Comprehensive genome-wide identification and functional characterization of non-specific lipid transfer proteins in Artemisia annua L.

Plant non-specific lipid transfer proteins (nsLTPs) are small secretory proteins that bind and transport hydrophobic molecules. nsLTPs are crucial players in various plant physiological processes, including lipid metabolism and defense. In Artemisia annua, glandular trichomes serve as the primary sites for artemisinin biosynthesis and storage. Although several nsLTPs have been reported in Artemisia annua, a comprehensive genome-wide analysis was previously lacking. In this study, 55 nsLTP genes were identified in the A. annua genome and classified into eight types (I, II, III, IV, V, VI, VII, and IX) through phylogenetic analysis. Notably, expression profiling revealed that certain AaLTP genes, especially AaLTP1 and AaLTP2, exhibit glandular trichome-specific expression, as confirmed by promoter::GUS assays. Overexpression of AaLTP1 significantly increased artemisinin content by 1.5-fold (p < 0.05), suggesting its direct role in facilitating artemisinin accumulation. Furthermore, an HD-ZIP transcription factor, AaHD8, was confirmed as a positive regulator of AaLTP1 and AaLTP2 through direct promoter binding. Overall, this study provides the first comprehensive characterization of the nsLTP gene family in A. annua and establishes a functional link between nsLTPs, glandular trichome biology, and artemisinin biosynthesis, offering new insights for metabolic engineering to enhance artemisinin production.