Production Of Plant Secondary Metabolites Research Articles

Novel Differentially Expressed LncRNAs Regulate Artemisinin Biosynthesis in Artemisia annua

Long non-coding RNAs (lncRNAs) are crucial in regulating secondary metabolite production in plants, but their role in artemisinin (ART) biosynthesis, a key anti-malarial compound from Artemisia annua, remains unclear. Here, by investigating high-artemisinin-producing (HAP) and lowartemisinin-producing (LAP) genotypes, we found that the final artemisinin content in A. annua is influenced by the quantity of the precursor compounds. We report on RNA deep sequencing in HAP and LAP genotypes. Based on the application of a stringent pipeline, 1419 novel lncRNAs were identified. Moreover, we identified 256 differentially expressed lncRNAs between HAP and LAP. We then established correlations between lncRNAs and artemisinin biosynthesis genes in order to identify a molecular framework for the differential expression of the pathway between the two genotypes. Three potential lncRNAs (MSTRG.33718.2, MSTRG.30396.1 and MSTRG.2697.4) linked to the key artemisinin biosynthetic genes (ADS: Amorpha-4,11-diene synthase, DXS: 1-deoxy-D-xylulose-5-phosphate synthase, and HMGS: 3-hydroxyl-3-methyglutaryl CoA synthase) were detected. Importantly, we observed that up-regulation of these lncRNAs positively modulates the target artemisinin biosynthetic genes, potentially leading to high artemisinin biosynthesis in HAP. In contrast, BAS (beta-amyrin synthase), which is involved in the artemisinin competing pathway, was strongly down-regulated in HAP compared to LAP, in line with the expression pattern of the linked lncRNA MSTRG.30396.1. By identifying and characterizing lncRNAs that are potentially linked to the regulation of key biosynthetic genes, this work provides new insights into the complex regulatory networks governing artemisinin production in A. annua. Such findings could pave the way for innovative approaches in metabolic engineering, potentially enhancing artemisinin yields and addressing challenges in sustainable production.

Characterization and quantification of peptaibol produced by novel Trichoderma spp: Harnessing their potential to mitigate moisture stress through enhanced biochemical and physiological responses in black pepper (Piper nigrum L.).

Trichoderma spp. is primarily applied to manage biotic stresses in plants. Still, they also can mitigate abiotic stresses by the stimulation of antioxidative protective mechanisms and enhanced synthesis of secondary metabolites. The study optimized the conditions to enhance peptaibol production by novel Trichoderma spp, characterized and quantified peptaibol- alamethicin using HPLC and LC MS-MS. The present study investigated these isolates efficacy in enhancing growth and the associated physio-biochemical changes in black pepper plants under moisture stress. Under in vitro conditions, out of 51 isolates studied, six isolatesviz., T. asperellum (IISR NAIMCC 0049), T. erinaceum (IISR APT1), T. harzianum (IISR APT2), T. harzianum (IISR KL3), T. lixii (IISR KA15) and T. asperellum (IISR TN3) showed tolerance to low moisture levels (5, 10 and 20%) and higher temperatures (35 and 40°C). In vivo evaluation on black pepper plants maintained under four different moisture levels (Field capacity [FC]; 75%, 50%, and 25%) showed that the plants inoculated with Trichoderma accumulated greater quantities of secondary metabolites viz., proline, phenols, MDA and soluble proteins at low moisture levels (50% and 25% FC). In the present study, plants inoculated with T. asperellum and T.harzianum showed significantly increased growth compared to uninoculated plants. The shortlisted Trichoderma isolates exhibited differences in peptaibol production and indicated that the peptide might be the key factor for their efficiency as biocontrol agents. The present study also demonstrated that Trichoderma isolates T. harzianum and T. asperellum (IISR APT2 & NAIMCC 0049) enhanced the drought-tolerant capabilities of black pepper by improving plant growth and secondary metabolite production.

Transcriptomic and metabolomic investigations of methyl jasmonate-mediated enhancement of low temperature stress resistance in Cassia obtusifolia L.

