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1.
Plant Cell Physiol ; 60(8): 1747-1760, 2019 Aug 01.
Article in English | MEDLINE | ID: mdl-31076768

ABSTRACT

Artemisinin, the frontline drug against malaria, is a sesquiterpenoid extracted from Artemisia annua. Light has been proposed to play an important role in the activation of artemisinin biosynthesis. Here, we report the basic leucine zipper transcription factor (TF) AaHY5 as a key regulator of light-induced biosynthesis of artemisinin. We show that AaHY5 transcription overlaps with that of artemisinin biosynthesis genes in response to light and in A. annua tissues. Analysis of AaHY5 overexpression and RNAi-suppression lines suggests that AaHY5 is a positive regulator of the expression of artemisinin biosynthesis genes and accumulation of artemisinin. We show that AaHY5 complements the hy5 mutant in Arabidopsis thaliana. Our data further suggest that AaHY5 interacts with AaCOP1, the ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1 in A. annua. In yeast one-hybrid and transient expression assays, we demonstrate that AaHY5 acts via the TF GLANDULAR TRICHOME-SPECIFIC WRKY 1 (AaGSW1) in artemisinin regulation. In summary, we present a novel regulator of artemisinin gene expression and propose a model in which AaHY5 indirectly controls artemisinin production in response to changing light conditions.


Subject(s)
Artemisia annua/metabolism , Artemisinins/metabolism , Light , Artemisia annua/radiation effects , Gene Expression Regulation, Plant/radiation effects , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/metabolism , Plants, Genetically Modified/radiation effects , Transcription Factors
2.
Planta ; 249(2): 497-514, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30267151

ABSTRACT

MAIN CONCLUSION: UV-B-induced flavonoid biosynthesis is epigenetically regulated by site-specific demethylation of AaMYB1, AaMYC, and AaWRKY TF-binding sites inAaPAL1promoter-causing overexpression ofAaPALgene inArtemisia annua. The present study was undertaken to understand the epigenetic regulation of flavonoid biosynthesis under the influence of ultraviolet-B radiation using Artemisia annua L. as an experimental model. In-vitro propagated and acclimatized plantlets were treated with UV-B radiation (2.8 W m-2; 3 h), which resulted in enhanced accumulation of total flavonoid and phenolics content as well as eleven individual flavonoids measured through HPLC-DAC. Expression of eight genes (phenylanaline ammonia lyase, cinnamate-4-hydroxylase, 4-coumarate: CoA ligase; chalcone synthase, chalcone isomerase, cinnamoyl reductase, flavonoid-3'-hydroxylase, and flavones synthase) from upstream and downstream flavonoid biosynthetic pathways was measured through RT-PCR and RT-Q-PCR and all were variably induced under UV-B irradiation. Among them, AaPAL1 transcript and its protein were most significantly upregulated. Global DNA methylation analysis revealed hypomethylation of genomic DNA in A. annua. Further epigenetic characterization of promoter region of AaPAL1 revealed cytosine demethylation at five sites, which in turn caused epigenetic activation of six transcription factor-binding sites including QELEMENT, EBOXBNNAPA/MYCCONSENSUSAT, MYBCORE, MYBCOREATCYCB1, and GCCCORE. MYB transcription factors are positive regulators of flavonoid biosynthesis. Epigenetic activation of transcription-enhancing cis-regulatory elements in AaPAL1 promoter and subsequent overexpression of AaMYB1 and AaMYC and AaWRKY transcription factors under UV-B irradiation may probably be the reason for higher AaPAL1 expression and hence greater biosynthesis of flavonoids in A. annua L. The present study is the first report that provides mechanistic evidence of epigenetic regulation of flavonoid biosynthesis under UV-B radiation in A. annua L.


Subject(s)
Artemisia annua/radiation effects , Epigenesis, Genetic , Flavonoids/metabolism , Ultraviolet Rays , Acyltransferases/metabolism , Artemisia annua/chemistry , Artemisia annua/genetics , Artemisia annua/metabolism , Blotting, Western , Chromatography, High Pressure Liquid , DNA Demethylation/radiation effects , Flavonoids/analysis , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/radiation effects , Lyases/metabolism , Metabolic Networks and Pathways , Promoter Regions, Genetic/genetics , Real-Time Polymerase Chain Reaction , Transcription Factors/metabolism , Transcriptome
3.
Molecules ; 23(6)2018 May 31.
Article in English | MEDLINE | ID: mdl-29857558

ABSTRACT

Artemisinin, which has been isolated from Artemisiaannua L., is the most effective antimalarial drug and has saved millions of lives. In addition, artemisinin and its derivatives have anti-tumor, anti-parasitic, anti-fibrosis, and anti-arrhythmic properties, which enhances the demand for these compounds. Improving the content of artemisinin in A.annua is therefore becoming an increasing research interest, as the chemical synthesis of this metabolite is not viable. Ultraviolet B and C irradiation have been reported to improve the artemisinin content in A.annua, but they are harmful to plant growth and development. Therefore, we screened other light sources to examine if they could promote artemisinin content without affecting plant growth and development. We found that red and blue light could enhance artemisinin accumulation by promoting the expression of the genes that were involved in artemisinin biosynthesis, such as amorpha-4,11-diene synthase (ADS) and cytochrome P450 monooxygenase (CYP71AV1) genes. Thus, in addition to being the main light sources for photosynthesis, red and blue light play a key role in plant secondary metabolism, and optimizing the combination of these light might allow for the productionof artemisinin-rich A.annua.


