Green leaf volatile sensory calcium transduction in Arabidopsis

Aratani, Yuri; Uemura, Takuya; Hagihara, Takuma; Matsui, Kenji; Toyota, Masatsugu

doi:10.1038/s41467-023-41589-9

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Published: 17 October 2023

Green leaf volatile sensory calcium transduction in Arabidopsis

Nature Communications volume 14, Article number: 6236 (2023) Cite this article

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Abstract

Plants perceive volatile organic compounds (VOCs) released by mechanically- or herbivore-damaged neighboring plants and induce various defense responses. Such interplant communication protects plants from environmental threats. However, the spatiotemporal dynamics of VOC sensory transduction in plants remain largely unknown. Using a wide-field real-time imaging method, we visualize an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in Arabidopsis leaves following exposure to VOCs emitted by injured plants. We identify two green leaf volatiles (GLVs), (Z)-3-hexenal (Z-3-HAL) and (E)-2-hexenal (E-2-HAL), which increase [Ca²⁺]_cyt in Arabidopsis. These volatiles trigger the expression of biotic and abiotic stress-responsive genes in a Ca²⁺-dependent manner. Tissue-specific high-resolution Ca²⁺ imaging and stomatal mutant analysis reveal that [Ca²⁺]_cyt increases instantly in guard cells and subsequently in mesophyll cells upon Z-3-HAL exposure. These results suggest that GLVs in the atmosphere are rapidly taken up by the inner tissues via stomata, leading to [Ca²⁺]_cyt increases and subsequent defense responses in Arabidopsis leaves.

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Introduction

Plants emit an array of volatile organic compounds (VOCs), including green leaf volatiles (GLVs), terpenoids, and amino acid derivatives, in response to wounding and herbivore attack^1,2. These VOCs exert multiple protective effects, such as directly repelling herbivores and attracting natural enemies of the hervibores^3,4,5. Neighboring intact plants perceive these VOCs as danger cues to trigger defense responses⁶ or prime themselves to respond to upcoming stresses in a timely manner⁷. Therefore, these VOCs serve as interplant airborne signals. Such interplant interaction mediated by volatile cues is known as plant–plant communication or plant eavesdropping¹. VOC perception in plants was first reported in the early 1980s. Two individual groups demonstrated that Sitka willow (Salix sitchensis) and poplar (Populus x euramericana) trees exhibited increased antiherbivore properties when grown near damaged plants^8,9. Such VOC-mediated interplant signaling has been reported in more than 30 plant species, including lima bean⁶, tobacco¹⁰, tomato¹¹, sagebrush¹², and Arabidopsis¹³.

GLVs, comprising six-carbon (C6) compounds, including alcohols, aldehydes, and esters, are among the most rapidly and abundantly produced VOCs in the plant kingdom, being generated within seconds after wounding¹⁴. Upon mechanical damage, GLV biosynthesis is immediately initiated by a lipoxygenase (LOX)-mediated dioxygenase reaction on fatty acids to yield fatty acid/lipid hydroperoxides. These derivatives are cleaved to form (Z)-3-hexenal (Z-3-HAL), a C6 volatile aldehyde, by hydroperoxide lyase (HPL), a key enzyme for GLV formation¹⁴. Arabidopsis accession Col-0 exhibits low GLV production because of a 10-bp deletion in HPL¹⁵. Z-3-HAL can be isomerized to (E)-2-hexenal (E-2-HAL) by an isomerase^16,17. Alternatively, Z-3-HAL is quickly reduced to the C6 alcohol (Z)-3-hexenol (Z-3-HOL), which can be further converted to (Z)-3-hexenyl acetate (Z-3-HAC) by an acetyl transferase. GLVs elicit a wide range of defense signals in plants. GLV exposure in plants induces the accumulation of the stress-related phytohormone jasmonic acid (JA)⁷ and expression of JA-dependent defense genes¹⁸ or plants prime themselves for rapid response to subsequent herbivory damage¹⁹. GLVs also trigger the induction of abiotic stress-related defense genes, including heat- and oxidative stress-responsive genes²⁰. Indeed, treating Arabidopsis with GLVs induces enhanced heat stress tolerance²¹.

Plant VOC-dependent defense responses are initiated following VOC entry into inner tissues²². Exogenous VOC uptake by plants is facilitated during the light period, in which stomata are opened and CO₂ exchange is enhanced²³. Furthermore, changes in the uptake rate of aldehydes and ketones can be coupled with those of stomatal conductance²⁴, suggesting that stomata play a critical role in VOC perception. In tobacco, TOPLESS-like protein, a transcriptional corepressor for JA signaling, was revealed to interact with the sesquiterpene β-caryophyllene²⁵. Upon binding to β-caryophyllene, the TOPLESS-like protein-mediated transcriptional suppression of defense-related genes is inhibited, thereby accelerating defense gene expression²⁵. However, the molecular mechanisms underlying the perception of VOCs, especially GLVs, are poorly understood.

Cytosolic Ca²⁺ plays crucial roles in a wide array of plant stress responses^26,27. Downstream signaling activation systems via cytosolic calcium ion concentration ([Ca²⁺]_cyt) increases have been extensively studied, including the identification of critical components linking stress perception to the formation of Ca²⁺ signals, as well as the mechanism of Ca²⁺ signal implementation²⁸. Using an electrophysiological technique and a Ca²⁺-sensitive fluorescent dye, membrane potential depolarization and [Ca²⁺]_cyt increases after exposure to several GLVs were observed in tomato leaves²⁹. In addition, [Ca²⁺]_cyt increases were detected in detached Arabidopsis leaves upon VOC exposure using a Ca²⁺-sensitive luminescent protein³⁰. These findings suggest that Ca²⁺ signaling is involved in an early process leading to downstream defense responses. However, little is known about the spatiotemporal dynamics of GLV sensory transduction because of technical limitations in real-time monitoring of GLV-induced Ca²⁺ signals in intact living plants.

