Changes in Physiological Traits, Gene Expression and Phytochemical Profile of Mentha piperita in Response to Elicitor.

Peppermint (Mentha piperita) is a perennial medicinal plant containing active ingredients that can be used for treating liver and prostate cancers, acute respiratory infections, allergies, digestive problems, neuralgia, and migraines. The objective of this research is to investigate the expression of essential genes in the menthol pathway of Mentha piperita, including Pulegone reductase (Pr), Menthofuran synthase (Mfs), and limonene synthase (Ls) using qPCR, physiological analysis and essential oil composition in response to methyl jasmonate (MeJA) (0.5 mM) elicitation. Physiological analysis showed that 0.5 mM MeJA triggers defensive responsiveness in Mentha piperita by increasing superoxide dismutase (SOD) and Peroxidase (POD) enzymes activity. The highest transcript levels of Pr and Mfs genes were observed during 8 and 12 h after treatment respectively, but following 24 h, they were down-regulated. Essential oil analysis indicated that the percentage of constituents in the essential oil was changed using MeJA at 48 h and 96 h after post-treatment. Effective antimicrobial compounds, α-pinene, ß-pinene, linalool and methyl acetate, were induced after 48 h. A non-significant positive relationship was detected between menthol content, and expression of the Pr and Mfs genes. Due to the significant change in the expression of Pr and Mfs genes in the menthol pathway, role of Pr gene in directing the pathway to the valuable compound menthol and deviation of the menthol pathway to the menthofuran as an undesirable component of essential oil by Mfs gene, it can be deduced that they are the most critical genes in response to MeJA treatment, which are appropriate candidates for metabolite engineering. In addition, MeJA improved defensive responsiveness and percentage of some constituents with antimicrobial properties in Mentha piperita.

Transcriptomic data reveals the dynamics of terpenoids biosynthetic pathway of fenugreek.

Medicinal plants are rich sources for treating various diseases due their bioactive secondary metabolites. Fenugreek (Trigonella foenum-graecum) is one of the medicinal plants traditionally used in human nutrition and medicine which contains an active substance, called diosgenin, with anticancer properties. Biosynthesis of this important anticancer compound in fenugreek can be enhanced using eliciting agents which involves in manipulation of metabolite and biochemical pathways stimulating defense responses. Methyl jasmonate elicitor was used to increase diosgenin biosynthesis in fenugreek plants. However, the molecular mechanism and gene expression profiles underlying diosgening accumulation remain unexplored. In the current study we performed an extensive analysis of publicly available RNA-sequencing datasets to elucidate the biosynthesis and expression profile of fenugreek plants treated with methyl jasmonate. For this purpose, seven read datasets of methyl jasmonate treated plants were obtained that were covering several post-treatment time points (6-120 h). Transcriptomics analysis revealed upregulation of several key genes involved in diosgenein biosynthetic pathway including Squalene synthase (SQS) as the first committed step in diosgenin biosynthesis as well as Squalene Epoxidase (SEP) and Cycloartenol Synthase (CAS) upon methyl jasmonate application. Bioinformatics analysis, including gene ontology enrichment and pathway analysis, further supported the involvement of these genes in diosgenin biosynthesis. The bioinformatics analysis led to a comprehensive validation, with expression profiling across three different fenugreek populations treated with the same methyl jasmonate application. Initially, key genes like SQS, SEP, and CAS showed upregulation, followed by later upregulation of Δ24, suggesting dynamic pathway regulation. Real-time PCR confirmed consistent upregulation of SQS and SEP, peaking at 72 h. Additionally, candidate genes Δ24 and SMT1 highlighted roles in directing metabolic flux towards diosgenin biosynthesis. This integrated approach validates the bioinformatics findings and elucidates fenugreek's molecular response to methyl jasmonate elicitation, offering insights for enhancing diosgenin yield. The assembled transcripts and gene expression profiles are deposited in the Zenodo open repository at https://doi.org/10.5281/zenodo.8155183 .

Improvement of Panax notoginseng saponin accumulation triggered by methyl jasmonate under arbuscular mycorrhizal fungi.

