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. 2025 Dec 1;15:42959. doi: 10.1038/s41598-025-26633-6

Methyl hexenoate and linalool emitted by hot peppers repel western flower thrips (Frankliniella occidentalis) and attract their predator (Orius laevigatus)

Mojtaba Esmaeily 1, Akhtar Ayoobi 1, Gahyeon Jin 1, Eeshita Mandal 1, Niayesh Shahmohammadi 1, Chulyoung Kim 1, Keono Kim 2, Hyunje Park 1, Falguni Khan 1, Daehong Lee 3, Hee-Jin Kim 1, Eunhye Ham 4, Hyong Woo Choi 1, Yongho Jeon 1, Jeehye Sung 2, Yonggyun Kim 1,✉
PMCID: PMC12673103  PMID: 41326439

Abstract

A plant emits diverse volatile compounds and communicates with other organisms at different trophic levels. Hot pepper, Capsicum annuum, leaves produced different compositions of 41 organic compounds upon an infestation by the western flower thrips, Frankliniella occidentalis. These compounds induced a non-preference behavior to the thrips while they attracted a natural enemy, Orius laevigatus. Among the differentially emitted compounds, Z-3-methyl hexenoate (MeH) and linalool repelled the herbivore but attracted the predator. MeH or linalool biosynthesis was suppressed by virus-induced gene silencing of hydroperoxide lyase (HPL) or linalool synthase (LS) expression in the hot pepper and led to significant malfunction in the tri-trophic communication. This communication was mediated by jasmonic acid (JA), but not salicylic acid, signal in the hot pepper. Plant growth-promoting rhizobacteria also induced the antixenosis of hot peppers through JA signaling. Hot peppers resistant against the thrips were positively correlated with HPL or LS expression levels.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-26633-6.

Subject terms: Ecology, Ecology, Plant sciences, Zoology

Introduction

The hot pepper, Capsicum annuum (L), is cultivated worldwide as an indispensable condiment for various foods because of its typical color, pungency, taste, and distinct aroma1. In Korea, nearly 80 different plant pathogens and insect pests have been identified as causing economic damage to hot pepper cultivation2. Among these, the western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is one of the most severe insect pests. It transmits the tomato spotted wilt virus (TSWV) to the hot peppers and also directly causes feeding damage3. Several chemical insecticides including spinosad have been effective in killing thrips4. However, the cryptic behavior of thrips between leaves and flowers enables them to avoid exposure to sprayed insecticides5. This challenge has led to an increase in the frequency and dosage of insecticide spraying, which may select for thrips resistant to chemical insecticides. Consequently, this facilitates the rapid buildup of insecticide resistance in thrips. For example, frequent spraying of spinosad has selected for point-mutated individuals in the acetylcholine receptor gene, which become over 3,000-fold more tolerant to spinosad compared to the susceptible strain6. To effectively control this insect pest, alternative control measures should be developed. As an alternative, a biological control strategy has been designed and implemented to control F. occidentalis using seasonal inoculative releases of a predatory bug, Orius laevigatus (Fieber) (Hemiptera: Anthocoridae)7. However, to successfully control the thrips, the biological control method occasionally needs supplementary applications of chemical insecticides, where the insecticide-resistant O. laevigatus would be optimal to minimize toxicant damage8,9.

Plant breeding resistant to insect pests has been regarded as an alternative insect pest control strategy because it minimizes chemical spraying and the associated labor, ideally reducing hazards to humans and the environment10. The insect resistance includes antixenosis, which deters herbivores by causing their non-preference to the plants; antibiosis, which delivers direct toxicants to herbivores after feeding; and tolerance of the plants to herbivore damage. Indeed, this host plant resistance against thrips has successfully been applied to prevent TSWV infection in peanuts, Arachis hypogaea, exhibiting both antixenosis and antibiosis11. Alternatively, TSWV reduces the host plant resistance to increase the accessibility of its vector thrips to the host plants for viral transmission12. In addition to direct insect resistance of host plants, a tri-trophic interaction can be exploited to recruit natural enemies to attack herbivores by releasing plant odors known as volatile organic compounds (VOCs) upon herbivore damage13. This chemical communication led us to hypothesize that hot peppers at the primary trophic level infested by F. occidentalis at the secondary trophic level release VOCs to repel thrips and attract the natural enemy, O. laevigatus, at the tertiary trophic level.

This study aimed to determine the compounds modulating the behaviors of F. occidentalis and O. laevigatus in response to thrips-infested hot peppers. Differential behaviors between the herbivore and the predator were elucidated by analyzing volatile compounds derived from the host plant. The influences of salicylic acid (SA) and jasmonic acid (JA) on inducing insect behaviors and the volatile compound production were evaluated based on plant defense signaling pathways. The functional VOCs associated with JA signal were tested by silencing gene expressions related to VOC biosynthesis through virus-induced gene silencing (VIGS) followed by behavioral assays. Finally, the study established a correlation between the levels of VOC-biosynthetic gene expression and hot pepper resistance to F. occidentalis.

Results

Damaged hot pepper repels herbivore but attracts predator through JA signaling

When the hot peppers were infested by F. occidentalis, the peppers became significantly less preferred than uninfested control plants (Fig. 1A). Conversely, the damaged plants were favored by their predator, O. laevigatus. These contrasting behaviors were time-dependent (Fig. 1B). Repellency against F. occidentalis was rapidly induced, reaching maximum levels 6 h post-infestation. Simultaneously, attraction towards O. laevigatus was also induced.

Fig. 1.

Fig. 1

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Modifications in host preference behaviors of F. occidentalis (‘Fo’) and O. laevigatus (‘Ol’) towards hot peppers following thrips damage. (A) Choice test comparison between damaged and undamaged hot peppers using a tunnel assay. (B) Contrasting responses of prey (= Fo) and predator (= Ol) to damaged hot peppers over time, analyzed using the tunnel assay. (C) Effects of methyl jasmonic acid (‘Me-JA’, 10 µM) and salicylic acid (‘SA’, 10 µM) on these behaviors, evaluated with a Y-tube choice test. (D) Impact of Me-JA treatment on hot peppers regarding these behaviors, exploring different times and concentrations using a Y-tube test. Each Y-tube test trial involved 20 adults of either F. occidentalis or O. laevigatus. Each trial in the tunnel assay used 50 adults of F. occidentalis or O. laevigatus. Every experiment was replicated three times. An asterisk (*) denotes a significant difference compared to the control at Type I error = 0.05 (t-test), while ‘ns’ indicates no significant difference. Attraction or repellency was calculated by percentage of the migrated individuals to or against the treatment, respectively, among a total of 20 for Y-tube test or a total of 50 for tunnel assay. ‘NR’ stands for no response in the choice tests. Different letters above standard error bars signify significant differences among means at Type I error = 0.05 (LSD test).