In this study, we observed that a plant-wide Ca²⁺ signature rapidly occurs in response to exposure to VOCs released by damaged plants using transgenic Arabidopsis expressing a green fluorescent protein-based Ca²⁺ biosensor and a wide-field real-time fluorescence microscope. We found that Z-3-HAL and E-2-HAL elicited rapid increases in [Ca²⁺]_cyt in Arabidopsis leaves, in which defense responses were activated. Tissue-specific high-resolution Ca²⁺ imaging and stomatal mutant analysis also clarified the spatiotemporal dynamics of VOC sensing and signal transduction networks in plants.

Results

VOCs released from plants trigger [Ca²⁺]_cyt increases in Arabidopsis

Using transgenic Arabidopsis expressing the Ca²⁺ biosensor GCaMP3³¹, we observed [Ca²⁺]_cyt changes in intact plants following exposure to VOCs emitted by damaged plants in real time. We used Arabidopsis accession No-0 and tomato as emitter plants because they emit various VOCs, including GLVs, in response to wounding^29,32,33. First, we monitored [Ca²⁺]_cyt increases in Arabidopsis (receiver) following exposure to VOCs emitted from Arabidopsis plants (source of VOCs) fed on by the common cutworm (Spodoptera litura; Fig. 1a, b and Supplementary Movie 1). This Ca²⁺ signal was rapidly transmitted to all parts of the plant within 20 min (Fig. 1b, c). Similar results were obtained when receiver Arabidopsis was exposed to VOCs released by tomato leaves (source of VOCs) consumed by S. litura (Fig. 1b, c and Supplementary Movie 2). Further, upon placing either homogenized Arabidopsis or tomato leaves (Source of VOCs) near receiver Arabidopsis (Fig. 2a), [Ca²⁺]_cyt increases were observed in several leaves (Fig. 2b, Supplementary Movies 3 and 4). In a time-course analysis of [Ca²⁺]_cyt changes in leaf 1 (L1), we found that VOCs emitted by homogenized tomato leaves caused larger signal changes than those emitted by Arabidopsis (Fig. 2c, d). These results suggest that VOCs emitted by damaged Arabidopsis and tomato plants caused [Ca²⁺]_cyt changes in neighboring intact Arabidopsis plants.

**Fig. 1: Exposure to VOCs emitted by leaves consumed by the herbivore *S. litura* induces [Ca²⁺]_cyt increases in receiver *Arabidopsis* leaves.**

**Fig. 2: Exposure to VOCs emitted by homogenized leaves induces [Ca²⁺]_cyt increases in receiver *Arabidopsis* leaves.**

Z-3-HAL and E-2-HAL induce [Ca²⁺]_cyt increases in Arabidopsis

To identify the VOCs responsible for triggering [Ca²⁺]_cyt changes in Arabidopsis, we examined five GLVs [Z-3-HAL, E-2-HAL, n-hexanal (n-HAL), Z-3-HOL, and Z-3-HAC], three terpenes (α-pinene, β-pinene, and β-caryophyllene), and methyl jasmonate (MeJA) because they have been reported to induce defense responses in receiver plants^2,11,29. We measured time-course changes in [Ca²⁺]_cyt in three regions, i.e., “tip,” “base,” and the midpoint between them (“mid”), in L1 of Arabidopsis after each VOC solution was placed in close proximity (Fig. 3a). Of these, Z-3-HAL immediately increased [Ca²⁺]_cyt at the tip within 30 s (Fig. 3b, c and Supplementary Movie 5). Subsequently, the signal was propagated to the mid and base regions within 1 and 2 min, respectively, and it persisted for over 15 min. Although exposing Arabidopsis to E-2-HAL also caused a [Ca²⁺]_cyt increase, the increase was weaker and slower than that induced by Z-3-HAL (Fig. 3b–d and Supplementary Movie 6). By measuring the velocities of Z-3-HAL- and E-2-HAL-induced Ca²⁺ transmission, we revealed that the signal transmission induced by Z-3-HAL (0.24–0.30 mm/s; N = 10) was faster than that induced by E-2-HAL (0.01–0.02 mm/s; N = 10; Fig. 3e). In contrast, no [Ca²⁺]_cyt increases were observed in leaves exposed to other VOCs (Fig. 3f). These results indicate that Z-3-HAL and E-2-HAL are key triggers of rapid [Ca²⁺]_cyt increases in Arabidopsis. Based on these observations, we focused on Z-3-HAL and E-2-HAL in subsequent analyses.

**Fig. 3: Z-3-HAL and E-2-HAL trigger [Ca²⁺]_cyt increases in *Arabidopsis*.**

To confirm that GLVs induce [Ca²⁺]_cyt increases upon exposure to VOCs emitted by homogenized leaves (Fig. 2), we monitored [Ca²⁺]_cyt changes in leaves following exposure to VOCs emitted by homogenized Arabidopsis that harbors inactive HPL and emits little GLVs³⁴. VOCs emitted by homogenized Arabidopsis carrying Col-0-derived HPL (hpl1 mutant) did not induce Ca²⁺ signals in receiver leaves (Supplementary Fig. 1). Using gas chromatography-mass spectrometry (GC-MS) and MonoTrap RGPS TD, which is a high-quality adsorbent, we further analyzed the VOC components rapidly produced by homogenized tomato and Arabidopsis leaves. Tomato leaves emitted significantly higher amounts of Z-3-HAL and E-2-HAL than wild-type (WT) Arabidopsis leaves upon homogenization (Supplementary Fig. 2). Moreover, the levels of these compounds emitted by homogenized hpl1 leaves were remarkably low (<25% of that emitted by WT leaves; Supplementary Fig. 2), consistent with the results of Ca²⁺ signature levels. Altogether, these results suggest that interplant airborne signaling that induces [Ca²⁺]_cyt changes is dependent on HPL-mediated GLV formation in Arabidopsis.