Low temperature stress negatively impacts plant growth and development, reducing crop yields by disrupting metabolite production and accumulation. Cassia obtusifolia L., a widely cultivated medicinal plant in subtropical regions, is particularly vulnerable to such stress. Methyl jasmonate (MeJA) regulates plant growth, defense mechanisms, and secondary metabolite production. This study investigated how C. obtusifolia responds to low temperature stress under MeJA treatment using transcriptomic and metabolomic approaches. MeJA treatment upregulated several transcription factor families, including APE/ERF-ERF, WRKY, NAC, and MYB-related, which modulated the plant's response to cold stress. These transcription factors influenced the expression of genes involved in glycolysis, sugar metabolism, and amino acid pathways, contributing to the plant's adaptive responses. Differentially expressed genes were associated with key metabolic processes such as flavonoid biosynthesis, ribosome function, glycolysis/gluconeogenesis, and sugar metabolism. Using H-NMR and LC-MS techniques, we observed that MeJA induced the accumulation of sugars and amino acids in the leaves of C. obtusifolia seedlings under cold stress, while levels of three flavonoid compounds Isoliquiritigenin, Kaempferol-7-O-glucoside, and Phloretin significantly decreased. Enrichment analysis indicated that MeJA modulates the biosynthetic pathways of these compounds in a dose-dependent manner. Integrating metabolomic and transcriptomic data further elucidated alterations in the flavonoid biosynthetic network and other metabolic pathways under low temperature stress. These findings offer insights into the molecular mechanisms underlying flavonoid synthesis and other metabolic adjustments in C. obtusifolia under MeJA treatment.

Unravelling the synergistic effects of arbuscular mycorrhizal fungi and vermicompost on improving plant growth,nutrient absorption, and secondary metabolite production in ginger (Zingiber officinale Rosc.)

Unravelling the synergistic effects of arbuscular mycorrhizal fungi and vermicompost on improving plant growth,nutrient absorption, and secondary metabolite production in ginger (Zingiber officinale Rosc.)

Construction of a beneficial microbes-enriched rhizosphere system assists plants in phytophagous insect defense: current status, challenges and opportunities.

The construction of a plant rhizosphere system enriched with beneficial microbes (BMs) can efficiently help plants defend against phytophagous insects. However, our comprehensive understanding of this approach is still incomplete. In this review, we methodically analyzed the progress made over the last decade, identifying both challenges and opportunities. The main methods for developing a BMs-enriched rhizosphere system include inoculating exogenous BMs into plants, amending the existing soil microbiomes with amendments, and utilizing plants to shape the soil microbiomes. BMs can assist plants in suppressing phytophagous insects across many orders, including 13 Lepidoptera, seven Homoptera, five Hemiptera, five Coleoptera, four Diptera, and one Thysanoptera species by inducing plant systemic resistance, enhancing plant tolerance, augmenting plant secondary metabolite production, and directly suppressing herbivores. Context-dependent factors such as abiotic and biotic conditions, as well as the response of insect herbivores, can affect the outcomes of BM-assisted plant defense. Several challenges and opportunities have emerged, including the development of synthetic microbial communities for herbivore control, the integration of biosensors for effectiveness assessment, the confirmation of BM targets for phytophagous insect defense, and the regulation of outcomes via smart farming with artificial intelligence. This study offers valuable insights for developing a BM-enriched rhizosphere system within an integrated pest management approach. © 2024 Society of Chemical Industry.

Not just candy: A herbivore‐induced defence‐related plant protein in honeydew enhances natural enemy fitness