Panax notoginseng is a highly valued perennial medicinal herb plant in Yunnan Province, China, and the taproots are the main medicinal parts that are rich in active substances of P. notoginseng saponins. The main purpose of this study is to uncover the physiological and molecular mechanism of Panax notoginseng saponin accumulation triggered by methyl jasmonate (MeJA) under arbuscular mycorrhizal fungi (AMF) by determining physiological indices, high-throughput sequencing and correlation analysis. Physiological results showed that the biomass and saponin contents of P. notoginseng, the concentrations of jasmonic acids (JAs) and the key enzyme activities involved in notoginsenoside biosynthesis significantly increased under AMF or MeJA, but the interactive treatment of AMF and MeJA weakened the effect of AMF, suggesting that a high concentration of endogenous JA have inhibitory effect. Transcriptome sequencing results indicated that differential expressed genes (DEGs) involved in notoginsenoside and JA biosynthesis were significantly enriched in response to AMF induction, e.g., upregulated genes of diphosphocytidyl-2-C-methyl-d-erythritol kinases (ISPEs), cytochrome P450 monooxygenases (CYP450s)_and glycosyltransferases (GTs), while treatments AMF-MeJA and salicylhydroxamic acid (SHAM) decreased the abundance of these DEGs. Interestingly, a high correlation presented between any two of saponin contents, key enzyme activities and expression levels of DEGs. Taken together, the inoculation of AMF can improve the growth and saponin accumulation of P. notoginseng by strengthening the activities of key enzymes and the expression levels of encoding genes, in which the JA regulatory pathway is a key link. This study provides references for implementing ecological planting of P. notoginseng, improving saponin accumulation and illustrating the biosynthesis mechanism.

Genomic characterization of WRKY transcription factors related to secoiridoid biosynthesis in Gentiana macrophylla.

Gentiana macrophylla is one of Chinese herbal medicines in which 4 kinds of iridoids or secoiridoids, such as loganic acid, sweroside, swertiamarin, and gentiopicroside, are identified as the dominant medicinal secondary metabolites. WRKY, as a large family of transcription factors (TFs), plays an important role in the synthesis of secondary metabolites in plants. Therefore, WRKY genes involved in the biosynthesis of secoiridoids in G. macrophylla were systematically studied. First, a comprehensive genome-wide analysis was performed, and 42 GmWRKY genes were identified, which were unevenly distributed in 12 chromosomes. Accordingly, gene structure, collinearity, sequence alignment, phylogenetic, conserved motif and promoter analyses were performed, and the GmWRKY proteins were divided into three subfamilies based on phylogenetic and multiple sequence alignment analyses. Moreover, the enzyme-encoding genes of the secoiridoid biosynthesis pathway and their promoters were then analysed, and the contents of the four secoiridoids were determined in different tissues. Accordingly, correlation analysis was performed using Pearson's correlation coefficient to construct WRKY gene-enzyme-encoding genes and WRKY gene-metabolite networks. Meanwhile, G. macrophylla seedlings were treated with methyl jasmonate (MeJA) to detect the dynamic change trend of GmWRKYs, biosynthetic genes, and medicinal ingredient accumulation. Thus, a total of 12 GmWRKYs were identified to be involved in the biosynthesis of secoiridoids, of which 8 (GmWRKY1, 6, 12, 17, 33, 34, 38 and 39) were found to regulate the synthesis of gentiopicroside, and 4 (GmWRKY7, 14, 26 and 41) were found to regulate the synthesis of loganic acid. Taken together, this study systematically identified WRKY transcription factors related to the biosynthesis of secoiridoids in G. macrophylla, which could be used as a cue for further investigation of WRKY gene functions in secondary metabolite accumulation.

Genome-wide identification and integrated analysis of TCP genes controlling ginsenoside biosynthesis in Panax ginseng.

