Enhancing artemisinin content in and delivery from Artemisia annua: a review of alternative, classical, and transgenic approaches.

MAIN CONCLUSION: This review analyses the most recent scientific research conducted for the purpose of enhancing artemisinin production. It may help to devise better artemisinin enhancement strategies, so that its production becomes cost effective and becomes available to masses. Malaria is a major threat to world population, particularly in South-East Asia and Africa, due to dearth of effective anti-malarial compounds, emergence of quinine resistant malarial strains, and lack of advanced healthcare facilities. Artemisinin, a sesquiterpene lactone obtained from Artemisia annua L., is the most potent drug against malaria and used in the formulation of artemisinin combination therapies (ACTs). Artemisinin is also effective against various types of cancers, many other microbes including viruses, parasites and bacteria. However, this specialty metabolite and its derivatives generally occur in low amounts in the source plant leading to its production scarcity. Considering the importance of this drug, researchers have been working worldwide to develop novel strategies to augment its production both in vivo and in vitro. Due to complex chemical structure, its chemical synthesis is quite expensive, so researchers need to devise synthetic protocols that are economically viable and also work on increasing the in-planta production of artemisinin by using various strategies like use of phytohormones, stress signals, bioinoculants, breeding and transgenic approaches. The focus of this review is to discuss these artemisinin enhancement strategies, understand mechanisms modulating its biosynthesis, and evaluate if roots play any role in artemisinin production. Furthermore, we also have a critical analysis of various assays used for artemisinin measurement. This may help to develop better artemisinin enhancement strategies which lead to decreased price of ACTs and increased profit to farmers.

Molecular Mechanisms of Nitric Oxide (NO) Signaling and Reactive Oxygen Species (ROS) Homeostasis during Abiotic Stresses in Plants.

Abiotic stressors, such as drought, heavy metals, and high salinity, are causing huge crop losses worldwide. These abiotic stressors are expected to become more extreme, less predictable, and more widespread in the near future. With the rapidly growing human population and changing global climate conditions, it is critical to prevent global crop losses to meet the increasing demand for food and other crop products. The reactive gaseous signaling molecule nitric oxide (NO) is involved in numerous plant developmental processes as well as plant responses to various abiotic stresses through its interactions with various molecules. Together, these interactions lead to the homeostasis of reactive oxygen species (ROS), proline and glutathione biosynthesis, post-translational modifications such as S-nitrosylation, and modulation of gene and protein expression. Exogenous application of various NO donors positively mitigates the negative effects of various abiotic stressors. In view of the multidimensional role of this signaling molecule, research over the past decade has investigated its potential in alleviating the deleterious effects of various abiotic stressors, particularly in ROS homeostasis. In this review, we highlight the recent molecular and physiological advances that provide insights into the functional role of NO in mediating various abiotic stress responses in plants.

Insights into strigolactone (GR24) mediated regulation of cadmium-induced changes and ROS metabolism in Artemisia annua.

Agricultural soil contamination and subsequently crops still require alternative solutions to reduce associated environmental risks. The effects of strigolactones (SLs) in alleviating cadmium (Cd) phytotoxicity in Artemisia annua plants were investigated during this study. Strigolactones play a vital role during plant growth and development due to their complex interplay during a plethora of biochemical processes. However, information on the potential of SLs to elicit abiotic stress signaling and trigger physiological modifications in plants is limited. In order to decipher the same, A. annua plants were exposed to different concentrations of Cd (20 and 40 mg kg-1), with or without the supplementation of exogenous SL (GR24, a SL analogue) at 4 µM concentration. Under Cd stress, excess Cd accumulation resulted in reduced growth, physio-biochemical traits, and artemisinin content. However, the follow-up treatment of GR24 maintained a steady state equilibrium between reactive oxygen species and antioxidant enzymes, improved chlorophyll fluorescence parameters such as Fv/Fm, ФPSII, and ETR for improved photosynthesis, enhanced chlorophyll content, maintained chloroplast ultrastructure, improved the glandular trichome (GT) attributes and artemisinin production in A. annua. Moreover, it also resulted in improved membrane stability, reduced Cd accumulation, and regulated the behaviour of stomatal apertures for better stomatal conductance under Cd stress. The results of our study suggest that GR24 could be highly effective in alleviating Cd-induced damages in A. annua. It acts via the modulation of the antioxidant enzyme system for redox homeostasis, protection of the chloroplasts and pigments for improved photosynthetic performance, and improved GT attributes for enhanced artemisinin production in A. annua.

