Companion basil plants prime the tomato wound response through volatile signaling in a mixed planting system.

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Riichiro Yoshida, Shoma Taguchi, Chihiro Wakita, Shinichiro Serikawa, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4314608/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Jul, 2024 Read the published version in Plant Cell Reports → Version 1 posted You are reading this latest preprint version Abstract Within mixed planting systems, companion plants can promote growth or enhance stress responses in target plants. However, the mechanisms underlying these effects remain poorly understood. To gain insight into the molecular nature of the effects of companion plants, we investigated the effects of basil plants (Ocimum basilicum var. minimum) on the wound response in tomato plants (Solanum lycopersicum cv. ‘Micro Tom’) within a mixed planting system. The results showed that the expression of Pin2, which specifically responds to mechanical wounding, was induced more rapidly and more strongly in the leaves of tomato plants cultivated with companion basil plants. This wound response priming effect was replicated through the exposure of tomato plants to an essential oil (EO) prepared from basil leaves. Tomato leaves pre-exposed to basil EO showed enhanced expression of genes related to jasmonic acid, mitogen-activated protein kinase (MAPK), and reactive oxygen species (ROS) signaling after wounding stress. Basil EO also enhanced ROS accumulation in wounded tomato leaves. The wound response priming effect of basil EO was confirmed in wounded Arabidopsis plants. Loss-of-function analysis of target genes revealed that MAPK genes play pivotal roles in controlling the observed priming effects. Spodoptera litura larvae fed tomato leaves pre-exposed to basil EO showed reduced growth compared with larvae fed control leaves. Thus, mixed planting with basil may enhance defense priming in both tomato and Arabidopsis plants through the activation of volatile signaling. Companion plants priming tomato volatile wound signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Key Message Volatile compounds released from basil prime the tomato wound response by promoting jasmonic acid, mitogen-activated protein kinase, and reactive oxygen species signaling. Introduction Agricultural systems worldwide are dominated by industrial approaches that produce single crops under the control of chemical fertilizers and pesticides (Horrigan et al. 2002). However, these systems carry a substantial risk of degrading topsoil, which is required for crop growth, and produce large amounts of greenhouse gases, which accelerate global warming (Gao et al. 2022). Regenerative agriculture, which aims to restore the natural environment while improving the soil for crop growth, has been proposed to ensure global food security while mitigating these problems (Giller et al. 2021). One step toward regenerative agriculture is the implementation of mixed planting, rather than monocultures. Companion planting, in which compatible crops of different species are grown together, originated in the USA, where Indigenous Americans planted a mixture of corn, pumpkins, and beans known as the “three sisters” (Pleasant 2016). Companion planting is generally considered beneficial to plants because of its ability to control pests and diseases (Finch et al. 2003; Parker et al. 2013; George et al. 2013; Fu et al. 2015), optimize soil nutrient supply (Mengel et al. 2001), and improve growing space efficiency (Bomford 2014). However, the specific effects of companion plants remain unclear. A typical example of companion planting is a “push–pull” system, in which natural plant–insect communication is harnessed to reduce herbivory by insects (Pickett et al. 2014). In this system, volatiles released from plants repel or disturb feeding insects while attracting their natural enemies. For instance, volatiles released from companion plants have been reported to effectively protect target plants against aphid or whitefly damage under greenhouse conditions (Ben-Issa et al. 2017a, b; Conboy et al. 2019). Thus, companion plants can help to enhance the defense systems of a target plant species. For example, volatiles released by mint plants were reported to increase pest resistance in soybean or Brassica rapa plants within mixed planting systems (Sukegawa et al. 2018), and volatiles released from injured Solidago canadensis were able to inhibit root nodule symbiosis by nitrogen-fixing bacteria on soybean roots (Takahashi et al. 2021). These findings indicate that volatile signaling is strongly involved in the effects of companion planting on target plants. However, few studies have comprehensively investigated this phenomenon, and molecular research is needed to elucidate its underlying mechanism. An understanding of the mechanisms that drive this effect may help to promote the widespread implementation of companion planting for sustainable agriculture and maximal effectiveness within mixed planting systems. Therefore, the objective of this study was to clarify the molecular basis for the effects of companion planting on target plants in a tomato–basil mixed planting system, primarily focusing on interplant communication. The results showed that basil plants induced a priming effect on the tomato wound response; experimental analysis showed that this effect was mediated by volatiles released from basil leaves. Gene expression analysis revealed that wound response priming by basil volatiles was attributable to the enhanced expression of genes related to jasmonic acid (JA), mitogen-activated protein kinase (MAPK), and reactive oxygen species (ROS) signaling in tomato plants. In a subsequent experiment, Spodoptera litura larvae fed tomato leaves pre-exposed to basil aroma exhibited reduced growth. The priming effect of basil volatiles was also observed in the model plant Arabidopsis thaliana ; loss-of-function analysis using a gene knockout mutant revealed that the priming effect may be partly attributed to MAPK genes. These results provide scientific evidence that this beneficial effect of companion planting is mainly driven by interplant communication via plant volatile signaling. Materials and methods Plant materials and growth conditions Tomato ( Solanum lycopersicum cv. ‘Micro Tom’), basil ( Ocimum basilicum var. minimum ), and Arabidopsis (Columbia ecotype) were used in this study. All plants were grown in soil consisting of a 1:1 ratio of Metro-Mix (Sun Gro Horticulture, Agawam, MA, USA) to vermiculite within a controlled environment at 23°C under a 12-h/12-h light/dark photoperiod. A mixed planting system was established by transplanting germinated tomato and basil seeds into 9-cm-diameter pots. Insect culture and feeding experiments Larvae hatched from eggs were reared on artificial feed and grown to the second instar stage at 25°C under a 14-h/10-h light/dark photoperiod. The larvae were fed untreated and basil EO-exposed tomato leaves at 23°C under a 14-h/10-h light/dark photoperiod, then weighed after 3 days to evaluate inhibitory effects on Spodoptera litura growth. Thirty larvae were included in each treatment. Basil EO and volatile compound treatments Tomato and Arabidopsis plants grown in 5-cm-diameter pots were placed inside a plant box (7 cm length × 7 cm width × 10 cm height); cotton swabs soaked with basil EO or one of the four tested volatile compounds (linalool, α-terpineol, chavicol, or eugenol) were attached to the bottom of the lid, and the box was closed. After 15 h of exposure, plants were removed from the box and desensitized for 1 h. Then, leaves of each plant were wounded with scissors in one area on each side of the leaf, bordering the main leaf vein. Basil EO was extracted by using steam distillation method (Tongnuanchan and Benjakul, 2014) Quantitative polymerase chain reaction (qPCR) analysis Total RNA was isolated