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Comparative transcriptome and tissue-specific expression analysis of genes reveal tissue-cultured plants as an alternative source for phenylethanoids and phenylpropanoids in Rhodiola imbricata (Edgew.)
Abstract
Rhodiola imbricata (Crassulaceae) is a traditional trans-Himalayan endangered medicinal herb with immense therapeutic applications. Over the years, over-exploitation, un-managed harvesting, and lack of captive cultivation procedures led to the huge market gap. Therefore, plant tissue culture and RNA-Seq-based molecular bioprospection of key regulatory genes will aid the understanding of molecular dynamics involved in specialized metabolites (phenylethanoids and phenylpropanoids) biosynthesis and its sustainable production. Hence, comparative transcriptomic analysis was performed using leaf and root tissues from the wild and tissue-cultured plants, revealing tissue-specific production of salidroside and rosavin. The transcriptome profiling resulted in 345 million raw reads yielding 92,380 unique transcripts with an N50 of 1260 bp. Tissue-specific gene expression analysis revealed that both phenylethanoids and phenylpropanoids biosynthesis are predominantly associated with the shikimate pathway. In addition to RNA-Seq data, the downstream biosynthesis pathways genes viz., phospho-2-dehydro-3-deoxyheptonate aldolase (DAHPS), 3-dehydroquinate synthase (DHQS), shikimate kinase (SK), chorismate mutase (CM), arogenate dehydrogenase (TYRAAT), aromatic-L-amino-acid decarboxylase (TDC), phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4-CL), cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD) showed higher expression pattern in wild plant tissues compared to tissue-cultured plants. The transcript fold expression determined by RT-qPCR results followed similar patterns as those observed in RNA-seq and targeted metabolite profiling data. Salidroside and rosavin content in wild plants were observed 2.40 folds and 1.77 folds increase compared to the tissue-cultured plant. The present investigation explained the tissue and condition-specific significant differences between the expression of proposed biosynthetic pathway genes and salidroside and rosavin content. Additionally, NAC, bHLH, and ARF were the most abundant transcription factor families found in the transcriptomic analysis of R. imbricata. The generated transcriptome dataset provides a valuable gene(s)/transcription factors hub that can be used for the sustainable production of salidroside and rosavin in R. imbricata under tissue culture conditions.
... Plant metabolomics is a crucial analytical tool that has been extensively utilized to assess the dynamic alterations of metabolites across various tissues, species, and developmental stages [50]. RNA-seq-based molecular bioprospecting approaches enable the identification of regulatory genes and account for variations in the expression levels of biosynthetic genes in relation to metabolite content [51]. A combined analysis of transcriptome data and metabolic profiles facilitates the identification of functional genes and the elucidation of primary and secondary metabolic pathways in plants [52,53]. ...
The resinous stem of Aquilaria sinensis (Lour.) Gilg is the sole legally authorized source of agarwood in China. However, whether other tissue parts can be potential substitutes for agarwood requires further investigation. In this study, we conducted metabolic analysis and transcriptome sequencing of six distinct tissues (root, stem, leaf, seed, husk, and callus) of A. sinensis to investigate the variations in metabolite distribution characteristics and transcriptome data across different tissues. A total of 331 differential metabolites were identified by chromatography–mass spectrometry (GC-MS), of which 22.96% were terpenoids. The differentially expressed genes (DEGs) in RNA sequencing were enriched in sesquiterpene synthesis via the mevalonate pathway. The present study establishes a solid foundation for exploring potential alternatives to agarwood.
... Biotechnological production of specialized metabolites using in vitro plant cultures, including culturing of plant tissues, organs, or cells, has the potential to overcome the limitations related to whole-plant growth (Grech-Baran et al. 2015;Rattan et al. 2022b). Plant cell culture recommends a promising alternative and efficient platform for producing a wide range of important specialized metabolites (Partap et al. 2020;Rattan et al. 2021a). ...
Precursor feeding is a well-known strategy to enhance the commercial production of bioactive compounds in plant cell culture systems. For the first time, an effective in vitro platform was established for the enhanced production of phenylpropanoids with a precursor feeding approach in the cell suspension culture derived from friable leaf callus of Rhodiola imbricata. Precursor, phenylalanine feeding of 0.5 mM, 1 mM, 2 mM, and 3 mM concentrations were added to the cell suspension of R. imbricata and further, it was evaluated for the cell growth and production of phenylpropanoids (rosavin, rosarin, and p-coumaric acid). The suspension culture showed maximum cell growth (FW; 117 g/L, DW; 5.73 g/L) on day 15 of the incubation period in precursor untreated cells. Furthermore, cell suspension cultures treated with 1 mM phenylalanine accumulated the maximum biomass (FW; 136 g/L, DW; 6.70 g/L) on day 15. The enhanced rosavin (1.57 mg/g DW) and rosarin (0.78 mg/g DW) content were achieved on day 12 in 1 mM precursor concentration. Similarly, p-coumaric acid was detected maximum (2.50 mg/g DW) on day 12 in 3 mM precursor-treated suspension cultures of R. imbricata. The total phenolic content was improved significantly (43.72 mg/g DW) on day 12 in 2 mM phenylalanine treatment. Whereas, total flavonoid content was observed maximum (24.50 mg/g DW) on day 12 with 2 mM precursor concentration. The present work concluded that the precursor-mediated approach enhanced the overall phenylpropanoids production in the cell suspension culture of R. imbricata.
Phenylalanine is an aromatic amino acid that plants synthesize, then metabolize, to make other chemical structures, defense mechanisms, and yet undiscovered functions via multiple genes. In the early vegetative stages of life, phenylalanine plays important roles in germination, organelle development, tissue and cell development, and function. The family of Phenylalanine Ammonia-Lyase (PAL) enzymes also play roles in the seed-to-seedling transition, although, while being the first enzyme (but not the sole enzyme) in the phenylpropanoids pathway, not all roles have been elucidated. In this review of recent literature on phenylalanine and PAL’s impact on seedlings and seedling establishment, it is clear from the last 5–10 years, in particular, that major advances have been made in chemistry, molecular, protein studies, and in silico studies. It is exciting that many chemical and molecular advances have been applied to plants of economic importance including agriculture, horticulture, and in medicinal chemical production utilizing the actions of phenylalanine and PAL.
Rhodiola imbricata is a rare and endangered plant of the Trans-Himalayan region having important medicinal properties. It holds immense therapeutic value against a wide range of diseases and health problems including hypoxia, cancer, stress, anxiety, fatigue, and gastrointestinal problems. The plant which is normally propagated through seeds suffers from drawbacks such as limited seed availability, low seed viability, and germination, limited geographical distribution, slow growth, and slow accumulation of secondary metabolites. Owing to the growing demand for this plant, a novel, highly efficient liquid culture system using meta-Topolin (mT) was developed for its rapid multiplication and continuous production of important bioactive compounds. A comparative analysis was conducted to evaluate the response of shoot multiplication, rooting, and secondary metabolite content in the solid and liquid culture media. In vitro seedlings were inoculated on Murashige and Skoog's (MS) (1962) medium supplemented with different concentrations of cytokinins. Among the tested cytokinins, the maximum number of shoots were observed in 20 mL of liquid MS medium supplemented with mT (5.0 µM). While Indole-3-butyric acid (IBA) (10.0 µM) exhibited the highest rooting response (95%). Mass propagation of microshoots was achieved using a specialized box, resulting in an improved survival rate of 85% during the subsequent hardening process. The secondary metabolite content, including rosavin, salidroside, tyrosol, total polyphenolic content (TPC), and antioxidant properties were estimated for shoots grown in both agar-gelled solid and liquid culture media. Overall, liquid MS medium supplemented with mT (5.0 µM) was found to be the optimum medium for secondary metabolites production in comparison to solid medium. Further, the genetic and phytochemical stability of the prolong culture of this plant under in vitro conditions were confirmed. This system facilitates large scale production of in vitro plants as well as secondary metabolites throughout the year, which is crucial for various industrial applications. Key message Rhodiola imbricata is an important, rare and endangered medicinal plant. A novel and efficient protocol for improved shoot proliferation and secondary metabolite production was developed using meta-Topolin and liquid culture.
