Artemisia annua ZFP8L regulates glandular trichome development.

Trichomes are known to be important biofactories that contribute to the production of secondary metabolites, such as terpenoids. C2H2-zinc finger proteins (C2H2-ZFPs) are vital transcription factors of plants' trichome development. However, little is known about the function of Artemisia annua C2H2-ZFPs in trichome development. To explore the roles of this gene family in trichome development, two C2H2-ZFP transcription factors, named AaZFP8L and AaGIS3, were identified; both are hormonally regulated in A. annua. Overexpression of AaZFP8L in tobacco led to a significant increase in the density and length of glandular trichomes, and improved terpenoid content. In contrast, AaGIS3 was found to positively regulate non-glandular trichome initiation and elongation, which reduces terpenoid accumulation. In addition, ABA contents significantly increased in AaZFP8L-overexpressing tobacco lines and AaZFP8L also can directly bind the promoter of the ABA biosynthesis genes. This study lays the foundation for further investigating A. annua C2H2-ZFPs in trichome development and terpenoid accumulation.

Identification and the potential involvement of miRNAs in the regulation of artemisinin biosynthesis in A. annua.

Micro RNAs (miRNAs) play crucial regulatory roles in multiple biological processes. Recently they have garnered the attention for their strong influence on the secondary metabolite production in plants. Their role in the regulation of artemisinin (ART) biosynthesis is, however, not fully elucidated. ART is a potent anti-malarial compound recommended by WHO for the treatment of drug-resistant malaria. It is produced by Artemisia annua (A. annua). The lower in planta content of ART necessitates a deep understanding of regulatory mechanisms involved in the biosynthesis of this metabolite. In this study, using modern high throughput small RNA-sequencing by Illumina Nextseq 500 platform for identification and stem-loop RT PCR for validation, miRNAs were identified in the leaf sample of A. annua plant. Here, we report a total of 121 miRNAs from A. annua that target several important genes and transcription factors involved in the biosynthesis of ART. This study revealed the presence of some important conserved miRNA families, miR396, miR319, miR399, miR858, miR5083 and miR6111 not identified so far in A. annua. The expression patterns and correlation between miRNAs and their corresponding targets at different developmental stages of the plant using real-time PCR indicate that they may influence ART accumulation. These findings thus, open new possibilities for the rational engineering of the secondary metabolite pathways in general and ART biosynthesis in particular.

Salicylic acid restrains arsenic induced oxidative burst in two varieties of Artemisia annua L. by modulating antioxidant defence system and artemisinin production.

Arsenic is a harmful and toxic substance to the growth and development of plants. Salicylic acid (SA) acts as a signaling molecule, plays pivotal roles in the overall growth and development of plants under various environmental stresses. Artemisinin extracted from the leaves of A. annua helps in malarial treatment. The present investigation is aimed to find out the possible ameliorative role of exogenously-applied salicylic acid (SA) on two varieties of Artemisia annua L., namely 'CIM-Arogya' and 'Jeevan Raksha' under arsenic (As) stress conditions. For this, growth, physiological and biochemical characterization, and artemisinin production was assessed. The various treatments applied on the plants were Control, 10-6 M SA, 10-5 M SA, 45 mg kg-1As, 45 mg kg-1 As + 10-6 M SA, and 45 mg kg-1 As + 10-5 M SA. Arsenic at 45 mg kg-1 of soil, reducing the overall performance of both varieties at 90 and 120 DAP. However, the levels of antioxidants were enhanced in As-stressed plants, and the supplementation of SA further increased these antioxidants in SA-treated plants. It has been observed that minimum reduction in growth and yield occurs with enhanced production of artemisinin in the case of 'CIM-Arogya' compared to 'Jeevan Raksha' under As stress (45 mg kg-1 of soil). Leaf-applied SA significantly increased the content (49.0% & 43.4%) and yield (53.3% & 46.3%) of artemisinin in both tolerant and sensitive varieties as compared to their respective controls. Thus, the variety 'CIM-Arogya' showed tolerant behavior over 'Jeevan Raksha' and is much adapted to higher As stress.

