Airborne signals from salt-stressed Arabidopsis plants trigger salinity tolerance in neighboring plants

Abstract

Plants have evolved sophisticated defense mechanisms to overcome their sessile nature. One remarkable strategy is the inter-plant communication mediated by volatile organic compounds (VOCs). Quantity and quality of plant VOCs are intricately regulated by biotic and abiotic stresses, and the alterations facilitate plant community to optimize their growth, development, and endogenous physiology to environmental fluctuations. Here, we report that Arabidopsis thaliana plants that experience high salinity emit VOCs and trigger induction of high salt resistance in neighboring plants. VOC emission of emitter plants is likely correlative to the plant damages to high salt, and VOC-fumigated receiver plants acquire high salt tolerance. The VOC-induced stress tolerance is independent of conventional abscisic acid (ABA) and salt stress signaling pathways. Together, this study demonstrates that salt-induced Arabidopsis VOCs are relevant in priming stress tolerance in neighboring plants. In addition, it also provides insight into how VOCs elicit stress responses in plant community.

Keywords: :

• Arabidopsis
• chemical ecology
• priming
• salt stress
• volatile organic compound

Plants are exposed to adverse abiotic and biotic stresses, such as low temperature, drought, high salinity, wounding, and pathogen attack. They have evolutionarily evolved a variety of defense mechanisms to cope with the environmental challenges. Conventional adaptive mechanisms include intra-plant communication that activates and primes plant tolerance to environmental stresses, establishing systemic acquired resistance (SAR).^1,2,3 Intriguingly, plants have also developed inter-plant communication through volatile signals to share awareness of the environmental changes.^4,5,6

More than 100,000 chemicals are synthesized in plants,⁷ and at least 1700 of which are released into the atmosphere in significant amounts.⁸ In particular, a significant step forward has been recently made in understanding ecological and physiological roles of volatile organic compounds (VOCs) that are defined as organic lipophilic compounds with high vapor pressure,⁹ including methanol, ethanol, formaldehyde, acetaldehyde, methane, ethylene, amino acid-derived products, green leaf volatiles (GLVs), and terpenes (isoprenoids).^10,11

The emission of VOCs is elaborately controlled by environmental factors and mediates a variety of interactions between plants and other organisms in ecosystems.^8,11 Primary function of VOCs is to facilitate plant community to defend against herbivores and microorganisms.^12,13 Indeed, the VOC exposure primes defense responses in neighboring plants, incurring fitness costs.^14,15 It also enables indirect defense by attracting predators or parasitoids that increase predation pressure on herbivores.¹⁶ In addition, particular emphasis has been placed on the role of VOCs in attracting beneficial insects or microbes, such as pollinators and symbiotic organisms,^17,18 demonstrating that plant VOCs are crucial for plant survival and reproduction.

Abiotic stresses, such as drought, high salinity, reduced nutrient availability, temperature extremes, and UV radiation, are the most limiting factors for plant growth, development, and productivity. Several lines of evidence have illustrated that some VOC signals are potentially engaged in eliciting priming plant responses to abiotic stresses in order to minimize their damages.^8,11 Nonetheless, the biological relevance and mechanism of inter-plant communication about the presence of upcoming environmental stress are still largely elusive.

We wanted to know whether abiotic stress induces emission of plant VOCs to alert neighboring plants and elicit their subsequent stress responses. To examine this, we applied salt stress, one of pervasive environmental stress, to emitter plants (emitters) and investigated the effect of airborne signals on stress adaptation of neighboring plants (receivers). We used two-compartment-dish plates for germinating Arabidopsis wild-type (Columbia-0) seeds on one compartment containing half-strength Murashige and Skoog (MS)-agar and the second compartment containing half-strength MS-agar with high salt (50, 100, or 150 mM NaCl) (Fig. 1A). The plates that allow to exchange VOCs between compartments were sealed and incubated at 23 °C for 2 wk under long day (16-light/8-dark) conditions (120 μmol m⁻² s⁻¹ white light).