Abstract Herbivore feeding often increases secondary metabolite production in plants. These herbivore‐induced plant proteins might end up in honeydew excreted by phloem‐feeding insects. This is important because honeydew is one of the most abundant and accessible carbohydrate sources for natural enemies in many agroecosystems and these proteins can thus mediate many tri‐trophic interactions. Here, we hypothesized that defensive metabolites induced in the phloem by herbivory accumulate in the honeydew excreted by phloem‐feeding insects and, consequently, affect the fitness of the herbivores' natural enemies that feed on it. We used a tri‐trophic system consisting of citrus plants, the mealybug Planococcus citri and its primary parasitoid Anagyrus vladimiri. First, we assessed A. vladimiri fitness when fed on P. citri honeydew. We then collected honeydew of seven phloem‐feeding insects, including P. citri, and analysed their protein content. Finally, we analysed the effect of superoxide dismutase (SOD), an antioxidant enzyme associated with plant defences that was commonly found in the analysed honeydews, on A. vladimiri fitness. The fitness of A. vladimiri increased when fed on honeydew compared to a sucrose‐based diet, demonstrating that honeydew can contain compounds that benefit natural enemies. Proteomic analyses showed that defence‐related plant proteins were present in honeydew of seven phloem‐feeding insects analysed. Among these, the enzyme SOD was present in honeydew of all of them. Moreover, the levels of SOD were 10‐fold higher in the phloem of plants infested by P. citri than in that of uninfested plants. SOD was also actively excreted in P. citri honeydew, and we proved that it increases the fecundity of the parasitoid A. vladimiri. We conclude that enzymatic proteins induced by herbivory in the phloem and involved in plant defence, accumulate in the honeydew excreted by phloem‐feeding insects and, contrary to the current paradigm, at least some of these can have positive effects on the third trophic level. Read the free Plain Language Summary for this article on the Journal blog.

Recent advances in paclitaxel biosynthesis and regulation.

Paclitaxel (PTX) is a high value plant natural product (PNP) derived from Taxus (yew) species. This plant secondary metabolite (PSM) and its derivatives constitute a cornerstone for the treatment of an increasing variety of cancers. New applications for PTX also continue to emerge, further promoting demand for this WHO designated essential medicine. Here we review recent advances in our understanding of PTX biosynthesis and its cognate regulation, which have been enabled by the development of transcriptomic approaches and the recent sequencing and annotation of three Taxus genomes. Collectively, this has resulted in the elucidation of two functional gene sets for PTX biosynthesis, unlocking new potential for the use of heterologous hosts to produce PTX. Knowledge of the PTX pathway also provides a valuable resource for understanding the regulation of this key PSM. Epigenetic regulation of PSM in plant cell culture (PCC) is a major concern for PTX production, given the loss of PSM production in long-term cell cultures. Recent developments aim to design tools for manipulating epigenetic regulation, potentially providing a means to reverse the silencing of PSM caused by DNA methylation. Exciting times clearly lie ahead for our understanding of this key PSM and improving its production potential.

Biotechnological approaches in the production of plant secondary metabolites for treating human viral diseases: Prospects and challenges

Over the last couple of decades, three highly pathogenic and deadly human coronaviruses have posed dreadful impacts on human life, namely SARS-CoV, MERS-CoV and SARS-CoV-2. In addition, many other viral diseases also affect various aspects of human well-being such as lifestyles and socio-economic conditions. Therefore, research on developing antiviral drugs should be progressively carried out to rapidly prevent or reduce the severity of infection for the current and future emerging infection with pandemic potential. Conventional antiviral drugs have been associated with high cost, toxicity and resistance issues. The use of medicinal plants and their secondary metabolites (SMs) with potential anti-inflammatory properties garners attention as a safer alternative for treating viral diseases. This review discussed various plant SMs with therapeutic potential for treating viral diseases. Due to various factors including low concentrations of the phytocompounds produced, relying on natural systems for producing medically important SMs is not sustainable. Thus, several biotechnology strategies such as metabolic engineering and genetic modification, in vitro production as well as gene editing to enhance the phytochemical compounds are also highlighted. Future perspectives on the potential of plant-based antiviral drugs as well as other strategies to treat viral diseases in humans are also elaborated. Altogether, this review gives a comprehensive view of the pharmaceutical importance of plant SMs as an alternative approach against viral diseases.

Exploring low-dose gamma radiation effects on monoterpene biosynthesis in Thymus vulgaris: insights into plant defense mechanisms.