Elicitation has been shown to be a very effective tool for improving bioactive molecule accumulation in various cell culture systems. The current study observed the effects of salicylic acid (SA) and methyl jasmonate (MeJA) on the increased accretion of salidroside and rosavin, as well as the transcriptional response of their biosynthetic genes in Rhodiola imbricata cell suspension culture. Suspension-cultured cells were treated with different concentrations of elicitors (SA and MeJA; 25, 50, 75, and 100 μM) on the 16th day of the incubation period and evaluated for growth biomass, metabolite content, and gene expression analysis after 24, 48, 72, and 96 h of elicitor treatment. The enhanced salidroside (7.71 mg/g DW) and rosavin (0.99 mg/g DW) contents were achieved in 50 μM SA elicitation. Similarly, MeJA elicitation improved salidroside (1.85 mg/g DW) and rosavin (0.67 mg/g DW) content in 100 μM concentration. In 50 μM SA and 100 μM MeJA elicitation, total phenolics were significantly increased (16.11 mg/g DW; 7.69 mg/g DW). Total flavonoids were detected at their highest levels (9.78 mg/g DW; 6.69 mg/g DW) in 50 μM SA and 100 μM MeJA elicitation, respectively. Furthermore, after SA and MeJA treatment, transcript level gene expression revealed a higher expression pattern of aromatic-L-amino-acid decarboxylase (TDC) and Uridine 5′ -diphospho-glucuronosyltransferase (UGT 73 C) genes in salidroside biosynthesis. While in rosavin biosynthesis, SA and MeJA elicitation significantly increased the expression levels of cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR). Overall, the study suggests that elicitation of SA and MeJA improved specialized metabolite accumulation and expression of key pathway genes in R. imbricata suspension-cultured cells.
This study aimed to establish a novel method for catalysing salidroside synthesis by immobilized β-D-glucosidase with deep eutectic solvents (DESs) as a new green non-aqueous medium. To this end, the catalytic properties of β-D-glucosidase (i.e. activity, stability, and enzymatic properties) were analysed within sixteen different DESs systems. Although most DESs significantly improved enzyme stability, they also reduced enzymatic activity. This was verified at the molecular level by employing fluorescence spectroscopy to evaluate conformational changes in β-D-glucosidase molecules. Subsequently, chitosan microspheres were used to immobilize β-D-glucosidase; the structures of the microspheres and immobilized enzyme were observed by scanning electron microscopy (SEM). Through single factor experiments and application of a response surface design, the optimal conditions for production of salidroside by the reaction of tyrosol with immobilized β-D-glucosidase were established. These conditions comprised 80 vol% ChCl/G (1:2), 50 °C reaction temperature, 5.8 pH, 100 h reaction time, 45 U/mL enzyme dosage, and a tyrosol/D-glucose molar ratio of 10. Under these conditions, the average conversion rate of the tyrosol substrate was 31.6%. Moreover, a salidroside crude product was obtained with a purity >70% (g/g) and a substrate conversion rate of immobilized β-D-glucosidase >50% of its initial value after five repeated uses. Collectively, this study establishes a novel green reaction system for the efficient preparation of salidroside and other glycoside compounds, while also providing a theoretical foundation for the analysis of β-D-glucosidase catalytic reactions in DESs systems.
Precursor feeding is a potential strategy for increasing specialized metabolite production in plant cell culture systems. In the present study, cell suspension cultures were developed and subsequently evaluated for precursor feeding investigations. Cell suspension cultures were established in Murashige and Skoog (MS) medium containing 0.5 mg/L thidiazuron (TDZ) + 1 mg/L α-naphthalene acetic acid (NAA). The growth biomass and metabolite pattern were analyzed to identify specific culture days required for prolific biomass production. The maximum cell dry weight (DW) was observed in leaf cell suspension (1.22 g/100 mL) and root cell suspension culture (1.12 g/100 mL) on day 21. Afterward, the effect of precursor concentrations (tyrosol; 0.5, 1, 2, and 3 mM) along with two light regimes, photoperiod (16L/8D h, 70 µmol/m²/s) and dark (24 h), was evaluated for cell growth and metabolite accumulation. The results revealed that leaf cell suspension treated with 3 mM tyrosol concentration detected maximum salidroside content (26.05 mg/g DW) on day 15, incubated under photoperiod (16L/8D h) condition. Similarly, under photoperiod (16L/8D h), root cell suspension treated with 3 mM tyrosol produced maximum salidroside content (26.62 mg/g DW) on day 12. Moreover, the total phenolics content increased significantly (44.21 mg/g DW) on day 12 in 3 mM tyrosol treatment under photoperiod (16L/8D h). However, precursor concentrations did not influence the total flavonoids content. The present investigation suggests that the immediate pathway precursor, tyrosol, has a strong effect on enhanced production of salidroside, irrespective of explant type and light regimes.
Medicinal plants of the NorthWestern Himalayan region are known for their unprecedented biodiversity and valuable secondary metabolites that are unique to this dynamic geo-climatic region. From ancient times these medicinal herbs have been used traditionally for their therapeutic potentials. But from the last 2 decades increasing pharmaceutical demand, illegal and unorganized trade of these medicinal plants have accelerated the rate of over-exploitation in a non-scientific manner. In addition, climate change and anthropogenic activities also affected their natural habitat and driving most of these endemic plant species to critically endangered that foresee peril of mass extinction from this eco-region. Hence there is an urgent need for developing alternative sustainable approaches and policies to utilize this natural bioresource ensuring simultaneous conservation. Hither, arise the advent of sequencing-based transcriptomic studies significantly contributes to better understand the background of important metabolic pathways and related genes/enzymes of high-value medicinal herbs, in the absence of genomic information. The use of comparative transcriptomics in conjunction with biochemical techniques in NorthWestern Himalayan medicinal plants has resulted in significant advances in the identification of the molecular players involved in the production of secondary metabolic pathways over the last decade. This information could be used to further engineer metabolic pathways and breeding programs, ultimately leading to the development of in vitro systems dedicated to the production of pharmaceutically important secondary metabolites at the industrial level. Collectively, successful adoption of these approaches can certainly ensure the sustainable utilization of Himalayan bioresource by reducing the pressure on the wild population of these critically endangered medicinal herbs. This review provides novel insight as a transcriptome-based bioresource repository for the understanding of important secondary metabolic pathways genes/enzymes and metabolism of endangered high-value NorthWestern Himalayan medicinal herbs, so that researchers across the globe can effectively utilize this information for devising effective strategies for the production of pharmaceutically important compounds and their scale-up for sustainable usage and take a step forward in omics-based conservation genetics.
Plantagos are important economical and medicinal plants that possess several bioactive secondary metabolites, such as phenolics, iridoids, triterpenes, and alkaloids. Triterpenoids are the ubiquitous and dynamic secondary metabolites that are deployed by plants for chemical interactions and protection under biotic/abiotic stress. Plantago ovata, a cultivated species, is the source of psyllium, while Plantago major, a wild species, has significant therapeutic potential. Wild species are considered more tolerant to stressful conditions in comparison to their cultivated allies. In view of this, the present study aimed to decipher the terpenoid biosynthetic pathway operative in P. ovata and P. major using a comparative transcriptomics approach. Majority of terpenoid biosynthetic genes were observed as upregulated in P. major including rate limiting genes of MVA (HMGR) and MEP (DXR) pathways and genes (α-AS, BAS, SM, and CYP716) involved in ursolic acid biosynthesis, an important triterpenoid prevalent in Plantago species. The HPLC output further confirmed the higher concentration of ursolic acid in P. major as compared to P. ovata leaf samples, respectively. In addition to terpenoid biosynthesis, KEGG annotation revealed the involvement of differentially expressed unigenes in several metabolic pathways, aminoacyl-tRNA biosynthesis, biosynthesis of antibiotics, and biosynthesis of secondary metabolites. MYB was found as the most abundant transcription factor family in Plantago transcriptome. We have been able to generate valuable information which can help in improving terpenoid production in Plantago. Additionally, the present study has laid a strong foundation for deciphering other important metabolic pathways in Plantago.
The plant cell culture provides an efficient technique for growth and production kinetics studies of bioactive compounds present in plants. Therefore, the present study investigated, for the first time, to identify the specific culture days required for higher metabolite yield in Rhodiola imbricata. A transcript-level gene expression study was conducted to understand the molecular cues associated with metabolite biosynthesis. The leaf callus cell line showed an optimum growth rate (FW: 20 g/100 mL, DW: 1.21 g/100 mL), growth index (19.00), salidroside (3.68 mg/g DW, rosavin (0.21 mg/g DW and rosarin (0.08 mg/g DW) on 24th day of the incubation period. However, salidroside, rosavin, and rosarin metabolite yield followed the growth-dependent manner in both callus cell lines, while tyrosol and p-coumaric acid showed growth independent accumulation pattern. Moreover, the antioxidant study displayed maximum phenolic content (8.07 mg/g DW) on day 18, flavonoid content (9.73 mg/g DW) on day 3, and DPPH activity (IC50; 0.79 mg/mL) on day 24 in leaf callus cell line. The RT-qPCR analysis showed upregulation of PAL, 4-HPAAS, 4-HPAAR, and UDPGT genes associated with metabolite accumulation. Expression analysis revealed a positive correlation with the metabolites yield. Furthermore, the optimized parameter could be exploited for a higher yield of specific metabolite and sequential scale-up studies.
