Linalool Activates Oxidative and Calcium Burst and CAM3-ACA8 Participates in Calcium Recovery in Arabidopsis Leaves

Plants produce linalool to respond to biotic stress, but the linalool-induced early signal remains unclear. In wild-type Arabidopsis, plant resistance to diamondback moth (Plutella xylostella) increased more strongly in a linalool-treated group than in an untreated control group. H2O2 and Ca2+, two important early signals that participated in biotic stress, burst after being treated with linalool in Arabidopsis mesophyll cells. Linalool treatment increased H2O2 and intracellular calcium concentrations in mesophyll cells, observed using a confocal microscope with laser scanning, and H2O2 signaling functions upstream of Ca2+ signaling by using inhibitors and mutants. Ca2+ efflux was detected using non-invasive micro-test technology (NMT), and Ca2+ efflux was also inhibited by NADPH oxidase inhibitor DPI (diphenyleneiodonium chloride) and in cells of the NADPH oxidase mutant rbohd. To restore intracellular calcium levels, Ca2+-ATPase was activated, and calmodulin 3 (CAM3) participated in Ca2+-ATPase activation. This result is consistent with the interaction between CAM7 and Ca2+-ATPase isoform 8 (ACA8). In addition, a yeast two-hybrid assay, firefly luciferase complementation imaging assay, and an in vitro pulldown assay showed that CAM3 interacts with the N-terminus of ACA8, and qRT-PCR showed that some JA-related genes and defense genes expressions were enhanced when treated with linalool in Arabidopsis leaves. This study reveals that linalool enhances H2O2 and intracellular calcium concentrations in Arabidopsis mesophyll cells; CAM3-ACA8 reduces intracellular calcium concentrations, allowing cells to resume their resting state. Additionally, JA-related genes and defense genes’ expression may enhance plants’ defense when treated with linalool.


Introduction
During plant defense, calcium functions as a crucial early signal. When plants perceive external signals of a pathogen attack, such as oral elicitors from insects or bacterial flagella, Ca 2+ ions immediately flow into the cytoplasm from extracellular calcium stores (in the interstitial spaces between the cell wall and plasmids or between cell walls) or intracellular calcium stores (in the vacuole, Golgi apparatus, or endoplasmic reticulum) [1,2]. Once Ca 2+ has completed its function as a second messenger, it is returned to the calcium stores via Ca 2+ -ATPases and the Ca 2+ /proton antiporter system [3]. H 2 O 2 , a reactive oxygen species (ROS) that also participates in many defense mechanisms of plants, has a close relationship with Ca 2+ . Ca 2+ and H 2 O 2 signals can be induced by exogenous polyamine and jasmonic acid (JA) treatment [4] and are also elicited by herbivore feeding [5]. Thus, Ca 2+ and H 2 O 2 participate in many resistance mechanisms in plants. 20 µM. In addition, the fresh/dry weight ratio can also reflect the growth status of plants. We detected the fresh/day weight in the WT control group (use ethyl alcohol) and WT linalool group (20 µM linalool final fumigation concentration). Additionally, there was no significant difference between the two groups ( Figure 1b). The two experiments' results indicated that the 20 µM linalool final fumigation concentration did not affect the growth of Arabidopsis plants and could be used for subsequent experiments.
groups. The Fv/Fm value increased slightly in the lowest linalool concentration group, the 10 μM linalool group (about 0.57), when compared with that in the 0 μM control group (about 0.47). Additionally, the 20 μM linalool group (about 0.47) and 50 μM linalool group (about 0.45) were closer to the 0 μM control group. Thus, we selected the second linalool concentration, 20 μM. In addition, the fresh/dry weight ratio can also reflect the growth status of plants. We detected the fresh/day weight in the WT control group (use ethyl alcohol) and WT linalool group (20 μM linalool final fumigation concentration). Additionally, there was no significant difference between the two groups ( Figure 1b). The two experiments' results indicated that the 20 μM linalool final fumigation concentration did not affect the growth of Arabidopsis plants and could be used for subsequent experiments.  Error bars denote ± SEM, n = 6~8, p < 0.05, Dunnett's C (variance not neat). (b) The ratio of fresh weight to dry weight. In the WT control group, Arabidopsis plants were treated with ethyl alcohol, and in the WT linalool group, Arabidopsis plants were treated with linalool at 20 µM final concentration. Error bars denote ± SEM (standard error of mean), n = 6~8, p < 0.05, Student's t-test. (c,d) Larval body length and weight gain were measured 7 days after inoculation. Every pot had 3 Arabidopsis plants, 3 larvae were put in each plant, and each group (WT control and WT + linalool) contained approximately 25-40 larvae. Error bars denote ± SEM, n ≥ 25, and columns labeled with different letters are significantly different at p < 0.05, Student's t-test. (e) Larvae phenotypes of the WT control (left) and WT + linalool (right). (f) Conditions of plants after 7 days of larval feeding. The plants were grown for 4 weeks (10 leaves). The scale bar represents 1 cm.
To confirm that linalool induces resistance to P. xylostella in Arabidopsis, we inoculated WT plants with P. xylostella larvae after 24 h of linalool treatment; the control group was treated with ethyl alcohol. After 7 days, we measured the length and weight of P. xylostella larvae. Both growth indicators were significantly lower in linalool-treated plants than in control plants (Figure 1c-e). In addition, the plant leaf damage was also more severe in the control group than in the linalool group (Figure 1f). These results indicate that linalool induces Arabidopsis plant defense against P. xylostella. Early signals are the first response of plants to external stimuli [24]. To further investigate the early signals, we conducted the following experiments.