Thymus vulgaris, commonly known as thyme, is a plant renowned for producing monoterpenes. This study aimed to understand the effects of low-dose gamma radiation, specifically in the range of 1-5Gy, on various traits of Thymus vulgaris, providing context on its importance in agricultural and medicinal applications. The research explored morpho-physiological, biochemical, and gene-expression responses in thyme plants under no gamma- and gamma-ray exposure conditions. The study revealed complex relationships between gamma-ray doses and plant characteristics. In particular, shoot and root lengths initially increased with low doses (1-3Gy) but decreased at higher doses (5Gy), suggesting a dose-dependent threshold effect. Similarly, shoot and root fresh weights displayed an initial increase followed by a decline with increasing doses. Biochemical parameters showed dose-dependent responses, with low to moderate doses (1-3Gy) stimulating enzyme activities and high doses (5Gy) inhibiting them. Gene expression analysis was focused on the following specific genes: thymol synthase, γ-terpinene synthase, and carvacrol synthase. Low to moderate doses increased the expression of these genes, resulting in increased production of bioactive compounds. However, higher doses had diminished effects or suppressed gene expression. Metabolite analysis demonstrated dose-dependent responses, with moderate doses enhancing secondary metabolite production, while higher doses provided limited benefits. These findings underscore the implications of using gamma radiation to enhance secondary metabolite production in plants and its potential applications in agriculture, medicine, and environmental science. The study emphasizes the potential of gamma radiation as an external stressor to influence plant responses and highlights the importance of understanding such effects in various fields.

Effects of leaf herbivory and autumn seasonality on plant secondary metabolites: A meta-analysis.

Plant secondary metabolites (PSMs) are produced by plants to overcome environmental challenges, both biotic and abiotic. We were interested in characterizing how autumn seasonality in temperate and subtropical climates affects overall PSM production in comparison to herbivory. Herbivory is commonly measured between spring to summer when plants have high resource availability and prioritize growth and reproduction. However, autumn seasonality also challenges plants as they cope with limited resources and prepare survival for winter. This suggests a potential gap in our understanding of how herbivory affects PSM production in autumn compared to spring/summer. Using meta-analysis, we recorded overall production of 22 different PSM subgroups from 58 published papers to calculate effect sizes from herbivory studies (absence to presence) and temperate to subtropical seasonal studies (summer to autumn), while considering other variables (e.g., plant type, increase in time since herbivory, temperature, and precipitation). We also compared production of five phenolic PSM subgroups - hydroxybenzoic acids, flavan-3-ols, flavonols, hydrolysable tannins, and condensed tannins. We wanted to detect a shared response across all PSMs and found that herbivory increased overall PSM production in herbaceous plants. Herbivory was also found to have a positive effect on individual PSM subgroups, such as flavonol production, while autumn seasonality was found to have a positive effect on flavan-3-ol and condensed tannin production. We discuss how these responses might stem from plants producing some PSMs constitutively, whereas others are induced only after herbivory, and how plants produce metabolites with higher costs only during seasons when other resources for growth and reproduction are less available, while other phenolic PSM subgroups serve more than one function for plants and such functions can be season dependent. The outcome of our meta-analysis is that autumn seasonality changes some PSM production differently from herbivory, and we see value in further investigating seasonality-herbivory interactions with plant chemical defense.

Biochar and vermicompost modulated Pb toxicity in summer savory (Satureja Hortensis L.) plants through inducing physiological and biochemical changes