Numerous secondary metabolites from plants are important for their medicinal, nutraceutical or sensory properties. Recently, significant progress has been made in the identification of the genes and enzymes of plant secondary metabolic pathways. Hence, there is interest in using synthetic biology to enhance the production of targeted valuable metabolites in plants. In this article, we examine the contribution that metabolic flux analysis will have on informing the rational selection of metabolic engineering targets as well as analysis of carbon and energy efficiency. Compared to microbes, plants have more complex tissue, cellular and subcellular organization, making precise metabolite concentration measurements more challenging. We review different techniques involved in quantifying flux and provide examples illustrating the application of the techniques. For linear and branched pathways that lead to end products with low turnover, flux quantification is straightforward and doesn't require isotopic labeling. However, for metabolites synthesized via parallel pathways, there is a requirement for isotopic labeling experiments. If the fed isotopically labeled carbons don't scramble, one needs to apply transient label balancing methods. In the transient case, it is also necessary to measure metabolite concentrations. While flux analysis is not able to directly identify mechanisms of regulation, it is a powerful tool to examine flux distribution at key metabolic nodes in intermediary metabolism, detect flux to wasteful side pathways, and show how parallel pathways handle flux in wild-type and engineered plants under a variety of physiological conditions.
This is the first report on de novo transcriptome of Dactylorhiza hatagirea, a critically-endangered, terrestrial orchid of alpine Himalayas. The plant is acclaimed for medicinal properties but little is known about its secondary-metabolites profile or cues regulating their biosynthesis. De novo transcriptome analysis was therefore, undertaken to gain basic understanding on these aspects, while circumventing the acute limitation of plant material availability. 65,384 transcripts and finally, 37,371 unigenes were assembled de novo from a total of 236 million reads obtained from shoot, tuber and leaves of the plant. Dominance of differentially-expressing-genes (DEGs) related to cold-stress-response and plant-hormone-signal-transduction; and those involved in photosynthesis, sugar-metabolism and secondary-metabolite-synthesis provided insights into carbohydrate-partitioning in the plant during its preparation for freezing winter at natural habitat. DEGs of glucomannan, ascorbic acid, carotenoids, phylloquinone/naphthoquinones, indole alkaloids, resveratrol and stilbene biosynthesis revealed the secondary-metabolite profile of D. hatagirea. UHPLC results confirmed appreciable amounts of resveratrol and trans-stilbene in D. hatagirea tubers, for the first time. Expression analysis of 15 selected genes including those of phenylpropanoid pathway confirmed the validity of RNA-seq data. Opportunistic growth, temperature- and tissue-specific-differential-expression of secondary metabolite biosynthesis and stress tolerant genes were confirmed using clonal plants growing at 8, 15 and 25 °C.
The production of secondary metabolites from medicinal plants, also called Plant-Derived Medicinal Compounds (PDMC), is gaining ground in the last decade. Concomitant to the increase in the knowledge about pharmacological properties of these compounds, horticultural plants are becoming the most important, sustainable and low-cost biomass source to obtain high-complex PDMCs to be used as medicaments. Biotechnological tools, including plant cell and tissue culture and plant genetic transformation, are increasingly being employed to produce high quality and rare PDMC under in vitro conditions. The proper use of these technologies requires studies in organogenesis to allow for better control of in vitro plant development and, thus, to the production of specific tissues and activation of biochemical routes that result in the biosynthesis of the target PDMCs. Either biotic or abiotic factors, called elicitors, are responsible for triggering the PDMC synthesis. In vitro techniques, when compared to the conventional cultivation of medicinal plants in greenhouse or in the field, have the advantages of (1) producing PDMCs in sterile and controlled environmental conditions, allowing better control of the developmental processes, such as organogenesis, and (2) producing tissues with high PDMC contents, due to the efficient use of different biotic and abiotic elicitors. Nevertheless, the process has many challenges, e.g., the establishment of step-by-step protocols for in vitro biomass and PDMC production, both involving and being affected by many factors. Other limitations are the high costs in opposition to the relatively cheaper alternative of growing medicinal plants conventionally. This paper aims to quickly review the general origin of plant secondary metabolites, the leading techniques and recent advances for PDMC in vitro production, and the challenges around the use of this promising technology.
Main conclusion
Plant tissue culture has been used for conservation, micropropagation, and in planta overproduction of some pharma molecules of medicinal plants. New biotechnology-based breeding methods such as targeted genome editing methods are able to create custom-designed medicinal plants with different secondary metabolite profiles.
For a long time, humans have used medicinal plants for therapeutic purposes and in food and other industries. Classical biotechnology techniques have been exploited in breeding medicinal plants. Now, it is time to apply faster biotechnology-based breeding methods (BBBMs) to these valuable plants. Assessment of the genetic diversity, conservation, proliferation, and overproduction are the main ways by which genetics and biotechnology can help to improve medicinal plants faster. Plant tissue culture (PTC) plays an important role as a platform to apply other BBBMs in medicinal plants. Agrobacterium-mediated gene transformation and artificial polyploidy induction are the main BBBMs that are directly dependent on PTC. Manageable regulation of endogens and/or transferred genes via engineered zinc-finger proteins or transcription activator-like effectors can help targeted manipulation of secondary metabolite pathways in medicinal plants. The next-generation sequencing techniques have great potential to study the genetic diversity of medicinal plants through restriction-site-associated DNA sequencing (RAD-seq) technique and also to identify the genes and enzymes that are involved in the biosynthetic pathway of secondary metabolites through precise transcriptome profiling (RNA-seq). The sequence-specific nucleases of transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, and clustered regularly interspaced short palindromic repeats-associated (Cas) are the genome editing methods that can produce user-designed medicinal plants. These current targeted genome editing methods are able to manage plant synthetic biology and open new gates to medicinal plants to be introduced into appropriate industries.
The present study focuses on development of a micropropagation protocol for true to type plants of Rhodiola imbricata, an endangered medicinal plant found in trans-Himalayan Leh-Ladakh region of India. It also aims at analyzing the pharmaceutically important secondary metabolites and antioxidant potential of in vitro and in vivo plants. Various cytokinins and auxins were tested for shoot proliferation and in vitro rooting of the microshoots, respectively. Random primers were used for checking genetic uniformity at different stages of micropropagation. Pharmaceutically important secondary metabolites of R. imbricata such as Rosavin, total polyphenols and free radical scavenging activity were analyzed by HPLC. Among different cytokinins used, BAP (5 µM) and TDZ (1 µM) were found to perform better in terms of shoot proliferation, shoot length and number of leaves as compared to other concentrations. For rooting of microshoots, a lower concentration of NAA (0.5 µM) yielded more efficient rooting of micro shoots (17.33 roots per micro shoot). In vitro rooted microshoots were hardened and showed 60% survival rate. The content of gallic acid, chlorogenic acid and 4-hydroxybenzoic acid was higher in the in vivo plant. The amount of ferulic acid was higher in the in vitro raised plant when compared to field grown plant. Furthermore, caffeic acid and p-coumaric acid were higher in the in vitro raised plants as compared to field grown plants. This work will facilitate in conservation of this endangered herb and provide necessary plant materials for various biotechnological and pharmaceutical applications.
Rhodiola imbricata is a rare medicinal plant of the trans-Himalayan region of Ladakh. It is used for the treatment of numerous health ailments. Compact callus aggregate (CCA) suspension cultures of Rhodiola imbricata were established to counter extinction threats and for production of therapeutically valuable phenolic compounds to meet their increasing industrial demands. The present study also investigated the effect of jasmonic acid (JA) on production of phenolic compounds and bioactivities in CCA suspension cultures. CCA suspension cultures established in an optimized Murashige and Skoog medium supplemented with 30 g/l sucrose, 3 mg/l NAA, and 3 mg/l BAP showed maximum biomass accumulation (8.43 g/l DW) and highest salidroside production (3.37 mg/g DW). Upon 100 μM JA treatment, salidroside production (5.25 mg/g DW), total phenolic content (14.69 mg CHA/g DW), total flavonoid content (4.95 mg RE/g DW), and ascorbic acid content (17.93 mg/g DW) were significantly increased in cultures. In addition, DPPH-scavenging activity (56.32%) and total antioxidant capacity (60.45 mg QE/g DW) were significantly enhanced upon JA treatment, and this was positively correlated with increased accumulation of phenolic compounds. JA-elicited cultures exhibited highest antimicrobial activity against Escherichia coli. This is the first report describing the enhanced production of phenolic compounds and bioactivities from JA-elicited CCA suspension cultures of Rhodiola imbricata.
WEGO (Web Gene Ontology Annotation Plot), created in 2006, is a simple but useful tool for visualizing, comparing and plotting GO (Gene Ontology) annotation results. Owing largely to the rapid development of high-throughput sequencing and the increasing acceptance of GO, WEGO has benefitted from outstanding performance regarding the number of users and citations in recent years, which motivated us to update to version 2.0. WEGO uses the GO annotation results as input. Based on GO's standardized DAG (Directed Acyclic Graph) structured vocabulary system, the number of genes corresponding to each GO ID is calculated and shown in a graphical format. WEGO 2.0 updates have targeted four aspects, aiming to provide a more efficient and up-to-date approach for comparative genomic analyses. First, the number of input files, previously limited to three, is now unlimited, allowing WEGO to analyze multiple datasets. Also added in this version are the reference datasets of nine model species that can be adopted as baselines in genomic comparative analyses. Furthermore, in the analyzing processes each Chi-square test is carried out for multiple datasets instead of every two samples. At last, WEGO 2.0 provides an additional output graph along with the traditional WEGO histogram, displaying the sorted P-values of GO terms and indicating their significant differences. At the same time, WEGO 2.0 features an entirely new user interface. WEGO is available for free at http://wego.genomics.org.cn.