Figure 1. Effects of VOCs emitted from Arabidopsis plants grown under high salinity on salt tolerance in neighboring plants. Arabidopsis wild-type seeds (Col-0) were germinated in two-compartment dish plates that have one compartment containing half-strength MS-agar and another compartment containing half-strength MS-agar with high salt (50, 100, or 150 mM NaCl) (A). Growth and development of two-week-old receiver plants grown together with emitter plants were compared (B). Two-week-old receiver plants grown with emitter plants exposed to different concentrations of NaCl were transferred to MS-agar supplemented with 150 mM NaCl (C). Biological triplicates were averaged. Different letters represent a significant difference at P < 0.05 (oneway ANOVA with Fisher’s post hoc test). Bars indicate standard error of the mean.

We first compared growth and development of receiver plants that were grown with emitter plants exposed to different concentration of high salt. While emitter plants were significantly influenced by high salt, growth patterns of receiver plants were not greatly altered (Fig. 1B). We then investigated whether VOCs released from emitter plants contribute to establishing induced resistance of receiver plants to high salinity. Two-week-old receiver plants were transferred to half-strength MS-agar plates supplemented with 150 mM NaCl. Receiver plants grown with emitter plants on 150 mM NaCl were more tolerant than those that have emitter plants on lower NaCl concentrations (Fig. 1C), indicating that salt-elicited VOCs are relevant in priming salt tolerance in neighboring plants and that emission of VOCs is probably correlative to high salt concentration.

To understand molecular mechanisms underlying the induced resistance of receiver plants to high salinity, we incubated 2-wk-old receiver plants in MS-liquid medium supplemented with 150 mM NaCl for up to 6 h and monitored expression of stress-responsive genes. ABA responses substantially overlap with responses to high salinity,¹⁹ but ABA signaling genes, such as ABA INSENSITIVE 1 (ABII) and RESPONSIVE TO DESICCATION 22 (RD22), were uninfluenced in receiver plants, which were exposed to a suite of salt-elicited VOCs, compared with wild-type plants (Fig. 2A). In addition, key signaling integrators for abiotic stress pathways, such as RD29B and KOLD-INDUCED 1 (KINI), were also unaffected (Fig. 2B). These results suggest that VOCs prime stress tolerance of receiver plants through as-yet-unidentified signaling pathways and mechanisms.

Figure 2. Effects of high salt-elicited plant VOCs on transcript accumulation of salt-responsive genes in neighboring plants. Two-week-old receiver plants grown with emitter plants exposed to different concentrations of high salt were transferred to MS-liquid medium supplemented with 150 mM NaCl and incubated for up to 6h. Transcript accumulation of ABA- (A) and salt-responsive (B) genes was analyzed by quantitative RT-PCR (qRT-PCR). Biological triplicates were averaged. Different letters represent a significant difference at P < 0.05 (oneway ANOVA with Fisher’s post hoc test). Bars indicate standard error of the mean.

It has been considered that Arabidopsis may not be a good species to study chemical exchanges between plants, because their VOCs are not abundant and colorful. Nonetheless, Arabidopsis is still beneficial considering the accessibilities of genomic information and diverse genetic resources. As an initial attempt, this study demonstrates that airborne signals emitted from Arabidopsis plants exposed to high salinity are biologically relevant in eliciting stress adaptive responses in neighboring plants yet experienced environmental stress. The VOCs seem to provoke priming responses, ensuring faster and stronger activation of physiological responses upon exposure to environmental challenges.^13,14