Impact of Long-Term Copper Exposure on Growth, Photosynthesis, Antioxidant Defence System and Artemisinin Biosynthesis in Soil-Grown Artemisia annua Genotypes.

The effects of copper (Cu) exposure on growth and physiological characteristics of three genotypes (CN-12, Cim-Sanjeevani and Cim-Arogya) of Artemisia annua L. were elucidated. The plants were grown under naturally illuminated greenhouse conditions and were harvested after physiological maturity (120 days after sowing). Results suggest that 10 mg kg- 1 Cu significantly enhanced the growth and physiological parameters like enzyme activities, photosynthesis. At higher concentrations, Cu inhibited the growth, biomass, photosynthetic parameters; while increased lipid peroxidation in all the genotypes. The activities of antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase were upregulated by the Cu stress. The highest applied concentration of Cu (60 mg kg- 1) proved most toxic for plants. Moreover, artemisinin content was increased upto 10 mg kg- 1 of Cu treatment, compared with control, however, the artemisinin accumulation decreased at higher doses of Cu in all the genotypes. On the basis of studied parameters, Cim-Arogya was found to be most tolerant among all for Cu toxicity.

Changes in the composition and structure of cell wall polysaccharides from Artemisia annua in response to salt stress.

Artemisia annua is cultivated mainly for isolation of artemisinin, a potent antimalarial compound. Moderate salt stress has been proved to increase the artemisinin synthesis by the plant. The aim of this study was to evaluate the influence of salt stress on physiological parameters and cell wall polysaccharides of A. annua. Plants subjected to salt stress displayed reduction in the biomass and length and showed high damage of cellular membranes. Cell wall polysaccharides extracted from aerial parts with hot water, EDTA and NaOH also exhibited modifications in the yield and monosaccharide composition. The main changes were found in the pectic polysaccharides: increase of homogalacturonan domain, increase of neutral side chains and increase in the methyl esterification. 1H NMR analyses of pectins indicated that for A. annua, arabinans have an important role in coping with salt stress. Hemicellulose domain was also modified under salt stress, with increased xylose contents. The results indicated adaptations in the cell wall of A. annua under salt stress.

A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua.

Glandular trichomes and cuticles are both specialized structures that cover the epidermis of aerial plant organs. The former are commonly regarded as 'biofactories' for producing valuable natural products. The latter are generally considered as natural barriers for defending plants against abiotic and biotic stresses. However, the regulatory network for their formation and relationship remains largely elusive. Here we identify a homeodomain-leucine zipper (HD-ZIP) IV transcription factor, AaHD8, directly promoting the expression of AaHD1 for glandular trichome initiation in Artemisia annua. We found that AaHD8 positively regulated leaf cuticle development in A. annua via controlling the expression of cuticle-related enzyme genes. Furthermore, AaHD8 interacted with a MIXTA-like protein AaMIXTA1, a positive regulator of trichome initiation and cuticle development, forming a regulatory complex and leading to enhanced transcriptional activity in regulating the expression of AaHD1 and cuticle development genes. Our results reveal a molecular mechanism by which a novel HD-ZIP IV/MIXTA complex plays a significant role in regulating epidermal development, including glandular trichome initiation and cuticle formation.

Jasmonate promotes artemisinin biosynthesis by activating the TCP14-ORA complex in Artemisia annua.