Main conclusion
Plant tissue culture as an important tool for the continuous production of active compounds including secondary metabolites and engineered molecules. Novel methods (gene editing, abiotic stress) can improve the technique.
Humans have a long history of reliance on plants for a supply of food, shelter and, most importantly, medicine. Current-day pharmaceuticals are typically based on plant-derived metabolites, with new products being discovered constantly. Nevertheless, the consistent and uniform supply of plant pharmaceuticals has often been compromised. One alternative for the production of important plant active compounds is in vitro plant tissue culture, as it assures independence from geographical conditions by eliminating the need to rely on wild plants. Plant transformation also allows the further use of plants for the production of engineered compounds, such as vaccines and multiple pharmaceuticals. This review summarizes the important bioactive compounds currently produced by plant tissue culture and the fundamental methods and plants employed for their production.
Plant secondary metabolites (SMs) are not only a useful array of natural products but also an important part of plant defense system against pathogenic attacks and environmental stresses. With remarkable biological activities, plant SMs are increasingly used as medicine ingredients and food additives for therapeutic, aromatic and culinary purposes. Various genetic, ontogenic, morphogenetic and environmental factors can influence the biosynthesis and accumulation of SMs. According to the literature reports, for example, SMs accumulation is strongly dependent on a variety of environmental factors such as light, temperature, soil water, soil fertility and salinity, and for most plants, a change in an individual factor may alter the content of SMs even if other factors remain constant. Here, we review with emphasis how each of single factors to affect the accumulation of plant secondary metabolites, and conduct a comparative analysis of relevant natural products in the stressed and unstressed plants. Expectantly, this documentary review will outline a general picture of environmental factors responsible for fluctuation in plant SMs, provide a practical way to obtain consistent quality and high quantity of bioactive compounds in vegetation, and present some suggestions for future research and development.
Salidroside is a bioactive tyrosine-derived phenolic natural product found in medicinal plants under the Rhodiola genus. In addition to their anti-fatigue and anti-anoxia roles in traditional medicine, Rhodiola total extract and salidroside have also displayed medicinal properties as anti-cardiovascular diseases and anti-cancer agents. The resulting surge in global demand of Rhodiola plants and salidroside has driven some species close to extinction. Here, we report the full elucidation of Rhodiola salidroside biosynthetic pathway utilizing the first comprehensive transcriptomics and metabolomics datasets for Rhodiola rosea. Unlike the previously proposed pathway involving separate decarboxylation and deamination enzymatic steps from tyrosine to the key intermediate 4-hydroxyphenylacetaldehyde (4-HPAA), Rhodiola contains a pyridoxal phosphate (PLP)-dependent 4-hydroxyphenylacetaldehyde synthase (4HPAAS) that directly converts tyrosine to 4-HPAA. We further identified genes encoding the subsequent 4-HPAA reductase (4HPAR) and tyrosol:UDP-glucose 8-O-glucosyltransferase (T8GT), respectively, to complete salidroside biosynthesis in Rhodiola. We show that heterologous production of salidroside can be achieved in yeast Saccharomyces cerevisiae as well as in plant Nicotiana benthamiana through transgenic expression of Rhodiola salidroside biosynthetic genes. This study provides new tools for engineering sustainable production of salidroside in heterologous hosts.
Biotechnology is playing a vital alternative role in the production of pharmaceutical plant secondary metabolites to support industrial production and mitigate over-exploitation of natural sources. High-value pharmaceuticals
that include alkaloids, flavonoids, terpenes, steroids, among others, are biosynthesized as a defensive strategy
by plants in response to perturbations under natural environmental conditions. However, they can also be produced using plant cell, tissue, and organ culture techniques through the application of various in vitro approaches and strategies. In the past decades, efforts were on the clonal propagation, biomass and secondary metabolites production in the in vitro cultures of medicinally important plants that produce these
molecules. In recent years, the effort has shifted towards optimizing culture conditions for their production through the application of cell line selection, elicitation, precursor feeding, two-phase co-culture among cell, tissue, and organ culture approaches. The efforts are made with the possibility to scale-up the production, meet pharmaceutical industry demand and conserve natural sources of the molecules. Applications of metabolic engineering and production from endophytes are also getting increasing attention but, the approaches are far from practical application in their industrial production.
Stevia is a natural source of commercially important steviol glycosides (SGs), which share biosynthesis route with gibberellic acids (GAs) through plastidal MEP and cytosolic MVA pathways. Ontogeny- dependent deviation in SGs biosynthesis is one of the key factor for global cultivation of Stevia, has not been studied at transcriptional level. To dissect underlying molecular mechanism, we followed a global transcriptome sequencing approach and generated more than 100 million reads. Annotation of 41,262 de novo assembled transcripts identified all the genes required for SGs and GAs biosynthesis. Differential gene expression and quantitative analysis of important pathway genes (DXS, HMGR, KA13H) and gene regulators (WRKY, MYB, NAC TFs) indicated developmental phase dependent utilization of metabolic flux between SGs and GAs synthesis. Further, identification of 124 CYPs and 45 UGTs enrich the genomic resources, and their PPI network analysis with SGs/GAs biosynthesis proteins identifies putative candidates involved in metabolic changes, as supported by their developmental phase-dependent expression. These putative targets can expedite molecular breeding and genetic engineering efforts to enhance SGs content, biomass and yield. Futuristically, the generated dataset will be a useful resource for development of functional molecular markers for diversity characterization, genome mapping and evolutionary studies in Stevia.
Trillium govanianum, an endangered medicinal herb native to the Himalaya, is less studied at the molecular level due to the non-availability of genomic resources. To facilitate the basic understanding of the key genes and regulatory mechanism of pharmaceutically important biosynthesis pathways, first spatial transcriptome sequencing of T. govanianum was performed. 151,622,376 (~11.5?Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 69,174 transcripts. Functional annotation with multiple public databases identified array of genes involved in steroidal saponin biosynthesis and other secondary metabolite pathways including brassinosteroid, carotenoid, diterpenoid, flavonoid, phenylpropanoid, steroid and terpenoid backbone biosynthesis, and important TF families (bHLH, MYB related, NAC, FAR1, bZIP, B3 and WRKY). Differentially expressed large number of transcripts, together with CYPs and UGTs suggests involvement of these candidates in tissue specific expression. Combined transcriptome and expression analysis revealed that leaf and fruit tissues are the main site of steroidal saponin biosynthesis. In conclusion, comprehensive genomic dataset created in the current study will serve as a resource for identification of potential candidates for genetic manipulation of targeted bioactive metabolites and also contribute for development of functionally relevant molecular marker resource to expedite molecular breeding and conservation efforts in T. govanianum.
Calotropis procera is a medicinal plant of immense importance due to its pharmaceutical active components, especially cardiac glycosides (CG). As genomic resources for this plant are limited, the genes involved in CG biosynthetic pathway remain largely unknown till date. Our study on stage and tissue specific metabolite accumulation showed that CG’s were maximally accumulated in stems of 3 month old seedlings. De novo transcriptome sequencing of same was done using high throughput Illumina HiSeq platform generating 44074 unigenes with average mean length of 1785 base pair. Around 66.6% of unigenes were annotated by using various public databases and 5324 unigenes showed significant match in the KEGG database involved in 133 different pathways of plant metabolism. Further KEGG analysis resulted in identification of 336 unigenes involved in cardenolide biosynthesis. Tissue specific expression analysis of 30 putative transcripts involved in terpenoid, steroid and cardenolide pathways showed a positive correlation between metabolite and transcript accumulation. Wound stress elevated CG levels as well the levels of the putative transcripts involved in its biosynthetic pathways. This result further validated the involvement of identified transcripts in CGs biosynthesis. The identified transcripts will lay a substantial foundation for further research on metabolic engineering and regulation of cardiac glycosides biosynthesis pathway genes.
