Jasmonate-Activated AaWRKY9-AabHLH93/AabHLH93-AaMYB7 Complexes Balance Artemisinin Biosynthesis in Artemisia annua

Artemisinin, an antimalarial sesquiterpene lactone, is biosynthesized in glandular trichomes of Artemisia annua. Although numerous transcription factors have been demonstrated to regulate artemisinin biosynthesis, the molecular mechanisms underlying the high expression of artemisinin-associated biosynthetic enzyme and transcription factor genes in young leaves and their marked decline during leaf maturation remain elusive. Here, we identify a trichome-enriched bHLH transcription factor, AabHLH93, through yeast two-hybrid screening using AaWRKY9 as bait. Yeast one-hybrid and electrophoretic mobility shift assays demonstrate that AabHLH93 directly binds to the AaCYP71AV1 promoter. Furthermore, overexpressing AabHLH93 elevates artemisinin levels, while its RNAi suppresses artemisinin biosynthesis. In young leaves, elevated JA represses AaMYB7 expression and triggers 26S proteasome-mediated degradation of AaJAZ9. AabHLH93 physically interacts with AaWRKY9 through its C-terminal domain to form the AaWRKY9-AabHLH93 complex, enhancing the activation of artemisinin biosynthetic genes. Conversely, age-dependent accumulation of the R2R3-MYB repressor AaMYB7 in mature trichomes disrupts this synergy. In mature leaves, age-dependent JA depletion permits AaMYB7 upregulation and AaJAZ9 accumulation. AaMYB7 directly binds to AabHLH93's C-terminal domain, displacing AaWRKY9 and hindering the formation of the AaWRKY9-AabHLH93 activation complex. These findings elucidate how antagonistic AabHLH93-AaWRKY9 and AabHLH93-AaMYB7 modules, coupled with JA and developmental cues, dynamically control artemisinin biosynthesis.