Panax ginseng is an important medicinal plant, and ginsenosides are the main bioactive molecules of ginseng. The TCP (TBI, CYC, PCF) family is a group of transcription factors (TFs) that play an important role in plant growth and development, hormone signalling and synthesis of secondary metabolites. In our study, 78 PgTCP transcripts were identified from the established ginseng transcriptome database. A phylogenetic tree analysis showed that the 67 PgTCP transcripts with complete open reading frames were classified into three subfamilies, including CIN, PCF, and CYC/TB1. Protein structure analysis showed that PgTCP genes had bHLH structures. Chromosomal localization analysis showed that 63 PgTCP genes were localized on 17 of the 24 chromosomes of the Chinese ginseng genome. Expression pattern analysis showed that PgTCP genes differed among different lineages and were spatiotemporally specific. Coexpression network analysis indicated that PgTCP genes were coexpressed and involved in plant activities or metabolic regulation in ginseng. The expression levels of PgTCP genes from class I (PCF) were significantly downregulated, while the expression levels of PgTCP genes from class II (CIN and CYC/TB1) were upregulated, suggesting that TCP genes may be involved in the regulation of secondary metabolism in ginseng. As the PgTCP26-02 gene was found to be related to ginsenoside synthesis, its predicted protein structure and expression pattern were further analysed. Our results provide new insights into the origin, differentiation, evolution and function of the PgTCP gene family in ginseng, as well as the regulation of plant secondary metabolism.

Integrative effects of phytohormones in the phenolic acids production in Salvia verticillata L. under multi-walled carbon nanotubes and methyl jasmonate elicitation.

Salvia verticillata L. is a well-known herb rich in rosmarinic acid (RA) and with therapeutic values. To better understand the possible roles of phytohormones in the production of phenolic acids in S. verticillata, in this work, we investigated some physiological and biochemical responses of the species to methyl jasmonate (MJ) and multi-walled carbon nanotubes (MWCNTs) as two effective elicitors. The leaves were sprayed with aqueous solutions containing 100 mg L-1 MWCNTs and 100 µM MJ and then harvested during interval times of exposure up to 96 h. The level of abscisic acid, as the first effective phytohormone, was altered in the leaves in response to MJ and MWCNTs elicitation (2.26- and 3.06-fold more than the control, respectively), followed by significant increases (P ˂ 0.05) detected in jasmonic acid and salicylic acid contents up to 8 h after exposure. Obtained data revealed that simultaneously with changes in phytohormone profiles, significant (P ˂ 0.05) rises were observed in the content of H2O2 (8.85- and 9.74-folds of control), and the amount of lipid peroxidation (10.18- and 17.01-folds of control) during the initial times after exposure to MJ and MWCNTs, respectively. Later, the content of phenolic acids increased in the elicited leaves due to changes in the transcription levels of key enzymes involved in their biosynthesis pathways, so 2.71- and 11.52-fold enhances observed in the RA content of the leaves after exposure to MJ and MWCNTs, respectively. It is reasonable to conclude that putative linkages between changes in some phytohormone pools lead to the accumulation of phenolic acids in the leaves of S. verticillata under elicitation. Overall, the current findings help us improve our understanding of the signal transduction pathways of the applied stimuli that led to enhanced secondary metabolite production in medicinal plants.

Phytohormone Response of Drought-Acclimated Illicium difengpi (Schisandraceae).

Illicium difengpi (Schisandraceae), which is an endemic, medicinal, and endangered species found in small and isolated populations that inhabit karst mountain areas, has evolved strategies to adapt to arid environments and is thus an excellent material for exploring the mechanisms of tolerance to severe drought. In experiment I, I. difengpi plants were subjected to three soil watering treatments (CK, well-watered treatment at 50% of the dry soil weight for 18 days; DS, drought stress treatment at 10% of the dry soil weight for 18 days; DS-R, drought-rehydration treatment at 10% of the dry soil weight for 15 days followed by rewatering to 50% of the dry soil weight for another 3 days). The effects of the drought and rehydration treatments on leaf succulence, phytohormones, and phytohormonal signal transduction in I. difengpi plants were investigated. In experiment II, exogenous abscisic acid (ABA, 60 mg L-1) and zeatin riboside (ZR, 60 mg L-1) were sprayed onto DS-treated plants to verify the roles of exogenous phytohormones in alleviating drought injury. Leaf succulence showed marked changes in response to the DS and DS-R treatments. The relative concentrations of ABA, methyl jasmonate (MeJA), salicylic acid glucoside (SAG), and cis-zeatin riboside (cZR) were highly correlated with relative leaf succulence. The leaf succulence of drought-treated I. difengpi plants recovered to that observed with the CK treatment after exogenous application of ABA or ZR. Differentially expressed genes involved in biosynthesis and signal transduction of phytohormones (ABA and JA) in response to drought stress were identified by transcriptomic profiling. The current study suggested that the phytohormones ABA, JA, and ZR may play important roles in the response to severe drought and provides a preliminary understanding of the physiological mechanisms involved in phytohormonal regulation in I. difengpi, an endemic, medicinal, and highly drought-tolerant plant found in extremely small populations in the karst region of South China.