Transcriptional regulation of picrosides biosynthesis, the iridoid glycosides of an endangered medicinal herb, Picrorhiza kurroa, is completely unknown. P. kurroa plants obtained from natural habitat accumulate higher picrosides than in-vitro cultured plants, which necessitates identification of transcription factors (TFs) regulating their differential biosynthesis. The current study investigates complete spectrum of different TF classes in P. kurroa transcriptomes and discerns their association with picrosides biosynthesis. Transcriptomes of differential picroside-I content shoots and picroside-II content roots were mined for seven classes of TFs implicated in secondary metabolism regulation in plants. Key TFs were identified through in silico transcript abundance and qPCR analysis was performed to confirm transcript levels of TFs under study in differential content tissues and genotypes. Promoter regions of key picrosides biosynthetic pathway genes were explored to hypothesize which TFs can possibly regulate target genes. A total of 131, 137, 107, 82 and 101 transcripts encoding different TFs families were identified in PKS-25, PKS-15, PKSS, PKR-25 and PKSR transcriptomes, respectively. ERF-18, bHLH-104, NAC-25, 32, 94 and SUF-4 showed elevated expression in roots (up to 37 folds) and shoots (up to 195 folds) of plants obtained from natural habitat, indicating their role as activators of picrosides biosynthesis whereas, elevated expression of WRKY-17, 40, 71 and MYB-4 in low picrosides content conditions suggested their down-regulatory role. In silico analysis of key picrosides biosynthetic pathway gene promoter regions revealed binding domains for ERF-18, NAC-25, WRKY-40 and MYB-4. Identification of candidate TFs contributing towards picrosides biosynthesis is a pre-requisite for designing appropriate metabolic engineering strategies aimed at enhancing picrosides content in vitro and in vivo.
Lonicera japonica is one of the most important medicinal plants with applications in traditional Chinese and Japanese medicine for thousands of years. Extensive studies on the constituents of L. japonica extracts have revealed an accumulation of pharmaceutically active metabolite classes, such as chlorogenic acid, luteolin and other flavonoids, and secoiridoids, which impart characteristic medicinal properties. Despite being a rich source of pharmaceutically active metabolites, little is known about the biosynthetic enzymes involved, and their expression profile across different tissues of L. japonica. In this study, we performed de novo transcriptome assembly for L. japonica, representing transcripts from nine different tissues. A total of 22 Gbps clean RNA-seq reads from nine tissues of L. japonica were used, resulting in 243,185 unigenes, with 99,938 unigenes annotated based on a homology search using blastx against the NCBI-nr protein database. Unsupervised principal component analysis and correlation studies using transcript expression data from all nine tissues of L. japonica showed relationships between tissues, explaining their association at different developmental stages. Homologs for all genes associated with chlorogenic acid, luteolin, and secoiridoid biosynthesis pathways were identified in the L. japonica transcriptome assembly. Expression of unigenes associated with chlorogenic acid was enriched in stems and leaf-2, unigenes from luteolin were enriched in stems and flowers, while unigenes from secoiridoid metabolic pathways were enriched in leaf-1 and shoot apex. Our results showed that different tissues of L. japonica are enriched with sets of unigenes associated with specific pharmaceutically important metabolic pathways and, therefore, possess unique medicinal properties. The present study will serve as a resource for future attempts for functional characterization of enzyme coding genes within key metabolic processes.
Advent of Next Generation Sequencing has led to possibilities of de novo transcriptome assembly of organisms without availability of complete genome sequence. Among various sequencing platforms available, Illumina is the most widely used platform based on data quality, quantity and cost. Various de novo transcriptome assemblers are also available today for construction of de novo transcriptome.
In this study, we aimed at obtaining an ameliorated de novo transcriptome assembly with sequence reads obtained from Illumina platform and assembled using Trinity Assembler. We found that, primary transcriptome assembly obtained as a result of Trinity can be ameliorated on the basis of transcript length, coverage, and depth and protein homology. Our approach to ameliorate is reproducible and could enhance the sensitivity and specificity of the assembled transcriptome which could be critical for validation of the assembled transcripts and for planning various downstream biological assays.
The Principal Component Analysis (PCA) is a widely used method of reducing the dimensionality of high-dimensional data, often followed by visualizing two of the components on the scatterplot. Although widely used, the method is lacking an easy-to-use web interface that scientists with little programming skills could use to make plots of their own data. The same applies to creating heatmaps: it is possible to add conditional formatting for Excel cells to show colored heatmaps, but for more advanced features such as clustering and experimental annotations, more sophisticated analysis tools have to be used. We present a web tool called ClustVis that aims to have an intuitive user interface. Users can upload data from a simple delimited text file that can be created in a spreadsheet program. It is possible to modify data processing methods and the final appearance of the PCA and heatmap plots by using drop-down menus, text boxes, sliders etc. Appropriate defaults are given to reduce the time needed by the user to specify input parameters. As an output, users can download PCA plot and heatmap in one of the preferred file formats. This web server is freely available at http://biit.cs.ut.ee/clustvis/.
© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
Rhodiola roseaL. (roseroot) has been categorized as an adaptogen and currently is one of the most studied medicinal plants for its enormous pharmaceutical significance and more reputably for its bioactive secondary metabolites, namely (rosin, rosavin, rosarin and salidroside). Roseroot is difficult to cultivate and develops very slowly in its natural environment, justifying development of new methods for production of its bioactive compounds. Different methods such as in vitro cell, tissue, and callus culture accompanied by some biotransformation trials have been carried out in the last two decades, but no feasible example has been developed for a large scale and industrial
production of roseroot glycosides. To achieve a clear understanding of how to design any of roseroot in vitro culture systems to maximize the content of its specific natural products, the knowledge of a real biosynthetic pathway of these glycosides is necessary and indispensible. In this review article we are going to summarize the attainments and
scientific results from molecular genetics of shikimate pathway in higher plants to give further clarification of salidroside and cinnamyl alcohol glycosides biosynthetic pathway. Considering all possibilities for these metabolites synthesis will help to think of alternative biotechnological methods for enhancing the biosynthesis of desired
compounds in plants and more specifically in Rhodiola rosea L.
Salidroside (1) is the most important bioactive component of Rhodiola (also called as "Tibetan Ginseng"), which is a valuable medicinal herb exhibiting several adaptogenic properties. Due to the inefficiency of plant extraction and chemical synthesis, the supply of salidroside (1) is currently limited. Herein, we achieved unprecedented biosynthesis of salidroside (1) from glucose in a microorganism. First, the pyruvate decarboxylase ARO10 and endogenous alcohol dehydrogenases were recruited to convert 4-hydroxyphenylpyruvate (2), an intermediate of L-tyrosine pathway, to tyrosol (3) in Escherichia coli. Subsequently, tyrosol production was improved by overexpressing the pathway genes, and by eliminating competing pathways and feedback inhibition. Finally, by introducing Rhodiola-derived glycosyltransferase UGT73B6 into the above-mentioned recombinant strain, salidroside (1) was produced with a titer of 56.9 mg/L. Interestingly, the Rhodiola-derived glycosyltransferase, UGT73B6, also catalyzed the attachment of glucose to the phenol position of tyrosol (3) to form icariside D2 (4), which was not reported in any previous literatures.
Rhodiola (Crassulaceae) an arctic-alpine plant, is extensively used in traditional folk medicine in Asian and European countries. A number of investigations have demonstrated that Rhodiola prep-arations exhibit adaptogenic, neuroprotective, anti-tumour, cardioprotective, and anti-depressant effects. The main compounds responsible for these activities are believed to be salidroside, rosin and its derivatives which became the target of biotechnological investi-gations. This review summarizes the results of the diverse biotechnological approaches undertaken to enhance the production of salidroside, rosin and its derivatives in callus, cell suspension and organ in vitro cultures of selected Rhodiola species. TyrDC Tyrosine decarboxylase UDP UDP-glucose:tyrosol glucosyltransferase UGT Uridine diphosphate dependent glucosyltransferase Introduction
With the aim to provide a resource for functional and evolutionary study of plant transcription factors (TFs), we updated
the plant TF database PlantTFDB to version 3.0 (http://planttfdb.cbi.pku.edu.cn). After refining the TF classification pipeline, we systematically identified 129 288 TFs from 83 species, of which 67 species
have genome sequences, covering main lineages of green plants. Besides the abundant annotation provided in the previous version,
we generated more annotations for identified TFs, including expression, regulation, interaction, conserved elements, phenotype
information, expert-curated descriptions derived from UniProt, TAIR and NCBI GeneRIF, as well as references to provide clues
for functional studies of TFs. To help identify evolutionary relationship among identified TFs, we assigned 69 450 TFs into
3924 orthologous groups, and constructed 9217 phylogenetic trees for TFs within the same families or same orthologous groups,
respectively. In addition, we set up a TF prediction server in this version for users to identify TFs from their own sequences.
