Funding
This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA23080603) and National Natural Science Foundation of China (Grant No. 31701956 ). The funder has no role in the design of the study and collection, analysis, and interpretation of data, or preparation of the manuscript.
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Figure 1 . Phylogenetic tree of candidate gene. The tree was drawn using the Neighbor-Joining method in the MEGA 7 program and were further plotted by web based iTOL (https://itol.embl.de). (a) The candidate TPSs genes were clustered into TPS-b which highlighted by an arrow in red, and genes in white font are all from lavenders (b) The target gene LaCYP71D582 was clustered into CYP 71 clan which was indicated by an arrow in red; CYPs are selected from 5 clans related to terpene metabolism according to Nelson and Werck-Reichhart (2011). The sequences used to construct the tree can be found in table S2 for TPSs and table S3 for CYPs.
Figure 2.Quantitative real-time PCR (qRT-PCR) analysis of LaTPS7, LaTPS8 andLaCYP71D582 expression in glandular trichome from different developmental stage. Each organ containing glandular trichome on flower and glandular trichome on leaf. The highest expression of three genes was at period of bud in glandular trichome on flower. Transcript levels were normalized to 18S rRNA genes. Results represent the mean ± SE of three repetitions.
Figure 3.Subcellular localization of LaTPS7 , LaTPS8 , and LaCYP71D582 inN. benthamiana. Observation under Confocal Fluorescence of cells of tobacco after injection for three days. Both La TPS7 andLa TPS8 were associated with chloroplasts, and La CYP71D582 was located in the endoplasmic reticulum (ER). Auto, chlorophyll autofluorescence; eGFP , enhanced Green Fluorescent Protein channel image; Light, light microscopy image; Merged, merged image between Auto and eGFP . Post processing of pictures were completed by image J (https://imagej.nih.gov/ij).
Figure 4. La TPS7 and La TPS8 enzymatic activity in vitro. Products catalyzed by La TPS7 and La TPS8 were shown. La TPS7-GPP: 1.α- Pinene, 2. Camphene, 3. Myrcene, 4. Limonene, 5. Terpinolene, 6. Linalool, 7. Terpineol; La TPS7-NPP: 1. α- Pinene, 2. Camphene, 3. Limonene, 4. Terpinolene, 5. Terpineol, 6. Nerolidol;La TPS8-GPP: 1. α -Pinene, 2. β -Pinene, 3. Sylvestrene, 4. Linalool, 5. Fenchol, 6. Geraniol; La TPS8-NPP: 1.α- Pinene, 2. Limonene, 3. Terpinolene, 4. Terpineol, 5. Nerolidol.
Figure 5.Enzyme activity in vivo . (a) Volatiles tested on tobacco leaves transfected with LaTPS7 or LaTPS8 respectively showing that La TPS7 only produced limonene from tobacco and La TPS8 produced α- pinene and sylvestrene. (b) Volatiles test on tobacco leaves co-expressed withLaCYP71D582-LaTPS7or with LaCYP71D582-LaTPS8. It indicated that limonene can be converted into carveol by La CYP71D582. (c) Limonene was hydroxylated at C6 by La CYP71D582 to form carveol.
Figure 6. GUS staining showing the expression pattern of LaTPS7 and LaTPS8 . (a) Histochemical stain of Pro -LaTPS7. 1-3, leaves; 4, flower; 5, trichome; 6, silique. The red arrows shows the wound on leaves indicating a wound-induced expression pattern of LaTPS7 . (b) Tissue stain of Pro -LaCYP71D582 . 1, leaf; 2, flower; 3, trichome; 4, silique.
Figure 7 . Behavioral responses of aphids and ladybugs toward tobacco leaves expressing LaTPS7 and co-expressed LaTPS7 and LaCYP71D582 . The percentage of insects choosing wild type leaves (blue bars) versus transfected leaves (green bars) are shown, which indicates that limonene and carveol are able to repel aphids and attract ladybugs. 90 aphids and 60 ladybugs were texted in total. Asterisks indicate a significant difference of choice towards different odor source using χ2 for ladubugs and variance (ANOVA) for aphids (***P < 0.05).
Figure 8 . A summary of the relationship of tritrophic interaction among lavender-aphid-ladybug. Volatiles including limonene, α -pinene catalyzed by terpene synthases in bud stage are able to defend plants against attack from aphids. Sequentially, CYP converts the limonene to carveol with stronger attraction to ladybugs. These regulatory networks work together to protect plants against herbivores.