Nitric oxide induces antioxidant machinery, PSII functioning and artemisinin biosynthesis in Artemisia annua under cadmium stress.

Soil contamination by heavy metals poses a significant environmental challenge, as the practical implementation of existing remediation technologies in the field has encountered numerous obstacles. This has necessitated the requirement of finding alternate solutions to reduce the harm caused to plants. In this study, nitric oxide (NO) was investigated for its potential to reduce cadmium (Cd) toxicity in A. annua plants. Although NO plays a vital role in the growth and development of plants, information on its role in reducing abiotic stress in plants is limited. A. annua plants were exposed to 20 and 40 mg/kg Cd regardless of the addition of exogenous sodium nitroprusside (SNP), a NO donor, at 200 µM concentration. Results showed that SNP treatment improved plant growth, photosynthesis, chlorophyll fluorescence, pigment content, and artemisinin production while reducing Cd accumulation and improving membrane stability in A. annua during Cd stress. The results demonstrated that NO can effectively reverse Cd-induced damage in A. annua by modulating the antioxidant system, maintaining redox homeostasis, and improving photosynthetic performance and different fluorescence parameters such as Fv/Fm, ФPSII, and ETR. The supplementation of SNP caused a substantial improvement in chloroplast ultrastructure, stomatal behavior, and different attributes relate to glandular secretory trichomes, which in turn increased artemisinin production; 14.11 % in plants exposed to Cd stress of 20 mg/kg. Our findings highlight that NO could be useful in mediating the repair of Cd-induced damage to A. annua, and suggest that it may play a critical role in plant signaling networks, improving plant adaptability to Cd stress. The results have important implications for developing new strategies to mitigate the negative impacts of environmental contaminants on plant health, and ultimately, the ecosystem.

Involvement of abscisic acid in silicon-mediated enhancement of copper stress tolerance in Artemisia annua.

Heavy metal (HM) toxicity is a well-known hazard which causes deleterious impact on the growth and development of plants. The impact of abscisic acid (ABA) in presence of silicon (Si) on plant development and quality traits has largely gone unexplored. The effects of ABA and Si on the growth, yield, and quality characteristics of Artemisia annua L. plants growing under copper (Cu) stress (20 and 40 mg kg-1) were investigated in a pot experiment. During this investigation, Cu stress caused severe damage to the plants but exogenous administration of Si and ABA ameliorated the harmful effects of Cu toxicity, and the plants displayed higher biomass and improved physio-biochemical attributes. Copper accumulated in the roots and shoots and its toxicity caused oxidative stress as demonstrated by the increased 2-thiobarbituric acid reactive substance (TBARS) content. It also resulted in the increased activity of antioxidant enzymes, however, the exogenous Si and ABA supplementation decreased the buildup of reactive oxygen species (ROS) and lipid peroxidation, alleviating the oxidative damage produced by HM stress. Copper toxicity had a considerable negative impact on glandular trichome density, ultrastructure as well as artemisinin production. However, combined Si and ABA enhanced the size and density of glandular trichomes, resulting in higher artemisinin production. Taken together, our results demonstrated that exogenous ABA and Si supplementation protect A. annua plants against Cu toxicity by improving photosynthetic characteristics, enhancing antioxidant enzyme activity, protecting leaf structure and integrity, avoiding excess Cu deposition in shoot and root tissues, and helping in enhanced artemisinin biosynthesis. Our results indicate that the combined application of Si and ABA improved the overall growth of plants and may thus be used as an effective approach for the improvement of growth and yield of A. annua in Cu-contaminated soils.