Tyrosine decarboxylase initializes salidroside biosynthesis. Metabolic characterization of tyrosine decarboxylase gene from Rhodiola crenulata (RcTYDC) revealed that it played an important role in salidroside biosynthesis. Recombinant 53 kDa RcTYDC converted tyrosine into tyramine. RcTYDC gene expression was induced coordinately with the expression of RcUDPGT (the last gene involved in salidroside biosynthesis) in SA/MeJA treatment; the expression of RcTYDC and RcUDPGT was dramatically upregulated by SA, respectively 49 folds and 36 folds compared with control. MeJA also significantly increased the expression of RcTYDC and RcUDPGT in hairy root cultures. The tissue profile of RcTYDC and RcUDPGT was highly similar: highest expression levels found in stems, higher expression levels in leaves than in flowers and roots. The gene expressing levels were consistent with the salidroside accumulation levels. This strongly suggested that RcTYDC played an important role in salidroside biosynthesis in R. crenulata. Finally, RcTYDC was used to engineering salidroside biosynthetic pathway in R. crenulata hairy roots via metabolic engineering strategy of overexpression. All the transgenic lines showed much higher expression levels of RcTYDC than non-transgenic one. The transgenic lines produced tyramine, tyrosol and salidroside at higher levels, which were respectively 3.21-6.84, 1.50-2.19 and 1.27-3.47 folds compared with the corresponding compound in non-transgenic lines. In conclusion, RcTYDC overexpression promoted tyramine biosynthesis that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside.
De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms. We also present Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes. In the procedure, we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sourceforge.net. The run time of this protocol is highly dependent on the size and complexity of data to be analyzed. The example data set analyzed in the procedure detailed herein can be processed in less than 5 h.
Rhodiola imbricata Edgew. (Rose root or Arctic root or Golden root or Shrolo), belonging to the family Crassulaceae, is an important food crop and medicinal plant in the Indian trans-Himalayan cold desert. Chemometric profile of the n-hexane, chloroform, dichloroethane, ethyl acetate, methanol, and 60% ethanol root extracts of R. imbricata were performed by hyphenated gas chromatography mass spectrometry (GC/MS) technique. GC/MS analysis was carried out using Thermo Finnigan PolarisQ Ion Trap GC/MS MS system comprising of an AS2000 liquid autosampler. Interpretation on mass spectrum of GC/MS was done using the NIST/EPA/NIH Mass Spectral Database, with NIST MS search program v.2.0g. Chemometric profile of root extracts revealed the presence of 63 phyto-chemotypes, among them, 1-pentacosanol; stigmast-5-en-3-ol, (3β,24S); 1-teracosanol; 1-henteracontanol; 17-pentatriacontene; 13-tetradecen-1-ol acetate; methyl tri-butyl ammonium chloride; bis(2-ethylhexyl) phthalate; 7,8-dimethylbenzocyclooctene; ethyl linoleate; 3-methoxy-5-methylphenol; hexadecanoic acid; camphor; 1,3-dimethoxybenzene; thujone; 1,3-benzenediol, 5-pentadecyl; benzenemethanol, 3-hydroxy, 5-methoxy; cholest-4-ene-3,6-dione; dodecanoic acid, 3-hydroxy; octadecane, 1-chloro; ethanone, 1-(4-hydroxyphenyl); α-tocopherol; ascaridole; campesterol; 1-dotriacontane; heptadecane, 9-hexyl were found to be present in major amount. Eventually, in the present study we have found phytosterols, terpenoids, fatty acids, fatty acid esters, alkyl halides, phenols, alcohols, ethers, alkanes, and alkenes as the major group of phyto-chemotypes in the different root extracts of R. imbricata. All these compounds identified by GC/MS analysis were further investigated for their biological activities and it was found that they possess a diverse range of positive pharmacological actions. In future, isolation of individual phyto-chemotypes and subjecting them to biological activity will definitely prove fruitful results in designing a novel drug.
Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site residues.
Background: Plant arylalkylamine and aldehyde synthesizing aromatic amino acid decarboxylases (AAAD) are effectively indistinguishable.
Results: Mutagenesis of a single AAAD residue enables the interconversion of activities.
Conclusion: A single residue is primarily responsible for differentiating plant AAAD decarboxylase and aldehyde synthase activities.
Significance: AAAD activity differentiation enables primary sequence activity identification, generation of unusual AAAD enzyme products, and AAAD mechanistic insights.
The sequence of a cDNA clone that includes the complete coding region of tryptophan decarboxylase (EC 4.1.1.28, formerly EC 4.1.1.27) from periwinkle (Catharanthus roseus) is reported. The cDNA clone (1747 base pairs) was isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA found in developing seedlings of C. roseus. The clone hybridized to a 1.8-kilobase mRNA from developing seedlings and from young leaves of mature plants. The identity of the clone was confirmed when extracts of transformed Escherichia coli expressed a protein containing tryptophan decarboxylase enzyme activity. The tryptophan decarboxylase cDNA clone encodes a protein of 500 amino acids with a calculated molecular mass of 56,142 Da. The amino acid sequence shows a high degree of similarity with the aromatic L-amino acid decarboxylase (dopa decarboxylase) and the alpha-methyldopa-hypersensitive protein of Drosophila melanogaster. The tryptophan decarboxylase sequence also showed significant similarity to feline glutamate decarboxylase and mouse ornithine decarboxylase, suggesting a possible evolutionary link between these amino acid decarboxylases.
The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies.
We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs) from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted) proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs) include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens), one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe), and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or approximately 54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of approximately 20% of the KOG set. This conserved portion of the KOG set is much greater than the ubiquitous portion of the COG set (approximately 1% of the COGs). In part, this difference is probably due to the small number of included eukaryotic genomes, but it could also reflect the relative compactness of eukaryotes as a clade and the greater evolutionary stability of eukaryotic genomes.
The updated collection of orthologous protein sets for prokaryotes and eukaryotes is expected to be a useful platform for functional annotation of newly sequenced genomes, including those of complex eukaryotes, and genome-wide evolutionary studies.
Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) is a critically endangered medicinal herb of immense importance due to its pharmaceutical bioactive compound, especially sipeimine, used for the treatment of chronic respiratory disorders. However, the industrial demand for sipeimine solely depends on its endangered natural habitat. Therefore; there is an utmost need for its biodiversity conservation as well as for the sustainable utilization of phytochemicals. Plant cell culture and transcriptomics-based molecular bioprospection of key regulatory genes involved in sipeimine biosynthesis as such will play a crucial role in exploring the unexplored traits, that are in supply crisis or nearly in extinction stage. De novo comparative transcriptome sequencing of the bulb (in vivo), callus, and regenerated plantlets (in vitro) resulted in more than 150 million high-quality paired-end clean reads that assembled into final 31,428 transcripts. Functional annotation and unigenes classification with multiple public databases such as KEGG, Refseq, Uniprot, TAIR, GO, and COG, etc. along with chemical structures and functional biocatalytic activity analysis of different steroidal alkaloids facilitated the identification of 30 unigenes specific to sipeimine biosynthesis. Additionally, ABC transporters and TFs like bHLH, MYC, MYB, and WRKY suggests their possible role in metabolite translocation and regulation in vivo as well as in vitro tissues. Differential gene expression and quantitative analysis revealed that the MVA pathway probably the predominant route for 5C intermediate (IPP & DMAPP) biosynthesis. Further, the genes involved in the downstream biosynthesis pathway viz. SQLE, CAS1, SMT1, SMO1, SMO2, SC5DL, DHCR7, DHCR24, CYP710A, 3β-HSD, CYP90D2, and CYP374A6 shown similar expression pattern with RNA-Seq and qRT-PCR findings. The positive correlation between higher expression of proposed biosynthetic pathway genes and relatively higher accumulation of sipeimine in differentiated naturally grown bulb tissues (in vivo), undifferentiated cells (callus), and de-differentiated tissues i.e. regenerated plantlets (in vitro) has been evident from the present study. Comprehensive genomic resources created in F. cirrhosa will provide strong evidence of bulb derived in vitro culture as an alternative promising source for steroidal alkaloids biosynthesis and metabolite upscaling through genetic and metabolic engineering.
Salidroside and rosavins have been found as the most potent ingredients used in Rhodiola based herbal formulations. Rhodiola imbricata is a medicinal herb of the trans-Himalayan Ladakh region of India. Since, the natural supply of this herb is rapidly decreasing, due to its over-exploitation, high altitude region, and traditional usage in the Amchi system of medicine. In the present study, friable callus culture was developed for the first time from leaf and root explants of R. imbricata. Furthermore, callus cell lines were also evaluated for rapid growth rate and high metabolite accumulation. The results revealed that callus induction frequency observed in juvenile leaves (100 %) and roots (87.50 %) in MS medium enriched with 0.5 mg/L TDZ and 1 mg/L NAA. Scanning electron microscopy (SEM) analysis was done for histological observations of the callus surface. A selective and efficient ultra-performance liquid chromatography with PDA detector (UPLC-PDA) method was developed and validated for the quantification of phenylethanoids, phenylpropanoids, and phenolic acids. The maximum salidroside was detected in leaf derived friable green calli (3.59 mg/g DW) subsequently followed by leaf derived friable white calli (2.31 mg/g DW). While rosavin and rosarin were detected maximum in root derived compact green calli (0.15 mg/g DW) and root derived friable green calli (0.07 mg/g DW). Genes encoding enzymes involved in salidroside and rosavins biosynthesis were also investigated for transcript abundance in wild as well as in vitro cultures using the RT-qPCR approach. The present study explained the friable callus culture as a potential alternative approach to obtain higher metabolite yield in R. imbricata. Further, it can be used for sequential scale-up of metabolite at bioreactor level to meet the industrial demand.