The defense system of hot peppers utilizing JA signaling was implicated to explain the behavioral changes of the thrips and their predators, as JA plays a critical role in plant defense against herbivore infestation14. Its impact on tri-trophic interactions was assessed in hot peppers by examining effects from JA or SA signaling pathways (Fig. 1C). Methyl JA (Me-JA) treatment on undamaged hot peppers repelled thrips while attracting the predator. However, SA treatment did not trigger these behaviors. Me-JA treatment prompted behavioral modifications in the thrips and its predator within a day, and the induction was dose-dependent (Fig. 1D). These results indicate that the thrips damage on the hot peppers triggers the JA signal to repel the thrips and attract its predator.

RNA-Seq analysis confirms that JA signaling is induced in hot peppers infested by F. occidentalis

To investigate the response of hot peppers to infestation by F. occidentalis, damaged and undamaged plants were compared in JA signaling using their comparative transcriptome analysis (Fig. 2). With the exception of one replication (‘R1’) in the ‘Damage’ treatment, more than 6.1 Gb of nucleotides were sequenced in each replication, and over 23,735 genes were predicted (Fig. 2A). RNA-Seq data from R1 of the ‘Damage’ treatment were excluded from further transcriptome analysis due to inadequate gene prediction. Using the predicted genes, their expression profiles were analyzed, revealing two distinct clusters between the damaged and undamaged hot peppers (Fig. 2B). Most (96.0%) of the genes were expressed in both treatments (Fig. 2C). Among these commonly expressed genes, 4,072 were differentially expressed (= DEG) by more than two-fold following thrips infestation (Fig. 2D). The up-regulated DEGs (= 2,584) due to thrips damage were further categorized by the number of fold changes, with 155 genes demonstrating more than 20-fold changes in their expression levels after thrips infestation (Fig. 2E). The highly expressed genes were functionally categorized into different stress responses including JA signaling and terpenoids (Fig. 2F). DEGs (= 63 genes) that exhibited over 40-fold changes are listed in Table S1. Notably, JA biosynthesis and signaling pathways are illustrated in Fig. S3A, where genes identified in the RNA-Seq analysis contributed to the synthetic and signaling components. Certain JA-associated genes were up-regulated in their expression levels following thrips infestation (Fig. 2G). Notably, the highly down-regulated DEGs include the growth-associated signal components mediated by auxin (Table S2). These DEG analyses suggest that thrips infestation induced terpenoid biosynthesis and JA signal in the hot peppers.

Fig. 2.

Fig. 2

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Up-regulation of jasmonic acid (‘JA’) signaling in hot peppers following infestation by F. occidentalis. (A) RNA-Seq summary for hot peppers that were either damaged or undamaged. Each plant, at the six-leaf stage, was exposed to 50 L2 larvae for 8 h. Three replicates (‘R1-R3’) were performed for each of two conditions (‘Undamaged’ and ‘Damaged’). (B) A heatmap analysis showing the differential transcriptomes between the two conditions across replicates. (C) Comparison of 22,801 gene contigs distributed between the two conditions in hot peppers. (D) Identification of differentially expressed genes (DEGs) in hot peppers in response to thrips infestation. DEGs were selected based on RPKM fold changes ≥ 2 among common contigs. (E) Distribution of up-regulated DEGs (= 2,584) classified by fold-changes. (F) Functional categorization of highly upregulated DEGs (= 155), focusing on plant defense signaling pathways using the KEGG mapper (https://www.genome.jp/kegg/mapper/search.html) and bioDBnet (Biological Database Network, https://biodbnetabcc.ncifcrf.gov/db/db2dbRes.php) ID conversion tool. (G) A heatmap illustrating the enhanced JA signal components in hot peppers following thrips infestation. Asterisks (*) denote a significant difference at a Type I error rate of 0.05 (t-test) between the two conditions. ‘ns’ indicates no statistical difference. The right-hand bar shows expression levels, indicated by varying color intensities.

Plant-growth promoting rhizobacteria (PGPR) induce the antixenosis of hot peppers through JA signaling

Several PGPRs are known to enhance JA signaling in hot peppers by inducing the production of VOCs to deter herbivores15. To demonstrate the role of PGPR-mediated JA signaling in modulating insect behaviors, applications of five different PGPRs PGPR were administered to hot peppers because several PGPRs are known to enhance JA signaling in hot peppers, thereby inducing the production of VOCs to deter herbivores14. Of the five PGPRs tested, two (‘G-2273’ and ‘AK-0’) effectively repelled F. occidentalis from the treated hot peppers (Fig. S1A). This repellency occurred 12 h post-treatment with the PGPRs (Fig. S1B). The two PGPR treatments significantly (P < 0.05) attracted the predator, O. laevigatus (Fig. S1C). These PGPR treatments up-regulated genes associated with JA biosynthesis such as 13-lipoxygenase (13-LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC) (Fig. S1D). The expression profiles of hot peppers treated with the effective PGPRs (‘AK-0’ and ‘G-2273’) differed from those treated with the ineffective PGPR (‘G-2311’) and the control (‘TSB’) according to clustering analysis. These results further strengthen the importance of JA signal in its mediation of the tri-trophic interaction among the hot pepper, thrips, and its predator.

Functional compounds produced by the hot peppers infested by F. occidentalis

To identify the VOC(s) responsible for the tri-trophic interaction, the organic compounds of the hot peppers were collected using the headspace-solid phase microextraction (HS-SPME) technique (Fig. S2A). A total of 41 compounds in the ground up leaf tissue obtained were identified from both damaged and undamaged plants, with differential detection across the two treatments (Fig. S2B, Supplementary file #1). Over 70% of the compounds were commonly found in the leaves (or produced by the leaves) across both treatments (Fig. 3A). Eight compounds uniquely present in the leaves from the damaged plants included green leaf volatiles (GLVs), terpenoids, and others (Fig. 3B).

Fig. 3.