Systems biology of chromium-plant interaction: insights from omics approaches.

Plants are frequently subjected to heavy metal (HM) stress that impedes their growth and productivity. One of the most common harmful trace metals and HM discovered is chromium (Cr). Its contamination continues to increase in the environment due to industrial or anthropogenic activities. Chromium is severely toxic to plant growth and development and acts as a human carcinogen that enters the body by inhaling or taking Cr-contaminated food items. Plants uptake Cr via various transporters, such as sulfate and phosphate transporters. In nature, Cr is found in various valence states, commonly Cr (III) and Cr (VI). Cr (VI) is soil's most hazardous and pervasive form. Cr elevates reactive oxygen species (ROS) activity, impeding various physiological and metabolic pathways. Plants have evolved various complex defense mechanisms to prevent or tolerate the toxic effects of Cr. These defense mechanisms include absorbing and accumulating Cr in cell organelles such as vacuoles, immobilizing them by forming complexes with organic chelates, and extracting them by using a variety of transporters and ion channels regulated by various signaling cascades and transcription factors. Several defense-related proteins including, metallothioneins, phytochelatins, and glutathione-S-transferases aid in the sequestration of Cr. Moreover, several genes and transcriptional factors, such as WRKY and AP2/ERF TF genes, play a crucial role in defense against Cr stress. To counter HM-mediated stress stimuli, OMICS approaches, including genomics, proteomics, transcriptomics, and metallomics, have facilitated our understanding to improve Cr stress tolerance in plants. This review discusses the Cr uptake, translocation, and accumulation in plants. Furthermore, it provides a model to unravel the complexities of the Cr-plant interaction utilizing system biology and integrated OMICS approach.

Chromium in plant-soil nexus: Speciation, uptake, transport and sustainable remediation techniques.

Heavy metal (HM) pollution has become a serious global problem due to the non-biodegradable nature of the HMs and their persistence in the environment. Agricultural soil is a non-renewable resource that requires careful management so that it can fulfill the increasing demand for agricultural food production. However, different anthropogenic activities have resulted in a large-scale accumulation of HMs in soil which is detrimental to soil and plant health. Due to their ubiquity, increased bioavailability, toxicity, and non-biodegradable nature, HM contamination has formed a roadblock in the way of achieving food security, safety, and sustainability in the future. Chromium (Cr), specifically Cr(VI) is a highly bioavailable HM with no proven role in the physiology of plants. Chromium has been found to be highly toxic to plants, with its toxicity also influenced by chemical speciation, which is in turn controlled by different factors, such as soil pH, redox potential, organic matter, and microbial population. In this review, the different factors that influence Cr speciation were analyzed and the relationship between biogeochemical transformations of Cr and its bioavailability which may be beneficial for devising different Cr remediation strategies has been discussed. Also, the uptake and transport mechanism of Cr in plants, with particular reference to sulfate and phosphate transporters has been presented. The biological solutions for the remediation of Cr contaminated sites which offer safe and viable alternatives to old-style physical and chemical remediation strategies have been discussed in detail. This review provides theoretical guidance in developing suitable approaches for the better management of these remediation strategies.

Exogenous Strigolactone (GR24) Positively Regulates Growth, Photosynthesis, and Improves Glandular Trichome Attributes for Enhanced Artemisinin Production in Artemisia annua.

