Neha Pandey1, Shashi Pandey-Rai. 1. Laboratory of Morphogenesis, Department of Botany, Faculty of Science, Banaras Hindu University, Varanasi, 221005, Uttar Pradesh, India.
Abstract
MAIN CONCLUSION: 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.
MAIN CONCLUSION: 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.