Fig. 3

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Emission of green leaf volatiles (GLVs) from hot peppers infested by F. occidentalis and how they influence the behavior of thrips and the predator, O. laevigatus, towards the plants. (A) Volatile organic compounds (VOCs = 41) were collected from both damaged and undamaged hot peppers by F. occidentalis using HS-SPME (Fig. S1B, Supplementary file #1). (B) Categorization of the 8 VOCs specific to damaged hot peppers into three distinct chemical groups. (C) A Y-tube choice test involved thrips and predators choosing between hot peppers treated with the solvent (‘Hexane’) and methyl hexenoate (‘MeH’). For each Y-tube test, 20 adult specimens of F. occidentalis or O. laevigatus were utilized. All experiments were replicated three times. (D) Heatmap showing expression analyses of six genes involved in GLV biosynthesis in response to thrip infestation. qPCR was conducted 8 h post-infestation. (E) Up-regulation of gene expression levels of hydroperoxide lyase (HPL) following thrips infestation or effective PGPR treatments, analyzed 48 h after the application. Internal β-actin gene expression levels were used for normalization. Each treatment was repeated three times. An asterisk (*) indicates a significant difference compared to the control at a Type I error of 0.05 (t-test), while ‘ns’ signifies no significant difference. ‘NR’ represents no response in the choice tests. Distinct letters above standard error bars denote significant differences among means at a Type I error of 0.05 (LSD test).

Among the GLVs, Z−3-methyl hexenoate (MeH) was unique to the compounds of the damaged plants and was chosen to test its biological activity (Fig. 3C). MeH effectively repelled F. occidentalis and attracted O. laevigatus at higher concentrations above 0.25 ppm. The GLV biosynthetic pathway is depicted in Fig. S3B, where genes identified in the RNA-Seq analysis were discovered in the pathway components. Most of the genes associated with GLV biosynthesis were upregulated in their expression levels following thrips infestation (Fig. 3D). Notably, hydroperoxide lyase (HPL), which catalyzes the cleavage of C18 fatty acids to produce the six-carbon GLV skeleton, was significantly upregulated following thrips infestation or treatments with effective PGPRs (Fig. 3E).

Among terpenoids, linalool was specific to the compounds derived from the damaged plants and used to assess its biological activity against the thrips and their predator (Fig. 4A). Linalool also repelled F. occidentalis and attracted O. laevigatus (Fig. 4B). The terpenoid biosynthetic pathway is depicted in Fig. S3C, where genes identified in the RNA-Seq analysis were located in the pathway components. Most genes associated with terpenoid biosynthesis were upregulated in their expression levels following thrips infestation (Fig. 4C). Notably, linalool synthetase (LS) was significantly upregulated following thrips infestation or treatments with effective PGPRs (Fig. 4D). These VOC analyses indicates both MeH and linalool mediate the tri-trophic interaction among the hot pepper, thrips, and its predator.

Fig. 4.

Fig. 4

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Emission of a terpene, linalool, from hot peppers infested by F. occidentalis, influences the behaviors of the thrips and the predator, O. laevigatus, towards the plants. Y-tube choice test of the thrips (A) and the predator (B) on hot peppers, comparing the solvent (‘Hexane’) and linalool treatments. Each run of the Y-tube test utilized 20 adults of F. occidentalis or O. laevigatus. All experiments were replicated three times. (C) Heatmap showing the expression analyses of MEP and MVA component genes in response to thrips infestation. qPCR was conducted at 8 h post-infestation. (D) Up-regulation of linalool synthase (LS) gene expression following thrips infestation or effective PGPR treatments. In the PGPR treatments, gene expression was analyzed 48 h after application. Internal β-actin gene expression levels served as normalization controls. Each treatment was replicated three times. An asterisk (*) indicates a significant difference compared to the control at a Type I error rate of 0.05 (t-test), while ‘ns’ indicates no significant difference. ‘NR’ denotes no response in the choice tests. Different letters above standard error bars signify significant differences among means at a Type I error of 0.05 (LSD test).

Suppression of HPL or LS expression disrupts the tri-trophic interactions involving hot pepper-F. occidentalis-O. Laevigatus

To test the role of MeH and linalool, the genes (HPL or LS) involved in their biosynthetic pathways were specifically suppressed in the hot peppers by the virus-induced gene silencing (VIGS) technique (Fig. 5). Co-injection of two expression vectors (pTRV1 and pTRV2) successfully silenced a specific target gene, phytoene desaturase (PDS), causing the treated hot peppers to lose their green color in contrast to the control (Fig. 5A). When the recombinant pTRV2 containing the HPL or LS construct was administered, the VIGS treatments significantly suppressed the target genes even following thrips infestation. Conversely, the controls exhibited significant induction of the target genes following thrips infestation (Fig. 5B). Suppression of the target genes resulted in a marked reduction of MeH and linalool emitted by the hot peppers post-thrips infestation (Fig. 5C).

Fig. 5.

Fig. 5

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Functional assay of hydroperoxide lyase (HPL) and linalool synthase (LS) gene expression in the resistance of hot peppers to F. occidentalis. (A) Suppression of HPL or LS expression was achieved using the virus-induced gene silencing (VIGS) technique by infecting with dual vectors of pTRV1 and pTRV2. For treatment, dsRNA constructs specific to HPL or LS were cloned into the pTRV2, termed dsHPL or dsLS, respectively. To control RNAi efficiency, pTRV2 recombinant with dsRNA specific to the phytoene desaturase gene (‘dsPDS’) was injected into hot peppers. After 28 days, decoloration of leaves was observed compared with those injected with the control vector (‘dsCON’). (B) The effect of VIGS on HPL and LS expression levels, with or without thrips infestation, was examined. At 28 days post-VIGS treatment, the hot peppers were exposed to thrips. After 8 h, gene expression analysis was conducted with three replications per treatment, with internal β-actin gene levels used to normalize expression. (C) Changes in methyl hexanoate (‘MeH’) or linalool levels post-VIGS treatment were assessed. Following 28 days of VIGS treatment and subsequent thrips exposure after 8 h, HS-SPME was utilized to collect volatile organic compounds, with three replications per treatment. (D) A choice test assessing preference of thrips and the predator, O. laevigatus, between wild type (‘WT’) and VIGS-treated hot peppers was conducted using a tunnel assay. During each assay, 50 adults of F. occidentalis or O. laevigatus were used. Each choice test was repeated three times with independently prepared samples. An asterisk (*) signifies a significant difference compared to the control at a Type I error rate of 0.05 (t-test), ‘ns’ denotes no significant difference, and ‘NR’ indicates no response in the choice tests. Different letters above the standard error bars depict significant differences among means at a Type I error rate of 0.05 (LSD test).