Z-3-HAL and E-2-HAL elicit electrical signals and defense gene expression

Because changes in the membrane potential upon GLV exposure were observed in tomato leaves²⁹, we simultaneously recorded the changes in [Ca²⁺]_cyt and the leaf surface potential upon C6 aldehyde exposure in Arabidopsis. Z-3-HAL and E-2-HAL exposure in Arabidopsis leaves resulted in rapid changes in the leaf surface potential, which is spatiotemporally coupled with changes in [Ca²⁺]_cyt (Fig. 4a–d). Interestingly, detailed analysis of the timing of the initial detectable signal changes revealed that the surface potential change significantly preceded the onset of the Ca²⁺ signal in the plant GLV sensory transduction system (Fig. 4e).

**Fig. 4: Z-3-HAL and E-2-HAL trigger the leaf surface potential changes and defense-related gene expression in *Arabidopsis*.**

We next examined the accumulation of defense-related gene transcripts after Z-3-HAL and E-2-HAL exposure. Expression of the heat and oxidative stress response marker genes HSP90.1 and ZAT12 increased in leaves after 30 and 60 min of Z-3-HAL or E-2-HAL exposure (Fig. 4f). Similarly, JA-related genes, such as OPR3 and JAZ7, were upregulated by these C6 aldehydes (Fig. 4f). Furthermore, we used a pharmacological approach to assess the role of GLV-induced [Ca²⁺]_cyt increases in triggering transcript accumulation. Pretreating Arabidopsis seedlings with a Ca²⁺ channel blocker (LaCl₃) or calcium chelator (EGTA) prevented both [Ca²⁺]_cyt increases and marker gene expression (Supplementary Fig. 3). Furthermore, we conducted the washout assay to evaluate the reversibility of the Ca²⁺ signals in Arabidopsis, allowing the assessment of any potential negative effects of these pharmacological reagents on plant cell viability. After an additional incubation period of 24 h with a liquid medium lacking LaCl₃ and EGTA, we observed approximately 65–95% recovery of the Ca²⁺ signal at 500 s compared with that in Mock-pretreated Arabidopsis (Supplementary Fig. 3a, c). Therefore, the GLV response in plants pretreated with the reagents is reversible, pharmacologically suggesting that these chemicals can block Ca²⁺ signals without drastically affecting the plant cells themselves. Altogether, these results indicate that [Ca²⁺]_cyt increases are required for the induction of transcriptional changes related to defense responses in plants.

Z-3-HAL induces local Ca²⁺ signals in a concentration-dependent manner

To gain further insights into the physiological properties of C6 aldehydes as signaling molecules, we examined the concentration dependency of [Ca²⁺]_cyt increases induced by Z-3-HAL. Upon exposure to lower Z-3-HAL concentrations, concentration-dependent decreases of [Ca²⁺]_cyt responses were observed (Fig. 5a, b, and Supplementary Fig. 4). We quantified the amount of Z-3-HAL released from Z-3-HAL solution under this experimental condition. Z-3-HAL was adsorbed by adsorbents placed at a distance of 5 mm from the 0.03 M solution. Z-3-HAL adsorption reached saturation within 1 min (Supplementary Fig. 5), and 3.85 nmol Z-3-HAL was adsorbed over 30 s. Considering the volume of the adsorbents (58 μL), it can be estimated that the local concentration of Z-3-HAL at a distance of 5 mm from the 0.03 M solution was 0.07 mM. Based on this finding, L1 in receiver Arabidopsis was exposed to approximately 6.42–385 nmol (0.1–6.7 mM) Z-3-HAL over 30 s following exposure to 0.05–3.0 M Z-3-HAL solutions (Fig. 5a).

**Fig. 5: Z-3-HAL-induced Ca²⁺ signature occurs locally and in a concentration-dependent manner.**

As Ca²⁺ has been proposed to act as a long-distance signal traveling to systemic undamaged parts to activate defense responses at a whole-plant level in other stress responses^31,35, we investigated whether Z-3-HAL triggers long-distance intracellular Ca²⁺ signal propagation. L1 was spatially isolated from other parts of the plant (Fig. 5c), ensuring that only L1 was exposed to Z-3-HAL. Exposure of L1 to Z-3-HAL caused a local [Ca²⁺]_cyt increase; however, Ca²⁺ signal propagation to distal unstimulated parts (L3) was not observed (Fig. 5d–f and Supplementary Movie 7). These results indicate that Z-3-HAL elicits [Ca²⁺]_cyt increases locally following direct exposure but not long-distance Ca²⁺ signals traveling toward systemic leaves.

GLVs are rapidly perceived by guard and mesophyll cells

We generated Arabidopsis expressing GCaMP3 driven by tissue-specific promoters, such as GC1, RBCS1A, SULTR2;2, and ATML1, to selectively express GCaMP3 in guard³⁶, mesophyll³⁷, vasculature³⁸, and epidermal cells³⁹, respectively. These transgenic lines enable the visualization of [Ca²⁺]_cyt changes specifically occurring in each tissue, thereby aiding in the understanding of the spatiotemporal patterns of GLV-induced responses. Upon Z-3-HAL exposure, [Ca²⁺]_cyt increased rapidly in the tip region of pGC1::GCaMP3 and pRBCS1A::GCaMP3 leaves within 40 s, whereas it increased gradually in pSULTR2;2::GCaMP3 and pATML1::GCaMP3 leaves (Fig. 6 and Supplementary Movies 8–11). Quantitative analysis of the timing of signal increases revealed that [Ca²⁺]_cyt first increased in pGC1::GCaMP3 and pRBCS1A::GCaMP3 within 1 min (Fig. 6f, g). Interestingly, pSULTR2;2::GCaMP3 and pATML1::GCaMP3 responded slowly to Z-3-HAL (Fig. 6f, g).