Podophyllum hexandrum is the major source of podophyllotoxin (PTOX), a highly bioactive lignan of great pharmacological importance with anti-cancerous activities. The industrial demand of PTOX relies on the highly endangered natural resources only. It is therefore, desirable to elucidate global molecular processes and identify key genes for enhancing PTOX biosynthesis by overexpressing the targeted candidates. Transcriptome of leaf, rhizome, and stalk was generated to analyse the spatial regulation of PTOX biosynthesis in genetically diverse genotypes. Overall, 198 million high-quality paired-end reads were assembled into 85,531 transcripts. In addition, 32,341 transcripts were assigned gene ontologies with 6570 hits in distinct pathways and 15,886 transcription factors representing 70 families. Interestingly, comparative expression analyses revealed that 12 of 31 genes of PTOX biosynthesis were upregulated in rhizome. However, shikimate and phenylalanine pathways that generate PTOX precursors were abundantly upregulated in leaves. Thus, a further insight on the inducers of these genes can be extended to enrich the aerial tissues for downstream pathway through genetic manipulations. Additionally, higher expression of transcription factors WRKY, MYB, bZIP, bHLH, and AP2, transporters ABCB and ABCC, UGTs, CYP450s, and jasmonate pathway in rhizome supported the secondary metabolism. The comprehensive genomic resource created during this study will provide deeper understanding of lignan biosynthesis and its regulation. This will further enable selection of the elite genotypes and potential genes that can be directed to enhance PTOX production and yield at an industrial scale.
Bulbous Fritillaria roylei (known as Jangli lahsan) is an important critically endangered medicinal herb of Astavarga group and widely used in traditional medicinal system. Being a medicinal potential but endangered herb, biotechnological interventions are urgently needed for its restoration and production of bioactive compounds. In the present study, in vitro cultures were established from bulb scales as explant by using optimized sterilization protocol (79.67% survival). From different combinations and concentrations of plant growth regulator tested; high efficiency callus induction (88.89%) and in vitro plantlets regeneration (77.78%) response was obtained in Murashige and Skoog medium supplemented with 0.5 mg/L thidiazuron +2.0 mg/L picloram and 1.0 mg/L kinetin +0.5 mg/L naphthaleneacetic acid, respectively. For the quantification of steroidal alkaloids i.e. sipeimine and peimine; selective, sensitive, and rapid liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analytical method was developed and validated. Quantification of sipeimine and peimine was done for the first time in established in vitro cultures and naturally grown tissues of F. roylei i.e. bulbs, leaves, stem and flower bud. Among in vitro raised cultures, higher sipeimine content (1.98 μg/g) was detected in callus. Whereas, maximum sipeimine content (599.72 μg/g) was observed in floral bud compared to other wild tissues. Peimine content was detected in bulb (1.13 μg/g), leaf (0.78 μg/g) and stem (0.67 μg/g). Qualitative and quantitative phytochemical screening revealed potentiality of this important medicinal herb for commercial utilization in drug formulation to the industries. Maximum antioxidant activity (IC50) was exhibited in floral buds (0.11 mg/mL), while among in vitro raised cultures; regenerated plantlets (0.80 mg/mL) shown higher scavenging activity than callus (2.71 mg/mL). Higher total phenol content (42.86 μg gallic acid/mg) was observed in bulb whereas, flavonoids content is higher in leaves (68.36 μg quercetin/mg) followed by callus (58.22 μg quercetin /mg). Putative steroidal alkaloids gene expression studies using RT-qPCR showed positive correlation with metabolite content. For the first time, present investigation concluded that floral buds, leaves and stem could also be used as an alternative potential source for steroidal alkaloids and callus culture as sustainable approach for metabolite production to meet industrial standards and demands.
Rhodiola imbricata is a rare medicinal herb well-known for its adaptogenic and antioxidant properties due to the presence of a diverse array of secondary metabolites, including phenylethanoids and phenylpropanoids. These secondary metabolites are generating considerable interest due to their potential applications in pharmaceutical and nutraceutical industries. The present study investigated the influence of light quality on growth, production of industrially important secondary metabolites and antioxidant activity in callus cultures of Rhodiola imbricata. Callus cultures of Rhodiola imbricata were established under different light conditions: 100% red, 100% blue, 100% green, RGB (40% red: 40% green: 20% blue) and 100% white (control). The results showed that the callus cultures grown under red light accumulated maximum amount of biomass (7.43 g/L) on day 21 of culture, as compared to other light conditions. Maximum specific growth rate (0.126 days⁻¹) and doubling time (132.66 h) was observed in callus cultures grown under red light. Reverse phase-high performance liquid chromatographic (RP-HPLC) analysis revealed that the callus cultures exposed to blue light accumulated maximum amount of Salidroside (3.12 mg/g DW) on day 21 of culture, as compared to other light conditions. UV–Vis spectrophotometric analysis showed that the callus cultures exposed to blue light accumulated maximum amount of total phenolics (11.84 mg CHA/g DW) and total flavonoids (5.53 mg RE/g DW), as compared to other light conditions. Additionally, callus cultures grown under blue light displayed enhanced DPPH free radical scavenging activity (53.50%). Callus cultures grown under different light conditions showed no significant difference in ascorbic acid content (11.05–13.90 mg/g DW) and total antioxidant capacity (27.37–30.17 mg QE/g DW). The correlation analysis showed a positive correlation between total phenolic content and DPPH free radical scavenging activity in callus cultures (r = 0.85). Taken together, these results demonstrate the remarkable potential of light quality on biomass accumulation and production of industrially important secondary metabolites in callus cultures of Rhodiola imbricata. This study will open new avenues and perspectives towards abiotic elicitation strategies for sustainable growth and enhanced production of bioactive compounds in in-vitro cultures of Rhodiola imbricata.
Plants are unrivaled in the natural world in both the number and complexity of secondary metabolites they produce, and the ubiquitous phenylpropanoids and the lineage-specific glucosinolates represent two such large and chemically diverse groups. Advances in genome-enabled biochemistry and metabolomic technologies have greatly increased the understanding of their metabolic networks in diverse plant species. There also has been some progress in elucidating the gene regulatory networks that are key to their synthesis, accumulation and function. This review highlights what is currently known about the gene regulatory networks and the stable sub-networks of transcription factors at their cores that regulate the production of these plant secondary metabolites and the differentiation of specialized cell types that are equally important to their defensive function. Remarkably, some of these core components are evolutionarily conserved between secondary metabolism and specialized cell development and across distantly related plant species. These findings suggest that the more ancient gene regulatory networks for the differentiation of fundamental cell types may have been recruited and remodeled for the generation of the vast majority of plant secondary metabolites and their specialized tissues.
Molecular and biochemical studies have shown that gene contains single or combination of different cis-acting regulatory elements are actively controlling the transcriptional regulation of associated genes, downstream effects of these result in modulation of various biological pathways such as biotic/abiotic stress responses, hormonal responses to growth and development processes and secondary metabolite production. Therefore, the identification of promoters and their cis-regulatory elements is one of intriguing area to study the dynamic complex regulatory network of genes activities by integrating computational, comparative, structural and functional genomics. A variety of bioinformatics servers or database have been established to predict the cis-acting elements present in the promoter region of target gene and their association with the expression profiles in the TFs. The aim of this study is to predict possible cis-acting regulatory elements that have putative role in the transcriptional regulation of a dynamic network of metabolite gene activities controlling prenylflavonoid and bitter acids biosynthesis in hop (Humulus lupulus). Recent release of hop draft genome enabled us to predict the possible cis-acting regulatory elements by extracting 2 kbp of 5′ regulatory regions of genes important for lupulin metabolome biosynthesis, using Plant CARE, PLACE and Genomatix Matinspector professional databases. The result reveals the plausible role of cis-acting regulatory elements in the regulation of gene expression primarily involved in lupulin metabolome biosynthesis including under various stress conditions.
... Thomas D Schmittgen 1 & Kenneth J Livak 2 . ABSTRACT. ... N. Engl. J . Med. ... 32, e178 (2004). | Article | PubMed | ChemPort |; Livak , KJ & Schmittgen , TD Analysis of relative gene expression data using real - time quantitative PCR and the 2 (- Delta Delta C(T)) Method . ...
... Thomas D Schmittgen 1 & Kenneth J Livak 2 . ABSTRACT. ... N. Engl. J . Med. ... 32, e178 (2004). | Article | PubMed | ChemPort |; Livak , KJ & Schmittgen , TD Analysis of relative gene expression data using real - time quantitative PCR and the 2 (- Delta Delta C(T)) Method . ...