Linalool Increases H 2 O 2 Levels in Arabidopsis Mesophyll Cells
H 2 O 2 functions as an early signal in various signal transduction processes. We tested the variation in H 2 O 2 levels in Arabidopsis mesophyll cells by performing confocal laser scanning microscopy of mesophyll cells from linalool-treated versus untreated control plants. H 2 O 2 levels were strongly increased in mesophyll cells from linalool-treated WT plants compared to the control. Following linalool treatment, we photographed the plants every 3 min for 15 min (Figure 2a). High levels (increase about 50% above control) of H 2 O 2 were maintained in the WT linalool group throughout the 15 min period (Figure 2b). In contrast, in linalool-treated mesophyll cells pretreated with the NADPH oxidase inhibitor DPI (diphenyleneiodonium chloride) and in cells of the NADPH oxidase mutant rbohd, H 2 O 2 enhanced levels (about 20% above control) were lower than WT linalool group after linalool treatment (Figure 2b). H 2 O 2 levels (about 30-45% above control) in linalool-treated WT cells pretreated with ruthenium red (a Ca 2+ channel inhibitor that blocks the release of internal Ca 2+ ) [25,26] and tpc1 cells were similar to those of the WT linalool group (Figure 2b; data from controls are shown in Figure S1). These results indicate that RBOHD functions in linalool signal transduction and that changes in calcium concentrations have little effect on H 2 O 2 levels in mesophyll cells.

Linalool Increases Intracellular Calcium Levels in Arabidopsis Mesophyll Cells and Functions Downstream of H 2 O 2 Signaling Burst
To investigate the changes in intracellular calcium concentrations, we performed confocal laser scanning microscopy to detect Ca 2+ fluorescence. Ca 2+ fluorescence was higher in the WT linalool group versus the other groups from 30 s to 120 s (Figure 3a; data for the control groups are shown in Figure S2). We then calculated the increase in Ca 2+ fluorescence. When WT Arabidopsis mesophyll cells were treated with linalool, intracellular Ca 2+ fluorescence rapidly increased for 30 s (about 17% above control), remained at a similar level at 60 s, and then gradually decreased to the initial level ( Figure 3b). However, in ruthenium-red-treated WT cells, tpc1 cells, DPI-treated WT cells, and rbohd cells, only slight increases (about 8-10% above control) in calcium concentrations were detected during this period in response to linalool treatment (Figure 3a    To further explore the changes in calcium flux in Arabidopsis mesophyll cells, we detected calcium flux by NMT. The test period was divided into pre-linalool treatment (pre), the linalool-responsive peak (peak), and the post-linalool response (post) based on To further explore the changes in calcium flux in Arabidopsis mesophyll cells, we detected calcium flux by NMT. The test period was divided into pre-linalool treatment (pre), the linalool-responsive peak (peak), and the post-linalool response (post) based on the transient processing of Ca 2+ ions by linalool. The pre-period lasted for 2 min, and the peak period began in the second minute. In WT mesophyll cells, Ca 2+ flux peaked at 281 pmol cm −2 s −1 and then underwent a steady decline. By 2.5 min, Ca 2+ flux was stable and entered the post-period. During both the pre-and post-periods, Ca 2+ flux was approximately 58 pmol cm −2 s −1 (Figure 4a). When WT plants were pretreated with ruthenium red, the peak (104 pmol cm −2 s −1 ) was significantly reduced following linalool treatment compared to that in WT plants treated with linalool but not pretreated with ruthenium red. The peak of the tpc1 mutant (166 pmol cm −2 s −1 ) showed the same degree of decline as that of the WT ruthenium red group (Figure 4b). In both DPI-pretreated (102 pmol cm −2 s −1 ) and rbohd (73 pmol cm −2 s −1 ) mesophyll cells, the peak following linalool treatment was reduced to a level similar to that of the WT ruthenium red and tpc1 groups (Figure 4c). To further test the differences among these groups, we calculated the mean Ca 2+ flux, finding that the peak was significantly higher in the WT linalool group than in the other groups. The mean Ca 2+ flux was calculated and compared amongst experimental treatments to test the statistical differences among these groups (Figure 4e). The results showed that the mean Ca 2+ fluxes were reduced in the ruthenium red pretreated group, tpc1 mutant group, DPI-pretreated group, and rbohd mutant group. The result of Ca 2+ flux is consistent with that of calcium concentrations.