**Fig. 6: [Ca²⁺]_cyt increases in guard, mesophyll, and vasculature cells, and subsequently in epidermal cells upon Z-3-HAL exposure.**

To better understand GLV propagation pathways, we observed [Ca²⁺]_cyt changes at the cellular level using an upright confocal laser scanning microscope (Fig. 7a–c). A rapid [Ca²⁺]_cyt increase was first observed in guard cells (pGC1::GCaMP3) within 1 min of Z-3-HAL exposure (Fig. 7d, g, h, and Supplementary Movie 12). An increase in [Ca²⁺]_cyt was detected earlier in mesophyll cells (pRBCS1A::GCaMP3) than in epidermal cells (pATML1::GCaMP3) (Fig. 7e–h and Supplementary Movies 13–14). These observations were consistent with the results obtained using Arabidopsis expressing GCaMP3 driven by the 35S promoter (Supplementary Fig. 6 and Movie 15). Based on these findings, we hypothesized that plant GLV sensory transduction could be initiated by GLV flux into inner tissues via stomata, resulting in subsequent defense signaling activation.

**Fig. 7: Upright confocal laser scanning microscopy reveals instant Z-3-HAL-induced [Ca²⁺]_cyt increases in guard cells, followed by mesophyll cells.**

Stomata play a critical role in rapid Z-3-HAL perception

To examine the function of stomata in rapid GLV sensory transduction, we used the phytohormone abscisic acid (ABA) to induce stomatal closure⁴⁰, as well as stomatal mutants exhibiting abnormal stomatal movement phenotypes^41,42,43. Pretreatment of Arabidopsis WT leaves with ABA resulted in stomatal closure (Supplementary Fig. 7), and the Z-3-HAL-induced increase in [Ca²⁺]_cyt was delayed compared with that in leaves pretreated with a stomatal opening buffer solution (Mock, Fig. 8). Loss-of-function mutants of SLOW ANION CHANNEL-ASSOCIATED 1 (slac1) and OPEN STOMATA 1 (ost1) exhibit impaired stomatal closure in the presence of ABA^41,42 (Supplementary Fig. 7). Z-3-HAL-induced [Ca²⁺]_cyt signatures in ABA-treated slac1-2 and ost1-3 mutants expressing GCaMP3 driven by the 35S promoter were similar to those in Mock-treated leaves (Fig. 8). Moreover, a critical role of stomata in rapid Z-3-HAL-induced electrical signaling was confirmed via the time-course analysis of leaf surface potential changes using ABA-pretreated leaves (Supplementary Fig. 8). These results support our hypothesis that GLV uptake from the atmosphere into tissues via stomata is the main pathway of rapid GLV sensory transduction in plants, which in turn activates plant defense responses.

**Fig. 8: Stomatal opening plays a crucial role in [Ca²⁺]_cyt increases upon Z-3-HAL exposure.**

Discussion

We visualized Ca²⁺ signal transduction in intact Arabidopsis plants exposed to VOCs emitted by mechanically- and herbivore-damaged plants (Figs. 1 and 2) and found that Z-3-HAL and E-2-HAL function as airborne signaling molecules triggering [Ca²⁺]_cyt increases, electrical signals, and transcriptional changes in Arabidopsis leaves (Figs. 3, 4). Since VOC components other than these two C6 aldehydes were unable to elicit [Ca²⁺]_cyt changes in Arabidopsis (Fig. 3f), the aldehyde moiety in their structures appeared necessary for activating [Ca²⁺]_cyt-based defense signaling. E-2-HAL containing an α,β-unsaturated carbonyl moiety is a reactive electrophile species that causes cell damage because of its ability to form adducts with nucleophiles⁴⁴. Z-3-HAL, which has a β,γ-unsaturated carbonyl group, exerts physiological effects on various organisms^14,20. Surprisingly, n-HAL and Z-3-HAC failed to trigger [Ca²⁺]_cyt increases (Fig. 3f), whereas Ca²⁺ response to Z-3-HAC exposure was previously reported in tomatoes²⁹. These findings suggest that plants possess species-specific sophisticated VOC recognition systems in addition to a mechanism capable of recognizing structural differences among GLV components (such as specialized receptor proteins)^12,45. Indeed, some volatile-specific receptors in plants have been isolated, including those for ethylene⁴⁶ and β-caryophyllene²⁵.

In addition to the volatile-specific perception system, VOC uptake pathways involving metabolic processes have been proposed to act as other primary pathways for downstream VOC signaling activation²². Z-3-HOL accumulated in tomato cells is glycosylated, leading to enhanced resistance against S. litura via the insecticidal effects⁴⁷. However, considering that neither Z-3-HOL nor β-caryophyllene triggered [Ca²⁺]_cyt changes (Fig. 3f), plants might be equipped with specialized VOC perception systems that activate downstream signaling independently at different levels (for example, Ca²⁺ signals, transcriptional changes, and metabolism).

The C6 aldehyde group can prime/induce plant defense responses to abiotic stresses²⁰. For example, heat and photooxidative stresses elicit enhanced E-2-HAL production in Arabidopsis, tomatoes^21,48, and tobacco⁴⁹. GLV-exposed Arabidopsis seedlings exhibit enhanced heat tolerance²¹. In this study, oxidative stress- and heat-responsive genes were upregulated upon GLV exposure in a Ca²⁺-dependent manner (Fig. 4f and Supplementary Fig. 3b). Although the detailed molecular mechanism underlying stress-related signaling activation upon GLV exposure remains unclear, [Ca²⁺]_cyt increases could mediate both stress-responsive gene expression^31,50 and immediate GLV formation via Ca²⁺ binding to the PLAT domain in LOX, a key enzyme for GLV biosynthesis⁵¹. Different experimental approaches, such as transcriptome analysis, could provide new insights into the mechanism underlying enhanced stress tolerance through GLV-induced Ca²⁺ signals.

