Differentially expressed genes during contrasting growth stages of Artemisia annua for artemisinin content.

Abstract

Artemisia annua is the source of antimalarial phytomolecule, artemisinin. It is mainly produced and stored in the glandular secretory trichomes present in the leaves of the plant. Since, the artemisinin biosynthesis steps are yet to be worked out, in this investigation a microarray chip was strategized for the first time to shortlist the differentially expressing genes at a stage of plant producing highest artemisinin compared to the stage with no artemisinin. As the target of this study was to analyze differential gene expression associated with contrasting artemisinin content in planta and a genotype having zero/negligible artemisinin content was unavailable, it was decided to compare different stages of the same genotype with contrasting artemisinin content (seedling--negligible artemisinin, mature leaf--high artemisinin). The SCAR-marked artemisinin-rich (~1.2%) Indian variety 'CIM-Arogya' was used in the present study to determine optimal plant stage and leaf ontogenic level for artemisinin content. A representative EST dataset from leaf trichome at the stage of maximal artemisinin biosynthesis was established. The high utility small scale custom microarray chip of A. annua containing all the significant artemisinin biosynthesis-related genes, the established EST dataset, gene sequences isolated in-house and strategically selected candidates from the A. annua Unigene database (NCBI) was employed to compare the gene expression profiles of two stages. The expression data was validated through semiquantitative and quantitative RT-PCR followed by putative annotations through bioinformatics-based approaches. Many candidates having probable role in artemisinin metabolism were identified and described with scope for further functional characterization.