Biochemical changes and enhanced accumulation of phenolic compounds in cell culture of Perilla frutescens (L.) by nano-chemical elicitation.

Perilla frutescens (L.) Britt is a renowned medicinal plant with pharmaceutically valuable phenolic acids and flavonoids. The present study was aimed to study the eliciting effect of silver and copper nanoparticles (AgNPs and CuNPs, 50 and 100 mg/L), and methyl jasmonate (MeJa, 50 and 100 µM) on the biochemical traits, the accumulation of phenolic compounds and antioxidative capacity of P. frutescens cell suspension culture. Suspension cells were obtained from friable calli derived from nodal explants in Murashige and Skoog (MS) liquid medium containing 1 mg/L 2,4-D and 1 mg/L BAP. The 21 days old cell suspension culture established from nodal explant derived callus supplemented with 100 mg/L MeJa resulted in the highest activity of catalase and guaiacol peroxidase enzymes, and CuNPs 100 mg/L treated cells indicated the maximum content of total phenol, total anthocyanin, superoxide dismutase, malondialdehyde, and H2O2. Also, the highest content of ferulic acid (1.41 ± 0.03, mg/g DW), rosmarinic acid (19.29 ± 0.12, mg/g DW), and phenylalanine ammonia-lyase (16.81 ± 0.18, U/mg protein) were observed with 100 mg/L CuNPs, exhibiting a total increase of 1.58-fold, 2.12-fold, and 1.51-fold, respectively, higher than untreated cells. On the other hand, AgNPs 100 mg/L treated cells indicated the most amounts of caffeic acid (0.57 ± 0.03, mg/g DW) and rutin (1.13 ± 0.07, mg/g DW), as well as the highest scavenging potential of free radicals. Overall, the results of the present study can be applied for the large-scale production of valuable phenolic acids and flavonoids from P. frutescens through CuNPs and AgNPs 100 mg/L elicited cell suspension cultures.

Formation and isomerization of (Z)-methyl epijasmonate, the key contributor of the orchid-like aroma, during tea processing.

The elegant orchid-like fragrance of tea has always been tea processors and consumers' top priority. Controlling the production process is very important for tea aroma formation. This study aims to investigate the synthesis of (Z)-methyl epijasmonate (epi-MeJA), a key contributor to orchid-like aroma properties in tea, during tea processing. The changes in content of epi-MeJA were analysed during the processing of two tea varieties (Anxi Tieguanyin and Taiping Houkui) with typical orchid-like fragrance. It was found to be mainly synthesized and accumulated during tea processing, as fresh tea leaves contained little or even no epi-MeJA. Its content was positively correlated with the processing time in the enzyme active stages (before fixation). During the fixation stages, isomerization occurred due to high temperatures, with a degree of epimerization to the much less odor active isomer (Z)-methyl jasmonate. Isomerization could also occurred during the drying process, which is dominated by the drying temperature.

Genome-wide characterization, evolutionary analysis, and expression pattern analysis of the trihelix transcription factor family and gene expression analysis under MeJA treatment in Panax ginseng.

Panax ginseng is a well-known medicinal plant with several pharmacological uses in China. The trihelix family transcription factors, also known as GT factors, can be involved in the regulation of growth and developmental processes in plants. There have been no in-depth reports or systematic studies about the trihelix transcription factor in ginseng. In this study, the structure, chromosomal localization, gene duplication, phylogeny, functional differentiation, expression patterns and coexpression interactions of trihelix transcripts were analysed using bioinformatics methods based on the ginseng transcriptome database. Thirty-two trihelix transcription factor genes were identified in ginseng, and these genes were alternatively spliced to obtain 218 transcripts. These transcripts were unevenly distributed on different chromosomes of ginseng, and phylogenetic analysis classified the PgGT transcripts into five subgroups. Gene Ontology (GO) analysis classified PgGT transcripts into eight functional subclasses, indicating that they are functionally diverse. The expression pattern analysis of 218 PgGT transcripts revealed that their expression was tissue-specific and spatiotemporally-specific in 14 different tissues of 4-year-old ginseng, 4 different ages of ginseng roots, and 42 farmers' cultivars of 4-year-old ginseng roots. Despite the differences in the expression patterns of these transcripts, coexpression network analysis revealed that these transcripts could be expressed synergistically in ginseng. In addition, two randomly selected PgGT transcripts in each of the five different subfamilies were subjected to methyl jasmonate treatment at different times, and PgGT was able to respond to the regulation of methy1 jasmonate. These results provide a theoretical basis and gene resources for an in-depth study of the function of trihelix genes in other plants.