CAM3 Interacts with ACA8 to Transport Calcium Ions Outside of Mesophyll Cells
After the calcium signal is transferred, the calcium must be transported into extracellular or intracellular calcium stores. Since the overall transmembrane calcium flow rate of the cell population at the monitoring site where the electrode monitored by NMT is located is efflux, we speculated that Ca 2+ -ATPase might play an important role in this process. We pretreated WT leaves with the Ca 2+ -ATPase inhibitor Eosin B and examined calcium flux. The peak of calcium flux was lower in the Eosin B-pretreated WT + linalool group (about 109 pmol cm −2 s −1 ) than in the WT + linalool group (about 281 pmol cm −2 s −1 ) (Figure 4d), indicating that Ca 2+ -ATPase functions in calcium transport. ACA8, a Ca 2+ -ATPase, is located in the plasma membrane. Additionally, CAM7 was previously shown to interact with ACA8 [12]. Because CAM3 and CAM7 are CAM family members that share high sequence similarity (up to 98.9%), the peak of calcium flux was also about 100 pmol cm −2 s −1 in the cam3-1 + linalool group and about 19 pmol cm −2 s −1 in the cam3-2 + linalool group (Figure 4d). We predicted that CAM3 is also a binding partner of ACA8. To investigate this hypothesis, we conducted Y2H, LCI, and in vitro pulldown assays.
Since ACA8 is a transmembrane protein, and CAM7 interacts with the N-terminus of ACA8, we used the N-terminus (residues 1-180) of ACA8 as bait and CAM3 as the prey protein, finding that the competent yeast strain AH109 cells with co-transformed plasmid pairs CAM3-AD plus ACA8 (residues 1-180)-BK grew on SD/−Leu/−Trp and SD/−Ade/−His/−Leu/−Trp and grew blue colonies on SD/−Ade/−His/−Leu/−Trp with X-α-gal. This result indicated that CAM3 binds to the N-terminus of ACA8 in the Y2H assay ( Figure 5a). CAM3 also interacted with the N-terminus of ACA8 in a pulldown assay. In the anti-HIS pulldown line, CAM3-HIS was not detected in CAM3-HIS with GST group, but in CAM3-HIS with ACA8-GST group, CAM3-HIS was detected by immunoblot analysis using anti-HIS antibodies (Figure 5b). In LCI assay, N. benthamiana leaf parts injected with CAM3-Cluc/ACA8-Nluc showed stronger fluorescence than the other three (control) leaf parts (Cluc/Nluc, CAM3-Cluc/Nluc, and Cluc/ACA8-Nluc) (Figure 5c). These results demonstrate that CAM3 interacts with ACA8. In addition, we examined calcium flux in the cam3 mutant after linalool treatment and found that the peak flux was lower in cam3 than in WT plants following linalool treatment (Figure 4d). These results provide further evidence that CAM3 interacts with ACA8 to participate in calcium efflux.    In the Y2H and in vitro pulldown assays, because ACA8 is a membrane protein, the N-terminus (residues 1-180), an intracellular domain, was used to interact with CAM3.
(c) N. benthamiana leaves were divided into four parts: the bottom right part was an experimental group, and the others were control groups. Red represents the maximum fluorescence intensity.