Subject(s)
Artemisia annua/metabolism , Artemisia annua/radiation effects , Artemisinins/metabolism , Light , Photosynthesis , Artemisia annua/classification , Artemisia annua/genetics , Biosynthetic Pathways , Computational Biology/methods , DNA, Ribosomal Spacer , Gene Expression Profiling , Gene Expression Regulation, Plant , Molecular Sequence Annotation , Reproducibility of Results , Secondary Metabolism , Transcriptome
4.
J Photochem Photobiol B ; 140: 292-300, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25194528

ABSTRACT

Artemisinin, an endoperoxide sesquiterpene lactone, is an effective antimalarial drug isolated from Artemisia annua L. In this study, a low dose (1.44 kJm(-2)d(-1)) of UV-B radiation (280-320 nm) for short-term (1h per day for 10 days) was applied to A. annua seedlings to stimulate artemisinin production. UV-B treatment not only induced the generation of reactive oxygen species (ROS), enhanced peroxidase activity and endogenous content of abscisic acid (ABA), but stimulated the biosynthesis of artemisinin in the seedlings. Here, transcriptomic changes during UV-B radiation in A. annua were detected using an Agilent GeneChip with 43,692 probe sets. In total, 358 transcripts were identified as differentially expressed under UV-B stress, of which 172 transcripts increased and 186 transcripts decreased in abundance. In terms of biological processes, gene ontology (GO) terms including primary carbohydrate and nitrogen compound metabolic processes were enriched in UV-B-repressed genes. The up-regulated genes were enriched in response to stress, ROS generation, hormone (ethylene, ABA) stimulus and cell cycle control. The expression of key enzymes such as amorpha-4,11-diene synthase (ADS) and cytochrome P450 dependent monooxygenase/hydroxylase (CYP71AV1), and related WRKY transcription factors was up-regulated significantly for artemisinin biosynthesis. This profile of global gene expression patterns during UV-B stress will be valuable for further identification of the enzymes involved in artemisinin biosynthesis.


Subject(s)
Artemisia annua/genetics , Artemisia annua/metabolism , Artemisinins/metabolism , Transcriptome/radiation effects , Ultraviolet Rays , Artemisia annua/cytology , Artemisia annua/radiation effects , Cell Death/radiation effects , Gene Ontology , Molecular Sequence Annotation , Multigene Family/genetics , Oligonucleotide Array Sequence Analysis , Photosynthesis/radiation effects , Pigments, Biological/metabolism
5.
Planta ; 240(3): 611-27, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25023630

ABSTRACT

MAIN CONCLUSION: UV - B radiation exposure for upto 3 h did not cause direct damage to physiology, but adjusted secondary metabolism and metabolites accumulation as an effective acclimation mechanism to mitigate the adverse effects of radiation. Artemisia annua L. plants were irradiated with UV-B radiation (280-315 nm; 2.8 Wm(-2)) for different short-term (1, 2, 3 and 4 h) durations. UV-B irradiation of 3 h reduced the photosynthetic rate, stomatal conductance and transpiration rate. However, F v/F m, a sensitive indicator of photosynthetic inhibition, remained stable (0.78) upto 3 h, thereafter it declined sharply (0.72). Interestingly, transcript level of LHCB1, PSBA and PSBO genes related to photosystem II (PSII) were induced under UV-B exposure. In addition, genes coding for Rubisco small (RBCS1B) and large (RBCL) subunits were also upregulated upto 3 h. To mitigate the adverse effects of UV-B radiation, plants tremendously induced defense-related secondary metabolites such as antioxidative phenolics, UV-B absorbing flavonoids, anthocyanins and protective terpenes. The GC-MS analysis of essential oils revealed relatively higher production of monoterpenes over sesquiterpenes as well as 1.2-folds higher total oil yield under UV-B radiation. Owing to its diverse biological activities, the altered quantity and quality of essential oil of A. annua may contribute towards improving its therapeutic properties. The results suggest that UV-B irradiation upto 3 h reduced photosynthesis, probably due to stomatal limitations rather than any direct injury to photosynthetic apparatus as evident from stable F v/F m value, upregulated genes and greater accumulation of their corresponding proteins which gauge PSII health, elevated UV-B absorbing compounds and other protective metabolites. Correlation analysis indicates a significant positive correlation of photosynthetic rate with stomatal conductance while a negative correlation with anthocyanin and monoterpene contents under UV-B radiation. The present study provides first hand information regarding photosynthesis, related physiological parameters and essential oil profiling in response to UV-B radiation in A. annua.