Transcriptomics Reveals the Molecular Basis for Methyl Jasmonate to Promote the Synthesis of Monoterpenoids in Schizonepeta tenuifolia Briq.

BACKGROUND: Methyl jasmonate has an important effect on the synthesis of plant secondary metabolites. Schizonepeta tenuifolia Briq. has a wide range of pharmacological effects and the secondary metabolites are dominated by monoterpenes (pulegone, menthone). OBJECTIVE: It is essential to determine the changes in secondary metabolites in S. tenuifolia under methyl jasmonate treatment and to probe the molecular mechanism. This can improve the accumulation of secondary metabolites in the medicinal plant S. tenuifolia and enrich the information gene expression at different MeJA levels, which can help to elucidate the molecular mechanism of monoterpenoid synthesis in S. tenuifolia. METHODS: In this study, we determined the changes in the content of monoterpenoids in S. tenuifolia under methyl jasmonate treatment. Meanwhile, we established a transcriptome database of S. tenuifolia under methyl jasmonate level using high-throughput sequencing. RESULTS: A certain concentration of MeJA promoted the accumulation of monoterpenoids in S. tenuifolia. The transcriptome database of S. tenuifolia leaves under 0, 50, 100 and 250 µM MeJA treatment was established. We generated 88,373 unigenes with an N50 length of 2678 bp, of which 50,843 (57.53%) can be annotated in at least one database. Compared with the CK (0 µM) group, 12,557 (50 µM), 15,409 (100 µM) and 13,286 (250 µM) differentially expressed genes were identified. GO and KEGG enrichment analysis revealed that JA signal transduction and monoterpenoid synthesis were the two most significant enrichment pathways. The expression levels of related DEGs involved in JA signaling and monoterpenoid synthesis were significantly up-regulated by MeJA. In addition, our phenotypic and differentially expressed gene association analysis revealed that monoterpenoid biosynthesis in S. tenuifolia was more associated with genes involved in plant trichome branching, phytohormone signaling and transcriptional regulation. CONCLUSIONS: This study confirmed that methyl jasmonate significantly promoted monoterpenoid biosynthesis in S. tenuifolia. A large number of genes responding to methyl jasmonate were associated with JA signaling and monoterpenoid biosynthesis.

Methyl jasmonate and salicylic acid as powerful elicitors for enhancing the production of secondary metabolites in medicinal plants: an updated review.

Plant secondary metabolites are bioactive scaffolds that are crucial for plant survival in the environment and to maintain a defense mechanism from predators. These compounds are generally present in plants at a minimal level and interestingly, they are found to have a wide variety of therapeutic values for humans. Several medicinal plants are used for pharmaceutical purposes due to their affordability, fewer adverse effects, and vital role in traditional remedies. Owing to this reason, these plants are exploited at a high range worldwide and therefore many medicinal plants are on the threatened list. There is a need of the hour to tackle this major problem, one effective approach called elicitation can be used to enhance the level of existing and novel plant bioactive compounds using different types of elicitors namely biotic and abiotic. This process can be generally achieved by in vitro and in vivo experiments. The current comprehensive review provides an overview of biotic and abiotic elicitation strategies used in medicinal plants, as well as their effects on secondary metabolites enhancement. Further, this review mainly deals with the enhancement of biomass and biosynthesis of different bioactive compounds by methyl jasmonate (MeJA) and salicylic acid (SA) as elicitors of wide medicinal plants in in vitro by using different cultures. The present review was suggested as a significant groundwork for peers working with medicinal plants by applying elicitation strategies along with advanced biotechnological approaches.

Metabolomic analysis of methyl jasmonate treatment on phytocannabinoid production in Cannabis sativa.