Artemisia annua is a medicinal plant particularly known for the production of a sesquiterpene lactone artemisinin; a specialty metabolite known for its efficacy in the treatment of malaria by killing different strains of Plasmodium falciparum due to radicals released upon the cleavage of its endoperoxide motif. Considering these facts and the immense medicinal value of artemisinin, the enhancement of in planta production of artemisinin is highly desirable. As strigolactones are known to regulate various aspects of plant growth and development, the effects of foliar spray of different concentrations of synthetic strigolactone analog GR24 (0, 0.5, 1, 2, 4, and 8 µM) on A. annua were studied. As compared to the control group, the foliar application of GR24 had a positive impact on general growth, photosynthesis, and other physiological indices with 4 µM GR24 showing the best results. The results indicate that GR24 application increased the plant biomass and various attributes related to photosynthesis, like total chlorophyll content, chlorophyll fluorescence, stomatal conductance, internal CO2, and net photosynthetic rate. Moreover, the activity of various enzymes related to photosynthesis like carbonic anhydrase, nitrate reductase, and RuBisCO was escalated. The GR24 also improved certain attributes related to glandular trichomes, with a significant enhancement in content and yield of artemisinin as compared to untreated plants.

Salicylic acid-mediated alleviation of soil boron toxicity in Mentha arvensis and Cymbopogon flexuosus: Growth, antioxidant responses, essential oil contents and components.

Boron (B) toxicity is a notable abiotic hindrance that restricts crop productivity by disturbing several physiological and biochemical processes in plants. This study was aimed to elucidate the role of salicylic acid (SA) in conferring tolerance to B stress in Mentha arvensis and Cymbopogon flexuosus. Boron toxicity led to a considerable decrease in shoot height and root length, fresh and dry mass of shoot and root, and physiological and biochemical parameters. However, exogenously applied SA relieved the adverse effects caused by B toxicity and led to an increase in growth parameters under B stress and non-stress conditions. The treatment of B resulted in its increased accumulation in roots and shoots of both the plants which, in turn, caused oxidative damage as evident by increased content of malondialdehyde and catalase, peroxidase, superoxide dismutase and glutathione reductase enzyme activities. However, exogenous SA supply significantly affected antioxidant enzyme activities and protected the plants from excess B. Moreover, the essential oil content of two selected plants declined under B toxicity and significantly enhanced in SA-treated stressed plants. The contents of menthol and menthyl acetate in M. arvensis were lowered in B stressed plants which significantly improved in SA treated B-stressed and in their respective SA alone treatment. Similarly, citral-A and citral-B content of C. flexuosus declined under B toxicity, however, SA reversed the negative effects of B toxicity on essential oil components. This assessment stipulated the promising role of exogenously applied SA in alleviating B toxicity in M. arvensis and C. flexuosus by improving antioxidant machinery and limiting B uptake which protects the structural integrity of leaves and also helps in increasing essential oil content.

Exogenous abscisic acid mediates ROS homeostasis and maintains glandular trichome to enhance artemisinin biosynthesis in Artemisia annua under copper toxicity.

One of the major abiotic stresses that cause environmental pollution is heavy metal stress. In the present investigation, copper (Cu) toxicity caused morphological and cellular damages to the Artemisia annua L. plants but supplementation of abscisic acid (ABA) ameliorated the damaging effect of Cu. Copper toxicity significantly reduced the shoot and root lengths; fresh and dry weights of shoot. However, exogenous application of ABA to Cu-treated plants significantly attenuated the damaging effects on plants caused by Cu toxicity. Copper stress also reduced the physiological and biochemical parameters, but ABA application ameliorated the negative effects of Cu in the affected plant. Accumulation of Cu in plant tissues significantly increased the membrane damage and oxidative enzyme activities such as catalase (CAT), peroxidase (POX) and superoxide dismutase (SOD). Further, the impact of high concentration of Cu on density, area and ultrastructure of glandular trichomes and artemisinin content was studied. Moreover, the foliar application of ABA improved the area, density of glandular trichomes and secured the plant cells from Cu toxicity. Therefore, this investigation indicated that the exogenous application of ABA protects A. annua plant by increasing antioxidant enzymes activity, which helps in maintaining cell integrity of leaves and results in increased artemisinin production.