In wild-type plants, thrips-infested hot peppers (‘Damaged’) significantly enhanced repellency against thrips and attraction of predators (Fig. 5D) compared to undamaged plants. Under the VIGS treatment specific to HPL expression, the thrips infestation did not repel the thrips as effectively as in the control without infestation (‘Undamaged in dsCON vs. Damaged in dsRNA’). Instead, the thrips showed a preference for the dsRNA-treated hot peppers (‘Undamaged in dsCON vs. Undamaged in dsRNA’ or ‘Damaged in dsCON vs. Damaged in dsRNA’) over those treated with dsCON. However, adding MeH restored the repellency by inhibiting the thrips’ orientation towards the dsRNA treatment (‘Damaged in dsCON vs Damaged in dsRNA’). By contrast, the attraction of predators was significantly suppressed in HPL-silenced plants, whether damaged or undamaged. The HPL-silenced plants were unsuccessful in attracting predators compared to the control without infestation (‘Undamaged in dsCON vs. Damaged in dsRNA’). Instead, predators showed a preference for the control plant over the HPL-silenced plants (‘Undamaged in dsCON vs. Undamaged in dsRNA’ or ‘Damaged in dsCON vs. Damaged in dsRNA’) compared to dsRNA-treated hot peppers. However, the introduction of MeH restored the attractiveness by inhibiting the orientation of the predators towards the control plants (‘Damaged in dsCON vs. Damaged in dsRNA’). The VIGS treatments targeting LS expression in the hot peppers also resulted in similar behavioral changes in both thrips and predators, as observed in the VIGS treatments targeting HPL expression. Suppression of LS expression reduced both thrips repellency and attractiveness towards O. laevigatus in the hot peppers infested by F. occidentalis. Linalool treatment restored the normal responses of thrips and their predators to the LS-silenced plants. These VIGS analyses support the role of MeH and linalool in the insect resistance of the hot peppers damaged by the thrips.

Differential expression levels of HPL and LS influence the antixenosis of various hot pepper varieties

To test the variation of the expression levels of HPL and LS in the anxenosis, ten distinct hot pepper varieties were evaluated for their repellency against F. occidentalis (Fig. 6A). In a choice test using a two-choice tunnel, two varieties (‘KM’ and ‘TD’) were found to be less preferred by the thrips. Additionally, two varieties (‘KM’ and ‘MS’) were more preferred by O. laevigatus when infested by the thrips (Fig. 6B). The organic compounds were collected using HS-SPME from these 10 varieties and categorized into 74 different compounds, including GLVs, terpenoids, and others (Table S3, Supplementary file #2). These compounds were analyzed across the 10 hot pepper varieties, revealing no independent cluster of the three varieties (‘TD’, ‘MS’, and ‘KM’) (Fig. 6C). However, linalool was exclusively found in one of these resistant varieties (‘TD’) though MeH was detected in all varieties (Fig. 6D, Supplementary file #3).

Fig. 6.

Fig. 6

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Effect of methyl-hexenoate (‘MeH’) and linalool production on insect resistance in hot peppers against thrips. (A) Screening of ten hot pepper varieties in a tunnel choice test involving F. occidentalis (‘Fo’), with the susceptible Gguary variety as a reference. Varieties tested include ‘911’ for PR 911, ‘DG’ for Dabokhangajeong, ‘DK’ for PR Daekan, ‘KJ’ for Callazzang, ‘KM’ for Kaltanmiso, ‘KY’ for Kaltanyeonseung, ‘MK’ for Meaunkaltan, ‘MS’ for Meotjinsanai, ‘SB’ for Subicho, and ‘TD’ for Titan Daebak. (B) A similar screening involving the predator, O. laevigatus (‘Ol’), was conducted to assess preference among ten hot pepper varieties using a tunnel assay. In each tunnel assay run, 50 adults of F. occidentalis or O. laevigatus were used. The experiments were replicated three times. Different letters above the standard error bars indicate significant differences among means at a Type I error rate of 0.05 (LSD test). (C) A comparison of the volatile organic compounds (VOCs) emitted by the ten hot pepper varieties was performed (Supplementary file #2). VOC emission patterns were analyzed using a phylogenetic tree via the ClustVis online tool (https://biit.cs.us.ee/clustvis/). VOCs were categorized into three groups green leaf volatiles (‘GLV’), terpenes (‘Terpene’), and unclassified (‘UC’). (D) A comparison of individual compounds, specifically GLVs and terpenes, released from the ten hot pepper varieties was conducted. Thirteen GLVs are identified (Supplementary file #3), including methyl-hexenoate (‘MeH’), 1-hexanol (‘GLV2’), 2-ethyl-1-hexanol (‘GLV3’), 2-hexenal (‘GLV4’), (E)−2-hexenal (‘GLV5’), 2-hexenoic acid methyl ester (‘GLV6’), (E)−3-hexen-1-ol (‘GLV7’), (Z)−3-hexen-1-ol (‘GLV8’), (Z)−3-hexen-1-ol acetate (‘GLV9’), 3-hexenal (‘GLV10’), 3-hexenoic acid methyl ester (‘GLV11’), hexanal (‘GLV12’), and hexanoic acid (‘GLV13’). Three terpenes are categorized, including linalool, eucalyptol (‘Terpene 2’), and geraniol (‘Terpene 3’). (E) Induction of HPL and LS expression followed thrips infestation in ten hot pepper varieties. An asterisk (*) denotes a significant difference in the damaged treatment compared to the undamaged control at a Type I error rate of 0.05 (t-test). (F) A correlation study was conducted to evaluate the relationships between HPL or LS expression levels and the repellency against F. occidentalis in hot pepper varieties.

All ten hot pepper varieties up-regulated the gene expression levels of HPL and LS following thrips infestation, although the induction levels varied among the varieties (Fig. 6E). The expression levels of HPL across the 10 hot pepper varieties strongly correlated with their repellent activities against F. occidentalis (Fig. 6F). The expression levels of LS also correlated with their repellent activities against F. occidentalis.

Discussion

This study demonstrated the role of VOCs in mediating the tri-trophic interaction among hot peppers, one of its major pests, the western flower thrips, F. occidentalis, and the thrips’ predator, the minute pirate bug, O. laevigatus through VOCs produced by hot peppers in response to thrips infestation. To elucidate the role of VOCs in mediating the biological activities in this system, the behaviors of the herbivore and its predator toward the damaged plants were investigated. VOC production was validated by comparing the transcriptomes of undamaged and damaged host plants, and then monitoring gene expressions related to the biosynthetic pathways of the candidate VOCs. Ultimately, the functional VOCs were analyzed for the expression levels of the biosynthetic genes across different hot pepper varieties, highlighting how susceptibility to thrips varies. This approach demonstrated the pivotal roles of GLV and terpenes in the chemical communication within the tri-trophic interaction.