The local concentration of Z-3-HAL at a distance of 5 mm from the 3.0 M solution, which consistently induced stable [Ca²⁺]_cyt increases, was estimated at 6.7 mM (Supplementary Fig. 5). Similarly, 381 nmol Z-3-HAL was detected within 10 min of exposure to 0.5 g homogenized Arabidopsis leaf tissues (Supplementary Fig. 2), indicating that receiver Arabidopsis is exposed to approximately 6.6 mM Z-3-HAL in 30 s. Although the homogenization of Arabidopsis leaf tissues might be unrealistic in nature, these estimations indicate that the concentration of Z-3-HAL solution corresponds to that emitted by plants and that the experimental conditions used in this study are relevant to the potential exposure or emission of Z-3-HAL in a natural context.

Plants possess the ability to efficiently absorb a wide range of surrounding atmospheric VOCs and accumulate them into their tissues. For example, tomato can absorb a significant amount of atmospheric methacrolein, with estimates ranging from 33% to 41% of the total methacrolein content present in the air⁵². The minimum amount of Z-3-HAL required to induce detectable Ca²⁺ signals was 0.1 M (Fig. 5a). Considering that approximately 12.8 nmol Z-3-HAL was adsorbed by the adsorbents within 30 s of exposure to 0.1 M Z-3-HAL, Arabidopsis accumulates approximately 4.2 nmol Z-3-HAL at 33% of the VOC-adsorbing capacity of the adsorbent. Based on the previous calculation³⁴, a single injury (7.5 μg, 0.05 mm²) produces 12.9 and 2.9 pmol Z-3-HAL in tomatoes and Arabidopsis, respectively (for detail, see Supplementary Fig. 2 and Methods). If these estimations are accurate, the Z-3-HAL emitted following a single injury is unlikely to induce detectable Ca²⁺ signals. To induce the release of 12.8 nmol Z-3-HAL (the accumulation of 4.2 nmol Z-3-HAL in Arabidopsis leaves) and Ca²⁺ signals under our experimental conditions, approximately 148.8 (992.3 mm²) and 662.1 (4409.0 mm²) mg of leaves need to be injured in tomatoes and Arabidopsis, respectively. This size of agricultural damage might be realistic in nature⁵³.

Although it is unlikely that plants are continuously exposed to high GLV concentrations (e.g., 3.0 M Z-3-HAL) under natural conditions, it should be noted that GLVs do not easily diffuse because of their high molecular weight, which may lead to high local GLV concentrations around damaged plants^54,55. Considering these findings, the possibility that plant cells and tissues are temporarily exposed to high GLV concentrations under specific circumstances (e.g., when receiver plants are in close proximity to disrupted plants capable of emitting numerous GLVs, including Vigna radiata and Momordica charantia⁵⁶, in response to herbivory in nature) cannot be dismissed. In fact, we detected Ca²⁺ signals in response to VOCs released from plants consumed by herbivores (Fig. 1). To further clarify this phenomenon, it would be beneficial to employ advanced technologies, such as the real-time detection of atmospheric VOC concentrations, and precisely determine the VOC adsorption capacity and efficiency of plants⁵⁷.

Using real-time Ca²⁺ imaging combined with pharmacological and genetic approaches, we proposed a model for the spatiotemporal propagation pathways of VOCs in plants (Fig. 9) where stomata play a critical role in perceiving VOC cues. Some studies support our idea of the importance of stomata for VOC uptake, especially for the absorption of atmospheric gases such as CO₂, and air pollutants^23,58. For example, VOC uptake by plant tissues is efficiently facilitated when stomata are opened²³. Aldehyde compounds can also be absorbed into the leaf interior via stomata⁵⁸. Conversely, the delayed Ca²⁺ signals in epidermal cells could be explained by the presence of the cuticle, which functions as a permeability barrier. This idea is supported by a previous finding that O₃ deposition in cuticles was negligibly small⁵⁹. Taken together, it is possible that stomata serve as a plant gateway mediating rapid VOC entry into interspaces in tissues.

**Fig. 9: Volatile C6 aldehyde sensory transduction system in *Arabidopsis*.**

Two glutamate receptor-like genes (GLRs) that are localized in phloem and xylem contact cells in the vasculature are activated in response to wounds, resulting in the propagation of Ca²⁺ and electrical signals from the wound site to distant organs^31,60. Although Z-3-HAL-induced [Ca²⁺]_cyt increases were detected in the vasculature tissue (Fig. 6c, e), no Ca²⁺ signal propagation toward systemic leaves was observed (Fig. 5d). Given that the amplitude and propagation rate of the wound-triggered systemic signals depend on the type and intensity of the stimuli⁶¹, and the necessity of damaging the main vein of the leaf⁶², GLVs might not activate the key elements required for long-distance signal transmission, such as GLRs, or the local response might not reach the threshold required to allow the Ca²⁺/electrical signals to move out of the local leaf. Interestingly, changes in the leaf surface potential preceded changes in [Ca²⁺]_cyt upon GLV exposure (Fig. 4e), as systemic electrical signals preceded Ca²⁺ signals upon wounding^63,64. Membrane depolarization could be induced by the activation of ion channels, such as Cl⁻-permeable, ROS-sensitive, or ligand-gated channels, followed by [Ca²⁺]_cyt increases via Ca²⁺ influx and efflux through plasma- and endo-membranes, respectively^63,64.