... Thomas D Schmittgen 1 & Kenneth J Livak 2 . ABSTRACT. ... N. Engl. J . Med. ... 32, e178 (2004). | Article | PubMed | ChemPort |; Livak , KJ & Schmittgen , TD Analysis of relative gene expression data using real - time quantitative PCR and the 2 (- Delta Delta C(T)) Method . ...
Study on transcriptome, the entire pool of transcripts in an organism or single cells at certain physiological or pathological stage, is indispensable in unraveling the connection and regulation between DNA and protein. Before the advent of deep sequencing, microarray was the main approach to handle transcripts. Despite obvious shortcomings, including limited dynamic range and difficulties to compare the results from distinct experiments, microarray was widely applied. During the past decade, next-generation sequencing (NGS) has revolutionized our understanding of genomics in a fast, high-throughput, cost-effective, and tractable manner. By adopting NGS, efficiency and fruitful outcomes concerning the efforts to elucidate genes responsible for producing active compounds in medicinal plants were profoundly enhanced. The whole process involves steps, from the plant material sampling, to cDNA library preparation, to deep sequencing, and then bioinformatics takes over to assemble enormous—yet fragmentary—data from which to comb and extract information. The unprecedentedly rapid development of such technologies provides so many choices to facilitate the task, which can cause confusion when choosing the suitable methodology for specific purposes. Here, we review the general approaches for deep transcriptome analysis and then focus on their application in discovering biosynthetic pathways of medicinal plants that produce important secondary metabolites.
It is common perception that the content of secondary metabolites in pure lines of advanced generations of pedigree selections or intra- and inter-specific crosses should be stable like other qualitative and quantitative traits, but in fact this is usually not the case. In this review article, we try to give probable explanations on why the content of secondary metabolites fluctuates in subsequent generations of pure lines as well as advanced generations (F7 onward) of intra- and inter-specific crosses. Genetic, ontogenic, morphogenetic and environmental factors are extremely important in the biosynthesis and accumulation of secondary metabolites. The biosynthesis of PSMs depend upon these various factors, change in only one factor may alter the content of PSMs though other factors remain constant. We here present a detailed view of the possible roles of the various factors in secondary metabolite instability. A good understanding of the mechanisms involved in secondary metabolite synthesis, degradation and accumulation in plants is required for the future formulation of strategies for the genetic improvement of secondary metabolite production in plants.
The most important pharmaceutically active compounds of Rhodiola rosea are glycosides, which are accumulated in the rhizome of the plant. The aim of the study was to investigate the dynamics of the glycoside content of the rhizome and the leaves during the vegetation period with HPLC. Along with this the expression of the genes involved in the biosynthesis of the rosavins were studied with qPCR.
Expression analysis of the genes encoding for phenylalanine ammonia lyase (PAL), 4-coumarate-CoA ligase (4CL), cinnamoyl-CoA oxidoreductase (CCR), cinnamyl alcohol dehydrogenase (CAD) was done by qPCR. Results of the HPLC analysis showed that traces of the glycosides are present in the leaves, only, while in the rhizomes salidroside content was 0.5–4.5%, rosin content was 0.01-0.1%, rosavin content was 0.02-2.37% and rosarin content was 0.1-0.55% of the dry weight. The 4 biological repetitions showed very
big deviation, no clear trend could be observed. Statistical analysis of the relative gene expression values showed, that the expression of 4CL in the rhizome was the most similar in the 4 biological repetitions, showing a decrease during flowering and then rising to the initial level. In case of PAL the 4 repetitions had higher diversity in the rhizome samples, while in case of 4CL, CAD higher diversity was observed in case of the leaf samples. CCR was equally diverse both in the rhizome and in the leaf samples. Linear regression analysis showed that mostly the expression of CCR and to a lesser extent of CAD and PAL had effect on the content of the cinnamyl alcohol glycosides. Based on the results it is not possible to conclude the expression profiles of neither the genes nor the accumulation pattern of the glycosides. Environmental factors and plant age certainly have major effect.
Salidroside, the 8-O-β-D-glucoside of tyrosol, is the main bioactive component of Rhodiola species and is found mainly in the plant roots. It is well known that glucosylation of tyrosol is the final step in the biosynthesis of salidroside; however, the biosynthetic pathway of tyrosol and its regulation are less well understood. A summary of the results of related studies revealed that the precursor of tyrosol might be tyramine, which is synthesized from tyrosine. In this study, a cDNA clone encoding tyrosine decarboxylase (TyrDC) was isolated from Rhodiola sachalinensis A. Bor using rapid amplification of cDNA ends. The resulting cDNA was designated RsTyrDC. RNA gel-blot analysis revealed that the predominant sites of expression in plants are the roots and high levels of transcripts are also found in callus tissue culture. Functional analysis revealed that tyrosine was best substrate of recombinant RsTyrDC. The over-expression of the sense-RsTyrDC resulted in a marked increase of tyrosol and salidroside content, but the levels of tyrosol and salidroside were 274 and 412%, respectively, lower in the antisense-RsTyrDC transformed lines than those in the controls. The data presented here provide in vitro and in vivo evidence that the RsTyrDC can regulate the tyrosol and salidroside biosynthesis, and the RsTyrDC is most likely to have an important function in the initial reaction of the salidroside biosynthesis pathway in R. sachalinensis.
Salidroside, the 8-O-β-d-glucoside of tyrosol, is a novel adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor. Due to the scarcity of R. sachalinensis and its low yield of salidroside, there is great interest in enhancing production of salidroside by biotechnological manipulations. In this study, two putative UDP-glycosyltransferase (UGT) cDNAs, UGT72B14 and UGT74R1, were isolated from roots and cultured cells of methyl jasmonate (MeJA)-treated R. sachalinensis, respectively. The level of sequence identity between their deduced amino acid sequences was ca. 20%. RNA gel-blot analysis established that UGT72B14 transcripts were more abundant in roots, and UGT74R1 was highly expressed in the calli, but not in roots. Functional analysis indicated that recombinant UGT72B14 had the highest level of activity for salidroside production, and that the catalytic efficiency (V(max)/K(m)) of UGT72B14 was 620% higher than that of UGT74R1. The salidroside contents of the UGT72B14 and UGT74R1 transgenic hairy root lines of R. sachalinensis were also ∼420% and ∼50% higher than the controls, respectively. UGT72B14 transcripts were mainly detected in roots, and UGT72B14 had the highest level of activity for salidroside production in vitro and in vivo.
Tyrosine decarboxylase (TyrDC) is an important enzyme in the secondary metabolism of several plant species, and was hypothesized to play a key role in the biosynthesis of salidroside, a pharmacologically valuable compound of roseroot. A 1520bp cDNA was cloned and sequenced, and turned out to contain an ORF of 963bp, which encodes a protein of 320 amino acids. The expression of the gene was studied by real-time PCR from leaves and roots of both high and low salidroside producer genotype of roseroot. The gene expression analysis showed the gene to be expressed in leaves as well as in roots; however, the expression was significantly higher in roots, which coincides with the fact that salidroside accumulates preferentially in the underground parts of the plant. The expression was also higher in the line accumulating high levels of salidroside, compared to the line with lower salidroside content. The difference in the expression intensity suggests a decisive role for this enzyme in the salidroside biosynthesis.
A comprehensive survey of the extensive literature relevant to the evolution, physiology, biochemistry, regulation, and genetic engineering applications of plant aromatic L-amino acid decarboxylases (AADCs) is presented. AADCs catalyze the pyridoxal-5'-phosphate (PLP)-dependent decarboxylation of select aromatic L-amino acids in plants, mammals, and insects. Two plant AADCs, L-tryptophan decarboxylase (TDC) and L-tyrosine decarboxylase (TYDC), have attracted considerable attention because of their role in the biosynthesis of pharmaceutically important monoterpenoid indole alkaloids and benzylisoquinoline alkaloids, respectively. Although plant and animal AADCs share extensive amino acid homology, the enzymes display striking differences in their substrate specificities. AADCs from mammals and insects accept a broad range of aromatic L-amino acids, whereas TDC and TYDC from plants exhibit exclusive substrate specificity for L-amino acids with either indole or phenol side chains, but not both. Recent biochemical and kinetic studies on animal AADCs support basic features of the classic AADC reaction mechanism. The catalytic mechanism involves the formation of a Schiff base between PLP and an invariable lysine residue, followed by a transaldimination reaction with an aromatic L-amino acid substrate. Both TDC and TYDC are primarily regulated at the transcriptional level by developmental and environmental factors. However, the putative post-translational regulation of TDC via the ubiquitin pathway, by an ATP-dependent proteolytic process, has also been suggested. Isolated TDC and TYDC genes have been used to genetically alter the regulation of secondary metabolic pathways derived from aromatic amino acids in several plant species. The metabolic modifications include increased serotonin levels, reduced indole glucosinolate levels, redirected shikimate metabolism, increased indole alkaloid levels, and increased cell wall-bound tyramine levels.