based long-term storage of U18i-CPi-Fk in liquid nitrogen. Production of sesamin in U18i-CPi-Fk re-thawed after six-month cryopreservation was equivalent to that of non-cryopreserved U18i-CPi-Fk. These data warrant on-demand production of sesamin anytime and anywhere. Collectively, the present study provides evidence that U18i-CP-Fk is an unprecedented platform for efficient, stable, and sustainable production of sesamin, and shows that a transgenic and specific light-regulated Forsythia cell-based metabolic engineering is a promising strategy for the acquisition of rare and beneficial lignans.