The wide-field real-time imaging approach used in this study provided physiological insights with significantly higher resolution using intact Arabidopsis, revealing the details of Ca²⁺ signals in response to GLVs in a more comprehensive manner. This methodology allowed the assessment of spatial and temporal aspects of GLV perception pathways at the cellular level. Additionally, by integrating real-time imaging with other techniques, we achieved a deeper understanding of the comprehensive orchestration of GLV responses, including Ca²⁺ signals and other signaling mechanisms such as electrical signals. This approach can be further extended to investigate VOC signaling networks across plant taxa using mutants that are defective in the putative elements of VOC responses. Furthermore, this Ca²⁺ imaging method can serve as a robust tool for investigating the molecular basis of airborne plant signaling, both within (e.g., Arabidopsis to Arabidopsis) and between species (e.g., tomato to Arabidopsis) (Figs. 1 and 2).

Methods

Plant material and growth condition

The seeds of Arabidopsis thaliana (accessions Col-0, No-0 and hpl1 mutant in the Ler-0 background³⁴) were surface-sterilized and sown on sterile Murashige and Skoog (MS) agar medium [1× MS salts, 1% (w/v) sucrose, 0.01% (w/v) myoinositol, 0.05% (w/v) MES, and 0.5% (w/v) gellan gum; pH 5.7 adjusted with 1 N KOH]. After incubation in the dark at 4 °C for 2 days, the plates were placed horizontally at 22 °C in a growth chamber under continuous light (90–100 μmol/m²/s) for approximately 2 weeks before use. After 2 weeks, Arabidopsis leaves were numbered from oldest to youngest⁶². Two-week-old Arabidopsis (accession No-0 and Ler-0) plants were transferred to soil and grown at 22 °C in a growth chamber under short-day conditions (8-h light/16-h dark photoperiod, 90–100 μmol/m²/s) for approximately 2 months before use. Two-week-old (accession Col-0) plants were used for Ca²⁺ imaging. Two-month-old plants (accession No-0 and Ler-0) were used for preparation of homogenized leaf tissues and leaves that were subjected to feeding by S. litura. Surface-sterilized tomato (Solanum lycopersicum cv. Micro-Tom) seeds were incubated overnight at 4 °C and sown on MS agar medium. Two-week-old plants were transferred to soil and grown at 25 °C in a growth chamber under long-day conditions (14-h light/10-h dark photoperiod, 90–100 μmol/m²/s) for approximately 2 months before use. Tomato (Solanum lycopersicum cv. Minicarol) seedlings potted in soil were purchased from a home improvement store located in Saitama, Japan. These seedlings were cultivated in a temperature-controlled room under long-day conditions (14-h light/10-h dark photoperiod, 90–180 μmol/m²/s) at 25–29 °C for more than 12 days and then used to prepare leaves that were consumed by S. litura.

Insects

Eggs of S. litura (Fabricius) (Lepidoptera: Noctuidae) were purchased from Sumika Technoservice Co. Ltd. (https://www.chemtex.co.jp). The insects were reared on a homemade artificial diet in the laboratory at 25 °C. Fifth-instar S. litura larvae were used for Ca²⁺ imaging.

DNA cloning and transformation

For the GCaMP3 constructs used for tissue-specific expression, genomic sequences of the 5′ end of the open-reading frames of GC1 (1716 bp, At1g22690), RBCS1A (1976 bp, At1g67090), SULTR2;2 (2001 bp, At1g77990), and ATML1 (3378 bp, At4g21750) were amplified via polymerase chain reaction (PCR) targeting restriction enzyme sites from Arabidopsis genomic DNA and used as the promoter sequences. These fragments were digested and inserted into the corresponding sites of the pAN19 vector containing the GCaMP3 and nopaline synthase terminator (NOSt) sequence, resulting in construction of the vectors pGC1::GCaMP3 NOSt, pRBCS1A::GCaMP3 NOSt, pSULTR2;2::GCaMP3 NOSt, and pATML1::GCaMP3 NOSt. The entire cassettes of GCaMP3 sequences driven by tissue-specific promoters were isolated via NotI digestion and cloned into the NotI site of the plant binary vector pBIN42. All binary vectors were transformed into the Agrobacterium tumefaciens strain GV3101 via electroporation. Arabidopsis plants were transformed using the floral dip method⁶⁵. To establish Arabidopsis mutants expressing GCaMP3, the entire cassette of p35s::GCaMP3 NOSt³¹ was transformed into the slac1-2 and ost1-3 mutants. The primers used for cloning are detailed in Supplementary Table 1.

Real-time [Ca²⁺]_cyt imaging

Real-time [Ca²⁺]_cyt imaging of the entire plants was performed as described previously³¹. For Ca²⁺ imaging at the cellular level, GCaMP3 signals were acquired using an upright confocal laser scanning microscope (A1R, Nikon). GCaMP3 was excited using a 488-nm laser/488-nm dichroic mirror, and fluorescent signals were detected at 510–560 nm using the GaAsP detector of the microscope. NIS-Elements imaging software was used to analyze GCaMP3 signals over time at several regions of interest (ROI). To calculate fractional fluorescence changes (ΔF/F), the following equation was used: ΔF/F = (F−F₀) /F₀, where F₀ denotes the average baseline fluorescence determined by the average of F over the first 10 frames of the recording before treatment.

Volatile treatment

β-Pinene and n-HAL were purchased from Wako Pure Chemical Industries, Ltd. α-pinene and β-caryophyllene were obtained from Tokyo Chemical Industry Co., Ltd. MeJA, E-2-HAL, Z-3-HOL, and Z-3-HAC were purchased from Sigma-Aldrich Co. LLC. Z-3-HAL was obtained from Nihon Zeon Co., Ltd. Each chemical was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution of 3.0 M. To examine the concentration dependency of the responses, Z-3-HAL was dissolved in DMSO at concentrations of 0.05, 0.1, 0.25, 0.5, 0.75, and 1.0 M. A 0.2-mL plastic tube cut with a scissor at a height of 5 mm was placed 5 mm from the tip region of L1, and then 10 μl of each volatile solution was applied.

