Currently, agricultural systems worldwide produce greenhouse gases including CO2, N2O, and CH4, 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 CO2, 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 H2O2-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.