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.