To selectively treat L1 with Z-3-HAL, a hole was created on the side of a square plastic dish with a cutter, and L1 was spatially segregated from other parts of the plant by inserting it into the hole, as described in Fig. 5c. Then, the hole was filled with 0.2% agarose gel, and Ca²⁺ imaging was performed.

For treatment with VOCs emitted by homogenized plants, approximately 10 and 5 g of the aboveground parts of 2-month-old Arabidopsis and tomato, respectively, were excised with a scissor and homogenized with a pestle in a mortar. Disrupted leaf tissues were immediately transferred to 1.5-mL plastic tubes and placed in close proximity to receiver Arabidopsis expressing GCaMP3. Subsequently, the MS agar plate was closed with a clear plastic cover to increase the volatile concentration, and then Ca²⁺ imaging was performed.

To facilitate exposure to VOCs emitted from leaves consumed by S. litura, we established an experimental setup consisting of two plastic bottles, flow meters (NFM-V-P-A-1, TEKHNE Corp.), and air pump (HD-603, FEDOUR), as shown in Fig. 1a. Briefly, approximately 7 g of the aboveground parts of 2-month-old Arabidopsis and tomato plants was excised with scissors. The severed leaves and 50–75 fifth instar S. litura larvae were placed inside a plastic bottle (approximately 215 cm³). The bottle was sealed with parafilm to prevent VOC leakage and incubated for 30 min. Prior to the experiment, air was pumped into an empty plastic bottle at a rate of approximately 450 mL/min and was directed toward receiver Arabidopsis for 10 min through a cotton-filled tip. This step was crucial for facilitating the adaptation of plants to the experimental conditions. Following adaptation, exposure to VOCs emitted by leaves consumed by S. litura as well as Ca²⁺ imaging was initiated by connecting the bottle and switching the valve. The air was pumped into the bottle at a rate of approximately 200 mL/min.

Recording of surface potential

Surface potential changes were measured as described previously with minor modifications⁶⁶. Ag/AgCl recording electrodes with a diameter of 0.2 mm were prepared via chloridation with hypochlorous acid and fixed to electrode holders. The recording electrodes were fixed to the tip region of L1 of Arabidopsis plants with the application of 5-μL droplets of 10 mM KCl. The handmade Ag/AgCl wire with a diameter of 0.5 mm was used as a reference electrode and inserted into the agar growth medium. For measuring surface potential changes, an operational amplifier (Axopatch 200A, Axon Instruments), headstage amplifier (CV-201A headstage, Axon Instruments), digitizer (Digidata 1322A, Axon Instruments), and electrophysiology data acquisition software (Clampex 9.2, Axon Instruments) were used. Simultaneous measurements were performed using the SMZ25 microscope in a Faraday cage. Surface potential changes were sampled at 5 kHz. To compare the timing of the initial signal change with Ca²⁺ increases, each data point was subsequently extracted at a reduced frequency of 0.5 Hz. For calculating the surface potential changes (ΔV), the following equation was used: ΔV = V−V₀, where V denotes the potential difference (PD) between the recording and the reference electrodes at a certain time and V₀ denotes the averaged baseline PD determined by the mean of V over the first 10 frames of the recording before treatment.

ABA treatment

L1 of Arabidopsis plants was harvested from 2-week-old plants, floated in stomatal opening buffer solution (5 mM MES and 50 mM KCl; pH 6.1 adjusted with 1 N KOH) abaxial side down, and incubated under light (90–100 μmol/m² /s) for 2 h to open the stomata. Subsequently, ABA (20 μM) (Sigma-Aldrich) was added to the solution to induce stomatal closure. After 2 h of treatment, leaves were used for the subsequent experiments. For Ca²⁺ imaging and surface potential recording, detached leaves were transferred to MS agar medium and treated with 3.0 M Z-3-HAL as previously described. For stomatal aperture measurement, epidermal strips were prepared by peeling away the epidermal cell layer using a clear Scotch tape as described previously⁶⁷. The adaxial leaf surface was fixed with cover glass, and stomata were observed under an upright confocal laser scanning microscope (A1R, Nikon). The stomatal aperture was measured using NIS-Elements imaging software (Nikon).

Pharmacological treatment

LaCl₃·7H₂O (Wako) and EGTA (Dojindo Laboratories) were dissolved in liquid MS medium at a final concentration of 50 mM. Eight-day-old Arabidopsis seedlings were transferred into a Petri dish filled with liquid MS medium containing either 50 mM LaCl₃ or EGTA and incubated for 16 h prior to experiments. For the washout assay, Arabidopsis seedlings that had been treated with 50 mM LaCl₃ or EGTA were uprooted from MS medium and washed 5 times with liquid MS medium devoid of these inhibitors to remove any residual inhibitors. Subsequently, the seedlings were transferred to an inhibitor-free MS agar plate and incubated for 24 h before conducting Ca²⁺ imaging.

Total RNA isolation, cDNA synthesis, and quantitative PCR

To extract total RNA, L1 and aboveground parts were harvested from 2-week-old Arabidopsis plants and 8-day-old Arabidopsis seedlings, respectively (Fig. 4f and Supplementary Fig. 3b). The harvested tissues were immediately frozen using liquid nitrogen. Total RNA was extracted from flash-frozen leaf tissue using the Plant Total RNA Mini Kit (FAVORGEN) following the manufacturer’s instructions. The samples were further treated with RNase-free DNase I to remove any residual genomic DNA using the RNase-Free DNase Set (QIAGEN) according to the manufacturer’s instructions. First-strand cDNA was then synthesized from the total RNA (500 ng) in a 10-μL reaction (50 ng of total RNA/μL) with PrimeScript™ RT Master Mix (Perfect Real-Time) for RT-PCR (Takara). In a 96-well optical PCR plate (ABgene), cDNA proportional to 10 ng of starting total RNA was combined with 100 nM of each primer (Supplementary Table 1) and 7.5 μL of 2× Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies) to a final volume of 15 μL. Using Arabidopsis ACT8 as an internal reference for standardization, qPCR was performed using the CFX96 Touch Deep Well Real-Time PCR System as well as CFX Maestro Software (Bio-Rad) with the following cycling parameters: 95 °C for 3 min; 40 cycles of 95 °C for 5 s and 60 °C for 10 s; and 1 cycle of dissociation from 65 °C to 95 °C with 0.5 °C increments. The expression of the marker genes was quantified using the quantification cycle [Cq].