The primary environmental factor affecting the biochemical attributes of plants is soil pollution by heavy metals. The detrimental effects of heavy metals on plants can be lessened by soil amendments. The goal of the current research was to determine the potential of biochar (BC) and vermicompost (VC) as soil amendments in reducing Pb toxicity in summer savory (Satureja HortensisL.) though physiological and biochemical modifications. Therefore, the pot experiment was conducted with Pb toxicity (control (non-Pb), 300, and 600 mg kg soil-1) and soil amendments (control, 2% BC, 10% VC, and 1% BC+5% VC) based on a completely randomized design. The results showed that Pb toxicity at 600 mg kg soil-1 led to significant decreases in shoot weight (41%), root weight (25%), leaf relative water content (20%), and chlorophyll content (39%) compared to the control. However, it resulted in increases in malondialdehyde (61%) and electrolyte leakage (49%) when BC and VC were not applied. The results showed that Pb toxicity at 600 mg kg soil-1 led to decreases in shoot weight (41%), root weight (25%), leaf relative water content (20%), and chlorophyll content (39%), but increases in malondialdehyde (61%), and electrolyte leakage (49%) compared to the control in the treatments without BC and VC application. However, BC and VC, particularly their combination were more effective in improving plant growth. The interaction of Pb at 300 mg kg-1 and combined BC and VC resulted in higher total phenolic content, total flavonoid content, essential oil (EO) content, and EO yield with 29, 62, and 39, and 35% raises compared with the control. Agglomerative hierarchical clustering revealed that Pb at 300 and 600 mg kg soil-1 differed from the control, and that combined VC and BC significantly varied from their individual values. Combining BC and VC is more effective than using them separately in alleviating Pb toxicity, as it enhances plant growth and secondary metabolite production. The results have the potential to benefit the improvement of summer savory resistance in Pb-polluted soils.

Endophytic fungi from Himalayan silver birch as potential source of plant growth enhancement and secondary metabolite production.

Mountain biodiversity is under unparalleled pressure due to climate change, necessitating in-depth research on high-altitude plant's microbial associations which are crucial for plant survival under stress conditions. Realizing that high-altitude tree line species of Himalaya are completely unexplored with respect to the microbial association, the present study aimed to elucidate plant growth promoting and secondary metabolite producing potential of culturable endophytic fungi of Himalayan silver birch (Betula utilis D. Don). ITS region sequencing revealed that the fungal isolates belong to Penicillium species, Pezicula radicicola, and Paraconiothyrium archidendri. These endophytes were psychrotolerant in nature with the potential to produce extracellular lytic activities. The endophytes showed plant growth promoting (PGP) traits like phosphorus solubilization and production of siderophore, indole acetic acid (IAA), and ACC deaminase. The fungal extracts also exhibited antagonistic potential against bacterial pathogens. Furthermore, the fungal extracts were found to be a potential source of bioactive compounds including the host-specific compound-betulin. Inoculation with fungal suspension improved seed germination and biomass of soybean and maize crops under net house conditions. In vitro PGP traits of the endophytes, supported by net house experiments, indicated that fungal association may support the growth and survival of the host in extreme cold conditions.

Secondary Metabolite Production In Plants: In Response To Biotic And Abiotic Stress Factors

Secondary metabolites (SMs) play vital roles in plant defence mechanisms, adaptation to environmental conditions, and interactions with other organisms. Biotic and abiotic stress factors can significantly influence the production, accumulation, and composition of SMs in plants. Understanding the intricate relationship between stress and SM production is crucial for enhancing plant resilience, agricultural productivity, and the development of novel phytopharmaceuticals. This research provides current knowledge regarding the impact of biotic and also abiotic stress on SMs in plants. Biotic stress factors such as pathogen infection, and herbivore attacks, as well as abiotic stress factors like drought, along with temperature extremes, and also salinity, can profoundly influence the biosynthesis and accumulation of SMs in plants. We discussed the methodology based on secondary sources underlying physiological, biochemical, and molecular mechanisms involved in stress-induced SM synthesis and highlight the potential implications for plant biology, agriculture, and human health. The study also emphasizes the functions of SMs in plants including defence against herbivores, pathogens, and abiotic stresses. The mechanism by which thesecompounds act as allelochemicals and signalling molecules is also discussed.

Enhanced azadirachtin production in neem (Azadirachta indica) callus through NaCl elicitation: Insights into differential protein regulation via shotgun proteomics