Thrips infestation prompted hot peppers to employ antixenosis, enhancing repellency over the control plants, as a defense against herbivore attack. Intriguingly, the damaged plants attracted the predator, O. laevigatus. Augmentative biological control programs have employed the predator bug, O. laevigatus, particularly in greenhouses cultivating vegetable crops including hot peppers, to combat thrips pests16. Natural enemies are generally affected both directly and indirectly in tri-trophic interactions with host plants17. Direct interactions between natural enemies and host plants involve physical characteristics, such as leaf trichomes, waxy leaves, and leaf thickness18–20, and biochemical properties including exudates, nectars, and odors21. Indirect effects involve the herbivores’ biological traits, such as developmental time, size, weight, fecundity, survival, longevity, establishment, and digestibility17,22,23. These biological characteristics of herbivores are influenced by the host plants, illustrating functional tri-trophic interactions. The damaged hot peppers’ modulation of behaviors that repel F. occidentalis and attract O. laevigatus suggested a chemical communication across tri-trophic levels: host plant (hot pepper), herbivore (F. occidentalis), and predator (O. laevigatus). The targeted communication calling specific natural enemies by the infested host plants has been demonstrated in various plants, including corn and cabbage, depending on the herbivores present24,25. In cabbages, the damaged host plants released different VOCs depending on the herbivores’ species and infestation density26. The study reported an up-regulation of VOCs in damaged cabbages, including GLVs ((Z)−3-hexenol, n-heptanal, and (Z)−3-hexenyl acetate), terpenes (α-pinene, sabinene, myrcene, limonene, camphor, and (E)−4,8-dimethyl-1,3,7-nonatriene), and another hydrocarbon (α-copaene). Our current study identified the VOCs, including GLVs and terpenes, released from hot peppers infested by F. occidentalis upon capturing the volatile compounds using HS-SPME. Due to the limitation of relatively small sample sizes (2–3 replications), there may be more VOCs newly synthesized upon the thrips infestation.

The antixenosis induced by the functional VOCs is likely mediated by the JA signaling pathway when hot pepper was infested by F. occidentalis. In addition to the MeJA effect on the induction of antixenosis in hot peppers, DEG analysis between control and infested hot peppers showed that genes associated with JA biosynthesis and signaling components were significantly upregulated in response to thrips infestation, along with the increased expression of GLV and terpene biosynthetic genes. Notably, the expression levels of 13-LOX, which oxygenates α-linolenic acid in a regio- and stereo-selective manner to produce 13(S)-hydroperoxylinolenic acid in JA synthesis, increased more than 200-fold after thrips infestation. Indeed, disruption of 13-LOX expression suppressed the JA pathway in peppers, leading to enhanced susceptibility to F. occidentalis27. The significance of JA signal was demonstrated in the tri-trophic interaction among the cabbage, caterpillar, and its parasitoid, in which a specific inhibitor (= phenidone) to LOX abolished the damaged cabbage to repel the caterpillars and attract the parasitoid28. Moreover, among the five bacterial isolates tested, two PGPRs (‘AK-0’ and ‘G-2273’) effectively induced antixenosis in hot peppers against F. occidentalis and up-regulated the expression of 13-LOX and other JA-synthetic genes. Even though this current study did not measure the VOCs after the PGPR application, the up-regulation of the JA-biosynthetic gene expressions and the temporal behavioral change of the thrips and predator strongly support the VOC production and release from the hot peppers. These suggest there might be functional interactions between hot peppers and the bacteria to modulate the plant defense systems. It is well-known that PGPRs assist plants in defending against various pathogens and herbivores. For example, in cotton insect resistance, gossypol is a primary antibiosis factor against insect pests, and its production was stimulated by treatment with a PGPR, classified as a Bacillus sp., that enhanced JA biosynthesis29. Insect resistance can be induced by suppressing a plant factor negatively associated with insect resistance. For example, when Arabidopsis thaliana was treated with a PGPR, JA synthesis was enhanced30. This, in turn, suppressed a specific microRNA, mir814, which antagonizes the insect resistance of A. thaliana, which then lead to the induction of systemic resistance in the plant30.The increased JA synthesis by PGPRs suppressed a specific microRNA, mir814, which antagonizes the insect resistance of Arabidopsis thaliana following treatment with Bacillus amyloliquefasciens FZB42, leading to induced systemic resistance30. In our current study, the selected PGPRs activate JA synthesis, potentially causing antixenosis by producing VOCs that repel F. occidentalis and attract O. laevigatus.

In our study, Z−3-methyl hexenoate (MeH), a GLV produced by infested hot peppers, was particularly effective in repelling thrips and attracting predators. GLVs are widely produced by green plants and their production can be induced by abiotic stimuli31, herbivores32, or pathogens33 via the HPL branch of the oxylipin pathway, often within seconds of stress exposure. While GLV emission is typically short-lived, it can persist for days during herbivory or repeated wounding34. Furthermore, GLVs serve as semiochemicals that guide insects to locate food or prey35. MeH is synthesized from hexenol through JA signaling in a medicinal herb36, and this precursor is derived from C18 unsaturated fatty acids such as linoleic acid and linolenic acid, following catalytic cleavage by hydroperoxide lyase (HPL). Our VIGS analysis, which aimed to suppress HPL expression, corroborated the biosynthetic pathway and physiological role of MeH. Additionally, MeH was detected solely in hot pepper varieties that are resistant to F. occidentalis. The expression levels of HPL were positively correlated with the degree of repellency exhibited by F. occidentalis towards the hot peppers. This functional GLV may also prime hot pepper plants against further attacks from herbivores or pathogens. GLVs induce defense priming in plants, enabling them to respond faster or more robustly to insect herbivores and pathogens. For instance, a tomato (Solanum lycopersicum) infested by the whitefly, Bemisia tabaci, releases Z−3-hexenol through JA signaling, which increases the expression of defense genes and flavonoid levels37. This indicates that the production and release of other GLVs, as well as MeH, may be stimulated by JA signaling, contributing to antixenosis.

Linalool, a terpene produced by hot peppers infested by F. occidentalis, is catalyzed by LS, a terpene synthase. The suppression of gene expression by gene-specific VIGS treatment prevented the upregulation of linalool titers in the hot peppers following thrips damage. Indirectly, linalool may also prime the host plant to produce reactive oxygen species, as demonstrated in Arabidopsis infested by P. xylostella, causing oxidative damage to the herbivore38. The expression of terpene synthase genes is regulated by the MYC2 transcription factor under the JA immune signaling pathway in hot peppers39. This suggests that JA signaling initiates the synthesis of various terpenes, including linalool.