from tomato and Arabidopsis leaves using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using ReverTra Ace (Toyobo, Osaka, Japan), in accordance with the manufacturer’s instructions. qPCR was performed using the Eco Real-Time PCR System (Illumina, San Diego, CA, USA) using the KAPA SYBR FAST qPCR kit (Sigma-Aldrich, St. Louis, MO, USA). The qPCR cycling protocol consisted of 40 cycles of 95°C for 5 s and 60°C for 30 s. Primer sequences used in this analysis are listed in Table S1. Expression levels for each target gene were normalized to the levels of ACTIN (for tomato) and ACTIN2 (for Arabidopsis). ROS determination and quantification DAB staining was performed as previously described (Poster and Luna 2013). H 2 O 2 accumulation in leaves was quantified using GIMP2 software. Results Companion planting with basil induced a priming effect on the tomato wound response The positive effects of companion planting are not easily detected. In our experimental system, the appearances of tomato plants grown with basil were observed and compared with the appearances of tomatoes grown without basil (Fig. 1A). To understand the effects of companion basil plants on tomato plants, tomato leaves were subjected to wound stress, followed by analyses of the expression levels of the wound response gene Pin2 (Peña-Cortés et al. 1995). The results showed that tomato plants grown with basil rapidly exhibited higher Pin2 expression levels than tomato plants grown without basil (Fig. 1B). Essential oil (EO) prepared from basil leaves primed the wound response in wounded tomato leaves To determine whether the observed wound response priming effect was caused by volatiles released from the aboveground parts of basil plants, we exposed tomato plants to purified EO extracted from basil leaves. Tomato plants were placed in plant boxes (Fig. 2A) and exposed to basil EO for 15 h; their leaves were then subjected to wound stress. Next, we examined Pin2 gene expression in each leaf. Tomato plants that were exposed to basil EO exhibited similar wound response priming to the findings in tomato plants grown with basil plants (Fig. 2B). Effects of individual volatile compounds in basil EO on tomato wound response priming Next, we investigated which volatile components of the basil EO are involved in the induction of wound response priming. Tomato plants were exposed to four major volatile compounds—linalool, α-terpineol, chavicol, and eugenol—at various concentrations (Fig. 2A). Because a previous study showed that (Z)-3-hexenol induced a priming effect in corn seedlings attacked by insects (Engelberth et al. 2004), we included this compound in our experiments. The results showed that linalool, α-terpineol, and chavicol exhibited a priming effect on wound-induced Pin2 expression compared with the control (Fig. 3), whereas no significant priming effect was observed for eugenol or (Z)-3-hexenol. Basil EO strengthened JA signaling in tomato plants Because tomato Pin2 gene expression is controlled by JA (Peña-Cortés et al. 1995), we investigated whether basil EO affects the expression of JA-related genes during wound stress. In tomato plants with a loss-of-function mutation in JAI1 , the tomato homolog of Arabidopsis COI1 , we found that jai1-1 mutants showed strong inhibition of Pin2 expression after experiencing wound stress (Fig. 4). Next, we examined the effects of basil EO on the induction of JA synthesis genes during the short-term response to leaf wounding. We found that the expression of three essential genes, LYPOXYGENASE D ( LOXD ), ALLENE OXIDE SYNTHASE ( AOS ), and ALLENE OXIDE CYCLASE ( AOC ), were directly and substantially induced by basil EO (Fig. 5). Furthermore, a similar strong priming response was observed for the expression of MYC2 , a key factor in JA signaling (Boter et al. 2004; Du et al. 2014), as well as PSY , a precursor to the plant peptide hormone systemin (Ryan and Pearce 1998) (Fig. 6). Basil EO induced the expression of MAPK- and ROS-related genes MAPK is involved in plant wound signaling and controls endogenous JA levels (Seo et al. 2007). The accumulation of MAPK proteins in cells has been associated with priming induction in plant stress signaling (Conrath 2011). Therefore, we examined whether basil EO promotes expression of the tomato MAPK genes SIMPK1 , SIMPK2 , and SIMPK3 after wound stress. The results of this experiment did not confirm wound-related induction of SIMPK1 and SIMPK2 expression in control plants; however, the expression of these genes was significantly induced by pre-exposure to basil EO (Fig. 7). In contrast, SIMPK3 was transiently expressed after wounding, with a peak at 30 min, and a priming effect was observed in plants pre-exposed to basil EO. ROS have also been proposed as strong candidates for controlling priming responses in plants (Pastora et al. 2013). Therefore, we examined the effect of basil EO on ROS accumulation in tomato leaves under wound stress via 3,3′-diaminobenzidine (DAB) staining. The results showed that ROS accumulation was up to threefold higher in basil EO-exposed leaves than in control leaves (Fig. 8A, B). Higher expression levels of Wfi1 , a key gene for ROS production in tomatoes (Song et al. 2018), were also observed in basil EO-exposed leaves after wounding (Fig. 8C). Basil EO promoted the wound response in Arabidopsis leaves Because basil EO primed the tomato wound response, we investigated whether a similar effect could occur in Arabidopsis. We exposed Arabidopsis plants to basil EO and subjected them to wound stress. In Arabidopsis leaves exposed to basil EO, we observed enhanced expression of the wound response gene VSP2 (Fig. 9). We also found that loss-of-function mutations affecting the Arabidopsis MAPK genes AtMPK3 and AtMPK6 eliminated the priming effect of basil EO on the wound response (Fig. 9). Furthermore, basil EO did not appear to enhance ROS accumulation in wounded leaves of atmpk3 or atmpk6 plants (Fig. S1). Spodoptera litura larvae fed basil EO-exposed tomato leaves exhibited growth inhibition Next, we evaluated whether the higher expression of the wound response gene Pin2 induced by basil EO in tomatoes could promote plant resistance to insect feeding. In this experiment, young Spodoptera litura larvae were fed tomato leaves, and changes in their growth were measured after the feeding period. The results showed that larvae fed basil EO-exposed tomato leaves were smaller than larvae fed control leaves (Fig. 10A). The weight of larvae fed leaves pre-exposed to basil EO was approximately half of the control larvae weight (Fig. 10B). Discussion Currently, agricultural systems worldwide produce greenhouse gases including CO 2 , N 2 O, and CH 4 , which contribute to accelerated global warming (Gao et al. 2022). Additionally, excessive nitrogen and phosphorus inputs to agricultural lands pollute rivers and lakes, creating severe environmental problems worldwide (Moss 2008). To meet growing agricultural demands while preserving the global environment, there is a need to rapidly establish agricultural practices that conserve the environment. The transportation of agricultural products also emits large amounts of CO 2 , such that shortening the distances between agricultural production and consumption areas (i.e., reducing food mileage) is a critical challenge that requires an aggressive shift from conventional large-scale farming to small-scale farming. For vegetable production, it is also necessary to review subsistence-based production systems in private gardens and urban–suburban production systems. Furthermore, the growing health consciousness among consumers is leading to an expectation of safe and secure agricultural products through reduced chemical pesticide use. Companion planting, which exploits compatibility between plant species to increase productivity per unit area and adaptability to environmental stresses, is expected to offer sustainable agriculture with reduced