The ameliorative effects of exogenous inoculation of Piriformospora indica on molecular, biochemical and physiological parameters of Artemisia annua L. under arsenic stress condition.

Aim of the current study was to investigate the effect of exogenously inoculated root endophytic fungus, Piriformospora indica, on molecular, biochemical, morphological and physiological parameters of Artemisia annua L. treated with different concentrations (0, 50, 100 and 150 µmol/L) of arsenic (As) stress. As was significantly accumulated in the roots than shoots of P. indica-inoculated plants. As accumulation and immobilization in the roots is directly associated with the successful fungal colonization that restricts most of As as compared to the aerial parts. A total of 4.1, 11.2 and 25.6 mg/kg dry weight of As was accumulated in the roots of inoculated plants supplemented with 50, 100 and 150 µmol/L of As, respectively as shown by atomic absorption spectroscopy. P. indica showed significant tolerance in vitro to As toxicity even at high concentration. Furthermore, flavonoids, artemisinin and overall biomass were significantly increased in inoculated-stressed plants. Superoxide dismutase and peroxidase activities were increased 1.6 and 1.2 fold, respectively under 150 µmol/L stress in P. indica-colonized plants. Similar trend was followed by ascorbate peroxidase, catalase and glutathione reductase. Like that, phenolic acid and phenolic compounds showed a significant increase in colonized plants as compared to their respective control/un-colonize stressed plants. The real-time PCR revealed that transcriptional levels of artemisinin biosynthesis genes, isoprenoids, terpenes, flavonoids biosynthetic pathway genes and signal molecules were prominently enhanced in inoculated stressed plants than un-inoculated stressed plants.

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.

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.

Using ß-ocimene to increase the artemisinin content in juvenile plants of Artemisia annua L.

OBJECTIVE: The anti-malarial drug, artemisinin, is harvested from the leaves of adult Artemisia annua L. plants. As its concentration in juvenile plants is very low, the present study aimed to assess if the airborne signaling molecule, ß-ocimene, could be used to enhance artemisinin accumulation in juvenile A. annua plants. RESULTS: Application of exogenous ß-ocimene increased artemisinin accumulation in A. annua. Treatment with 10 µM ß-ocimene for 4 days resulted in juvenile plants accumulating artemisinin contents of up to 25 mg/g (2.5%) of dry weight. The expression levels of key genes encoding enzymes involved in both precursor biosynthetic pathways and artemisinin biosynthetic pathways induced by ß-ocimene were upregulated. Glandular secretory trichome (GST) size and density increased by 49.2% and 38.2%, respectively, along with the upregulation of genes associated with GST development. CONCLUSION: ß-ocimene enhances artemisinin accumulation in juvenile A. annua plants by modulating artemisinin biosynthetic pathways and GST development.

Jasmonic acid-responsive AabHLH1 positively regulates artemisinin biosynthesis in Artemisia annua.

Artemisia annua is the only natural source of the sesquiterpenoid artemisinin, which is widely used to treat malaria. The phytohormone jasmonic acid (JA) can significantly promote artemisinin biosynthesis in A. annua. AabHLH1 can bind and activate artemisinin biosynthetic genes, such as AaADS and AaCYP71AV1. In this study, we proved that AabHLH1 was responsive to MeJA treatment and highly expressed in glandular trichome-enriched tissues, and that its expression profile was similar to that of AaADS. Yeast two-hybrid assays showed that AabHLH1 interacted with all nine AaJAZ proteins in A. annua. Functional analysis with transgenic plants showed that several artemisinin biosynthetic genes were upregulated in AabHLH1-OE transgenic A. annua lines and downregulated in AabHLH1-EAR lines; furthermore, the artemisinin content was increased in the AabHLH1-OE lines and decreased in the AabHLH1-EAR lines. These results demonstrate that the JA-induced AabHLH1 positively regulates artemisinin biosynthesis by regulating the biosynthetic genes, and thus provide new insight into the regulatory mechanism of JA-induced artemisinin biosynthesis in A. annua.

Enhanced arsenic tolerance and secondary metabolism by modulation of gene expression and proteome profile in Artemisia annua L. after application of exogenous salicylic acid.