With their remarkable bioactivity and evolving commercial importance, plant secondary metabolites (PSMs) have gained significant research interest in recent years. Plant tissue culture serves as a credible tool to examine how abiotic stresses modulate the production of PSMs, enabling clear insights into plant stress responses and the prospects for controlled synthesis of bioactive compounds. Azadirachta indica, or neem has been recognized as a repository of secondary metabolites for centuries, particularly for the compound named azadirachtin, due to its bio-pesticidal and high antioxidant properties. Introducing salt stress as an elicitor makes it possible to enhance the synthesis of secondary metabolites, specifically azadirachtin. Thus, in this research, in vitro callus cultures of neem were micro-propagated and induced with salinity stress to explore their effects on the production of azadirachtin and identify potential proteins associated with salinity stress through comparative shotgun proteomics (LCMS/MS). To induce salinity stress, 2-month-old calli were subjected to various concentrations of NaCl (0.05–1.5%) for 4 weeks. The results showed that the callus cultures were able to adapt and survive in the salinity treatments, but displayed a reduction in fresh weight as the NaCl concentration increased. Notably, azadirachtin production was significantly enhanced in the salinity treatment compared to control, where 1.5% NaCl-treated calli produced the highest azadirachtin amount (10.847 ± 0.037 mg/g DW). The proteomics analysis showed that key proteins related to primary metabolism, such as defence, energy, cell structure, redox, transcriptional and photosynthesis, were predominantly differentially regulated (36 upregulated and 93 downregulated). While a few proteins were identified as being regulated in secondary metabolism, they were not directly involved in the synthesis of azadirachtin. In conjunction with azadirachtin elicitation, salinity stress treatment could therefore be successfully applied in commercial settings for the controlled synthesis of azadirachtin and other plant-based compounds. Further complementary omics approaches can be employed to enhance molecular-level modifications, to facilitate large-scale production of bioactive compounds in the future.

UHPLC-Q-Orbitrap-HRMS-based untargeted metabolomics reveal metabolites change in Justicia gendarussa and its antioxidant capacity at different doses of nitrogen fertilizer.

This study delves into the antioxidant potential of Justicia gendarussa, commonly known as gendarussa, and its response to varying doses of nitrogen fertilizer. Gendarussa exhibits the potential for antioxidant activity. The diverse ecological conditions in which it thrives may influence its biological activity and lead to inconsistent production of secondary metabolites. Nitrogen, a pivotal factor in secondary metabolite production in plants, has become a focal point of this research. This research aims to determine the optimal nitrogen fertilizer dose on gendarussa antioxidant capacity and metabolites using a metabolomics approach. Employing a randomized block design for cultivation, the investigation revealed that a maximum harvest weight of 10.9 g/aerial parts of the plant was achieved with 270 kg/ha of nitrogen fertilizer. This study explored the DPPH, ABTS, FRAP, and CUPRAC assays of antioxidant effect, and found insignificant differences between the various nitrogen treatments. UHPLCQ-Orbitrap HRMS was employed to identify 30 metabolites in positive and 18 in negative ionization modes. Gendarusin A, a major metabolite in gendarussa, is identified in both positive and negative ionization. PCA and heatmap analysis successfully categorized these metabolites in the aerial parts of gendarussa at different nitrogen fertilizer dosages. Based on the metabolomics approach, variations in nitrogen fertilizer made metabolites at doses of 90 kg/ha had higher relative concentrations of metabolites compared to doses of 180 kg/ha and 270 kg/ha. So, 90 kg/ha are the optimal nitrogen fertilizer dose for cultivation and utilization strategies.

Antioxidant and Secondary Metabolite Responses in Wheat under Cereal Leaf Beetle (Oulema melanopus L.) Infestation

AbstractWheat is one of the most important cereal crops grown in the Western Himalayas of India but its production is challenged by the insect “cereal leaf beetle (CLB)”. This study explores the impact of domestication and modern crop improvement on wheat's defense mechanisms against the CLB, a global threat to wheat cultivation. Sixteen diverse wheat genotypes having different ploidy levels were investigated, including wild wheat, landraces, mutants, advanced breeding lines, commercial varieties, and a Rye grass genotype. Genotypes with resistance genes, landraces, and wild wheat exhibited the lowest CLB infestation and oxidative damage. These genotypes displayed enhanced antioxidant enzyme activity, such as peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), and polyphenol oxidase (PPO), reduced reactive oxygen species (ROS) production, and increased plant secondary metabolite (phenol and tannin) production upon CLB infestation. Resistant durum wheat demonstrated moderate responses and exhibited higher levels of flavonoid production. On the other hand, susceptible durum wheat genotypes, commercial variety Shalimar Wheat‐02 (SW‐2), showed high CLB infestation, ROS production, reduced antioxidant enzyme and secondary metabolite production. This may be because modern varieties like SW‐2 were not selected for insect resistance. These findings emphasize the significance of preserving genetic diversity from wild wheat and landraces to bolster wheat breeding programs against pests such as CLB. This study underscores the need to harness ancestral wheat lines to enhance insect pest resistance in modern wheat cultivars.