environmental impact. However, many studies have failed to produce results demonstrating these benefits, possibly due to the lack of scientific data supporting the effectiveness of companion plants, or the lack of well-established conditions and methods to detect their effectiveness. Clarification of the scientific basis for the benefits of companion plants is needed to establish effective strategies for their use in agricultural production. In the present study, we established a mixed planting system consisting of tomato and basil plants to elucidate the molecular basis underlying the beneficial effects of companion plants on target plants. This experimental system showed that basil companion plants significantly enhanced the wounding response in tomato plants, which has previously been described as a priming effect (Mauch-Mani et al. 2017). Prior studies have suggested that both above- and belowground parts of basil plants are involved in this effect; in the present study, we focused on plant–plant interactions through volatiles released from aboveground parts. Subsequently, we demonstrated that an EO prepared from basil leaves could prime the wounding response in tomato plants. In plants, energy allocation to growth and stress responses typically follows a trade-off relationship, such that the induction of stress adaptation actively suppresses plant growth (Karasov et al. 2017). However, stress response priming induction has minimal effects on plant growth; it allows rapid and decisive responses to irregularly encountered stresses (Frost et al. 2008). Several molecular mechanisms are involved in the induction of plant stress response priming (Pastora et al. 2013). Our experiments showed that basil volatiles induce MAPK expression and ROS production, both of which constitute essential mediators of plant stress signaling (Meng and Zhang. 2013). In Arabidopsis, benzothiadiazole activates plant stress responses by inducing the expression of AtMPK3 and AtMPK6 , leading to enhanced expression of downstream disease resistance genes (Beckers et al. 2009). Additionally, thiamine (i.e., vitamin B1) enhances the accumulation of ROS and callose during pathogen infection, resulting in H 2 O 2 -dependent induction of defense gene expression (Ahn et al. 2007). These chemicals may promote the accumulation of intracellular signaling factors and enhance downstream signaling (Pastora et al. 2013). The observed priming effect of basil volatiles, which enhanced the tomato wound response, is presumably driven by a similar mechanism. Basil volatiles promoted the expression of JA-related genes after wounding. Because MAPKs reportedly function as essential signal mediators in wound and JA-related responses (Seo et al. 2007; Takahashi et al. 2007), it is reasonable to speculate that basil volatiles activate or enhance MAPK-mediated JA signaling. Our findings suggest that ROS also function as critical mediators of volatile signaling. Several studies have demonstrated that ROS function both upstream and downstream of MAPKs (e.g., Jalmi and Sinha 2015). We observed a similar priming effect in Arabidopsis exposed to basil EO. Loss-of-function analysis of Arabidopsis MAPKs strongly suggested that AtMPK3 and AtMPK6 are involved in basil EO-dependent defense priming. Although this effect was less pronounced than the effect observed in tomato plants, we detected a slight increase in ROS among wounded Arabidopsis leaves exposed to basil EO. This increase was not observed in atmpk3 and atmpk6 mutants, suggesting that MAPKs function upstream of ROS. We attempted to analyze the effects of basil EO on ROS accumulation in atrborD:atrborF , a double loss-of-function mutant of NADPH oxidoreductase; however, unfavorable growth conditions prevented us from completing the experiment. Further analyses of MAPK- and ROS-mediated pathways, including MAPK activation, are required. Although the involvement of other mechanisms for wound response priming has not been investigated, basil is expected to play a role in inducing this priming effect by amplifying intracellular signaling factors (e.g., MAPKs or ROS) in tomato plants through the release of volatiles. The mechanism by which plants recognize volatiles as signals (i.e., their specific receptors) remains poorly understood. Thus far, ethylene is the only volatile compound that has been confirmed to act as a plant signal (Lacey and Binder 2014). However, beginning with studies of the poplar eavesdropping effect (Baldwin and Schultz 1983), various studies have revealed the potential for plant-derived volatile compounds to function as specific chemical signals. Recent studies have demonstrated that β-caryophyllene, released from insect-damaged plants, specifically binds to the transcriptional regulatory protein TOPLESS in tobacco cells and induces the expression of stress response-related genes (Nagashima et al. 2019). Intriguingly, plants may recognize the volatile signal as a blend of multiple compounds, rather than as a single compound (Kikuta et al. 2011). In the present study, we confirmed that four volatile compounds contained in basil EO play roles in the induction of wound response priming in tomato plants. In a future study, we will examine how different combinations of these four compounds influence wound responses in tomato and Arabidopsis plants. Although we focused on volatile compounds released from aboveground plant parts to explore the scientific basis of companion planting in the present study, we previously reported that belowground interactions may also be involved in the enhancement of stress responses (Fig. S2). Therefore, we are conducting experiments to investigate the effects of interactions between companion plants and soil microorganisms on stress responses in target plants. Our preliminary results indicate that mixed planting with basil substantially increases the symbiosis of mycorrhizal fungi in tomato plant roots (data not shown). Several studies have revealed that mycorrhizal fungi can prime disease resistance in plants (Pozo and Azcón-Aguilar 2007; Sabine et al. 2012). Interplant networks composed of mycorrhizal fungi mycelia are also suspected to function as communication tools in salicylic acid and JA signaling (Song et al. 2010; Song et al. 2014). The ability of companion planting to enhance plant stress adaptation through mycorrhizal fungi requires further study. Elucidation of the molecular origins of both above- and belowground interplant communication would substantially contribute to the global implementation of companion planting and future development of sustainable agriculture. Declarations Acknowledgments This work was supported in part by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (JSPS-KAKENHI; grant number 15K07294). Author contributions ST and RY conceived and designed the project. ST, CW, SS, HM and MI performed experiments. ST, CW, SS, HM, MI and RY analyzed the data. RY wrote the manuscript, with contributions from all authors. Funding The Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (JSPS-KAKENHI; grant number 15K07294). 