Artemisia annua produces the valuable medicinal component, artemisinin, which is a sesquiterpene lactone widely used in malaria treatment. AaORA, a homolog of CrORCA3, which is involved in activating terpenoid indole alkaloid biosynthesis in Catharanthus roseus, is a jasmonate (JA)-responsive and trichome-specific APETALA2/ETHYLENE-RESPONSE FACTOR that plays a pivotal role in artemisinin biosynthesis. However, the JA signaling mechanism underlying AaORA-mediated artemisinin biosynthesis remains enigmatic. Here, we report that AaORA forms a transcriptional activator complex with AaTCP14 (TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTOR 14), which is also predominantly expressed in trichomes. AaORA and AaTCP14 synergistically bind to and activate the promoters of two genes, double bond reductase 2 (DBR2) and aldehyde dehydrogenase 1 (ALDH1), both of which encode enzymes vital for artemisinin biosynthesis. AaJAZ8, a repressor of the JA signaling pathway, interacts with both AaTCP14 and AaORA and represses the ability of the AaTCP14-AaORA complex to activate the DBR2 promoter. JA treatment induces AaJAZ8 degradation, allowing the AaTCP14-AaORA complex to subsequently activate the expression of DBR2, which is essential for artemisinin biosynthesis. These data suggest that JA activation of the AaTCP14-AaORA complex regulates artemisinin biosynthesis. Together, our findings reveal a novel artemisinin biosynthetic pathway regulatory network and provide new insight into how specialized metabolism is modulated by the JA signaling pathway in plants.

Prolonged exposure to salt stress affects specialized metabolites-artemisinin and essential oil accumulation in Artemisia annua L.: metabolic acclimation in preferential favour of enhanced terpenoid accumulation accompanying vegetative to reproductive phase transition.

Artemisia annua accumulates substantial quantities of unique and highly useful antimalarial sesquiternoid artemisinin and related phytomolecules as well as its characteristic essential oil in its glandular trichomes. The phytomolecules are mainly produced in its leaves and inflorescences. Artemisia annua plants were grown under NaCl salinity (50, 100 and 200 mM) stress conditions imposed throughout the entire life cycle of the plant. Results revealed that specialized metabolites like artemisinin, arteannuin-B, artemisinic acid + dihydroartemisinic acid and essential oil accumulation were positively modulated by NaCl salinity stress. Interestingly, total content of monoterpenoids and sesquiterpenoids of essential oil was induced by NaCl salinity treatment, contrary to previous observations. Production of camphor, the major essential oil constituent was induced under the influence of treatment. The metabolic acclimation and manifestations specific to terpenoid pathway are analysed vis-a-vis vegetative to reproductive periods and control of the modulation. WRKY and CYP71AV1 play a key role in mediating the responses through metabolism in glandular trichomes. The distinctness of the salinity induced responses is discussed in light of differential mechanism of adaptation to abiotic stresses and their impact on terpenoid-specific metabolic adjustments in A. annua. Results provide potential indications of possible adaptation of A. annua under saline conditions for agrarian techno-economic benefaction.

Cumulative role of bioinoculants on growth, antioxidant potential and artemisinin content in Artemisia annua L. under organic field conditions.

Artemisia annua L. is mostly known for a bioactive metabolite, artemisinin, an effective sesquiterpene lactone used against malaria without any reputed cases of resistance. In this experiment, bioinoculants viz., Streptomyces sp. MTN14, Bacillus megaterium MTN2RP and Trichoderma harzianum Thu were applied as growth promoting substances to exploit full genetic potential of crops in terms of growth, yield, nutrient uptake and particularly artemisinin content. Further, multi-use of the bioinoculants singly and in combinations for the enhancement of antioxidant potential and therapeutic value was also undertaken which to our knowledge has never been investigated in context with microbial application. The results demonstrated that a significant (P < 0.05) increase in growth, nutrient uptake, total phenolic, flavonoid, free radical scavenging activity, ferric reducing antioxidant power, reducing power and total antioxidant capacity were observed in the A. annua treated with a combination of bioinoculants in comparison to control. Most importantly, an increase in artemisinin content and yield by 34 and 72 % respectively in the treatment having all the three microbes was observed. These results were further authenticated by the PCA analysis which showed positive correlation between plant macronutrients and antioxidant content with plant growth and artemisinin yield of A. annua. The present study thus highlights a possible new application of compatible bioinoculants for enhancing the growth along with antioxidant and therapeutic value of A. annua.

Artemisinin production by plant hairy root cultures in gas- and liquid-phase bioreactors.