Linalool Activates JA-Related Gene Expression
JA is an important hormone that functions in plant defense. To further explore the roles of JA in linalool-induced signaling, we measured the expression of important genes related to JA pathways, including LOX6, MYC2, JAZ4, and JAZ8, in WT plants following linalool treatment for 5 min, 0.5 h, and 2 h. In addition, we measured the expression of defense genes PDF1. 2 and VSP2 (about 5-fold) gene expression was at 0.5 h (Figure 6g,h). Overall, these results indicate that linalool can induce JA-related gene and defense gene expression. In the Y2H and in vitro pulldown assays, because ACA8 is a membrane protein, the N-terminus (residues 1-180), an intracellular domain, was used to interact with CAM3. (c) N. benthamiana leaves were divided into four parts: the bottom right part was an experimental group, and the others were control groups. Red represents the maximum fluorescence intensity.

Linalool Activates JA-Related Gene Expression
JA is an important hormone that functions in plant defense. To further explore the roles of JA in linalool-induced signaling, we measured the expression of important genes related to JA pathways, including LOX6, MYC2, JAZ4, and JAZ8, in WT plants following linalool treatment for 5 min, 0.5 h, and 2 h. In addition, we measured the expression of defense genes PDF1.2, THI2.1, VSP1, and VSP2 in WT plants following linalool treatment for 0.5 h, 2 h, and 8 h. Almost all genes are expressed in the WT (Figure 6

Discussion
In this study, we demonstrated that linalool, a volatile released from plants, enhances H 2 O 2 and intracellular calcium concentrations in Arabidopsis mesophyll cells; CAM3-ACA8 reduces intracellular calcium concentrations, allowing cells to resume their calcium resting state. In addition, the observation that linalool treatment enhances plant defense may be due to JA-related genes' expression and defense genes' expression.
When Arabidopsis recognizes linalool, H 2 O 2 levels in leaf mesophyll cells rapidly increase. We found that this H 2 O 2 burst was inhibited in the leaves of WT plants pretreated with the NADPH oxidase inhibitor DPI and in leaves of the NADPH oxidase mutant rbohd (Figure 2). Similarly, linalool treatment resulted in rapid increases in the intracellular Ca 2+ concentration and Ca 2+ flux, and both of these effects were inhibited in DPI-treated and rbohd mesophyll cells (Figures 3 and 4). Increases in intracellular Ca 2+ concentrations and Ca 2+ flux were also inhibited when we used the intracellular calcium store inhibitor ruthenium red and in tpc1, a mutant of TPC1 (a depolarization-activated Ca 2+ channel located in the vacuolar membrane). Therefore, we measured H 2 O 2 levels in ruthenium-redpretreated wild-type and tpc1 leaves following linalool treatment. The H 2 O 2 burst was not inhibited in these cells, indicating that the linalool-induced H 2 O 2 burst occurs before the increase of Ca 2+ levels in Arabidopsis leaf mesophyll cells. (E)-2-hexenal can also increase the Ca 2+ concentration in leaves of Arabidopsis thaliana. When leaves were pretreated with ruthenium red, calcium ions could not be induced by (E)-2-hexenal, suggesting that intracellular calcium store is involved in the increase in the (E)-2-hexenal-induced intracellular Ca 2+ concentration [27].
Ca 2+ and H 2 O 2 function together in two processes in plants: Ca 2+ -induced ROS production and ROS-induced Ca 2+ release [6]. Here, we demonstrated that linalool-induced Ca 2+ and H 2 O 2 signaling function in ROS-induced Ca 2+ release. The recognition of different stimuli occurs via different processes during early signaling, making the sequence of early signaling events a special 'language' in plant resistance. TPC1 activation is regulated by many factors. Ca 2+ binding to the TPC1 cytosolic EF-hand domain triggers conformational changes to activate TPC1 [28]. Additionally, ABA, ATP, cAMP, and Ca 2+ treatment increase vacuolar Ca 2+ release, while vacuolar Ca 2+ efflux was strongly suppressed by H 2 O 2 in an earlier study [29]. In our study, linalool-induced H 2 O 2 burst is upstream of the enhancement of calcium concentration in Arabidopsis mesophyll cells. Therefore, there may be other ways to slightly increase intracellular calcium concentration in the cytoplasm before linalool-induced calcium is released from the intracellular calcium store, thereby activating TPC1 and releasing large amounts of calcium from the intracellular calcium store into the cytoplasm. At the same time, a more detailed study of how TPC1 is activated with linalool treatment should be undertaken in a separate investigation.
Ca 2+ plays an important role in many signaling transduction processes, but high concentrations of calcium in the cytoplasm are toxic to cells. After calcium has functioned as a second messenger, it is transported into calcium stores. The P-type ATPase, Ca 2+ -ATPase, located in the plasma membrane and the intracellular calcium store membrane, participates in the process of calcium efflux. Studies have shown that only II B Ca 2+ -ATPase can bind to CAMs. When the N-terminal of Ca 2+ -ATPase binds to Ca 2+ , CAM also binds to the N-terminal CAM binding site [30]. By examining Ca 2+ flux, we showed that linalooltreated mesophyll cells exhibit strong Ca 2+ efflux. Therefore, we investigated whether the Ca 2+ -ATPase inhibitor Eosin B would inhibit this Ca 2+ efflux. Our findings indicate that Ca 2+ -ATPase participates in linalool-induced Ca 2+ efflux in mesophyll cells and that Ca 2+ efflux is inhibited in cam3 plants (Figure 4d). Since CAM7 interacts with ACA8 [12], CAM3 and CAM7 share high sequence similarity (up to 98.9%), so we suggested that CAM3 may also interact with ACA8. Y2H, LCI, and in vitro pulldown assays indicated that CAM3 binds to the N-terminus of ACA8 and functions in the Ca 2+ efflux process (Figures 4d and 5).
Early signals are the beginning of a series of responses that begin in plants when they feel external stimulations [24]. Plants that receive VOCs influence gene regulation, metabolism, phenotype, responses to stress, and behavioral choices [31]. In our study, linalool-treated plants can be seen as 'receivers' that change in gene regulation and responses to stress (linalool-treated plants are more resistant to insects) to improve the ability of plants to cope with biotic stress. In WT plants treated with linalool, the expression of almost all genes related to the JA pathway and defense genes increased compared to those in the untreated control ( Figure 6). These results indicate that the linalool-induced upregulation of JA-related genes and defense genes made plants in a more stress-resistant state to enhance plant defense.
Notably, changes in intracellular Ca 2+ concentrations regulate the JA biosynthesis pathway. When plants are wounded by insects, CML37 and CML42 influence the JA biosynthesis pathway [32,33]. Further studies could uncover the Ca 2+ sensors involved in linalool-induced plant defense. In addition, much remains to be learned about the messages encoded in different VOCs. This knowledge could lay the foundation for the strategies plants use to defend themselves against insects.