This study was designed to investigate the effect of exogenous application of salicylic acid (SA) on proteins pattern and secondary metabolites in arsenic (As) stressed Artemisia annua. A. annua was treated by As 100 µM, SA 100 µM and combined treatment of SA 100 µM + As 100 µM upto 3 days. Significant accumulation of As was observed in roots than shoots at As 100 µM treatment. Under As treatment, oxidative stress was induced as indicated by increased TBARS content. Biomass, carotenoid, flavonoids were enhanced whereas total chlorophyll pigment was reduced under As treatment. Combined treatment of SA 100 µM + As 100 µM was more effective for increment of biomass, total chlorophyll content, and flavonoids as compared to As 100 µM treatment. Protein profiling revealed 20 differentially abundant proteins by 2-DE PAGE and MALDI-TOF-MS analysis. Identified proteins were related to photosynthesis, energy metabolism, transcriptional regulators, secondary metabolism, lipid metabolism, transport proteins and unknown/hypothetical proteins. All identified proteins were significantly increased in abundance under combined treatments of SA 100 µM + As 100 µM. The expression analysis of key genes involved in biosynthesis of lipid metabolism, signal molecule, transcriptional regulators, artemisinin biosynthetic genes, isoprenoids pathway, terpenes and flavonoids pathway were significantly upregulated under combined treatments of SA 100 µM + As 100 µM, suggesting a fine linkage in regulation of primary and secondary metabolism to modulate tolerance capacity and to improve phytoremediation property of A. annua against arsenic toxicity.

Exogenous salicylic acid-mediated modulation of arsenic stress tolerance with enhanced accumulation of secondary metabolites and improved size of glandular trichomes in Artemisia annua L.

The present study was undertaken to find out individual and interactive effects of arsenic (As) and salicylic acid (SA) on an important medicinal plant, Artemisia annua. As uptake and its accumulation was detected and found to be maximum in roots at higher As concentration (150 µM). Under As treatments, H2O2 and MDA content were induced. Biomass and chlorophyll content were negatively affected under As treatments. Furthermore, enzymatic (SOD, CAT, APX, and GR) and non-enzymatic antioxidants were also enhanced under As treatments. Exogenous application of SA reduced the extent of H2O2 and O2- generation and lipid peroxidation, while reverted biomass and chlorophyll content to overcome oxidative stress. Simultaneous application of SA with As increased endogenous SA level, artemisinin, and dihydroartemisinic acid as compared with individual As treatment and pre-application of SA with As treatments. The expression of four key artemisinin biosynthetic pathway genes, i.e., ADS, CYP71AV1, DBR2, and ALDH1 were upregulated at a maximum in plants simultaneously treated with SA and As. Similar pattern of artemisinin accumulation and glandular trichome size was observed which attest that SA has a stimulatory impact on artemisinin biosynthesis under As stress. Our study suggests that exogenous application of SA and As together induced more tolerance in A. annua than a comparable dose of SA pre-treatment. The study may provide a platform with dual benefits by developing As-tolerant plants to be used for phytoremediation of arsenic from As-contaminated soil and obtaining high artemisinin-producing A. annua plants.

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.

HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua.

Glandular trichomes are generally considered biofactories that produce valuable chemicals. Increasing glandular trichome density is a very suitable way to improve the productivity of these valuable metabolites, but little is known about the regulation of glandular trichome formation. Phytohormone jasmonate (JA) promotes glandular trichome initiation in various plants, but its mechanism is also unknown. By searching transcription factors regulated by JA in Artemisia annua, we identified a novel homeodomain-leucine zipper transcription factor, HOMEODOMAIN PROTEIN 1 (AaHD1), which positively controls both glandular and nonglandular trichome initiations. Overexpression of AaHD1 in A. annua significantly increased glandular trichome density without harming plant growth. Consequently, the artemisinin content was improved. AaHD1 interacts with A. annua jasmonate ZIM-domain 8 (AaJAZ8), which is a repressor of JA, thereby resulting in decreased transcriptional activity. AaHD1 knockdown lines show decreased sensitivity to JA on glandular trichome initiation, which indicates that AaHD1 plays an important role in JA-mediated glandular trichome initiation. We identified a new transcription factor that promotes A. annua glandular trichome initiation and revealed a novel molecular mechanism by which a homeodomain protein transduces JA signal to promote glandular trichome initiation. Our results also suggested a connection between glandular and nonglandular trichome formations.

Molecular cloning and characterization of a flavanone 3-Hydroxylase gene from Artemisia annua L.