Cannabis sativa is a multi-use and chemically complex plant which is utilized for food, fiber, and medicine. Plants produce a class of psychoactive and medicinally important specialized metabolites referred to as phytocannabinoids (PCs). The phytohormone methyl jasmonate (MeJA) is a naturally occurring methyl ester of jasmonic acid and a product of oxylipin biosynthesis which initiates and regulates the biosynthesis of a broad range of specialized metabolites across a number of diverse plant lineages. While the effects of exogenous MeJA application on PC production has been reported, treatments have been constrained to a narrow molar range and to the targeted analysis of a small number of compounds. Using high-resolution mass spectrometry with data-dependent acquisition, we examined the global metabolomic effects of MeJA in C. sativa to explore oxylipin-mediated regulation of PC biosynthesis and accumulation. A dose-response relationship was observed, with an almost two-fold increase in PC content found in inflorescences of female clones treated with 15 mM MeJA compared to the control group. Comparison of the inflorescence metabolome across MeJA treatments coupled with targeted transcript analysis was used to elucidate key regulatory components contributing to PC production and metabolism more broadly. Revealing these biological signatures improves our understanding of the role of the oxylipin pathway in C. sativa and provides putative molecular targets for the metabolic engineering and optimization of chemical phenotype for medicinal and industrial end-uses.

The Influence of Methyl Jasmonate on Expression Patterns of Rosmarinic Acid Biosynthesis Genes, and Phenolic Compounds in Different Species of Salvia subg. Perovskia Kar L.

Salvia yangii B.T. Drew and Salvia abrotanoides Kar are two important fragrant and medicinal plants that belong to the subgenus Perovskia. These plants have therapeutic benefits due to their high rosmarinic acid (RA) content. However, the molecular mechanisms behind RA generation in two species of Salvia plants are still poorly understood. As a first report, the objectives of the present research were to determine the effects of methyl jasmonate (MeJA) on the rosmarinic acid (RA), total flavonoid and phenolic contents (TFC and TPC), and changes in the expression of key genes involved in their biosynthesis (phenylalanine ammonia lyase (PAL), 4-coumarate-CoA ligase (4CL), and rosmarinic acid synthase (RAS)). The results of High-performance liquid chromatography (HPLC) analysis indicated that MeJA significantly increased RA content in S. yungii and S. abrotanoides species (to 82 and 67 mg/g DW, respectively) by 1.66- and 1.54-fold compared with untreated plants. After 24 h, leaves of Salvia yangii and Salvia abrotanoides species treated with 150 M MeJA had the greatest TPC and TFC (80 and 42 mg TAE/g DW, and 28.11 and 15.14 mg QUE/g DW, respectively), which was in line with the patterns of gene expression investigated. Our findings showed that MeJA dosages considerably enhanced the RA, TPC, and TFC contents in both species compared with the control treatment. Since increased numbers of transcripts for PAL, 4CL, and RAS were also detected, the effects of MeJA are probably caused by the activation of genes involved in the phenylpropanoid pathway.

Heterologous expression of Chlorophytum borivilianum Squalene epoxidase in tobacco modulates stigmasterol production and alters vegetative and reproductive growth.

KEYMESSAGE: CbSE overexpression increased stigmasterol levels and altered plant morphology. The genes upstream and downstream of CbSE were found to be upregulated, which confirms its regulatory role in the saponin biosynthetic pathway. Chlorophytum borivilianum is a high-value medicinal plant with many promising preclinical applications that include saponins as a major active ingredient. Squalene epoxidase (SE) is one of the major rate-limiting enzymes of the saponin biosynthetic pathway. Here, we functionally characterized C. borivilianum SE (CbSE) by over-expressing heterologously in Nicotiana tabacum. The heterologous expression of CbSE resulted in stunted pant growth with altered leaf and flower morphology. Next, RT-qPCR analysis of transgenic plants overexpressing CbSE revealed increased expression levels of Cycloartenol synthase (CAS), Beta amyrin synthase (ßAS), and cytochrome P450 monooxygenase 51 (CYP51) (Cytochrome P450), which encode key enzymes for triterpenoid and phytosterol biosynthesis in C. borivilianum. Further, Methyl Jasmonate (MeJa) treatment upregulated Squalene synthase (SQS), SE, and Oxidosqualene cyclases (OSCs) to a significant level. GC-MS analysis of the leaf and hairy roots of the transformants showed an increased stigmasterol content (0.5-1.0 fold) compared to wild type (WT) plants. These results indicate that CbSE is a rate-limiting gene, which encodes an efficient enzyme responsible for phytosterol and triterpenoid production in C. borivilianum.