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J Food Sci 79:1231-1249. https://doi.org/10.1111/1750-3841.12492 Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 21 Jul, 2024 Read the published version in Plant Cell Reports → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4314608","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":295570396,"identity":"72488e3d-5093-4af6-8565-c04a41454f11","order_by":0,"name":"Riichiro Yoshida","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABP0lEQVRIie3RPWuDQBgH8EcEXY6m40kCfoULghlCmrFfQxF0uSGQJdAh2VwCrm75CkrBWXBwcS4ZOhgKThksgZJChz4aUmg0hW6F+j/hjpPfPfcC0KXLH40ITIReNTKwYaRTV4dcJ8rqd6RSMZzq1OSnjHwrOcxmY1VL06cyB1tV790i3/E7XETclTB4viSDrW33feYMo4zPfQP4MMyyETMjC4mkUSDFJaGU6yJhiRDF3MJtLYTQ5xI1I3EZxKDjWZIWoh2QTB+9fU2moe8USJZYRX67QlgfiRlQJ0HCzQ01dCQJEtJehRQ2Esfyt3sRDGZbAckqkoKSkDk1Ws4i442Rj/HE85wX4biwJhvXLZT36AFuUjcsX9eNG4Nb4zwiDKD64vMEvheY67hBel9Tcl536urb/2OTdOnSpct/yyfoOG01UK6CtwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4688-4046","institution":"Kagoshima University Faculty of Agriculture Graduate School of Agriculture: Kagoshima Daigaku Nogakubu Daigakuin Nogaku Kenkyuka","correspondingAuthor":true,"prefix":"","firstName":"Riichiro","middleName":"","lastName":"Yoshida","suffix":""},{"id":295570397,"identity":"2cb1ecc6-0604-4239-9020-d21cb3442ae3","order_by":1,"name":"Shoma Taguchi","email":"","orcid":"","institution":"Kagoshima University Faculty of Agriculture Graduate School of Agriculture: Kagoshima Daigaku Nogakubu Daigakuin Nogaku Kenkyuka","correspondingAuthor":false,"prefix":"","firstName":"Shoma","middleName":"","lastName":"Taguchi","suffix":""},{"id":295570398,"identity":"bf702848-d715-4deb-b782-2da1fd373409","order_by":2,"name":"Chihiro Wakita","email":"","orcid":"","institution":"Kagoshima University Faculty of Agriculture Graduate School of Agriculture: Kagoshima Daigaku Nogakubu Daigakuin Nogaku Kenkyuka","correspondingAuthor":false,"prefix":"","firstName":"Chihiro","middleName":"","lastName":"Wakita","suffix":""},{"id":295570399,"identity":"5c1c8183-53da-4507-b818-680f67f50c4e","order_by":3,"name":"Shinichiro Serikawa","email":"","orcid":"","institution":"Kagoshima University Faculty of Agriculture Graduate School of Agriculture: Kagoshima Daigaku Nogakubu Daigakuin Nogaku Kenkyuka","correspondingAuthor":false,"prefix":"","firstName":"Shinichiro","middleName":"","lastName":"Serikawa","suffix":""},{"id":295570400,"identity":"992f8419-2a47-4f51-9862-0f5bc287cadf","order_by":4,"name":"Hiroyuki Miyaji","email":"","orcid":"","institution":"Kagoshima University Faculty of Agriculture Graduate School of Agriculture: Kagoshima Daigaku Nogakubu Daigakuin Nogaku Kenkyuka","correspondingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Miyaji","suffix":""}],"badges":[],"createdAt":"2024-04-24 00:28:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4314608/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4314608/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00299-024-03285-w","type":"published","date":"2024-07-22T00:33:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55727969,"identity":"18438bec-673b-40b9-88f0-ea91db217d68","added_by":"auto","created_at":"2024-05-02 10:34:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":219206,"visible":true,"origin":"","legend":"\u003cp\u003eMixed planting with basil enhanced \u003cem\u003ePin2\u003c/em\u003e expression in tomato plants under wounding stress. (A) Tomato plants were grown for 3 weeks with or without basil companion plants. (B) Effects of basil on expression of the wound response gene \u003cem\u003ePin2\u003c/em\u003e in tomato leaves. Leaves were wounded on both sides with scissors, sampled at the indicated times, and then subjected to quantitative polymerase chain reaction (qPCR) analysis. Bars represent means ± standard deviations (SDs) from three independent experiments. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, one-way analysis of variance [ANOVA] followed by Tukey’s test; n = 3).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4314608/v1/9a914a088449f00c6289447b.png"},{"id":55727968,"identity":"b8eeec41-3b1e-44df-bbdd-fdee2a41ce46","added_by":"auto","created_at":"2024-05-02 10:34:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":84470,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of basil essential oil (EO) on the wounding response in tomato plants. (A) Schematic representation of the experimental setup. Plants were placed inside a box; cotton swabs soaked with 5 mL of basil EO (5 mL of water for controls) were attached to the bottom of the lid, and the box was closed. After 15 h of exposure, the box was opened, and the plant leaves were wounded with scissors after a desensitization period. (B) Effects of basil EO on \u003cem\u003ePin2\u003c/em\u003egene expression in tomato leaves. Tomato leaves were sampled at the indicated times and subjected to qPCR analyses. Bars represent means ± SDs from three independent experiments. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, one-way ANOVA followed by Tukey’s test; n = 3).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4314608/v1/6a82eeebb141194263d07a8f.png"},{"id":55727967,"identity":"642b697c-e2bc-47f1-b8e6-47767a89d93f","added_by":"auto","created_at":"2024-05-02 10:34:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":25887,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of five volatile compounds on the wound response in tomato plants. Tomato plants were pre-exposed to each compound at the indicated concentrations for 15 h, then wounded with scissors. At 10 h after wounding, tomato leaves were sampled and subjected to qPCR analysis. Bars represent means ± SDs from three independent experiments. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, one-way ANOVA followed by Tukey’s test; n = 3). Leaves were sampled and their \u003cem\u003ePin2\u003c/em\u003e transcript levels were analyzed by qPCR.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4314608/v1/4969b797d20d1959b1d13256.png"},{"id":55727970,"identity":"280be407-cf90-4d59-8308-fd106e9993e9","added_by":"auto","created_at":"2024-05-02 10:34:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16377,"visible":true,"origin":"","legend":"\u003cp\u003eBasil EO promotes jasmonic acid (JA) signaling in tomato plants. Effect of JA-insensitive\u003cem\u003e jai1-1\u003c/em\u003e mutation on wound-induced \u003cem\u003ePin2\u003c/em\u003eexpression. Wild-type (WT) and \u003cem\u003ejai1-1\u003c/em\u003e mutant plants were pre-exposed to basil EO for 15 h, then wounded with scissors. Tomato leaves were sampled at the indicated times, then subjected to qPCR analysis. Bars represent means ± SDs from three independent experiments. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, one-way ANOVA followed by Tukey’s test; n = 3).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4314608/v1/6a3b7ce2b48df91b9d267fcc.png"},{"id":55727973,"identity":"7f71b2db-ca7f-4cdf-a559-2a74beeb8029","added_by":"auto","created_at":"2024-05-02 10:34:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26359,"visible":true,"origin":"","legend":"\u003cp\u003eBasil EO enhances wound-induced expression of JA biosynthesis-related genes in tomato plants. Effects of basil EO on the wound-induced expression of JA biosynthesis-related genes \u003cem\u003eLYPOXYGENASE D\u003c/em\u003e (\u003cem\u003eLOXD\u003c/em\u003e), \u003cem\u003eALLENE OXIDE SYNTHASE\u003c/em\u003e (\u003cem\u003eAOS\u003c/em\u003e), and \u003cem\u003eALLENE OXIDE CYCLASE \u003c/em\u003e(\u003cem\u003eAOC\u003c/em\u003e). WT plants were pre-exposed to basil EO for 15 h, then wounded with scissors. Tomato leaves were sampled at the indicated times, then subjected to qPCR analysis. Bars represent means ± SDs from three independent experiments. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, one-way ANOVA followed by Tukey’s test; n = 3).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4314608/v1/ff23178612a4e442754169de.png"},{"id":55727972,"identity":"fc11f77e-0ad5-4e5c-a1e8-62c5cc0b4f61","added_by":"auto","created_at":"2024-05-02 10:34:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":20398,"visible":true,"origin":"","legend":"\u003cp\u003eBasil EO enhances wound-induced expression of JA signaling-related genes in tomato plants. Effects of basil EO on the wound-induced expression of JA signaling-related genes \u003cem\u003ePROSYSTEMIN\u003c/em\u003e (\u003cem\u003ePSYS\u003c/em\u003e) and \u003cem\u003eMYC2\u003c/em\u003e in tomato plants. WT plants were pre-exposed to basil EO for 15 h, then wounded with scissors. Tomato leaves were sampled at the indicated times, then subjected to qPCR analysis. Bars represent means ± SDs from three independent experiments. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, one-way ANOVA followed by Tukey’s test; n = 3).