KEY MESSAGE: Alternative biotechnological protocol for large-scale artemisinin production was established. It featured enhanced growth and artemisinin production by cultivation of hairy roots in nutrient mist bioreactor (NMB) coupled with novel cultivation strategies. Artemisinin is used for the treatment of cerebral malaria. Presently, its main source is from seasonal plant Artemisia annua. This study featured investigation of growth and artemisinin production by A. annua hairy roots (induced by Agrobacterium rhizogenes-mediated genetic transformation of explants) in three bioreactor configurations-bubble column reactor, NMB and modified NMB particularly to establish their suitability for commercial production. It was observed that cultivation of hairy roots in a non-stirred bubble column reactor exhibited a biomass accumulation of 5.68 g/l only while batch cultivation in a custom-made NMB exhibited a higher biomass concentration of 8.52 g/l but relatively lower artemisinin accumulation of 0.22 mg/g was observed in this reactor. A mixture of submerged liquid-phase growth (for 5 days) followed by gas-phase cultivation in nutrient mist reactor operation strategy (for next 15 days) was adopted for hairy root cultivation in this investigation. Reasonably, high (23.02 g/l) final dry weight along with the artemisinin accumulation (1.12 mg/g, equivalent to 25.78 mg/l artemisinin) was obtained in this bioreactor, which is the highest reported artemisinin yield in the gas-phase NMB cultivation.

[Influence of continuous cropping on growth of Artemisia annua and bacterial communities in soil].

In this study, several types of Artemisia annua in soil, including the soil which had not been planted, or planted for one year, or continuously planted for three or five years were collected, in order to study the influences of continuous cropping on the growth of A. annua, content of artemisinin, available nutrient of soil, and bacterial community structure through adopting routine analysis and Illumina MiSeq high-throughput sequencing. The results showed that continuous cropping inhibited significantly the growth of A. annua and reduced leaf biomass, content and yield of artemisinin, with the maximum decreasing amplitude of 30.20%, 7.70% and 35.58% respectively. The content of soil organic matter, available nitrogen, available phosphorus and 16S rRNA sequence number were increased to different extents after continuous cropping of A. annua. According to the results of high-throughput sequencing, 634-812 types of common bacteria belonged to 21 categories were planted in different soil of A. annua with different planting years, which represented that the distribution distance of the point of bacterial community with different years among coordinate system of principal component was relative distant, and community structure had significant changes (P<0.05). As the planting years increased, the abundance of Actinobacteria, Chloroflexi, Gemmatimonadetes decreased in contrast to Proteobacteria, Acidobacteria and Verrucomicrobia. In the top 20 types of predominant bacteria,Nitrospira japonica and Nitrospira disappeared, among which, only Gemmatimonadaceae, Micromonosporaceae, Nitrosomonadaceae, Xanthobacteraceae, and unculture bacterium JG30-KF-AS9 were similar, indicating that the planting and continuous cropping of A. annua selectively inhibited the growth and reproduction of soil bacteria, and influenced the supply and transform of soil nutrient, leading to a poor growth and resulting in reduction of artemisinin content and yield. Therefore, it is necessary to advocate crop rotation in the process of planting A. annua.

[Spatial Distribution and Global Potential Suitability Regions of Artemisia annua].

OBJECTIVE: To study the spatial distribution and potential climatic suitability regions of Artemisia annua around the world. METHODS: The spatial distribution and climatic characteristics were researched by factor analysis based on Global Biodiversity Information Facility Database and World Climate Database. The global potential suitability regions of Artemisia annua were analyzed by ArcGIS. RESULTS: Artemisia annua distributed in three longitude zones, including 90. 55 °W - 77. 14 °W, 2. 03 °E - 11. 75 °E and 98. 27 °E - 111. 05 °E,which were respectively in North America, Europe and Asia. The latitude range was mainly 29. 15 °N - 51. 36 ° N. 80% of Artemisia annua were in the regions which elevation range was 22. 00 - 491. 00 m, annual precipitation was 492. 30 ~ 1 366. 70 mm, annual average temperature was from 8. 10 to 17. 27 °C. The potential suitability regions of Artemisia annua with 95% ~ 100% climate similarity were mainly in 30 °S and 30 °N regions, centered around the equator axis. Conclusion: Latitude is closely related to the distribution of Artemisia annua, the key affecting climatic factors are annual precipitation, the wettest season precipitation, the warmest season precipitation and the highest temperature in the warmest month, the average temperature of the warmest season, as well as the average temperature of the wettest season. The potential suitability regions of Artemnisia annua are in eastern North America, western Europe and eastern Asia.

Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: engineering attempts using a modified SNARE protein.

Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking. The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.

Type 2C phosphatase 1 of Artemisia annua L. is a negative regulator of ABA signaling.

The phytohormone abscisic acid (ABA) plays an important role in plant development and environmental stress response. Additionally, ABA also regulates secondary metabolism such as artemisinin in the medicinal plant Artemisia annua L. Although an earlier study showed that ABA receptor, AaPYL9, plays a positive role in ABA-induced artemisinin content improvement, many components in the ABA signaling pathway remain to be elucidated in Artemisia annua L. To get insight of the function of AaPYL9, we isolated and characterized an AaPYL9-interacting partner, AaPP2C1. The coding sequence of AaPP2C1 encodes a deduced protein of 464 amino acids, with all the features of plant type clade A PP2C. Transcriptional analysis showed that the expression level of AaPP2C1 is increased after ABA, salt, and drought treatments. Yeast two-hybrid and bimolecular fluorescence complementation assays (BiFC) showed that AaPYL9 interacted with AaPP2C1. The P89S, H116A substitution in AaPYL9 as well as G199D substitution or deletion of the third phosphorylation site-like motif in AaPP2C1 abolished this interaction. Furthermore, constitutive expression of AaPP2C1 conferred ABA insensitivity compared with the wild type. In summary, our data reveals that AaPP2C1 is an AaPYL9-interacting partner and involved in the negative modulation of the ABA signaling pathway in A. annua L.

Enhanced production of artemisinin by hairy root cultivation of Artemisia annua in a modified stirred tank reactor.

Artemisinin is an important drug commonly used in the treatment of malaria as a combination therapy. It is primarily produced by a plant Artemisia annua, however, its supply from plant is significantly lower than its huge demand and therefore alternative in vitro production routes are sought. Hairy root cultivation could be one such alternative production protocol. Agrobacterium rhizogenes was used to induce hairy roots of A. annua. Statistical optimization of media was thereafter attempted to maximize the biomass/artemisinin production. The growth and product formation kinetics and the significant role of O2 in hairy root propagation were established in optimized media. Mass cultivation of hairy roots was, thereafter, attempted in a modified 3-L Stirred Tank Bioreactor (Applikon Dependable Instruments, The Netherlands) using optimized culture conditions. The reactor was suitably modified to obtain profuse growth of hairy roots by segregating and protecting the growing roots from the agitator rotation in the reactor using a perforated Teflon disk. It was possible to produce 18 g biomass L(-1) (on dry weight basis) and 4.63 mg L(-1) of artemisinin in 28 days, which increased to 10.33 mg L(-1) by the addition of elicitor methyl jasmonate.

Effect of irradiated sodium alginate and phosphorus on biomass and artemisinin production in Artemisia annua.

It is now being realized that irradiation products of natural bioactive agents can also be beneficially utilized to impart value addition in agriculture by converting these bioactive agents into more useful form. Polysaccharides, such as sodium alginate, have proven to be wonderful growth promoting substances in their depolymerized form for various plants. Artemisinin has been increasingly popular as an effective and safe alternative therapy against malaria; also proved effective against the highly adaptable malaria parasite, which has already become resistant to many other drugs. The drug artemisinin can be extracted from the leafy tissues of Artemisia annua. Therefore, experiments were conducted with an aim to evaluate artemisinin production and overall plant development though depolymerized sodium alginate application and nutrient supply. In the present study, sodium alginate, irradiated by Co-60 gamma rays together with various phosphorus doses, was used to study their effect on growth, physiological and biochemical processes and production of artemisinin in A. annua. Among various applied doses of phosphorus fertilizer, P40 (40 kg Pha(-1)) together with ISA80 (80 mg L(-1)) significantly improved all the parameters studied. Increase in plant height as well as weight was noted at this treatment. Dry leaf yield, artemisinin concentration in leaves and artemisinin yield was also significantly enhanced by the treatment.