Subject(s)
Acclimatization , Artemisia annua/radiation effects , Oils, Volatile/metabolism , Pigments, Biological/metabolism , Secondary Metabolism , Artemisia annua/metabolism , Gene Expression Regulation, Plant/radiation effects , Phenols/metabolism , Photosynthesis , Plant Leaves/metabolism , Ultraviolet Rays
6.
J Photochem Photobiol B ; 105(3): 216-25, 2011 Dec 02.
Article in English | MEDLINE | ID: mdl-22019553

ABSTRACT

Present study was undertaken to investigate if short-term UV-B (4.2 kJ m(-2) day(-1)) and UV-C (5.7 kJ m(-2) day(-1)), pre-treatments can induce artemisinin biosynthesis in Artemisia annua. Twenty-one day old Artemisia seedlings were subjected to short-term (14 days) UV pre-treatment in an environmentally controlled growth chamber and then transplanted to the field under natural conditions. Treatment of A. annua with artificial UV-B and UV-C radiation not only altered the growth responses, biomass, pigment content and antioxidant enzyme activity but enhanced the secondary metabolites (artemisinin and flavonoid) content at all developmental stages as compared to non-irradiated plants. The extent of oxidative damage was measured in terms of the activities of enzymes such as catalase, superoxide dismutase and ascorbate peroxidase. Reinforcement in the antioxidative defense system seems to be a positive response of plants in ameliorating the negative effects of UV-B and UV-C radiations. While the carotenoid content was elevated, the chlorophyll content decreased under UV-B and UV-C pre-treatments. The reverse transcription PCR analysis of the genes associated in artemisinin/isoprenoid biosynthesis like 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), cytochrome P450 oxidoreductase (CPR) and amorpha-4,11-diene synthase (ADS) genes at different growth stages revealed UV induced significant over-expression of the above protein genes. UV-B and UV-C pre-treatments, led to an increase in the concentrations of artemisinin at full bloom stage by 10.5% and 15.7% than that of the control respectively. Thus, the result of our study suggests that short term UV-B pre-treatment of seedlings in greenhouse prior to transplantation into the field enhances artemisinin production with lesser yield related damages as compared to UV-C radiation in A. annua.


Subject(s)
Antimalarials/metabolism , Artemisia annua/metabolism , Artemisia annua/radiation effects , Artemisinins/metabolism , Ultraviolet Rays/adverse effects , Antioxidants/metabolism , Artemisia annua/enzymology , Artemisia annua/physiology , Biomarkers/metabolism , Flavonoids/metabolism , Gene Expression Regulation, Plant/radiation effects , Hydrogen Peroxide/metabolism , Oxidative Stress/radiation effects , Photosynthesis/radiation effects , Pigments, Biological/metabolism , Proline/metabolism , Seedlings/enzymology , Seedlings/metabolism , Seedlings/physiology , Seedlings/radiation effects , Stress, Physiological/radiation effects , Thiobarbituric Acid Reactive Substances/metabolism , Time Factors
7.
Int J Radiat Biol ; 84(11): 878-84, 2008 Nov.
Article in English | MEDLINE | ID: mdl-19016136

ABSTRACT

PURPOSE: This research work aimed to create in vitro plantlet variants of Artemisia annua through gamma irradiation. The obtained variants were then evaluated for the correlation between their artemisinin content and enzyme activity of amorpha-4,11-diene synthase (ADS), the first enzyme of the artemisinin pathway. MATERIALS AND METHODS: Shoot tips from plantlets of A. annua were exposed to gamma rays at a low-dose range and transferred to hormone-free MS medium for in vitro cultivation. A dose-response curve and the value of 50% of lethal dose (LD50) were then obtained. This LD50 dose of gamma rays was used for treating another batch of shoot tips. The surviving plantlets after four subsequent subcultures were evaluated for their ability to accumulate artemisinin in correlation with the enzyme activity of ADS. RESULTS: The dose-response curve showed that the LD50 value was at 8 Gray (Gy). The surviving irradiated plantlets from this dose treatment had artemisinin content ranging from 0.03-0.70% (w/w) of dry weight, comparing with only 0.18% present in the original non-irradiated samples. Their correlation coefficient between the ADS activity and the artemisinin content appeared to be R2 = 0.090 for all the 18 samples tested, although, selectively, more than half of these (11 samples) showed their R2 value of as high as 0.851. CONCLUSIONS: There was no correlation between the artemisinin content and ADS activity found in the whole population, but the correlation was observed in the main subpopulation of the irradiated A. annua plantlets.


Subject(s)
Alkyl and Aryl Transferases/metabolism , Artemisia annua/metabolism , Artemisia annua/radiation effects , Artemisinins/metabolism , Gamma Rays , Artemisia annua/enzymology , Artemisia annua/growth & development , Dose-Response Relationship, Radiation
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