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4314608/v1/ac42f25e32e5e6bc7400d7d0.png"},{"id":55727971,"identity":"22c57309-24a2-4fce-bda5-ab52f6d94fea","added_by":"auto","created_at":"2024-05-02 10:34:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":23756,"visible":true,"origin":"","legend":"\u003cp\u003eBasil EO induces and promotes the expression of mitogen-activated protein kinase (MAPK) genes in tomato plants. Effects of basil EO on the wound-induced expression of three MAPK genes (\u003cem\u003eSIMPK1\u003c/em\u003e, \u003cem\u003eSIMPK2\u003c/em\u003e, and \u003cem\u003eSIMPK3\u003c/em\u003e) in tomato plants. WT plants were pre-exposed to basil EO for 15 h, then wounded with scissors. Tomato leaves were sampled at the indicated times, then subjected to qPCR analysis. Bars represent means ± SDs from three independent experiments. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, one-way ANOVA followed by Tukey’s test; n = 3).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4314608/v1/0643263afc12375144409442.png"},{"id":55727977,"identity":"4f7617da-5ce1-4023-b6b2-de3450a6db41","added_by":"auto","created_at":"2024-05-02 10:34:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":122945,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of basil EO on wound-induced reactive oxygen species (ROS) accumulation in tomato leaves. (A) DAB (3,3′-diaminobenzidine) staining of wounded tomato leaves. Tomato plants were pre-exposed to basil EO for 15 h, then wounded with scissors. (B) DAB staining intensity in wounded leaves was quantified using GIMP software. Blue and orange bars represent control and basil EO treatments, respectively. (C) qPCR analysis of transcript levels of the tomato NADPH-oxidase gene \u003cem\u003eWfi1\u003c/em\u003e in wounded tomato leaves. Tomato plants were pre-exposed to basil EO for 15 h, then wounded with scissors. Tomato leaves were sampled at the indicated times, then subjected to qPCR analysis. Bars represent means ± SDs from three independent experiments. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, one-way ANOVA followed by Tukey’s test; n = 3).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4314608/v1/8f656f1ab54c2c666536a907.png"},{"id":55727974,"identity":"0c5933d7-b4be-43a8-8c59-37509647dc9c","added_by":"auto","created_at":"2024-05-02 10:34:58","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":26242,"visible":true,"origin":"","legend":"\u003cp\u003eBasil EO promotes the wound response in Arabidopsis through a mechanism mediated by MAPK genes. Effects of basil EO on expression of the wound response gene \u003cem\u003eVSP2\u003c/em\u003e in Arabidopsis. WT, \u003cem\u003eatmpk3\u003c/em\u003e, and \u003cem\u003eatmpk6\u003c/em\u003e plants were pre-exposed to basil EO for 15 h, then wounded with scissors. Arabidopsis leaves were sampled at the indicated times, then subjected to qPCR analysis. Bars represent means ± SDs from three independent experiments. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, one-way ANOVA followed by Tukey’s test; n = 3).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4314608/v1/de3aa93439c2cf3bf24115af.png"},{"id":55728826,"identity":"cbb83242-1454-4450-93c9-9116dbf9ca39","added_by":"auto","created_at":"2024-05-02 10:42:58","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":95799,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of basil EO on the growth of \u003cem\u003eSpodoptera litura\u003c/em\u003e larvae. (A) Second instar larvae of \u003cem\u003eSpodoptera litura\u003c/em\u003ewere fed (a) control or (b) basil EO-exposed tomato leaves. (B) Larval weights were determined at the end of the feeding trial. Bars represent means ± SDs. Significant differences were evaluated using Student’s t-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4314608/v1/791260b6844a098f005be338.png"},{"id":60858370,"identity":"2c742382-99fb-4faf-865d-d778ecc05575","added_by":"auto","created_at":"2024-07-23 00:33:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1140340,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4314608/v1/4c917c14-6288-4588-8b7f-d8c38bf4aa7d.pdf"},{"id":55727978,"identity":"33635149-3dd5-4d73-8c2d-c0fe3c7907fa","added_by":"auto","created_at":"2024-05-02 10:34:58","extension":"docx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":1745761,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4314608/v1/bbba1d535be701264d909804.docx"}],"financialInterests":"","formattedTitle":"Companion basil plants prime the tomato wound response through volatile signaling in a mixed planting system.","fulltext":[{"header":"Key Message","content":"\u003cp\u003eVolatile compounds released from basil prime the tomato wound response by promoting jasmonic acid, mitogen-activated protein kinase, and reactive oxygen species signaling.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAgricultural systems worldwide are dominated by industrial approaches that produce single crops under the control of chemical fertilizers and pesticides (Horrigan et al. 2002). However, these systems carry a substantial risk of degrading topsoil, which is required for crop growth, and produce large amounts of greenhouse gases, which accelerate global warming (Gao et al. 2022). Regenerative agriculture, which aims to restore the natural environment while improving the soil for crop growth, has been proposed to ensure global food security while mitigating these problems (Giller et al. 2021). One step toward regenerative agriculture is the implementation of mixed planting, rather than monocultures. Companion planting, in which compatible crops of different species are grown together, originated in the USA, where Indigenous Americans planted a mixture of corn, pumpkins, and beans known as the \u0026ldquo;three sisters\u0026rdquo; (Pleasant 2016). Companion planting is generally considered beneficial to plants because of its ability to control pests and diseases (Finch et al. 2003; Parker et al. 2013; George et al. 2013; Fu et al. 2015), optimize soil nutrient supply (Mengel et al. 2001), and improve growing space efficiency (Bomford 2014). However, the specific effects of companion plants remain unclear. A typical example of companion planting is a \u0026ldquo;push\u0026ndash;pull\u0026rdquo; system, in which natural plant\u0026ndash;insect communication is harnessed to reduce herbivory by insects (Pickett et al. 2014). In this system, volatiles released from plants repel or disturb feeding insects while attracting their natural enemies. For instance, volatiles released from companion plants have been reported to effectively protect target plants against aphid or whitefly damage under greenhouse conditions (Ben-Issa et al. 2017a, b; Conboy et al. 2019).\u003c/p\u003e\n\u003cp\u003eThus, companion plants can help to enhance the defense systems of a target plant species. For example, volatiles released by mint plants were reported to increase pest resistance in soybean or \u003cem\u003eBrassica rapa\u003c/em\u003e plants within mixed planting systems (Sukegawa et al. 2018), and volatiles released from injured \u003cem\u003eSolidago canadensis\u003c/em\u003e were able to inhibit root nodule symbiosis by nitrogen-fixing bacteria on soybean roots (Takahashi et al. 2021). These findings indicate that volatile signaling is strongly involved in the effects of companion planting on target plants. However, few studies have comprehensively investigated this phenomenon, and molecular research is needed to elucidate its underlying mechanism. An understanding of the mechanisms that drive this effect may help to promote the widespread implementation of companion planting for sustainable agriculture and maximal effectiveness within mixed planting systems.