Various hot pepper varieties differ in their susceptibility to F. occidentalis infestation as well as in their ability to produce GLVs and terpenes. Even without damage, hot peppers emit a range of VOCs. For instance, the Habanero pepper (Capsicum chinense (Jacq.)), known for its intense pungency and fruity aroma, releases 66 volatile compounds, including multiple short-chain hydrocarbons such as 6-methyl-(E)−4-heptenyl 3-methylbutanoate, 6-methyl-(E)−4-heptenyl 2-methylpropanoate, 6-methyl-(E)−4-heptenyl 2-methylbutanoate, 2-methylbutanoate, and 6-methyl-(E)−4-heptenol40. However, it is primarily the various GLVs and terpenes that characterize the VOCs of damaged hot peppers. Our current data indicate that the JA signaling pathway, which is triggered by thrips infestation, upregulates biosynthetic gene expressions such as HPL and LS, and these are positively correlated with antixenosis resistance to F. occidentalis. In other words, hot pepper varieties expressing high levels of HPL and LS genes tend to be resistant to F. occidentalis. This positive correlation provides crucial information for the breeding of resistant hot peppers by gene introgression using these insect-resistant markers against F. occidentalis, a significant insect pest that transmits TSWV to high-value crops3. However, functional validation of these breeding markers (HPL and LS) through transgenic overexpression or deletion mutagenesis is necessary before they can be deemed reliable for marker-assisted selection.

The tri-trophic interactions might be varied in field conditions because of other biological factors. Besides VOCs, the established tri-trophic interactions include the relationship among hot peppers, F. occidentalis, and O. laevigatus, which is mediated by the aggregation pheromone released by F. occidentalis. This pheromone facilitates chemical communication among the thrips, but functions as a kairomone for both adult and nymph stages of O. laevigatus, particularly in its active components mixture of (R)-lavandulyl acetate and neryl (S)−2-methylbutanoate41. Conversely, tri-trophic interactions may be compromised by viral infections such as TSWV that reduce predator access to the plants, thus promoting the virus’ horizontal transmission. F. occidentalis larvae thrive better on TSWV-infected host plants than on non-infected hosts and evade predation by predatory mites42. This complexity in tri-trophic interactions have led to the identification of chemical signals originating from the primary trophic level, which are interpreted differently by the herbivore and its predator.

Altogether, this study identified two functional VOCs of MeH and linalool, which repel the thrips and attract the predator. It also highlights resistant genetic markers like HPL and LS that deter thrips from hot peppers, beneficial for future breeding programs.

Methods

Insect rearing

Adults of F. occidentalis were collected from a hot pepper field in Andong, Korea, and reared on sprouted bean seed kernels under specific laboratory conditions: a constant temperature of 25 ± 1 °C, a photoperiod of 16:8 h (L: D), and a relative humidity of 60 ± 5%. Under these conditions, the thrips underwent two instars, prepupa, and pupa before maturing into adults. A colony of the predator, O. laevigatus, donated by Oal, Inc. (Seoul, Korea), and was maintained on hot pepper plants with larvae of F. occidentalis provided as food.

Plant growth-promoting rhizobacteria (PGPR)

Five different PGPR bacteria, namely Paenibacillus polymyxa GYUN-2273 (NCBI accession number: OR883773), Bacillus subtilis GYUN-2311 (KACC accession number: 92471), B. velezensis AK-0 (KACC accession number: 92099), B. tequilensis G-300 (KACC accession number: 81153), and Brevibacillus halotolerans B-4359 (NCBI GenBank number: CP139435), were isolated from the rhizosphere of agricultural regions in Andong or donated by the Nakdonggang National Institute of Biological Resources, Sangju, Korea. These bacteria were inoculated in a single colony into 100 mL of tryptic soy broth (TSB: Difco, Sparks, MD, USA) and cultured at 28 °C with an agitation of 180 rpm. After 48 h culture, 10 mL of the broth was evenly applied to the soil in a pot (10 cm diameter and 10 cm height) containing young hot pepper plants at the six-leaf stage. At various times, the effects of the PGPR on the insect resistance of the hot peppers were evaluated by insect behaviors using Y-tube olfactometer and analyzing expressions of three genes: 13-LOX, AOS, and AOC.

Cultivating different hot pepper varieties

All hot pepper varieties were cultivated in a nursery under conditions of 25 ± 1 °C, a 12:12 h (L: D) photoperiod, and approximately 60% RH. Biological activities were evaluated using the Gguari variety (Hanlim, Seoul, Korea) as a susceptible reference. The hot pepper varieties used in the tests included PR Daekan (Jenong S&T, Jeju, Korea), Dabokhangajeong (Sakata Korea, Seoul, Korea), PR 911 (Hana Seed, Anseong, Korea), Kaltanmiso (Seedland, Cheongju, Korea), Meotjinsanai (Syngenta Korea, Seoul, Korea), Subicho (Yeongyang, Korea), Meaunkaltan (Pepper & Breeding Institute, Gimje, Korea), Callazzang (Nongwoobio, Suwon, Korea), Titan Daebak (Farmhannong, Seoul, Korea), and Kaltanyeonseung (Nongwoobio, Suwon, Korea). The assays were conducted using 6-week-old plants at the six-leaf stage to ensure uniformity and developmental consistency.

Chemicals

Methyl hexenoate (99%), linalool (97%), salicylic acid (99%), methyl jasmonate (95%), and cyclopentanone (99%) were purchased from Sigma Aldrich Korea (Seoul, Korea). These compounds were subsequently dissolved in dimethyl sulfoxide (DMSO) to prepare the test solutions. DNA Taq polymerase, RT Premix, and Power SYBR Green PCR Master Mix were sourced from GeneALL Biotechnology Company (Seoul, Korea), Intron Biotechnology (Seoul, Korea), and Life Technologies (Carlsbad, CA, USA), respectively. Furthermore, the restriction enzymes (BamHI and XbaI) were sourced from Takara (Kyoto, Japan).

Insect damage treatment to hot peppers with F. occidentalis

Leaves of the young hot pepper plants at the six-leaf stage were infested with 50 L2 larvae of F. occidentalis per plant for 8 h under controlled culturing conditions. Afterwards, the insects were manually removed with brush, and the cleaned plants were immediately used for further experiments.

Behavioral bioassay using Y-tube olfactometer

To assess the behavioral responses to chemicals, a Y-tube olfactometer was utilized as described by Khan et al.43 with some modification. Briefly, the transparent Y-tube glass consisted of a 100 mm stem with two 80 mm arms separated by a 45° angle. The internal diameter of the Y-tube was 10 mm. Humidified air filtered through activated charcoal was supplied into the two arms. All behavioral assays were performed in a dark room under an infrared light (1,000 lx illumination) at 25 ± 1 °C and 65% relative humidity. In each trial, 20 male and female adults of F. occidentalis or O. laevigatus were placed at the bottom of the stem tube of the Y-tube for observation. Test insects or compounds were installed at one side of the arm tube. The running time of each run was 10 min. Positive responses included insects trapped on a sticky card (2 × 2 cm) or observed feeding on pepper plants. Those that did not cross the fork were recorded as no response (‘NR’). All trials were conducted in darkness at 25 ± 1 °C and 65% RH. Each treatment consisted of four replications while changing the installation position of the test samples between two arms in each replication.

