Diversified quantity, gene structure, and expression profile of OPR gene family of A. annua

Artemisia annua, the source of artemisinin production, is a traditional herb used for treating malaria for thousand years. The genetic background is of high heterozygosity and traits (plant height, biomass, artemisinin content, etc.) are diverse across different germplasms. Unraveling the key genes associated with growth and secondary metabolism is essential for the efficient production of artemisinin. The 12-oxo-phytodienoic acid reductase (OPR) genes, crucial for plant growth and development and stress resistance, remain unexplored in A. annua. In this study, nine OPR genes (named as AaOPR1 to AaOPR9) were identified in A. annua, including two pairs of genes formed from recent tandem duplications. The number of OPRs varied among different haplotype genomes, and each OPR gene exhibiting distinct expression pattern. Moreover, the OPR family displayed evolutionarily activity with significant variations in numbers and gene structures observed across different plant species. Widespread gene duplication of OPRs, observed in the majority of analyzed plant genomes, brought evolutionary potential. DBR2, a member of AaOPRs involved in artemisinin biosynthesis, had two copies (AaOPR1/DBR2.1 and AaOPR2/DBR2.2) with different expression patterns, one of which was a recently generated copy with a significant 7-amino acids truncation. Heterologous protein expression and functional characterization of the two copies of DBR2 yielded multiple isomers with identical molecular weights but different arrangements, indicating neofunctionalization of the newly generated copy. The polymorphism within the OPR gene family merely scratches the surface of the genetic diversity in A. annua, and further investigation of genetic features is needed for the screening of elite germplasm resources.