\u003c/p\u003e\n\u003cp\u003eTherefore, the objective of this study was to clarify the molecular basis for the effects of companion planting on target plants in a tomato\u0026ndash;basil mixed planting system, primarily focusing on interplant communication. The results showed that basil plants induced a priming effect on the tomato wound response; experimental analysis showed that this effect was mediated by volatiles released from basil leaves. Gene expression analysis revealed that wound response priming by basil volatiles was attributable to the enhanced expression of genes related to jasmonic acid (JA), mitogen-activated protein kinase (MAPK), and reactive oxygen species (ROS) signaling in tomato plants. In a subsequent experiment, \u003cem\u003eSpodoptera litura\u0026nbsp;\u003c/em\u003elarvae fed tomato leaves pre-exposed to basil aroma exhibited reduced growth. The priming effect of basil volatiles was also observed in the model plant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e; loss-of-function analysis using a gene knockout mutant revealed that the priming effect may be partly attributed to MAPK genes. These results provide scientific evidence that this beneficial effect of companion planting is mainly driven by interplant communication via plant volatile signaling.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e cv. \u0026lsquo;Micro Tom\u0026rsquo;), basil (\u003cem\u003eOcimum basilicum\u003c/em\u003e var. \u003cem\u003eminimum\u003c/em\u003e), and Arabidopsis (Columbia ecotype) were used in this study. All plants were grown in soil consisting of a 1:1 ratio of Metro-Mix (Sun Gro Horticulture, Agawam, MA, USA) to vermiculite within a controlled environment at 23\u0026deg;C under a 12-h/12-h light/dark photoperiod. A mixed planting system was established by transplanting germinated tomato and basil seeds into 9-cm-diameter pots.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eInsect culture and feeding experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLarvae hatched from eggs were reared on artificial feed and grown to the second instar stage at 25\u0026deg;C under a 14-h/10-h light/dark photoperiod. The larvae were fed untreated and basil EO-exposed tomato leaves at 23\u0026deg;C under a 14-h/10-h light/dark photoperiod, then weighed after 3 days to evaluate inhibitory effects on \u003cem\u003eSpodoptera litura \u003c/em\u003egrowth. Thirty larvae were included in each treatment.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eBasil EO and volatile compound treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTomato and Arabidopsis plants grown in 5-cm-diameter pots were placed inside a plant box (7 cm length \u0026times; 7 cm width \u0026times; 10 cm height); cotton swabs soaked with basil EO or one of the four tested volatile compounds (linalool, \u0026alpha;-terpineol, chavicol, or eugenol) were attached to the bottom of the lid, and the box was closed. After 15 h of exposure, plants were removed from the box and desensitized for 1 h. Then, leaves of each plant were wounded with scissors in one area on each side of the leaf, bordering the main leaf vein. Basil EO was extracted by using steam distillation method (Tongnuanchan and Benjakul, 2014)\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eQuantitative polymerase chain reaction (qPCR) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from tomato and Arabidopsis leaves using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using ReverTra Ace (Toyobo, Osaka, Japan), in accordance with the manufacturer\u0026rsquo;s instructions. qPCR was performed using the Eco Real-Time PCR System (Illumina, San Diego, CA, USA) using the KAPA SYBR FAST qPCR kit (Sigma-Aldrich, St. Louis, MO, USA). The qPCR cycling protocol consisted of 40 cycles of 95\u0026deg;C for 5 s and 60\u0026deg;C for 30 s. Primer sequences used in this analysis are listed in Table S1. Expression levels for each target gene were normalized to the levels of \u003cem\u003eACTIN\u003c/em\u003e (for tomato) and \u003cem\u003eACTIN2\u003c/em\u003e (for Arabidopsis).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eROS determination and quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDAB staining was performed as previously described (Poster and Luna 2013). H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation in leaves was quantified using GIMP2 software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCompanion planting with basil induced a priming effect on the tomato wound response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe positive effects of companion planting are not easily detected. In our experimental system, the appearances of tomato plants grown with basil were observed and compared with the appearances of tomatoes grown without basil (Fig. 1A). To understand the effects of companion basil plants on tomato plants, tomato leaves were subjected to wound stress, followed by analyses of the expression levels of the wound response gene \u003cem\u003ePin2\u003c/em\u003e (Pe\u0026ntilde;a-Cort\u0026eacute;s et al. 1995). The results showed that tomato plants grown with basil rapidly exhibited higher \u003cem\u003ePin2\u003c/em\u003e expression levels than tomato plants grown without basil (Fig. 1B).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEssential oil (EO) prepared from basil leaves primed the wound response in wounded tomato leaves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether the observed wound response priming effect was caused by volatiles released from the aboveground parts of basil plants, we exposed tomato plants to purified EO extracted from basil leaves. Tomato plants were placed in plant boxes (Fig. 2A) and exposed to basil EO for 15 h; their leaves were then subjected to wound stress. Next, we examined \u003cem\u003ePin2\u003c/em\u003e gene expression in each leaf. Tomato plants that were exposed to basil EO exhibited similar wound response priming to the findings in tomato plants grown with basil plants (Fig. 2B).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEffects of individual volatile compounds in basil EO on tomato wound response priming \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we investigated which volatile components of the basil EO are involved in the induction of wound response priming. Tomato plants were exposed to four major volatile compounds\u0026mdash;linalool, \u0026alpha;-terpineol, chavicol, and eugenol\u0026mdash;at various concentrations (Fig. 2A). Because a previous study showed that (Z)-3-hexenol induced a priming effect in corn seedlings attacked by insects (Engelberth et al. 2004), we included this compound in our experiments. The results showed that linalool, \u0026alpha;-terpineol, and chavicol exhibited a priming effect on wound-induced \u003cem\u003ePin2\u003c/em\u003e expression compared with the control (Fig. 3), whereas no significant priming effect was observed for eugenol or (Z)-3-hexenol.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eBasil EO strengthened JA signaling in tomato plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause tomato \u003cem\u003ePin2\u003c/em\u003e gene expression is controlled by JA (Pe\u0026ntilde;a-Cort\u0026eacute;s et al. 1995), we investigated whether basil EO affects the expression of JA-related genes during wound stress. In tomato plants with a loss-of-function mutation in \u003cem\u003eJAI1\u003c/em\u003e, the tomato homolog of Arabidopsis \u003cem\u003eCOI1\u003c/em\u003e, we found that \u003cem\u003ejai1-1\u003c/em\u003e mutants showed strong inhibition of \u003cem\u003ePin2\u003c/em\u003e expression after experiencing wound stress (Fig. 4). Next, we examined the effects of basil EO on the induction of JA synthesis genes during the short-term response to leaf wounding. We found that the expression of three essential genes, \u003cem\u003eLYPOXYGENASE D\u003c/em\u003e (\u003cem\u003eLOXD\u003c/em\u003e), \u003cem\u003eALLENE OXIDE SYNTHASE\u003c/em\u003e (\u003cem\u003eAOS\u003c/em\u003e), and \u003cem\u003eALLENE OXIDE CYCLASE \u003c/em\u003e(\u003cem\u003eAOC\u003c/em\u003e), were directly and substantially induced by basil EO (Fig. 5). Furthermore, a similar strong priming response was observed for the expression of \u003cem\u003eMYC2\u003c/em\u003e, a key factor in JA signaling\u003cem\u003e \u003c/em\u003e(Boter et al. 2004; Du et al. 2014), as well as \u003cem\u003ePSY\u003c/em\u003e, a precursor to the\u003cem\u003e \u003c/em\u003eplant peptide hormone systemin (Ryan and Pearce 1998) (Fig. 6).