UV-B-caused DNA hypomethylation and UV-B-mediated epigenetic activation of additional WRKY-binding site(s) in the DBR2 promoter may contribute to the overexpression of the DBR2 gene in Artemisia annua.
DNA methylation is one of the key mechanisms behind stress-induced transcriptional switch off/on. Here, we evaluate the DNA methylation level in response to UV-B radiation in Artemisia annua which produces artemisinin, a sesquiterpene that has been recommended by WHO for the frontline treatment of malaria. However, the drug is facing serious shortage due to its low concentration in plants. UV-B treatment (3 h) enhanced artemisinin concentration up to 1.91-fold as compared to control. A key regulatory gene of artemisinin biosynthesis, DBR2 was upregulated under UV-B. This study presents observations regarding contributions of DNA methylation to the gene regulation using DBR2 as an example. Restriction digestion of genomic DNA by isoschizomers (MspI and HpaII) suggested UV-B involvement in DNA hypomethylation in A. annua. The global level of DNA methylation (R) was 3.4 and 5.9 % for UV-B treated and control plants, respectively, attesting hypomethylation of DNA in response to UV-B. Further bisulfite sequencing PCR showed demethylation at two CHG sites in 18S rRNA gene. Similarly, bisulfite sequencing of promoter region of DBR2 has demonstrated demethylation at 4 CG-, 4 CHH- and 2 CHG-sites. In silico analysis revealed UV-B-mediated demethylation at seven putative transcription factor binding sites including WRKY, which are positive regulators of artemisinin biosynthesis. UV-B treatment has resulted in activation of additional WRKY-binding site in UV-B-treated plants compared with single active WRKY-binding site in control and this could be the probable reason for overexpression of DBR2. It is suggested that DNA demethylation is an important epigenetic response to UV-B radiation in A. annua that surely will provide new horizons to further elucidate the mechanistic evidence of plant’s responses to UV-B radiation.
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The authors are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, India, for financial assistance in the form of project. Authors would like to acknowledge the guidance and support of Dr. Manoj Prasad, National Institute of Plant Genome Research, New Delhi, India, during TLC-analysis. NP is thankful to University Grant Commission (UGC), New Delhi, India for financial assistance in the form of RFSMS (Research fellowship in science for meritorious students).
Conflict of interest
The authors declare that they have no conflict of interest.
N. Pandey and S. Pandey-Rai have contributed equally.
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Pandey, N., Pandey-Rai, S. Deciphering UV-B-induced variation in DNA methylation pattern and its influence on regulation of DBR2 expression in Artemisia annua L.. Planta 242, 869–879 (2015). https://doi.org/10.1007/s00425-015-2323-3
- DNA methylation
- Bisulfite sequencing PCR
- Artemisia annua
- DBR2 promoter