Identification and functional characterization of three cytochrome P450 genes for the abietane diterpenoid biosynthesis in Isodon lophanthoides.

MAIN CONCLUSION: We identify two ferruginol synthases and a 11-hydroxyferruginol synthase from a traditional Chinese medicinal herb Isodon lophanthoides and propose their involvement in two independent abietane diterpenoids biosynthetic pathways. Isodon lophanthoides is a traditional Chinese medicinal herb rich in highly oxidized abietane-type diterpenoids. These compounds exhibit a wide range of pharmaceutical activities, yet the biosynthesis is barely known. Here, we describe the screening and functional characterization of P450s that oxidize the abietane skeleton abietatriene. We mainly focused on CYP76 family and identified 12 CYP76AHs by mining the RNA-seq data of I. lophanthoides. Among the 12 CYP76AHs, 6 exhibited similar transcriptional expression features as upstream diterpene synthases, including root or leaf-preferential expression pattern and highly MeJA inducibility. These six P450s were considered as first-tier candidates and functionally characterized in yeast and plant cells. In yeast assays showed that both CYP76AH42 and CYP76AH43 were ferruginol synthases hydroxylating the C12 position of abietatriene, whereas CYP76AH46 was characterized as a 11-hydroxyferruginol synthase which catalyzes two successive oxidations at C12 and C11 of abietatriene. Heterologous expression of three CYP76AHs in Nicotiana benthamiana resulted in the formation of ferruginol. qPCR analysis showed CYP76AH42 and CYP76AH43 were mainly expressed in the root, which was consistent with the distribution of ferruginol in the root periderms. CYP76AH46 was primarily expressed in the leaves where barely ferruginol or 11-hydroxyferruginol was detected. In addition to distinct organ-specific expression pattern, three CYP76AHs exhibited different genomic structures (w or w/o introns), low protein sequence identities (51-63%) and were placed in separate subclades in the phylogenetic tree. These results suggest that the identified CYP76AHs may be involved in at least two independent abietane biosynthetic pathways in the aerial and underground parts of I. lophanthoides.

Biotechnological Approach to Increase Oxyresveratrol Production in Mulberry In Vitro Plants under Elicitation.

Morus alba L. is used for a range of therapeutic purposes in Asian traditional medicine, and its extracts are reported to be effective against lipidemia, diabetes, and obesity, as well as being hepatoprotective and tyrosinase-inhibitory. They are also included in cosmetic products as anti-aging and skin-whitening agents. Stilbenes, the major bioactive compounds found in M. alba, have received renewed attention recently because of their putative activity against COVID-19. In this study M. alba plants were established in vitro, and the effect of elicitation on plant growth and stilbene accumulation, specifically oxyresveratrol and trans-resveratrol, was investigated. Different concentrations of the elicitors including methyl jasmonate and cyclodextrins were applied, and stilbene levels were determined in leaves, roots, and the culture medium. Elicitation of the M. alba plants with 5 mM cyclodextrins, alone or in combination with 10 µM methyl jasmonate, significantly increased the total phenolic content in the culture medium and leaves after 7 days of treatment. The higher total phenolic content in the roots of control plants and those treated only with methyl jasmonate indicated that cyclodextrins promoted metabolite release to the culture medium. Notably, the cyclodextrin-treated plants with the highest levels of oxy- and trans-resveratrol also had the highest total phenolic content and antioxidant capacity. These results indicate that elicited M. alba in vitro plants constitute a promising alternative source of bioactive stilbenes to supply pharmaceutical and cosmeceutical industries.

Characterization of the WRKY Gene Family Related to Anthocyanin Biosynthesis and the Regulation Mechanism under Drought Stress and Methyl Jasmonate Treatment in Lycoris radiata.