Flavonoids were found to synergize anti-malaria and anti-cancer compounds in Artemisia annua, a very important economic crop in China. In order to discover the regulation mechanism of flavonoids in Artemisia annua, the full length cDNA of flavanone 3-hydroxylase (F3H) were isolated from Artemisia annua for the first time by using RACE (rapid amplification of cDNA ends). The completed open read frame of AaF3H was 1095 bp and it encoded a 364-amino acid protein with a predicted molecular mass of 41.18 kDa and a pI of 5.67. The recombinant protein of AaF3H was expressed in E. coli BL21(DE3) as His-tagged protein, purified by Ni-NTA agrose affinity chromatography, and functionally characterized in vitro. The results showed that the His-tagged protein (AaF3H) catalyzed naringenin to dihydrokaempferol in the present of Fe(2+). The Km for naringenin was 218.03 µM. The optimum pH for AaF3H reaction was determined to be pH 8.5, and the optimum temperature was determined to be 35 °C. The AaF3H transcripts were found to be accumulated in the cultivar with higher level of flavonoids than that with lower level of flavonoids, which implied that AaF3H was a potential target for regulation of flavonoids biosynthesis in Artemisia annua through metabolic engineering.

Branch Pathway Blocking in Artemisia annua is a Useful Method for Obtaining High Yield Artemisinin.

There are many biosynthetic pathways competing for the metabolic flux with the artemisinin biosynthetic pathway in Artemisia annua L. To study the relationship between genes encoding enzymes at branching points and the artemisinin biosynthetic pathway, ß-caryophyllene, ß-farnesene and squalene were sprayed on young seedlings of A. annua. Transient expression assays indicated that the transcription levels of ß-caryophyllene synthase (CPS), ß-farnesene synthase (BFS) and squalene synthase (SQS) were inhibited by ß-caryophyllene, ß-farnesene and squalene, respectively, while expression of some artemisinin biosynthetic pathway genes increased. Thus, inhibition of these genes encoding enzymes at branching points may be helpful to improve the artemisinin content. For further study, the expression levels of four branch pathway genes CPS, BFS, germacrene A synthase (GAS) and SQS were down-regulated by the antisense method in A. annua. In anti-CPS transgenic plants, mRNA levels of BFS and ADS were increased, and the contents of ß-farnesene, artemisinin and dihydroartemisinic acid (DHAA) were increased by 212, 77 and 132%, respectively. The expression levels of CPS, SQS, GAS, amorpha-4,11-diene synthase (ADS), amorphadiene 12-hydroxylase (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1) were increased in anti-BFS transgenic plants and, at the same time, the contents of artemisinin and DHAA were increased by 77% and 54%, respectively, and the content of squalene was increased by 235%. In anti-GAS transgenic plants, mRNA levels of CPS, BFS, ADS and ALDH1 were increased. The contents of artemisinin and DHAA were enhanced by 103% and 130%, respectively. In anti-SQS transgenic plants, the transcription levels of BFS, GAS, CPS, ADS, CYP71AV1 and ALDH1 were all increased. Contents of artemisinin and DHAA were enhanced by 71% and 223%, respectively, while ß-farnesene was raised to 123%. The mRNA level of artemisinic aldehyde Δ11(13) reductase (DBR2) had changed little in almost all transgenic plants.

Molecular cloning and promoter analysis of the specific salicylic acid biosynthetic pathway gene phenylalanine ammonia-lyase (AaPAL1) from Artemisia annua.

Phenylalanine ammonia-lyase (PAL) is the key enzyme in the biosynthetic pathway of salicylic acid (SA). In this study, a full-length cDNA of PAL gene (named as AaPAL1) was cloned from Artemisia annua. The gene contains an open reading frame of 2,151 bps encoding 716 amino acids. Comparative and bioinformatics analysis revealed that the polypeptide protein of AaPAL1 was highly homologous to PALs from other plant species. Southern blot analysis revealed that it belonged to a gene family with three members. Quantitative RT-PCR analysis of various tissues of A. annua showed that AaPAL1 transcript levels were highest in the young leaves. A 1160-bp promoter region was also isolated resulting in identification of distinct cis-regulatory elements including W-box, TGACG-motif, and TC-rich repeats. Quantitative RT-PCR indicated that AaPAL1 was upregulated by salinity, drought, wounding, and SA stresses, which were corroborated positively with the identified cis-elements within the promoter region. AaPAL1 was successfully expressed in Escherichia. coli and the enzyme activity of the purified AaPAL1 was approximately 287.2 U/mg. These results substantiated the involvement of AaPAL1 in the phenylalanine pathway.

[Stability analysis of reference gene based on real-time PCR in Artemisia annua under cadmium treatment].