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eBasil EO induced the expression of MAPK- and ROS-related genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMAPK is involved in plant wound signaling and controls endogenous JA levels (Seo et al. 2007). The accumulation of MAPK proteins in cells has been associated with priming induction in plant stress signaling (Conrath 2011). Therefore, we examined whether basil EO promotes expression of the tomato MAPK genes \u003cem\u003eSIMPK1\u003c/em\u003e, \u003cem\u003eSIMPK2\u003c/em\u003e, and \u003cem\u003eSIMPK3\u003c/em\u003e after wound stress. The results of this experiment did not confirm wound-related induction of \u003cem\u003eSIMPK1\u003c/em\u003e and \u003cem\u003eSIMPK2\u003c/em\u003e expression in control plants; however, the expression of these genes was significantly induced by pre-exposure to basil EO (Fig. 7). In contrast, \u003cem\u003eSIMPK3\u003c/em\u003e was transiently expressed after wounding, with a peak at 30 min, and a priming effect was observed in plants pre-exposed to basil EO.\u003c/p\u003e\n\u003cp\u003eROS have also been proposed as strong candidates for controlling priming responses in plants (Pastora et al. 2013). Therefore, we examined the effect of basil EO on ROS accumulation in tomato leaves under wound stress via 3,3\u0026prime;-diaminobenzidine (DAB) staining. The results showed that ROS accumulation was up to threefold higher in basil EO-exposed leaves than in control leaves (Fig. 8A, B). Higher expression levels of \u003cem\u003eWfi1\u003c/em\u003e, a key gene for ROS production in tomatoes (Song et al. 2018), were also observed in basil EO-exposed leaves after wounding (Fig. 8C).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eBasil EO promoted the wound response in Arabidopsis leaves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause basil EO primed the tomato wound response, we investigated whether a similar effect could occur in Arabidopsis. We exposed Arabidopsis plants to basil EO and subjected them to wound stress. In Arabidopsis leaves exposed to basil EO, we observed enhanced expression of the wound response gene \u003cem\u003eVSP2\u003c/em\u003e (Fig. 9). We also found that loss-of-function mutations affecting the Arabidopsis MAPK genes\u003cem\u003e AtMPK3\u003c/em\u003e and \u003cem\u003eAtMPK6\u003c/em\u003e eliminated the priming effect of basil EO on the wound response (Fig. 9). Furthermore, basil EO did not appear to enhance ROS accumulation in wounded leaves of \u003cem\u003eatmpk3\u003c/em\u003e or \u003cem\u003eatmpk6\u003c/em\u003e plants (Fig. S1).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSpodoptera litura\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e larvae fed basil EO-exposed tomato leaves exhibited growth inhibition \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we evaluated whether the higher expression of the wound response gene \u003cem\u003ePin2\u003c/em\u003e induced by basil EO in tomatoes could promote plant resistance to insect feeding. In this experiment, young \u003cem\u003eSpodoptera litura\u003c/em\u003e larvae were fed tomato leaves, and changes in their growth were measured after the feeding period. The results showed that larvae fed basil EO-exposed tomato leaves were smaller than larvae fed control leaves (Fig. 10A). The weight of larvae fed leaves pre-exposed to basil EO was approximately half of the control larvae weight (Fig. 10B).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCurrently, agricultural systems worldwide produce greenhouse gases including CO\u003csub\u003e2\u003c/sub\u003e, N\u003csub\u003e2\u003c/sub\u003eO, and CH\u003csub\u003e4\u003c/sub\u003e, which contribute to accelerated global warming (Gao et al. 2022). Additionally, excessive nitrogen and phosphorus inputs to agricultural lands pollute rivers and lakes, creating severe environmental problems worldwide (Moss 2008). To meet growing agricultural demands while preserving the global environment, there is a need to rapidly establish agricultural practices that conserve the environment. The transportation of agricultural products also emits large amounts of CO\u003csub\u003e2\u003c/sub\u003e, such that shortening the distances between agricultural production and consumption areas (i.e., reducing food mileage) is a critical challenge that requires an aggressive shift from conventional large-scale farming to small-scale farming. For vegetable production, it is also necessary to review subsistence-based production systems in private gardens and urban\u0026ndash;suburban production systems. Furthermore, the growing health consciousness among consumers is leading to an expectation of safe and secure agricultural products through reduced chemical pesticide use. Companion planting, which exploits compatibility between plant species to increase productivity per unit area and adaptability to environmental stresses, is expected to offer sustainable agriculture with reduced environmental impact. However, many studies have failed to produce results demonstrating these benefits, possibly due to the lack of scientific data supporting the effectiveness of companion plants, or the lack of well-established conditions and methods to detect their effectiveness. Clarification of the scientific basis for the benefits of companion plants is needed to establish effective strategies for their use in agricultural production.\u003c/p\u003e\n\u003cp\u003eIn the present study, we established a mixed planting system consisting of tomato and basil plants to elucidate the molecular basis underlying the beneficial effects of companion plants on target plants. This experimental system showed that basil companion plants significantly enhanced the wounding response in tomato plants, which has previously been described as a priming effect (Mauch-Mani et al. 2017). Prior studies have suggested that both above- and belowground parts of basil plants are involved in this effect; in the present study, we focused on plant\u0026ndash;plant interactions through volatiles released from aboveground parts. Subsequently, we demonstrated that an EO prepared from basil leaves could prime the wounding response in tomato plants.\u003c/p\u003e\n\u003cp\u003eIn plants, energy allocation to growth and stress responses typically follows a trade-off relationship, such that the induction of stress adaptation actively suppresses plant growth (Karasov et al. 2017). However, stress response priming induction has minimal effects on plant growth; it allows rapid and decisive responses to irregularly encountered stresses (Frost et al. 2008). Several molecular mechanisms are involved in the induction of plant stress response priming (Pastora et al. 2013). Our experiments showed that basil volatiles induce MAPK expression and ROS production, both of which constitute essential mediators of plant stress signaling (Meng and Zhang. 