Improvement of Oleanolic Acid Production in Saccharomyces Cerevisiae Based on OptKnock Framework.

Biosynthesis of plant-derived natural products in the eukaryotic microbe Saccharomyces cerevisiae often faces the issue of the inefficient production due to the poor compatibility between the heterologous genes and chassis cells. In order to improve the biosynthetic efficiency of heterologous production of plant secondary metabolites in S. cerevisiae, people usually do metabolic engineering in and around the heterologous metabolic pathways based on researchers' experience and mass of trials, which usually consumes a lot of manpower and financial resources. Herein, to further improve the heterologous production of oleanolic acid (OA), a pentacyclic triterpenoid in many plants with several promising pharmacological activities, in a genetically engineered, OA-producing strain S. cerevisiae OA07 effectively, a genome-scale metabolic model of the strain was developed, with the named as Yeast-OA07, and then OptKnock, a flux balance analysis-based pathway design algorithm with bilevel objectives, was utilized to develop in silico gene-knockout strategies to guide the molecular operations in S. cerevisiae OA07. Yeast8-OA07 contained 1133 genes, 2702 metabolites, and 3997 reactions. Five in silico gene-knockout strategies, which were expected to increase OA productivities, were obtained based on the metabolic flux analysis of Yeast8-OA07 through OptKnock. Afterwards, five mutant strains, named as LK1, LK2, LK3, LK4 and LK5, were constructed according to the in silico strategies. It was found that the mutant strain LK2, in which 2-amino-4-hydroxy-6-hydroxymethyl dihydropteridine diphosphokinase-encoding gene FOL1 and formate dehydrogenase-encoding gene FDH1 were deleted, had an OA yield of 125.04 mg·L-1, which was significantlyhigher than the original strain OA07 (89.50 mg·L-1), while the mutant strain LK5, which eliminated paminobenzoic acid synthase-encoding gene ABZ1 and glycine hydroxymethyl transferase-encoding gene SHM1, had an even higher OA yield of 207.37 mg·L-1. Nevertheless, strain LK6, which was developed by integrating the in silico gene-knockout strategies of LK2 and LK5, had a significant decrease of OA production than S. cerevisiae OA07, indicating that in silico knockout strategies do not fit to in vivo iteration directly. Our study provides a novel, efficient method to improve the heterologous production of plant metabolites in microbial cell factories.

Modulation of morpho-physiological attributes and in situ analysis of secondary metabolites using Raman spectroscopy in response to red and blue light exposure in Artemisia annua

Differential light conditions, such as variations in photoperiod, light quality, and light intensity, play a crucial role as external factors that can influence plant morphogenesis and secondary metabolism. However, there is still a lack of understanding of how Artemisia annua responds to monochromatic and dichromatic light regimes in terms of the different physiological mechanisms that control plant growth and secondary metabolite production. Therefore, the primary aim of this study was to investigate and assess the various physiological parameters, biochemical analysis, and molecular aspects in detail. Therefore, the plantlets were exposed to LED lighting such as monochromatic red light (R), monochromatic blue light (B), white light (W), and a combination of red and blue light (RB, 1:1) at a PPFD (photosynthetic photon flux density) of 200 mol. m−2. s−1 for 10 days. Our results indicate that exposure to RB light resulted in an immense increase in ROS accumulation, flavonoids, lignin, and artemisinin by 4.7-fold, 44%, and 53.4%, respectively, in contrast to W light. Whereas blue light led to increments of 160.2% in phenolic and 107.9% in anthocyanin content. RB and B light also influence the parameters of chlorophyll fluorescence, as well as leaf area, stomatal density, trichome size, antioxidant enzyme activity, and the production of more secondary metabolites to combat oxidative stress. Real-time PCR analysis of biosynthetic pathway-associated genes HMGR, DXR, DXS, FPS, ADS, CYP71AVI, DBR2, ALDH1, and flavonoid key biosynthesis pathway genes PAL, C4H, 4CL, CHS, and F3′H showed the highest increment under RB, light followed by B and R light exposure. Further, RB LED light has significant potential for enhancing natural bio-active compounds as revealed by Raman spectroscopy, such as camphor, limonene, terpene-4-ol, α pinene, 1,8-cineole, β-caryophyllene, artemisinin, kaempferol, luteolin, rutin, and caffeic acid in A. annua. Taken together, red and blue LEDs can serve as significant elicitors for the production of commercially important metabolites.