Plant Materials and Culture Conditions
Wild-type (Col-0) Arabidopsis, rbohd CS9555 (AT5G47910), tpc1 SALK_125658 (AT4G03560), cam3-1 SALK_149754C (AT3G56800), and cam3-2 SALK_042391C (AT3G56800) mutants were used in this study. The seeds were vernalized at 4 • C for 2 days in the dark. After vernalization, the seeds were surface-sterilized for 4 min in 75% ethyl alcohol, washed four times in sterile water, sown in the autoclaved soil mixture, and placed in an incubator (Percival model: I-36vl). Plants were grown in soil at 21-23 • C and 70% relative humidity with a light intensity of 80-110 µmol m −2 s −1 under long-day (16 h light/8 h dark) conditions. The plants used for insect inoculation were grown for 4 weeks, and the plants used in the other experiments were grown for 2 weeks before treatment.

Plutella xylostella Egg Hatching and Inoculation
Plutella xylostella (diamondback moth) eggs were hatched in an incubator at 25 ± 1 • C with a relative humidity of 50 ± 10%. After 1-2 days, larvae that were identical in length and weight were chosen for inoculation onto Arabidopsis plants; each plant was inoculated with three larvae. WT control and WT linalool-treated plants were prepared for analysis. Seven days later, the insects were removed, and their lengths and weights were measured. Each group contained 25-40 insects.

Chlorophyll Fluorescence Measurement
WT control and WT linalool-treated plants were used to measure leaf chlorophyll fluorescence. After 0.5 h dark adaption, the maximum photochemical efficiency Fv/Fm was of PSII, obtained with a modulated fluorometer IMAGING PAM (Zealquest Scientific Technology Co., Ltd., Shanghai, China). Every group contained 6-8 Arabidopsis plants.