2013). In Arabidopsis, benzothiadiazole activates plant stress responses by inducing the expression of \u003cem\u003eAtMPK3\u003c/em\u003e and \u003cem\u003eAtMPK6\u003c/em\u003e, leading to enhanced expression of downstream disease resistance genes (Beckers et al. 2009). Additionally, thiamine (i.e., vitamin B1) enhances the accumulation of ROS and callose during pathogen infection, resulting in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-dependent induction of defense gene expression (Ahn et al. 2007). These chemicals may promote the accumulation of intracellular signaling factors and enhance downstream signaling (Pastora et al. 2013). The observed priming effect of basil volatiles, which enhanced the tomato wound response, is presumably driven by a similar mechanism. Basil volatiles promoted the expression of JA-related genes after wounding. Because MAPKs reportedly function as essential signal mediators in wound and JA-related responses (Seo et al. 2007; Takahashi et al. 2007), it is reasonable to speculate that basil volatiles activate or enhance MAPK-mediated JA signaling. Our findings suggest that ROS also function as critical mediators of volatile signaling. Several studies have demonstrated that ROS function both upstream and downstream of MAPKs (e.g., Jalmi and Sinha 2015).\u003c/p\u003e\n\u003cp\u003eWe observed a similar priming effect in Arabidopsis exposed to basil EO. Loss-of-function analysis of Arabidopsis MAPKs strongly suggested that AtMPK3 and AtMPK6 are involved in basil EO-dependent defense priming. Although this effect was less pronounced than the effect observed in tomato plants, we detected a slight increase in ROS among wounded Arabidopsis leaves exposed to basil EO. This increase was not observed in \u003cem\u003eatmpk3\u003c/em\u003e and \u003cem\u003eatmpk6\u003c/em\u003e mutants, suggesting that MAPKs function upstream of ROS. We attempted to analyze the effects of basil EO on ROS accumulation in \u003cem\u003eatrborD:atrborF\u003c/em\u003e, a double loss-of-function mutant of NADPH oxidoreductase; however, unfavorable growth conditions prevented us from completing the experiment. Further analyses of MAPK- and ROS-mediated pathways, including MAPK activation, are required. Although the involvement of other mechanisms for wound response priming has not been investigated, basil is expected to play a role in inducing this priming effect by amplifying intracellular signaling factors (e.g., MAPKs or ROS) in tomato plants through the release of volatiles.\u003c/p\u003e\n\u003cp\u003eThe mechanism by which plants recognize volatiles as signals (i.e., their specific receptors) remains poorly understood. Thus far, ethylene is the only volatile compound that has been confirmed to act as a plant signal (Lacey and Binder 2014). However, beginning with studies of the poplar eavesdropping effect (Baldwin and Schultz 1983), various studies have revealed the potential for plant-derived volatile compounds to function as specific chemical signals. Recent studies have demonstrated that \u0026beta;-caryophyllene, released from insect-damaged plants, specifically binds to the transcriptional regulatory protein TOPLESS in tobacco cells and induces the expression of stress response-related genes (Nagashima et al. 2019). Intriguingly, plants may recognize the volatile signal as a blend of multiple compounds, rather than as a single compound (Kikuta et al. 2011). In the present study, we confirmed that four volatile compounds contained in basil EO play roles in the induction of wound response priming in tomato plants. In a future study, we will examine how different combinations of these four compounds influence wound responses in tomato and Arabidopsis plants. Although we focused on volatile compounds released from aboveground plant parts to explore the scientific basis of companion planting in the present study, we previously reported that belowground interactions may also be involved in the enhancement of stress responses (Fig. S2). Therefore, we are conducting experiments to investigate the effects of interactions between companion plants and soil microorganisms on stress responses in target plants. Our preliminary results indicate that mixed planting with basil substantially increases the symbiosis of mycorrhizal fungi in tomato plant roots (data not shown). Several studies have revealed that mycorrhizal fungi can prime disease resistance in plants (Pozo and Azc\u0026oacute;n-Aguilar 2007; Sabine et al. 2012). Interplant networks composed of mycorrhizal fungi mycelia are also suspected to function as communication tools in salicylic acid and JA signaling (Song et al. 2010; Song et al. 2014). The ability of companion planting to enhance plant stress adaptation through mycorrhizal fungi requires further study. Elucidation of the molecular origins of both above- and belowground interplant communication would substantially contribute to the global implementation of companion planting and future development of sustainable agriculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (JSPS-KAKENHI; grant number 15K07294).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eST and RY conceived and designed the project. ST, CW, SS, HM and MI performed experiments. ST, CW, SS, HM, MI and RY analyzed the data. RY wrote the manuscript, with contributions from all authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (JSPS-KAKENHI; grant number 15K07294).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhn I, Kim S, Lee Y, Suh S (2007) Vitamin B1-induced priming is dependent on hydrogen peroxide and the NPR1 gene in Arabidopsis. 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J Food Sci 79:1231-1249. https://doi.org/10.1111/1750-3841.12492\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Companion plants, priming, tomato, volatile, wound signaling","lastPublishedDoi":"10.21203/rs.3.rs-4314608/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4314608/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Within mixed planting systems, companion plants can promote growth or enhance stress responses in target plants. However, the mechanisms underlying these effects remain poorly understood. To gain insight into the molecular nature of the effects of companion plants, we investigated the effects of basil plants (Ocimum basilicum var. minimum) on the wound response in tomato plants (Solanum lycopersicum cv. ‘Micro Tom’) within a mixed planting system. The results showed that the expression of Pin2, which specifically responds to mechanical wounding, was induced more rapidly and more strongly in the leaves of tomato plants cultivated with companion basil plants. This wound response priming effect was replicated through the exposure of tomato plants to an essential oil (EO) prepared from basil leaves. Tomato leaves pre-exposed to basil EO showed enhanced expression of genes related to jasmonic acid, mitogen-activated protein kinase (MAPK), and reactive oxygen species (ROS) signaling after wounding stress. Basil EO also enhanced ROS accumulation in wounded tomato leaves. The wound response priming effect of basil EO was confirmed in wounded Arabidopsis plants. Loss-of-function analysis of target genes revealed that MAPK genes play pivotal roles in controlling the observed priming effects. Spodoptera litura larvae fed tomato leaves pre-exposed to basil EO showed reduced growth compared with larvae fed control leaves. Thus, mixed planting with basil may enhance defense priming in both tomato and Arabidopsis plants through the activation of volatile signaling.","manuscriptTitle":"Companion basil plants prime the tomato wound response through volatile signaling in a mixed planting system.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-02 10:34:53","doi":"10.21203/rs.3.rs-4314608/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"87cd681b-5a1d-4e7a-b065-909715585c5f","owner":[],"postedDate":"May 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-07-23T00:33:26+00:00","versionOfRecord":{"articleIdentity":"rs-4314608","link":"https://doi.org/10.1007/s00299-024-03285-w","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2024-07-22 00:33:26","publishedOnDateReadable":"July 22nd, 2024"},"versionCreatedAt":"2024-05-02 10:34:53","video":"","vorDoi":"10.1007/s00299-024-03285-w","vorDoiUrl":"https://doi.org/10.1007/s00299-024-03285-w","workflowStages":[]},"version":"v1","identity":"rs-4314608","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4314608","identity":"rs-4314608","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}