Lycoris radiata, belonging to the Amaryllidaceae family, is a well-known Chinese traditional medicinal plant and susceptible to many stresses. WRKY proteins are one of the largest families of transcription factors (TFs) in plants and play significant functions in regulating physiological metabolisms and abiotic stress responses. The WRKY TF family has been identified and investigated in many medicinal plants, but its members and functions are not identified in L. radiata. In this study, a total of 31 L. radiata WRKY (LrWRKY) genes were identified based on the transcriptome-sequencing data. Next, the LrWRKYs were divided into three major clades (Group I-III) based on the WRKY domains. A motif analysis showed the members within same group shared a similar motif component, indicating a conservational function. Furthermore, subcellular localization analysis exhibited that most LrWRKYs were localized in the nucleus. The expression pattern of the LrWRKY genes differed across tissues and might be important for Lycoris growth and flower development. There were large differences among the LrWRKYs based on the transcriptional levels under drought stress and MeJA treatments. Moreover, a total of 18 anthocyanin components were characterized using an ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis and pelargonidin-3-O-glucoside-5-O-arabinoside as well as cyanidin-3-O-sambubioside were identified as the major anthocyanin aglycones responsible for the coloration of the red petals in L. radiata. We further established a gene-to-metabolite correlation network and identified LrWRKY3 and LrWRKY27 significant association with the accumulation of pelargonidin-3-O-glucoside-5-O-arabinoside in the Lycoris red petals. These results provide an important theoretical basis for further exploring the molecular basis and regulatory mechanism of WRKY TFs in anthocyanin biosynthesis and in response to drought stress and MeJA treatment.

Exogenous methyl jasmonate and cytokinin antagonistically regulate lignin biosynthesis by mediating CsHCT expression in Camellia sinensis.

Tea plant, an important beverage crop, is cultivated worldwide. Lignification can improve the hardness of tea plant, which is of great significance for tea quality. Jasmonates (JAs) and cytokinin are plant hormones that control processes of plant development and secondary metabolite accumulation. Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) is primarily involved in lignin biosynthesis. The effects of exogenous application of JAs and cytokinin on lignin biosynthesis and related HCT gene expression profiles in tea plants are still unclear. In order to investigate the effects of exogenous JAs and cytokinin on lignin accumulation, anatomical structures, and CsHCT gene profiles in tea plants, we treated tea plants with methyl jasmonate (MeJA) and cytokinin (6-BA). MeJA and 6-BA treatments triggered the lignification at 6 and 12 d in tea leaves. The combined treatment resulted in an increase in lignin content at 6 d, which was 1.32 times of that at 0 d for 'Mengshan 9.' The CsHCTs in clade 2 (CsHCT5, CsHCT6, CsHCT7, and CsHCT8) were mainly expressed in leaves. We found that exogenous MeJA and cytokinin might be able to antagonistically regulate tea plant lignin accumulation through the mediation of CsHCT expression. This study revealed that HCTs play potential important roles involved in lignin biosynthesis of tea plant development and hormonal stimuli.

Transcriptome profiling of transcription factors in Ganoderma lucidum in response to methyl jasmonate.

Ganoderma lucidum is a traditional Chinese medicine and its major active ingredients are ganoderma triterpenoids (GTs). To screen for transcription factors (TFs) that involved in the biosynthetic pathway of GTs in G. lucidum, the chemical composition in mycelia, primordium and fruiting body were analyzed, and the transcriptomes of mycelia induced by methyl jasmonate (MeJA) were analyzed. In addition, the expression level data of MeJA-responsive TFs in mycelia, primordia and fruiting body were downloaded from the database, and the correlation analysis was carried out between their expression profiles and the content of total triterpenoids. The results showed that a total of 89 components were identified, and the content of total triterpenoids was the highest in primordium, followed by fruiting body and mycelia. There were 103 differentially expressed TFs that response to MeJA-induction including 95 upregulated and 8 downregulated genes. These TFs were classified into 22 families including C2H2 (15), TFII-related (12), HTH (9), fungal (8), bZIP (6), HMG (5), DADS (2), etc. Correlation analysis showed that the expression level of GL23559 (MADS), GL26472 (HTH), and GL31187 (HMG) showed a positive correlation with the GTs content, respectively. While the expression level of GL25628 (fungal) and GL26980 (PHD) showed a negative correlation with the GTs content, respectively. Furthermore, the over expression of the Glmhr1 gene (GL25628) in Pichia pastoris GS115 indicated that it might be a negative regulator of GT biosynthesis through decreasing the production of lanosterol. This study provided useful information for a better understanding of the regulation of TFs involved in GT biosynthesis and fungal growth in G. lucidum.