In this study, Actin, 18S rRNA, PAL, GAPDH and CPR of Artemisia annua were selected as candidate reference genes, and their gene-specific primers for real-time PCR were designed, then geNorm, NormFinder, BestKeeper, Delta CT and RefFinder were used to evaluate their expression stability in the leaves of A. annua under treatment of different concentrations of Cd, with the purpose of finding a reliable reference gene to ensure the reliability of gene-expression analysis. The results showed that there were some significant differences among the candidate reference genes under different treatments and the order of expression stability of candidate reference gene was Actin > 18S rRNA > PAL > GAPDH > CPR. These results suggested that Actin, 18S rRNA and PAL could be used as ideal reference genes of gene expression analysis in A. annua and multiple internal control genes were adopted for results calibration. In addition, differences in expression stability of candidate reference genes in the leaves of A. annua under the same concentrations of Cd were observed, which suggested that the screening of candidate reference genes was needed even under the same treatment. To our best knowledge, this study for the first time provided the ideal reference genes under Cd treatment in the leaves of A. annua and offered reference for the gene expression analysis of A. annua under other conditions.

Inducing effect of dihydroartemisinic acid in the biosynthesis of artemisinins with cultured cells of Artemisia annua by enhancing the expression of genes.

Artemisinin has been used in the production of "artemisinin combination therapies" for the treatment of malaria. Feeding of precursors has been proven to be one of the most effective methods to enhance artemisinin production in plant cultured cells. At the current paper, the biosynthesis of artemisinin (ART) and its four analogs from dihydroartemisinic acid (DHAA) in suspension-cultured cells of Artemisia annua were investigated. ARTs were detected by HPLC/GC-MS and isolated by various chromatography methods. The structures of four DHAA metabolites, namely, dihydro-epi-deoxyarteannuin B, arteannuin I, arteannuin K, and 3-ß-hydroxy-dihydro-epi-deoxyarteannuin B, were elucidated by physicochemical and spectroscopic analyses. The correlation between gene expression and ART content was investigated. The results of RT-PCR showed that DHAA could up-regulate expression of amorpha-4,11-diene synthase gene (ADS), amorpha-4,11-diene C-12 oxidase gene (CYP71AV1), and farnesyl diphosphate synthase gene (FPS) (3.19-, 7.21-, and 2.04-fold higher than those of control group, resp.), which indicated that biosynthesis processes from DHAA to ART were enzyme-mediated.

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.

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.

Studies on the expression of sesquiterpene synthases using promoter-ß-glucuronidase fusions in transgenic Artemisia annua L.

In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for ß-caryophyllene (CPS), epi-cedrol (ECS) and ß-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.

The effect of roots and media constituents on trichomes and artemisinin production in Artemisia annua L.

KEY MESSAGE : Rooting of Artemisia annua increases trichome size on leaves and helps drive the final steps of the biosynthesis of the sesquiterpene antimalarial drug, artemisinin. Artemisia annua produces the antimalarial drug, artemisinin (AN), which is synthesized and stored in glandular trichomes (GLTs). In vitro-grown A. annua shoots produce more AN when they form roots. This may be a function not of the roots, but rather media components such as the phytohormones, α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), or salts and sucrose used to maintain either rooted or unrooted shoot cultures. We investigated how three main media components altered artemisinic metabolite production, pathway gene transcripts, and GLT formation in both mature and developing leaves in rooted and unrooted cultures. Although transcript levels of AN biosynthetic genes were not altered, AN levels were significantly different, and there were major differences in both artemisinic metabolite levels and trichomes in mature versus developing leaves. For example, NAA induced higher AN production in rooted shoots, but only in mature leaves. In developing leaves, BAP increased GLT density on the leaf surface. When both phytohormones were present, GLTs were larger on young developing leaves, but smaller on mature leaves. Furthermore, although other media components increased GLT density, their size decreased on young leaves, but there was no effect on mature leaves. Roots also appeared to drive conversion of artemisinic precursors towards end products. These results suggest that, while the presence of roots affects AN and trichome production, phytohormones and other media constituents used for in vitro culture of A. annua also exert an influence.

Exogenous nitric oxide donor protects Artemisia annua from oxidative stress generated by boron and aluminium toxicity.

Nitric oxide (NO) is an important signal molecule modulating the response of plants to environmental stress. Here we report the effects of boron (B) and aluminium (Al) contamination in soil, carried out with or without application of exogenous SNP (NO donor), on various plant processes in Artemisia annua, including changes in artemisinin content. The addition of B or Al to soil medium significantly reduced the yield and growth of plants and lowered the values of net photosynthetic rate, stomatal conductance, internal CO(2) concentration and total chlorophyll content. The follow-up treatment of NO donor favoured growth and improved the photosynthetic efficiency in stressed as well as non-stressed plants. Artemisinin content was enhanced by 24.6% and 43.8% at 1mmole of soil-applied B or Al. When SNP was applied at 2mmole concentration together with either 1mmole of B and/or Al, it further stimulated artemisinin biosynthesis compared to the control. Application of B+Al+SNP proved to be the best treatment combination for the artemisinin content in Artemisia annua leaves.