Volatile analysis

VOCs emitted from homogenized leaves were identified and quantified as described previously with some modifications⁶⁸. Briefly, the aboveground parts of Arabidopsis (No-0 and Ler-0 accession) and tomato (Solanum lycopersicum cv. Micro-Tom) plants were excised with a razor blade and weighed. Five hundred milligrams of tissues were immediately placed into a mortar and homogenized using a pestle and liquid nitrogen. Then, these tissues were transferred to a grass vial (22 ml, Perkin Elmer). A MonoTrap cartridge (silica monolith matrix coated with octadecyl silyl group and activated carbon, RGPS TD, GL Sciences) was suspended in the headspace of the glass vial, allowing it to adsorb VOCs for 10 min. To quantify the volatilization of Z-3-HAL from DMSO solution, the MonoTrap cartridge was placed 5 mm from a 0.2-mL plastic tube containing 10 μL of 0.03 M Z-3-HAL solution in DMSO, and VOC adsorption was performed for 0.5, 1, 2 and 5 min (Supplementary Fig. 5). Volatiles collected by the cartridge were analyzed by a GC–MS system (GCMS QP2030, Shimadzu) equipped with a thermal desorption system (TQ8040-NX, Shimadzu). Volatiles were desorbed at 250 °C for 10 min with He gas flow (70 mL/min) and concentrated onto a trap set at −25 °C. Volatiles were desorbed again at 250 °C for 2 min and fractionated with a DB-WAX capillary column (30 m × 0.25 mm, 0.25 μm film thickness, Agilent). The GC oven program was maintained at an initial temperature of 40 °C (held for 5 min), followed by a ramp of 5.0 °C/min to a final temperature of 200 °C (held for 2 min). The electron ionization mode with an ionization voltage of 70 eV was used, and the m/z was recorded from 40 to 400. To identify each compound, we used the retention indices and MS profiles of the corresponding authentic specimens. To construct calibration curves for Z-3-HAL and E-2-HAL, a given amount of authentic compounds (generous gift from Zeon Co., and purchased from Fujifilm Wako Pure Chemicals, respectively) was directly injected onto the MonoTrap cartridge and analyzed as previously described.

To estimate Z-3-HAL production from a single injury, we followed a previously described method³⁴. This estimation was based on the detection of Z-3-HAL that elicits [Ca²⁺]_cyt increases in Arabidopsis following leaf homogenization as described in Supplementary Fig. 2. The total amounts of Z-3-HAL produced by homogenized Arabidopsis and tomato leaves were quantified as 380.9 and 1718.7 nmol/gFW, respectively. These values represent the maximum capacity of Z-3-HAL synthesis by the leaves. Based on previous research³⁴, the average weight of a leaf was defined as 150 µg/mm². Considering the estimated area of a single injury to be 0.05 mm² as previously described³⁴, we calculated that a single wound would result in the production of approximately 2.9 and 12.9 pmol of Z-3-HAL for Arabidopsis and tomatoes, respectively.

Statistical analysis

We performed Student’s t test for pairwise analysis and one-way analysis of variance followed by Bonferroni’s or Tukey’s post hoc tests using GraphPad Prism (GraphPad Software, Inc.) to compare multiple samples. Statistical significance was indicated by P < 0.05. All data are presented as the mean ± SE.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

The authors thank Juntaro Negi (Kyushu University) for providing slac1-2 and ost1-3 mutant seeds. This research was supported by grants from KAKENHI (18H05491) to M.T., (22KJ0451) to T.U., and by grants from A-STEP and Shiraishi Foundation of Science Development to M.T.

Author information

These authors contributed equally: Yuri Aratani, Takuya Uemura.

Authors and Affiliations

Department of Biochemistry and Molecular Biology, Saitama University, Saitama, 338-8570, Japan
Yuri Aratani, Takuya Uemura, Takuma Hagihara & Masatsugu Toyota
Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi, 753-8515, Japan
Kenji Matsui
Suntory Rising Stars Encouragement Program in Life Sciences (SunRiSE), Suntory Foundation for Life Sciences, Kyoto, 619-0284, Japan
Masatsugu Toyota
Department of Botany, University of Wisconsin, Madison, WI 53706, USA
Masatsugu Toyota

Authors

Yuri Aratani
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Takuya Uemura
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Takuma Hagihara
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Kenji Matsui
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Masatsugu Toyota
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Contributions

Y.A., T.U., T.H., and M.T. designed the study. Y.A., T.U., and T.H. performed Ca²⁺ imaging, electrophysiological measurements, qPCR analysis. K.M. performed volatile analysis. Y.A., T.U., T.H., and K.M. performed data analysis. Y.A., T.U., and M.T. wrote the manuscript. All authors discussed the results and contributed to the manuscript.

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Correspondence to Masatsugu Toyota.

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Aratani, Y., Uemura, T., Hagihara, T. et al. Green leaf volatile sensory calcium transduction in Arabidopsis. Nat Commun 14, 6236 (2023). https://doi.org/10.1038/s41467-023-41589-9

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Received: 16 January 2023
Accepted: 11 September 2023
Published: 17 October 2023
DOI: https://doi.org/10.1038/s41467-023-41589-9

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