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Figure 1. 

Alignment of multiple sequences of AaLTP proteins. The conserved cysteine residues are marked against red backgrounds.

Figure 2. 

Phylogenetic tree of AaLTPs together with motif analysis and gene structure analysis. The full length of mature protein sequences of nsLTPs from A. annua was used to construct the phylogenetic tree using a Maximum likelihood method. Motif analysis was performed using MEME, while domain analysis was performed using the batch Web CD-Search tool. All of the above results were visualized together with the gene annotation file.

Figure 3. 

Phylogenetic tree of nsLTPs in A. thaliana and A. annua. The full length of mature protein sequences of nsLTPs from A. thaliana and A. annua were used to construct the phylogenetic tree using a Maximum likelihood method. Different colors represent different clusters.

Figure 4. 

Expression analysis of AaLTPs. The heatmap shows expression comparison between six organs of AaLTPs. The data shown were the results of column scaling each gene by RNA-seq data using the Zero To One Scale Method. The color bar represents the relative expression level.

Figure 5. 

Volcano plot shows differentially expressed genes in AaHD8-RNAi. The red points indicate down-regulated genes, while the green ones indicate up-regulated genes. FC stands for fold change.

Figure 6. 

GUS expression (blue) in A. annua plants transformed with the 1391Z-proLTP1-GUS and 1391Z-proLTP2-GUS vector. (a), (b) Leaves of 1391Z-proLTP1-GUS plants. (c), (d) Leaves of 1391Z-proLTP2-GUS plants.

Figure 7. 

Expression levels of AaLTP1 in AaLTP1 over-expression plants measured by qRT-PCR. The average expression level of genes in WT was set as 1. Actin was used as internal control; the error bars show means ± SD from three technical repeats; Student's t-test ** p < 0.01, * p < 0.05.

Figure 8. 

Artemisinin contents in AaLTP1 over-expression lines were measured by HPLC. Error bars indicate ± SD of three biological replicates. ** p < 0.01, Student's t-test.

Figure 9. 

AaHD8 directly binds to the promoter of AaLTP1 and AaLTP2. (a), (b) Yeast one-hybrid assay of protein–DNA interaction. pB42AD is an empty vector used as a negative control. (c), (d) Probe and mutant probe. (e), (f) AaHD8 binds to the L1-box-like motif from AaLTP; 5×, 10×, and 20× indicate the dilution multiple of cold competitors relative to that of the labeled probe; mu: labeled mutated L1-probes were tested as negative controls; His-TF protein was used as a negative control.

Figure 10. 

Relative expression of AaHD8 and AaLTP in AaHD8 over-expression plants. The average expression level of genes in WT was set as 1. Actin was used as internal control; the error bars show means ± SD from three technical repeats; Student's t-test ** p < 0.01, * p < 0.05.