Environmental stresses increase abundance of VOC emission and alter quality of VOCs.⁸ Hence, one of the major challenges is to identify exchanged VOCs and elucidate roles of the individual compound. While high salinity-elicited chemicals have not been intensively identified, a few classes of VOCs are postulated to be involved in salt stress responses. A variety of terpene volatiles that are the major constituents of the plant volatilome, such as isoprenes/hemiterpenes (C₅), monoterpenes (C₁₀), and sesquiterpenes (C₁₅), play a particular role in plant protection to thermal and oxidative stresses in a range of plant species.⁹ Volatile terpene emission is stimulated by a wide range of environmental stresses.^8,11 Then they neutralize reactive oxygen species (ROS), possibly due to the presence of conjugated double bonds, and protect plants from negative effects caused by environmental stresses, indicating the antioxidant properties.^20,21 In agreement with this, inhibition of isoprene and monoterpene biosynthesis results in enhanced oxidative damages and reduced photosynthetic performance.²² By contrast, plants emitting abundant isoprene show enhanced tolerance to high light, heat stress, ozone, and other ROS.²³ Given that virtually all environmental stresses involve ROS accumulation, terpenes may also contribute to relieving damages to abiotic stresses, including high salt, in plant community.

In addition to their role as antioxidant agents, volatile terpenes also stabilize cellular membranes,²⁴ because of their lipophilic nature. They are efficiently incorporated into phospholipid bilayer of cellular membranes and ensure structural persistency of the membranes by enhancing hydrophobic interactions. Consistently, plants fumigated with isoprenes and monoterpenes are resistant to heat stress that disrupts cellular membranes.²⁵ In this regard, sustained volatile terpene synthesis under high salinity may also be relevant in priming salt tolerance in neighboring plants.²⁶

Oxylipins are another important chemical blend of VOCs. They are derived from polyunsaturated fatty acids through the action of lipoxygenase (LOX) enzymes.²⁷ The oxylipins are subsequently cleaved into GLVs (C6- or LOX-products) by 13-(S)-hydroperoxide lyase (HPL).⁸ GLVs include saturated or monounsaturated aldehydes, alcohols and esters, and their diverse configuration enable to have different sensory properties.⁸ Emission of GLVs is rapidly elicited upon mechanical damages and herbivory attack, which accompany membrane denaturations and/or damages.²⁸ Consistent with this, those are also released with significant level in response to drought and salt stresses that entails cellular damages,²⁹ supposing the role of GLVs in salt stress adaptation.

Transient induction of methanol and/or methane can be engaged in the plant-plant communication under high salinity. Both volatile compounds are derived from degradation of cell wall pectins. Methanol emission, which is catalyzed by pectin methylesterases (PMEs), is induced by cellular mechanical damages, such as herbivore feeding and wounding.³⁰ Methoxyl groups of pectin can be a source of aerobic methane. It has been shown that UV radiation induces plants to produce methane though ROS-mediated cell wall breakdown.³¹ Since environmental stresses also involve cellular damages as well as ROS accumulation, aerobic methanol and methane production can be speculated in salt-damaged plants.

In addition, the gaseous phytohormone ethylene can also be responsible for high salt adaptation. Ethylene is significantly induced by ABA, drought, and high salinity.³² The rate-limiting enzyme of ethylene synthesis, ACC synthase (ACS) that catalyzes production of ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), is also enzymatically activated in response to osmotic stress.³³ Transgenic plants overexpressing ethylene responsive transcriptional regulators exhibit increased salt tolerance,^34,35 supporting that ethylene signaling is intimately related to priming salt responses in neighboring plants.

While further investigations are required to elucidate volatile compounds responsible for the plant-plant communications, it is still obvious that VOCs emitted from plant exposed to environmental stresses are relevant in priming stress tolerance in neighboring plants. Metabolic analyses and genetic manipulation will provide comprehensive views of inter-plant communication and biological mechanisms underlying volatile signal perception and interpretation.

Abbreviations:
GLV	=	green leaf volatile
HPL	=	13-(S)-hydroperoxide lyase
LOX	=	lipoxygenase
PME	=	pectin methylesterase
ROS	=	reactive oxygen species
SAR	=	systemic acquired resistance
VOC	=	volatile organic compound

Disclosure of Potential Conflicts of Interest

No potential conflicts of interests were disclosed.

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A1004831). K.L. was supported by BK21 PLUS program in the Department of Bioactive Material Sciences.