Metabolic analysis of the increased adventitious rooting mutant of Artemisia annua reveals a role for the plant monoterpene borneol in adventitious root formation.

Adventitious root (AR) formation is a critical process for plant clonal propagation. The role of plant secondary metabolites in AR formation is still poorly understood. Chemical and physical mutagenesis in combination with somatic variation were performed on Artemisia annua in order to obtain a mutant with changes in adventitious rooting and composition of plant secondary metabolites. Metabolic and morphological analyses of the iar (increased adventitious rooting) mutant coupled with in vitro assays were used to elucidate the relationship between plant secondary metabolites and AR formation. The only detected differences between the iar mutant and wild-type were rooting capacity and borneol/camphor content. Consistent with this, treatment with borneol in vitro promoted adventitious rooting in wild-type. The enhanced rooting did not continue upon removal of borneol. The iar mutant displayed no significant differences in AR formation upon treatment with camphor. Together, our results suggest that borneol promotes adventitious rooting whereas camphor has no effect on AR formation.

Arsenic, chromium and NaCl induced artemisinin biosynthesis in Artemisia annua L.: a valuable antimalarial plant.

Effect of As(III), Cr(VI) and NaCl on plant growth, antioxidant enzymes, SOD, TBRAS, protein, cDNA amplification of key genes of artemisinin pathway and artemisinin biosynthesis have been investigated to explore the actual changes in total herb and artemisinin yield in a crop cycle of Artemisia annua. Enhanced TBARS and SOD activity (4 U mg⁻¹), decreased catalase activity and total cholorophyll content were observed under metal(loid) and NaCl stress. Accumulation of As (III; µg mg⁻¹ DW) was higher in roots (10.75±0.00) than shoot (0.43±0.00) at 10 µg ml⁻¹. While Cr(VI; µg ml⁻¹ DW) accumulated more in shoots (37±9.6, 41.1±7.2 and 52.71±19.6). cDNA template of these treated plants along with control were amplified with HMGR, ADS and CYP71AV1 genes (artemisinin biosynthetic pathway genes); showed very low expression with Cr(VI) while As(III) (5 and 7.5 µg ml⁻¹) showed higher expression than control. The results obtained from this study suggest that A. annua can grow well with favoring artemisinin biosynthesis with treatment of As(III) 5, 7.5 µg ml⁻¹ and NaCl, while 10 µg ml⁻¹ As(III) and all doses of Cr(VI) affect artemisinin synthesis. Finally some evidence also suggests that As(III), Cr(VI) and NaCl induces stress affects on total herb yield of plant.

Pyrolytic and kinetic analysis of two coastal plant species: Artemisia annua and Chenopodium glaucum.

The large amount of coastal plant species available makes them ideal candidates for energy production. In this study, thermogravimetric analysis was used to evaluate the fuel properties of two coastal plant species, and the distributed activation energy model (DAEM) was employed in kinetic analysis. The major mass loss due to devolatilization started at 154 and 162°C at the heating rate of 10°C min(-1) for Artemisia annua and Chenopodium glaucum, respectively. The results showed that the average activation energies of Artemisia annua and Chenopodium glaucum were 169.69 and 170.48 kJ mol(-1), respectively. Furthermore, the activation energy changed while the conversion rate increased, and the frequency factor k 0 decreased greatly while the activation energy decreased. The results also indicated that the devolatilization of the two coastal plant species underwent a set of first-order reactions and could be expressed by the DAEM. Additionally, a simplified mathematical model was proposed to facilitate the prediction of devolatilization curves.