Linalool Fumigation
Arabidopsis plants were treated with linalool (≥97%, a mixture of the two enantiomers of linalool, purchased from Sigma-Aldrich, Shanghai, China, CAS NO.: 78-70-6) via fumigation in glass bell jars (height: 12.5 cm; diameter: 15 cm) in insect growth test. Cotton balls 1 cm in diameter were soaked in linalool dissolved in ethyl alcohol (in insect inoculation and qRT-PCR experiments; linalool's final concentration was 20 µM) and hung in the bell jars; cotton balls soaked in the same volume of ethyl alcohol were used for the control. After adding the cotton balls, the bell jars were immediately sealed with Vaseline petroleum jelly.

Fresh/Dry Weight Measurements
Arabidopsis plants treated with ethyl alcohol and linalool (linalool's final concentration was 20 µM) were used to measure fresh weight. Additionally, the samples were then dried in an oven at 80 • C for 24 h to constant weight and weighed as dry weight. Every group contained 6-8 Arabidopsis plants.
The Ca 2+ concentration in the mesophyll cells was measured near the cells and 30 µm away from the cells at 0.2 Hz. Each leaf was tested for 2 min before linalool treatment (pre-linalool treatment; pre). After linalool was quickly added to the test solution to a final concentration of 100 µM, data were collected for approximately 2.5 min as the linalool response peak (peak) group. Data were then collected from the post-linalool response (post) group to indicate the end of the reaction. The final flux values are reported as the mean of eight individual plants per treatment. The Ca 2+ flux was calculated as: where J is the flux of Ca 2+ (pmol cm −2 s −1 ), D is the diffusion coefficient (0.79 × 10 −5 cm 2 s −1 ), ∆C is the difference between the concentrations near and far from the cells, and ∆X is 30 µm. Each group contained 6-8 replicates.

Ca 2+ Fluorescence Measurements in Mesophyll Cells
Before treatment with Fluo3-AM (10 µM), Arabidopsis leaves were soaked in a test solution (the same solution used for the Ca 2+ flux experiment). The leaves were then soaked in Fluo3-AM solution to label Ca 2+ ions for 1 h in the dark in an incubator. The leaves were washed and placed in the fresh test solution. Ca 2+ fluorescence was detected under a confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems, Wetzlar, Germany). The excitation wavelength was 488 nm, and the emission wavelength was 530 nm. Before linalool (a final concentration of 100 µM) addition (0 min), the basal level of Ca 2+ fluorescence in mesophyll cells was measured. Following the addition of linalool, fluorescence was measured every 30 sec for 150 s. Each group contained 20-40 cells.
The increase in Ca 2+ fluorescence = (X − a)/a × 100%, where X is the fluorescence of linalool-treated samples, and a is fluorescence at 0 s. The value for each group was calculated separately.

In Vitro Pulldown Assay
The genes encoding ACA8 (residues 1-180) were cloned into pGEX-4T-1, and the genes encoding CAM3 were cloned into pET28A. ACA8-GST and CAM3-HIS were transformed into E. coli Rosetta (DE3) cells for protein expression. Protein-GST was used as the bait protein. Glutathione beads containing 50 µg ACA8-GST or GST were incubated with 50 µg CAM3-HIS in pulldown buffer (1% NP40, 150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, pH 7.5) at 4 • C for 2 h, respectively. The protein beads complexes were washed with pulldown buffer, then centrifuged 500× g for 5 min, the supernatant was removed, and the whole process was repeated 4 times; at last, the SDS sample buffer was boiled for 5-10 min. The bindings of CAM3-HIS with ACA8-GST were detected by immunoblot analysis using anti-GST and anti-HIS antibodies.
Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA), and the 2 −∆∆Ct method was used to calculate relative gene expression levels [39].

Statistical Analysis
Student's t-test and Dunnett's C (variance not neat) at the level of p < 0.05 was significant. Error bars denote ± standard error of mean (SEM).

Conclusions
In this study, we revealed the early signaling process induced by linalool. H 2 O 2 burst mainly because of NADPH oxidase RBOHD, then calcium ions were released into the cytoplasm through intracellular calcium stores, and the excess calcium ions in the cytoplasm were pumped out through the interaction between CAM3 and ACA8. In addition, linalool may enhance insect resistance by activating the JA pathway in Arabidopsis plants, which requires further study.