Molecular Characterization of the 1-Deoxy-D-Xylulose 5-Phosphate Synthase Gene Family in Artemisia annua

Abstract

<i>Artemisia annua</i> produces artemisinin, an effective antimalarial drug. In recent decades, the later steps of artemisinin biosynthesis have been thoroughly investigated; however, little is known about the early steps of artemisinin biosynthesis. Comparative transcriptomics of glandular and filamentous trichomes and ¹³CO₂ radioisotope study have shown that the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, rather than the mevalonate pathway, plays an important role in artemisinin biosynthesis. In this study, we have cloned three <i>1-deoxy-D-xylulose 5-phosphate synthase</i> (<i>DXS</i>) genes from <i>A</i>. <i>annua</i> (<i>AaDXS1</i>, <i>AaDXS2</i>, and <i>AaDXS3</i>); the DXS enzyme catalyzes the first and rate-limiting enzyme of the MEP pathway. We analyzed the expression of these three genes in different tissues in response to multiple treatments. Phylogenetic analysis revealed that each of the three <i>DXS</i> genes belonged to a distinct clade. Subcellular localization analysis indicated that all three AaDXS proteins are targeted to chloroplasts, which is consistent with the presence of plastid transit peptides in their N-terminal regions. Expression analyses revealed that the expression pattern of <i>AaDXS2</i> in specific tissues and in response to different treatments, including methyl jasmonate, light, and low temperature, was similar to that of artemisinin biosynthesis genes. To further investigate the tissue-specific expression pattern of <i>AaDXS2</i>, the promoter of <i>AaDXS2</i> was cloned upstream of the <i>β-glucuronidase</i> gene and was introduced in arabidopsis. Histochemical staining assays demonstrated that <i>AaDXS2</i> was mainly expressed in the trichomes of Arabidopsis leaves. Together, these results suggest that <i>AaDXS2</i> might be the only member of the DXS family in <i>A. annua</i> that is involved in artemisinin biosynthesis.