Behavioral bioassay using a two-choice tunnel assay

A two-choice tunnel assay was employed to evaluate the behavioral choice between two hot pepper varieties for F. occidentalis and O. laevigatus44. The arena consisted of two identical size (25 × 25 × 25 cm) chambers for control and treatment. The connecting tube between the two chambers was 50 cm. In each test, 50 male and female adults of F. occidentalis or O. laevigatus were introduced into the tunnel’s midpoint through an entrance hole. Airflow from each chamber to the connecting tube was maintained at 400 mL/min by two inline flow meters (Cole Parmer Instrument, Chicago, IL, USA). All assessments were performed in the dark at 25 ± 1 °C and 65% RH, and each lasted 6 h because there was no more remarkable change in the choice of the thrips after 6 h. Positive responses were those where insects were trapped on a sticky card (24 cm × 14 cm) or seen feeding on the plants. More than 85% among the oriented thrips to either direction were collected to the sticky trap. After each session, the tunnel was cleaned thoroughly, with at least one hour between trials. Each treatment was replicated three times.

RNA extraction, cDNA synthesis, RT-PCR, and RT-qPCR

Plant leaves were collected and flash-frozen in liquid nitrogen before storage at −80 °C to prevent cross-contamination and RNA degradation. Each sample involved grinding 0.1 g of plant tissues thoroughly with a cold (4 °C) mortar and pestle, which had been preheated at 180 °C for 20 min to inactivate RNases. RNA was extracted using Trizol reagent according to the manufacturer’s instructions. After extraction and DNase treatment, RNA was resuspended in nuclease-free water, and its concentration was measured using a spectrophotometer (NanoDrop, Thermo Scientific, Wilmington, DE, USA). RNA was then used for cDNA synthesis with an RT Premix containing oligo-dT primer, following the manufacturer’s instructions. RT-PCR involved DNA Taq polymerase and proceeded under these conditions: initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at various temperatures (Table S4) for 1 min, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min using gene-specific primers. Each 20 µL RT-PCR reaction contained the cDNA template, dNTPs (each at 2 mM), 10 pmol of each forward and reverse primer, and 2 units/µL of Taq polymerase. Gene expression levels were assessed with a real-time PCR machine (Step One Plus Real-Time PCR System, Applied Biosystems, Singapore) using Power SYBR Green PCR Master Mix as per Bustin et al.45. The expression of β-actin served as a reference to normalize target gene expression levels under various treatments. Melting curve analysis confirmed the specificity of each PCR product. Quantitative analysis used the comparative CT (2−∆∆CT) method46, with each experiment replicated three times with independent cohorts or sample preparations.

RNA-Seq analysis of hot peppers after thrips damage

Hot peppers infested by F. occidentalis were used to extract RNA as previously described. Each treatment was replicated three times. The concentration and purity of the RNA samples were assessed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and the samples were sent to Macrogen (Seoul, Korea) for RNA library construction and next-generation sequencing using the Illumina NovaSeq 6000 system (Illumina, San Diego, CA, USA).

The sequenced FASTQ data were uploaded to the NCBI database under the project number PRJNA104655. Data analysis was conducted using the bioinformatics software CLC Genomic Workbench (version 22.0.1, Qiagen, Hilden, Germany). The raw sequenced data were trimmed to remove low-quality sequences and mapped to the C. annuum genome. RPKM (reads per kilobase per million mapped reads) values were used to calculate relative mRNA expression levels. Gene IDs were retrieved from the gene symbols with LOC numbers in the mapped reads, using the general feature format (GFF) file of the C. annuum genome.

Differentially expressed genes (DEGs) were identified based on a minimum two-fold change and a p-value of less than 0.05 by comparing RPKM values. Gene ontology (GO) analysis was conducted using Blast2GO 6.0 (BioBam, Valencia, Spain) to classify biological, cellular, and molecular functional categories of selected DEGs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to determine the functional categories of genes as defined in the KEGG database, using the KEGG mapper (https://www.genome.jp/kegg/mapper/search.html) and the BioDBnet (Biological Database Network, https://biodbnetabcc.ncifcrf.gov/db/db2dbRes.php) ID conversion tool.

Transcript analysis of the damaged hot peppers in the JA pathway

Genes involved in the JA biosynthetic pathway were selected based on the model proposed by Wang et al.47 and our current transcriptome data of hot peppers to compare expression levels across treatments. The analyzed genes include phospholipase A1 (PLA1), 13-LOX, AOS, AOC, JA-amino acid synthetase (JASSY), L-3-ketoacyl CoA thiolase (KAT), multifunctional protein (MCX), acyl-CoA oxidase (ACX), oxophytodienoic acid reductase 3 (OPR3), coronatine insensitive 1 (COI1), jasmonate zim domain (JAZ), and MYC2.

Genes involved in the terpene biosynthetic pathway were identified using the pathway outlined by Mani et al.48 from our current transcriptome of the hot peppers: acetyl-CoA acetyltransferase (AACT, XP_016580453.1), hydroxymethylglutaryl-CoA synthase (HMGS, XP_016551730.1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, XP_016557834.2), mevalonate kinase (MK, XP_016538569.2), phosphomevalonate kinase (PMK, XP_016574993.2), diphosphomevalonate decarboxylase (MPD, XP_016551358.2), farnesyl pyrophosphate synthase (FPS, NP_001311799.1), sesquiterpene synthase (STPS, XP_016566183.2), (-)-germacrene D synthase (GDS, XP_016564626.1), 1-deoxy-D-xylulose-5-phosphate synthase (DXS, XP_047256568.1), 1-deoxy-D-xylulose-5-phosphate reductase (DXR, XP_016563443.1), 2-methyl-D-erythritol 4-phosphate cytidylyltransferase (CMS, XP_016538918.1), 2-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS, XP_016566381.1), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS, XP_016547325.1), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR, XP_016539712.1), geranylgeranyl diphosphate synthase (GPPS, XP_016556802.1), and linalool synthase (LS, XP_016539311.2).