Investigating how nitrogen nutrition and pruning impacts on CBD and THC concentration and plant biomass of Cannabis sativa

Precise crop fertilization requires an in-depth understanding of plant uptake and utilisation to optimise sustainable production. This study investigated the influence of nitrogen (N) nutrition and pruning on the cannabinoid concentrations and biomass of a commercial cannabis cultivar; the rationale for this study is how N supply and pruning affect cannabinoid yields and concentration in a commercial setting. Clones of a Cannabis sativa L. (CBD-type) were grown in a controlled-environment glasshouse in pots with coarse sand. After five weeks of vegetative growth under 210 mg/L N and an 18 h light regime, rates of 30, 60, 210, and 500 mg/L N were applied to plants for twelve weeks and a light regime set at 12 h. Double stem pruning was applied as an additional treatment to investigate efficacy on biomass increase. Biomass, N concentrations, and cannabinoid concentrations were measured after the final harvest. Pruning treatment did not increase cannabinoid concentrations or affect biomass. It was coincidentally found that plants on the glasshouse edge with higher exposure to sunlight developed more biomass and higher cannabinoid concentrations. Only biomass in leaves was increased significantly via higher nitrogen nutrition. Cannabinoid concentration, as well as cannabinoid yield per plant were decreased with the increase in N supply. High rates of fertilizer are not recommended because of reduced cannabinoid concentration and biomass yield: the ideal N supply is likely to be between 60 and 210 mg/L. This research will benefit growers and advisors in understanding the complexity of effects of nitrogen fertiliser and pruning practices on plant biomass and secondary metabolite production in medicinal cannabis.

Evaluation of the artemisinin content of Artemisia annua L. grown in different Agro ecological zones of Cameroon

Artemisinin and its derivatives are potent antimalarials that have become essential components of Artemisinin Based Combination Therapies (ACTs) for malaria treatment. Production of artemisinin is less compared to its world demand and access to ACTs is still limited in worst hit countries. Secondary metabolite production in plants depends on a number of factors including agro ecology. This work sought to evaluate the growth characteristics and artemisinin content of Artemisia annua grown in four agro ecological zones of Cameroon. Experimental fields comprising of A annua were set up in the different agro-ecological zones, for growth and artemisinin assessments. Artemisinin detection and quantification was compared in leaves/flowers and stems of Artemisia annua from these agro-ecological zones, using High-Performance Liquid Chromatography. Soil provenance and other agro ecological factors significantly affected the growth parameters of A. annua as there were significant differences (p<0.05) in plant height, leaf area, number of branches, stem girth and number of leaves across the different study areas. There was significant variation (P<0.05) in the content of artemisinin from the plant in the different agro ecological zones. The mean levels of artemisinin in the samples was 0.34-0.80% in stem samples, and 0.18-0.61% for leaves/flowers. The highest artemisinin yield in leaf/flowers samples (0.61%) was recorded in samples from Bamenda, while that of stem samples (0.80%) was recorded in samples from Ngaoundere. The study confirms the effect of agro ecological factors on artemisinin content of A. annua in Cameroon and reveal that A. annua grown in most regions of the country contain commercially viable levels of artemisinin which can further be exploited for pharmaceutical purposes.