Genes associated with the GLV biosynthetic pathway were selected via the method proposed by Scala et al.35 from our current transcriptome of the hot peppers: lipoxygenase (LOX, NP_001311748.1), hydroperoxide lyase (HPL, NP_001311810.1), alcohol dehydrogenase (ADH, XP_016568293.2), aldo-keto reductase (AKR, XP_016543252.1), aldehyde reductase (ADR, XP_016578140.2), and alcohol acetyltransferase (AAT, XP_016541551.2). These genes were used to compare gene expression levels in the terpene pathway between damaged and undamaged hot pepper plants.

Virus-induced gene Silencing (VIGS)

Virus-induced gene silencing was performed following the methods of Zhang & Liu49 with slight modifications. Open reading frames (ORFs) of HPL (GenBank accession number: U51674.1) and LS (GenBank accession number: XM_047394954.1) genes were cloned into the pCR2.1-TOPO vector using the TOPO TA Cloning Kit (Invitrogen) with ORF primers (Table S4). The confirmed clones were cultured in LB medium containing ampicillin (100 µg/mL), and their plasmids were extracted using a spin column (Plasmid SV, GeneAll). Extracted pCR2.1-HPL and pCR2.1-LS plasmids were sequenced at Macrogen. Upon confirmation, the recombinant vectors were digested with BamHI and XbaI, and the resulting inserts were ligated to construct the recombinant pTRV2-HPL and pTRV2-LS expression vectors. These vectors were transformed into Escherichia coli (DH5α) competent cells and cultured in LB containing kanamycin (100 mg/mL). Finally, 10 µL of the validated recombinant plasmid of pTRV2-HPL and pTRV2-LS were transferred into Agrobacterium tumefaciens strain GV3101 via electroporation and cultured on LB with kanamycin (100 µg/mL) and rifampicin (50 µg/mL) at 28 °C for 72 h. Subsequent clones were resuspended in infiltration buffer (10 mM MES, 10 mM MgCl2, and 200 µM acetosyringone) to achieve a bacterial concentration between 1.3 and 1.5 at OD600. For injection, equal volumes of bacterial suspension containing pTRV1 and either pTRV2-HPL or pTRV2-LS were mixed and injected into the abaxial side of cotyledons using a needless syringe. The inoculated seedlings were then cultured under standard plant culturing conditions.

Analysis of the organic compounds produced by hot pepper leaves using headspace-solid phase Microextraction (HS-SPME)

The leaves treated with thrips or VIGS were harvested and pulverized using a mortar and pestle under liquid nitrogen. Two grams of the pulverized leaf samples were placed into a 20 mL headspace vial containing an internal standard (cyclohexanone, 8,000 ppm), which was subsequently stored at −80 °C until analysis. The samples were equilibrated at 40 °C for 10 min in a thermostatic autosampler tray, followed by a 40-min exposure to the divinylbenzene/carboxyl/polydimethylsiloxane (DVB/CAR/PDMS) fiber (50/30 µm, Supelco, Bellefonte, PA, USA). The fiber was then thermally desorbed in the GC injector port at 250 °C for 10 min (spitless). The GC–MS system (Agilent 8890 A/5977B MSD Series, Agilent Technologies, Santa Clara, CA, USA) was operated in the electron impact (EI, ionization energy: 70 eV) mode with a scan range of m/z 35–550. VOCs were separated on a DB-WAX capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness; Agilent). The temperature program for the GC oven started at 35 °C for 2 min, increased from 35 to 45 °C at a rate of 2 °C/min, then from 45 to 130 °C at 5 °C/min, and finally held at 225 °C at a rate of 10 °C/min for 5 min. Helium served as the carrier gas at a constant flow rate of 1.5 mL/min. The transfer line temperature was maintained at 250 °C. VOC identification was conducted by matching mass spectra against the standard National Institute of Standards and Technology (NIST library version 20). A series of n-alkane (C7-C30) standards was employed to determine the linear retention index (RI) of each compound (Supplementary files #1 and #2). The relative quantification of VOCs was performed by calculating the peak area ratio of each analyte to that of the internal standard in each sample. Quantification of the major compounds, methyl hexanoate and linalool, was based on calibration curves generated from authentic standards. Each experiment was replicated three times with individual sample preparation.

Classification of the compounds derived from hot peppers and pattern analysis

The compounds identified via GC-MS were systematically classified into distinct groups such as GLVs and terpenes, according to their chemical structures. GLVs, exemplified by compounds like hexanal and its derivatives, exhibit straight-chain and branched-chain hydrocarbons with a basic C6 skeleton50. In contrast, terpenes consisted of isoprene unit compounds, which included monoterpenes (C10) and sesquiterpenes (C15)51 in this study.

Statistical analysis

All tests involved three independent biological replicates. Means were compared using the least significant difference (LSD) test in a one-way analysis of variance (ANOVA) conducted with PROC GLM of the SAS program52. For comparisons between two groups, T-test was performed with the SAS program. Significant differences were established at a Type I error of 0.05. Pattern analysis involved generating a phylogenetic tree using the ClustVis online tool (https://biit.cs.us.ee/clustvis/), and the corresponding heatmap was visualized using Prism 10.0.1 (GraphPad Software, Boston, MA, USA).

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (49.9KB, xlsx)
Supplementary Material 2 (329.2KB, xlsx)
Supplementary Material 3 (14.2KB, xlsx)
Supplementary Material 4 (694.9KB, docx)

Author contributions

M.E., A.A., G.J., E.M., N.S., C.K., K.K., H.P., F.K., D.L., H.K., E.H., H.W.C., Y.J., J.S.: methodology, validation, formal analysis, investigation, writing. Y.K.: conceptualization, writing, writing—review and editing, supervision, project administration.

Funding

This work was supported by a grant (No. 2022R1A2B5B03001792) from the National Research Foundation (NRF), funded by the Ministry of Science, ICT and Future Planning, Republic of Korea. This study was also supported by a research grant from Gyeongkuk National University.

Data availability

RNA-Seq data in Fig. 2 were deposited to GenBank with an accession number of PRJNA104655. HS-SPME/GC-MS raw data in Fig 4 + Fig. S1, Fig. 7 C, and Fig. 7D are provided with three supplementary three Excel files. Other raw data in Results can be available from the corresponding author on request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (49.9KB, xlsx)
Supplementary Material 2 (329.2KB, xlsx)
Supplementary Material 3 (14.2KB, xlsx)
Supplementary Material 4 (694.9KB, docx)

Data Availability Statement

RNA-Seq data in Fig. 2 were deposited to GenBank with an accession number of PRJNA104655. HS-SPME/GC-MS raw data in Fig 4 + Fig. S1, Fig. 7 C, and Fig. 7D are provided with three supplementary three Excel files. Other raw data in Results can be available from the corresponding author on request.

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